Clinical Correlates of Vertebral Fracture in Children with Acute Lymphoblastic Leukemia.Kerry Siminoski*1, Celia Rodd2, Johannes Roth3, Andy Ni3, Nathalie Alos4, Stephanie Atkinson5, Robert Couch6, Elizabeth Cummings7, Francis H Glorieux8, Jacqueline Halton3, Brian Lentle9, Mary-Ann Matzinger3, Helen Nadel9, Nazih Shenouda3, Robert Stein10, David Stephure11, Shayne P Taback12, Frank Rauch8, Leanne M Ward3, and the Canadian STOPP Consortium13. 1University of Alberta, Edmonton, AB, Canada, 2McGill University, Montreal, QC, Canada, 3University of Ottawa, Ottawa, Ontario, Canada, 4Université de Montréal, Montréal, Quebec, Canada, 5McMaster University, Hamilton, Ontario, Canada, 6University of Alberta, Edmonton, Alberta, Canada, 7Dalhousie University, Halifax, Nova Scotia, Canada, 8McGill University, Montréal, Quebec, Canada, 9University of British Columbia, Vancouver, British Columbia, Canada, 10University of Western Ontario, London, Ontario, Canada, 11University of Calgary, Calgary, Alberta, Canada, 12University of Manitoba, Winnipeg, Manitoba, Canada, 13Canadian Pediatric Bone Health Working Group, Ottawa, Ontario, Canada
Vertebral fractures (VF) are present in some patients with childhood acute lymphoblastic leukemia (ALL), but spinal radiography is not routinely performed in these patients, so fractures go undetected. In this study we have explored the relationships of back pain and low BMD to VF in patients with newly diagnosed ALL. Subjects were 186 children (median age 5.3 years; 58% male) with ALL (90% precursor B cell, 10% T-cell), who were enrolled in a national bone health research program. Vertebral morphometry was performed from T4 to L4 using the Genant semi-quantitative method. Spine BMD was determined by DXA. One or more VF was present in 29 patients (16%). The average number of fractures among those with fractures was 2.6 with 71% in the thoracic spine and 29% lumbar. Fifty-seven percent of fractures were grade 1 (mild), 32% were grade 2 (moderate), and 11% were grade 3 (severe). Eighty-five percent were wedge fractures, 11% were crush, and 4% were endplate fractures. Subjects with VF had lumbar spine BMD Z-scores of −2.1 ± 1.5 (mean ± SD) compared to −1.1 ± 1.2 for those without (p<0.001).
Back pain was present in 25.3%. The presence of back pain had an odds ratio (OR) for VF of 4.7 (95% CI, 1.5–14.5, p<0.01) and gave a sensitivity of 55% (37–72%) and specificity of 80% (73–86%). In the entire population, the OR for fracture increased 1.8-fold (95% CI, 1.1–1.9) for every 1 SD reduction in BMD Z-score. The area under the receiver operating characteristics curve (ROC AUC) for detection of VF by BMD was 0.73 (0.66–0.78; p<0.001). Using Z-score < −2.0 as a detection threshold, sensitivity was 52% (34–69%) and specificity was 79% (72–85%). The OR rose faster among those with back pain as BMD decreased, illustrated in the figure (reference group is those with no back pain and BMD Z-score >0). Defining positive findings as either back pain or BMD Z-score < −2.0, the ROC AUC was 0.72 (0.54–0.85; p<0.01), sensitivity 69% (51–83%), specificity 64% (57–71%), positive predictive value 26% (17–38%), and negative predictive value 92% (85–96%).
Back pain and low BMD are clinical correlates of prevalent vertebral fracture in children with newly diagnosed ALL. Using a clinical approach of performing spine radiographs in ALL patients with either back pain or BMD Z-score <−2.0, radiographs would be performed in 41%, 69% of fractures would be detected, 26% of radiographs would reveal VF, and 92% of those not x-rayed would be fracture-free.
Disclosures: Kerry Siminoski, P&G, 8
25-Hydroxyvitamin D, Insulin-Like Growth Factor-I and Bone Mineral Accrual During Growth.Maria E. Breen*1, Emma Laing2, Daniel B. Hall1, Dorothy B. Hausman1, Ruth G. Taylor1, Carlos Isales3, Kehong Ding3, Norman Pollock4, Mark Hamrick4, Clifton Baile5, Richard Lewis2. 1The University of Georgia, Athens, USA, 2The University of Georgia, Athens, GA, USA, 3Medical College of Georgia, Augusta, GA, 4Medical College of Georgia, Augusta, GA, USA, 5University of Georgia, Athens, GA, USA
The extent to which 25-hydroxyvitamin D [25(OH)D] and insulin-like growth factor-I (IGF-I) influence bone mineral accrual from early to mid-puberty is unclear. This project sought to determine relationships among changes in plasma 25(OH)D, plasma IGF-I and bone mineral content (BMC) in prepubertal females (N = 76; aged 4 to 8 years at baseline), over a period of up to 7 years. BMC of the total body, total proximal femur, radius, and lumbar spine was measured using dual-energy X-ray absorptiometry (Hologic QDR-1000W). Plasma 25(OH)D and plasma IGF-I were assessed using quantikine enzyme-linked immunosorbent serologic assay (R&D Systems) and radioimmunoassay (DiaSorin, Inc.), respectively. Prior to the primary analyses, 25(OH)D and IGF-I were log-trans formed and the former variable was further adjusted for baseline dierences in season and race. Linear mixed modeling that included a random subject-specific intercept and a random subject-specific slope on age was employed to analyze the transformed 25(OH)D and IGF-I variables for the proportion of variance each explained on the four bone outcomes. IGF-I was more strongly associated with BMC accrual than 25(OH)D at the total body (R2 = 0.874 vs. 0.809), total proximal femur (R2 = 0.847 vs. 0.771), radius (R2 = 0.812 vs. 0.759), and lumbar spine (R2 = 0.759 vs. 0.698). At each skeletal site, the rate of BMC accrual was negatively associated with changes in 25(OH)D, but positively associated with changes in IGF-I. When IGF-I and 25(OH)D were included in the same regression equation, 25(OH)D did not have a significant predictive eect on BMC accrual at any site above and beyond that of IGF-I. These longitudinal data in early adolescent females indicate that both 25(OH)D and IGF-I have a significant impact on bone mineral accrual; however, the positive eect of IGF-I on BMC is greater than the negative influence from 25(OH)D.
Disclosures: Maria E. Breen, None.
A Cross-Over Study of BMD and Markers in Adolescent Users of Combined Oral Contraceptives with Different Estrogen Content.Jan Stepan*1, David Cibula2, Jana Skrenkova2, Martin Hill3. 1Institute of Rheumatology, Charles University, Prague, Czech Republic, 2Department of Obstetrics & Gynecology, Faculty of Medicine, Prague, Czech Republic, 3Institute of Endocrinology, Prague, Czech Republic
Background: Combined Oral Contraceptive (COC) use may adversely impact bone mineral density (BMD) in young women. Use of low-estrogen dose COC has recently been increasing, but conflicting data are available concerning an effect of COC on bone metabolism in adolescent age, which is characterized by fast increase of BMD. The aim of this study was to evaluate changes of BMD and biochemical markers of bone turnover in healthy adolescents and in users of COC with different estrogen content. Methods: Healthy adolescent girls (15–18 years) have been randomized to use two combined oral contraceptive pills with either 30 or 15 mcg of ethinyl-estradiol in combination with gestodene in cross-over design of 9 month intervention each in reverse order. BMD (DXA) measured at the hip, spine, distal forearm and whole body, and serum biochemical markers of bone turnover, intact N- terminal propeptide of type I procollagen (PINP), type 1 collagen cross-linked C-telopeptide (beta-CTX), and N-MID osteocalcin (OC) were assessed at the beginning and at the end of each intervention period. Healthy non-users of the same age were selected in random as controls. Results: In healthy COC non-users, BMD increase was observed at all measured locations during 18 months; it reached significance at the lumbar spine (2%) and distal radius (3%). In COC users, no significant BMD increase was observed, with the exception of lumbar spine BMD in the group using 30 mcg EE (p<0.05). In the cross-over design assessment, a significant difference between the low- and very low dose COC users was found in lumbar spine BMD changes (p<0.05), where BMD increase was prevented to the highest degree in the 15 mcg EE users. Serum concentrations of E2 were significantly lower in COC users in comparison with the controls (p<0.05). Serum markers decreased continuously (PINP by 40%). Increase of BMD at the lumbar spine, distal radius and total body was impaired namely in 15 mcg COC users. COC use was associated with a significant decrease in biochemical markers (PIPN by 40% during 9 months). Moreover, switching from 30 to 15 mcg after 9 months resulted in a significant increase in biochemical markers of bone turnover (PINP by 20%) and decrease in the lumbar spine BMD (by 1%). Conclusions: Physiological acquisition of lumbar spine BMD can be significantly hampered by the use of combined oral contraceptives, especially those with very low dose of ethinyl-estradiol (15 mcg per day).
Disclosures: Jan Stepan, None.
A Three Years School Curriculum Based Exercise Program During Early Adolescence Increase Bone Mineral Accrual and Bone Size But Do Not Reduce the Fracture Risk - Fracture Data in 2005 Children in the Prospective Controlled Paediatric Osteoporosis Prevention (POP) Study.Bjarne Lofgren*1, Susanna Stenevi-Lundgren2, Christian Linden3, Jan-Åke Nilsson4, Magnus Karlsson5. 1University Hospital Malmo Sweden, Lund. Sweden, 2Dep of Clin Science, Malmö University Hospital, Malmoe, Sweden, 3Malmo University Hospital, Malmo, Sweden, 4Dep of CLin Science, Malmö University Hospital, Malmö, Sweden, 5Clinical & Molecular Osteoporosis Research Unit, Malmö University Hospital, Malmo, Sweden
The longest reported prospective controlled exercise intervention study in children evaluating the effect on the skeleton span 20 months and no similar study have used fracture as endpoint. The purpose of this population based prospective controlled study was to follow the skeletal effect of an exercise intervention program within the school curriculum during three school years and evaluate if the program could reduce the incidence of fractures. The skeletal development was followed in 76 of the boys and 48 of the girls, at baseline aged 7–9 years, in the intervention school and in 55 of the age matched boys and 44 of the girls in three neighbour control schools. The physical educational classes were in the intervention group increased to 40 minutes/day whereas the controls continued with the Swedish mean of 60 minutes/week. Bone mineral density (BMD; g/cm2) was measured annually with dual X-ray absorbtiometry (DXA) at lumbar spine (LS) and femoral neck (FN). Fractures were registered for three years in 643 children in the intervention school (1611 person-years) and 1362 children in the control schools (3604 person-years). With 80% power and significance level of 0.05 the trial would find a risk reduction of 44%. Data is presented as mean ± SD. The annual gain (g/cm2) in LS BMD was greater in both boys and girls in the intervention group than in the controls (boys 0.028 ± 0.011 vs. 0.023 ± 0.009 and girls 0.038 ± 0.023 vs. 0.023 ± 0.012, both p<0.05 respectively). There occurred 30 fractures in the intervention group (18.6 events/1000 person-years) and 58 in the control group (16.1 events/1000 person-years), producing a rate ratio (RR) of 1.16 (95% CI 0.72, 1.81). There was slightly more fractures in boys than in girls, but the RR did not differ between the genders (data not shown).
A school based exercise intervention program during the three first school years increase the accrual of BMD in both genders but do not influence the fracture incidence. However, it must be emphasized that with our study design we could not capture a small fracture reduction. We could only conclude that there was no fracture reduction of more than 28%. Increased school physical education seems to have potential to increase peak bone mass. Future studies with a longer duration into the pubertal years must be conducted as to evaluate if an extended intervention program have fracture reductive effects.
Disclosures: Bjarne Lofgren, None.
Adiposity and Bone Structural Development in Young Adult Females: A 3-Year Follow-Up Study.Norman Pollock*1, Ashley J. Ferira2, Emma Laing3, Mark Hamrick1, Clifton Baile4, Richard Lewis3. 1Medical College of Georgia, Augusta, GA, USA, 2The University of Georgia, Athens, USA, 3The University of Georgia, Athens, GA, USA, 4University of Georgia, Athens, GA, USA
Cross-sectional data from our lab indicate that high levels of adiposity may be potentially harmful to the young adult skeleton. The purpose of this study was to compare 3-year changes in bone geometry, density and strength indices of the tibia and radius using peripheral quantitative computed tomography (pQCT) in adiposity groups that were originally defined as normal and high percentages of body fat. Seventy-one of the original 115 participants participated in this follow-up study [normal-fat (<32% body fat; n=59; aged 21.3 ± 0.4 years) and high-fat (>32% body fat; n=12; aged 21.3 ± 0.5 years)]. Fat-free soft tissue mass (FFST), fat mass (FM) and %fat were measured using dual-energy X-ray absorptiometry. Tibial and radial bone measurements were assessed by pQCT at the 4% and 20% sites from the distal metaphyses, which reflect trabecular bone and cortical bone, respectively. At the trabecular site of the tibia and radius, the following bone outcomes were assessed: total and trabecular volumetric BMD (vBMD), total bone cross-sectional area, bone strength index (BSI; total bone cross-sectional area x total vBMD2). At the radial and tibial cortical sites, cortical vBMD, cortical bone cross-sectional area, total bone cross-sectional area, cortical BMC, cortical thickness, periosteal circumference, endosteal circumference, and polar strength-strain index (SSI; integrated product of the section modulus and the density of cortical bone) were assessed. The Mann-Whitney U test was used to compare % changes in bone and body composition outcomes between adiposity groups. Over 3 years, no dierences were observed between groups for change in height, weight, FFST, FM, and %fat. At the trabecular site of the tibia and radius, no dierences in bone outcomes were observed over time. Cortical bone cross-sectional area and cortical thickness at the tibia and radius and cortical BMC at the tibia increased more in the high- vs. the normal-fat group, whereas there were no dierences for change in the other bone outcomes at the 20% site of the tibia and radius. Having high levels of body fat may be beneficial for cortical bone because of greater changes in cortical area and thickness, yet this did not reflect potentially stronger bone as evidenced by validated indices of bone strength. Future investigations are needed to determine why excess fat is associated with greater bone cortical size, but not increased bone strength.
Disclosures: Norman Pollock, None.
Body Composition and Calcium Retention in Adolescents.Kathleen Hill*1, Berdine Martin1, Linda McCabe2, George McCabe2, Connie Weaver1. 1Purdue University, West Lafayette, IN, USA, 2Purdue University, West Lafayette, USA
Adolescent overweight has almost tripled in the United States over the past 30 years. During the same time, incidence of distal forearm fractures in adolescents has increased by about 50 percent. Overweight in adolescents may exacerbate fracture risk due to higher force placed on bones during falls. Studies in both adolescents and adults have shown that body composition is associated with BMD where higher lean mass, but not fat mass, is positively associated with BMD. We have shown that the relationship between calcium intake and calcium retention in adolescents is dependent on body mass index (BMI)-for-age; overweight adolescents have a greater increase in calcium retention than normal weight adolescents as calcium intake increases. Here we report the additional contribution of body composition to the relationship between Ca intake, BMI, and Ca retention. Pooled data from a series of Ca metabolic balance studies in adolescents conducted between 1990 and 2007 were analyzed to determine the relationship between Ca intake, BMI, body composition (percent fat and percent lean mass), and Ca retention in adolescents. Total subjects included were 283 (206 female) adolescents age 10–16y. Races (African-American, White, and Asian-American) were analyzed both together and separately. BMI ranged from 11.6–42.7 kg/m2. During the studies, subjects were fed controlled diets with calcium intakes ranging from 663–2195 mg/d. Twenty-four hour fecal and urine samples were collected. Ca retention was determined by Ca intake minus fecal and urinary Ca losses. Post-menarcheal age (PMA) in girls and serum IGF-1 in boys, which we have previously shown are predictors of calcium retention in adolescents, were used as surrogate sexual maturation measures. Regression and model building techniques were used to determine the relationship between Ca intake, BMI-for-age, body composition, and Ca retention using Statistical Analysis Software (SAS Institute, Inc., Cary, NC). When Ca intake, sexual maturation, and BMI-for-age are included in the model for Ca retention in adolescents, body composition variables do not contribute to the variability in Ca retention. Our results indicate that adolescents with higher BMI-for-age, regardless of body composition, may be capabale of retaining more Ca if dietary Ca intake is increased, which may reduce the risk for fractures in overweight adolescents.
Disclosures: Kathleen Hill, Wyeth, 6; Pharamavite, 6
Lower bone mass and adiponectin in overweight adolescents with cardiometabolic risk factors.Norman Pollock*1, Yanbin Dong2, Bernard Gutin2, Mark Hamrick1. 1Medical College of Georgia, Augusta, GA, USA, 2Medical College of Georgia, Augusta, Georgia, USA
The effect of excess adiposity on bone development is not well understood, because studies to date have not taken into account the metabolic disturbances usually associated with obesity. Since cardiometabolic risk factors occur in ∼30% of overweight adolescents, it is vital to understand their impact on the relationship between fat and bone. The objectives of this study were: 1) to compare total bone mineral content (TBBMC) and bone area (TBBA) in overweight/obese adolescents (N=107; 16.1 ± 1.2 years of age, BMI-for-age percentile ≥85, 64% black, and 52% female) among 2 groups (either with or without cardiometabolic risk factors), and 2) to evaluate adipocyte-derived inflammatory factors (i.e., leptin, adiponectin and resistin) between these 2 groups. Cardiometabolic risk factors included central obesity (waist circumference ≥88 cm and ≥102 cm for males and females, respectively), prediabetes (fasting plasma glucose ≥100 mg/dL), poor lipid metabolism (triglycerides ≥ 150 mg/dL and HDL cholesterol <50 mg/dL and <40 mg/dL for females and males, respectively), and prehypertension [defined as an average BP ≥90th and <95th percentile of systolic (SBP) or diastolic (DBP) blood pressure according to age, sex, and height, or a SBP/DBP ≥120/80 mm/Hg]. Total body BMC and TBBA were measured by DXA (Hologic QDR-4500). Leptin was measured by ELISA from R&D Systems, and both adiponectin and resistin were assayed by ELISA from Linco Research. Independent samples t-tests and MANCOVA were employed to evaluate bone and adipocyte-derived inflammation outcomes between groups. Unadjusted bone outcomes were not different between groups; however, after differences in age, sex, race, maturation, height, and lean mass were controlled, overweight/obese adolescents with cardiometabolic risk factors had significantly lower TBBMC and TBBA compared to the group without risk factors (all P<0.05). While leptin and resistin were not different between groups, adiponectin was significantly lower in the overweight/obese group with cardiometabolic risk factors (P=0.04). In conclusion, our data indicate that bone mass and size may be negatively influenced by the metabolic disturbances applied to the skeleton of overweight adolescents, and adiponectin appears to play an important role in this process.
Disclosures: Norman Pollock, None.
Periosteal response to hypothalamic suppression during growth is maturity dependent.Vanessa Yingling*1, Elle Saine2. 1Temple University. Philadelphia, PA, USA, 2Temple University, Philadelphia, USA
Periosteal apposition is key to bone strength development during growth since the bending strength of bone is exponentially related to bone diameter. Estrogen levels act as a constraint to periosteal bone formation at puberty, therefore, lower estrogen levels may be beneficial to periosteal size. However, investigators have identified bone densities in young athletic women with decreased estrogen levels similar to those of 51-year-old women. The periosteal response to exercise is maturity dependent, thus the response to low estrogen levels may be as well. The purpose of this study was to determine the effect of hypothalamic suppression on the periosteal surface in post-pubescent female rats. At 23 days of age, female rats were assigned to a baseline group (BL65) (n=10) sacrificed on day 65, a control group (C) (n=15) sacrificed on day 90, and an experimental group (Ex) (n=9) sacrificed on day 90 that received daily injections of gonadotropin releasing hormone antagonist. Injections were given for a 25 day period from 65 to 90 days of age (2.5 mg/kg/dose). Differences in body weights, histomorphometry, trabecular u-CT, estradiol and IGF-1 serum levels were detected using a one-way ANOVA (p < 0.05). The study was approved by the IACUC. Body weights were similar on day 65 however on day 90, the Ex group was significantly heavier than C (17%). IGF-1 levels were 24 % higher than C and similar to BL65 levels. Estradiol levels were suppressed by 36% in Ex compared to C and 26% to BL65. Yet femoral cortical strength was similar between the Ex and C groups. We found a significant increase in endocortical area (13.7%) and trabecular bone volume was significantly decreased in the Ex group (51.5%) compared to C and 46.8% lower than the BL65. No change in cortical bone area but a significant increase in endocortical bone formation rate (49.4%) occurred. Similar mineral apposition rate between groups was found but the percent labeled surface increased 34.6% due to a shift to increased double labeled surface (57.1%) and lower single labeled surface (58.9%). These data suggest a typical response to suppressed estradiol was achieved in the endosteal envelope; increased resorption and formation. However, there was no change in periosteal bone formation rate with suppressed estradiol. The limited periosteal response during adolescence may rescue cortical bone strength in the short term but may conceal the detrimental effects of long term estradiol suppression.
Disclosures: Vanessa Yingling, None.
Early Skeletal Changes in Children with Newly Diagnosed Glucocorticoid-treated Nephrotic Syndrome: Results of a National Multicenter Prospective Study.Johannes Roth1, Celia Rodd*2, Andy Ni1, Nathalie Alos3, Stephanie Atkinson4, Robert Couch5, Elizabeth Curnmings6, Janusz Feber1, Francis H Glorieux7, Brian Lentle8, Mary-Ann Matzinger1, Helen Nadel8, Frank Rauch9, Nazih Shenouda1, Robert Stein10, David Stephure11, Shayne Taback12, Kerry Siminoski13, Leanne M. Ward1, and the Canadian STOPP Consortium14. 1University of Ottawa, Ottawa, Ontario, Canada, 2McGill University, Montreal, QC, Canada, 3Université de Montréal, Montréal, Quebec, Canada, 4McMaster University, Hamilton, Ontario, Canada, 5University of Alberta, Edmonton, Alberta, Canada, 6Dalhousie University, Halifax, Nova Scotia, Canada, 7McGill University, Montréal, Quebec, Canada, 8University of British Columbia, Vancouver, British Columbia, Canada, 9McGill University, Montréal, Quebec, Canada, 10University of Western Ontario, London, Ontario, Canada, 11University of Calgary, Calgary, Alberta, Canada, 12University of Manitoba, Winnipeg, Manitoba, Canada, 13University of Alberta, Edmonton, AB, Canada, 14Canadian Pediatric Bone Health Working Group, Ottawa, Ontario, Canada
Glucocorticoids (GC) are known for their adverse eect on bone health; however, little is known about their skeletal eects following short-term, systemic use in children. The purpose of this study was to evaluate the skeletal status in children with nephrotic syndrome (NS) within 30 days of GC initiation (time A) and 3 months later (time B). Sixty-three children (39 boys) with newly diagnosed idiopathic NS (median age 3.9 years) were studied. Lumbar spine areal bone mineral density (BMD), second metacarpal percent cortical area (CtA) on left hand radiograph and vertebral fracture status on lateral thoracolumbar spine were assessed at time A; spine BMD was repeated 3 months later at time B. GC exposure was calculated as cumulative dose up to and including time A (which corresponded with the time of the first spine BMD, taken within 30 days of GC initiation), and up to and including time B (the time of the second spine BMD, 3 months later). Despite normal height Z-scores, the mean spine BMD Z-score was reduced compared to the healthy average at time A (−0.55 ± 0.99, p<0.0001) and B (−0.52 ± 1.08, p=0.001). Cumulative GC exposure was 0.96 ± 0.6 g/m2 at time A, increasing to 3.8 ± 1.1 g/m2 at time B. Multivariate analysis showed that the cumulative GC dose was inversely associated with spine BMD Z-score at time A (−0.36, 95%, 95% CI −0.78 to 0.06; p=0.09). This relationship was persistent 3 months later: for every 1 g/m2 increase in cumulative GC dose over the first 3 months, spine BMD Z-score declined by 0.14 (95% CI −0.26 to −0.02; p=0.023). Mild vertebral fractures were evident in 3 children at time A, while mean CtA Z-score was above average (0.20 ± 0.8, p=0.05). Spine BMD is already reduced within 30 days of GC initiation, remains low during the first 3 months of therapy, and can be associated with mild vertebral fractures. On the other hand, second metacarpal percent CtA (a cortical site) is preserved. These results suggest that even short-term GC administration can have an adverse eect on spine health in children with NS.
Disclosures: Celia Rodd, P&G, 8
Comparative Analysis Of The Effects Of Pharmacological Inhibition Vs Genetic Reduction of DPP-4 Activity On Murine Bone Quality.Kimberly A. Kyle*1, Thomas L. Willett2, Laurie L. Baggio2, Daniel J. Drucker2, Marc Grynpas3. 1University of TorontoSamuel Lunenfeld Research Institute, Toronto, ON, Canada, 2Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada, 3Samuel Lunenfeld Research Institute, Toronto, ON, Canada
Potentiation of gut hormone action (GIP, GLP-1, GLP-2) has been shown to cause increased bone mass over time in rodents. These gut hormones are inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4). Drugs that inhibit DPP-4 activity are currently used for the treatment of type II diabetes mellitus. Although DPP-4 is a widely expressed enzyme, little information is known regarding its role in the regulation of bone. The objective of this study was to investigate the consequences of reduced DPP-4 activity on bone quality in two mouse models. The first model sets out to investigate pharmacological inhibition with a selective DPP-4 inhibitor, sitagliptin, in high fat fed (HFF) mice. The second model sets out to investigate the consequences of genetic ablation of the DPP-4/CD26 gene via analysis of CD26 knockout (KO) mice. We analyzed differences in bone mineral density (BMD) by dual energy x-ray absortiometry (DXA) and Micro-Computed Tomography (MicroCT). Three-point bending and femoral neck fracture of the femur were used to determine differences in cortical bone mechanical properties. Trabecular mechanical properties were determined using vertebral compression. Groups include male CD26 KO and littermate +/+ mice and wild-type (WT) mice treated with or without sitagliptin (n=9–12/group, 4g drug/kg chow, 12 weeks). DXA revealed a significant increase in vertebral BMD (p=0.040) in sitagliptin-treated male mice but no significant changes in femoral BMD. DXA did not reveal any significant differences in BMD for CD26 KO mice. Similarly, microCT did not reveal significant changes in BMD for both models. No changes, relative to control HFF mice, were seen in sitagliptin-treated male mice with three-point bending, femoral neck fracture testing and vertebral compression. Similarly, no changes were seen in CD26 KO male mice with three-point bending and femoral neck fracture testing. However, vertebral compression did reveal significant decreases in ultimate stress (p=0.023), failure stress (p=0.022) and Yong's Modulus (p=0.011) in CD26 KO male mice. The results suggest that chemical inhibition of DPP-4 activity in HFF mice does not produce an overall effect on bone. However, genetic ablation of the DPP-4 gene does produce a significantly negative effect on trabecular bone mechanical properties. These findings raise the possibility that complete germline elimination of the DPP-4 gene produces different consequences for bone relative to suppression of DPP-4 activity.
Disclosures: Kimberly A. Kyle, Merck Frosst, 5
This study received funding from: Merck Frosst Canada Ltd.
Osteoblast in Vivo IGF-I Production Regulates Tissue Level Properties in Mice.Patrick Ammann*1, Tara C Brennan2, René Rizzoli2. 1Division of Bone Diseases, Geneva, Switzerland, 2Division of Bone Diseases, Geneva, Switzerland
Bone strength is determined by bone mass, geometry and microarchitecture as well as by material tissue properties. This latter is highly influenced by ovariectomy and by agents used for osteoporosis treatment. Tissue material changes may be due to modification of the organic matrix or of its degree of mineralisation. The aim of the present study was to investigate whether material tissue properties can be influenced by locally produced IGF-I. In previous studies, we have demonstrated that isocaloric reduced protein intake is associated with depressed plasma IGF-I levels and by osteoblastic IGF-I production. To investigate this issue, 6 month-old transgenic male mice over-expressing IGF-I in osteoblasts under the control of collagen type 1 promoter (TG-IGF) and wild type mice(WT), were fed a normal or an isocaloric low protein diet for 8 weeks. After sacrifice, tibias were tested using nano-indentation technique for bone tissue quality at the level of the proximal tibia. Modulus, hardness and working energy of the trabecular bone were determined on hydrated bone tissue samples. Blood was also collected for IGF-I determination. Plasma IGF-I was similar in WT and TG-IGF-I and equally decreased by the low protein diet. In WT the low protein intake was associated with alteration of bone tissue quality as attested by decreased modulus, hardness and working energy. In contrast, in TG-IGF-I submitted to a low protein diet, the over-express ion of bone IGF-I was able to maintain normal bone material level properties despite low circulating levels of IGF-I. On the other hand over-expression under a regular diet did not influence nano-indention values characterizing properties of the material level. The absence of eect on nanoindentation values under a regular diet may indicate that bone IGF-I production plays a role mainly when circulating levels of IGF-I are low. In conclusion high levels of locally produced bone IGF-I was able to overcome the deleterious eects on material tissue properties of protein deficiency.
Disclosures: Patrick Ammann, None.
Phenotypic co-variation in the tibiae of four young adult populations.Charles Negus*1, Rachel Evans2, Karl Jepsen3, Brad Nindl4, William Kraemer5, Dani Moran6. 1L-3 Applied Technologies, San Diego, CA, USA, 2U.S. Army Research Institute of Environmental Science, Natick, MA, 3Mount Sinai School of Medicine, New York, NY, 4U.S. Army Research Institute of Environmental Medicine, Natick, MA, USA, 5University of Connecticut, Storrs, CT, USA, 6Heller Institute of Medical Research, Sheba Medical Center, Tel Hashomer, Israel
Recent studies have demonstrated that long bones co-vary morphological and compositional traits during growth in response to external bone size and daily loading. Specifically, cortical tissue-mineral density (Ct.Dn) and relative cortical area (Ct.Ar/Tt.Ar) have been shown to be higher in individuals whose diaphyseal diameter is small relative to their tibial length. In some people, this co-adaptation during growth leads to adult trait sets that may increase fracture risk, particularly under extreme loading conditions. The purpose of this study was to investigate how morphological and compositional traits compensate for relative bone size in four separate healthy, young adult populations.
Peripheral QCT images collected at 66% of tibial length (from the distal endplate) were analyzed from four cohorts: 59 American college women (age 20.4 ± 1.8), 97 female Israeli military recruits (age 18.3 ± 0.6), 16 male Israeli military recruits (age 18.5 ± 0.6), and 72 males from an Israeli combat unit (age 18.5 ± 0.8). Body weight (BW) and tibial length (Le) were recorded for each subject and cortical area (Ct.Ar), total area (Tt.Ar), marrow area (Ma.Ar) and Ct.Dn were calculated using image analysis software. Linear regression analyses examined bi-variate relationships among traits. Path Analysis was conducted to quantify the functional interactions among robustness (Tt.Ar/Le), Ct.Dn (a measure of tissue-quality compensation), and relative cortical area (a measure of morphological compensation).
Significant functional interactions among traits were confirmed for all four cohorts (Table 1). Ct.Dn and Ct.Ar/Tt.Ar both correlated inversely with robustness in all cohorts and in both men and women. All groups also achieved a similar Ct.Ar for their BW, independent of external bone size, though the Israeli cohort did so with greater robustness (p<0.04), lower Ct.Ar/Tt.Ar (p<0.0001), and lower mineralization (p<0.0001), indicating greater sub-periosteal expansion in response to weight-bearing loads compared to the American cohort. In contrast, the male cohorts had greater robustness (p<0.0001) combined with the expected lower Ct.Dn (p<0.0001) compared to the two female groups. However, the combat soldiers were leaner compared to the male Israeli recruits, but had more robust tibiae (p<0.0001) with lower Ct.Dn (p<0.0001) and lower Ct.Ar/Tt.Ar (p<0.0001). This multivariate analysis showed that different cohorts of young adult men and women used different phenotypic co-variation strategies to adapt bone strength to applied loads.
Disclosures: Charles Negus, None.
The High Bone Mass phenotype is charcterised by increased trabecular density and reduced endosteal expansion.Celia Gregson*1, Joern Rittweger2, Victor Lazar3, Sue Steel3, Jon Tobias4. 1University of Bristol, Bristol, United Kingdom, 2Bone Metabolism Unit, Institute for Biophysical & Clinical Research into Human Movement, Manchester Metropolitan University, Manchester, United Kingdom, 3Hull Royal Infirmary, Hull, United Kingdom, 4Academic Rheumatology, Clinical Science at North Bristol, Bristol University, Bristol, United Kingdom
High Bone Mass (HBM) is an uncommon sporadic finding of raised bone density on DXA scanning in individuals who are otherwise asymptomatic. Some cases have been reported in association with activating LRP5 gene mutations, which enhance osteoblast activity. X-rays of such cases have shown widened long bones and cortices1, in-keeping with increased bone formation. We aimed to perform the first systematic evaluation of the skeletal phenotype of HBM individuals, by carrying out peripheral Quantitative Computed Tomography (pQCT) measurements in a unique collection of these cases.
169 HBM index cases were identified by screening a hospital DXA database (n=96,377). HBM was defined as a) L1 Z score of ≥ +3.5 plus total hip Z score of ≥ + 1.2 or b) total hip Z score ≥ +3.5. Cases with significant osteoarthritis and/or other causes of raised BMD were excluded. First-degree relatives and spouses were recruited, in whom HBM affectation status was defined as L1 Z plus total hip Z scores of ≥ +3.5. Controls comprised unaffected relatives and spouses. pQCT was performed at the 4 & 66% radius and 4 & 66% tibia, using a Stratec XCT2000L. Cases were compared with controls using linear regression, adjusting for age, sex, weight & height.
89 HBM cases (66 index cases & 23 affected relatives) (82% female) and 55 controls (49% female) underwent pQCT; mean age 62 & 57 years, height 166 & 172 cm, weight 85 & 83 kg respectively; all Caucasian. As shown in the table below, compared with controls, HBM cases had considerably higher trabecular BMD at both the 4% radius and tibia, as well as greater cortical BMD at the 66% radius and tibia. Whilst total bone area in HBM cases was similar to that of controls, cortical bone area was higher at both the 66% radius and tibia, with correspondingly greater percentage cortical bone area.
In summary, reduced endosteal expansion leading to increased cortical area, and greater cortical and particularly trabecular density contribute to the greater bone mass of HBM individuals. In contrast, total bone area, an indicator of periosteal apposition, is unaffected. Taken together, our results suggest that over-suppression of bone resorption may underlie most cases of HBM, contrary to previous case reports indicating a primary role of osteoblast activation.
Boyden 2002 NEJM 16;346:1513
Babij 2003 JBMR18;6:960
Disclosures: Celia Gregson, None.
This study received funding from: The Wellcome Trust, NIHR CRN
High Resolution Peripheral Quantitative Computed Tomography (HRpQCT) Measurements Predict Central and Peripheral Fragility Fractures in Postmenopausal Women.Emily Stein1, Thomas Nickolas2, Adi Cohen2, Valerie Thomas3, Halley Rogers3, Felicia Cosman4, Geri Nieves5, Marcella Walker6, Donald McMahon1, Elizabeth Shane*1. 1Columbia University College of Physicians & Surgeons, New York, NY, USA, 2Columbia University Medical Center, New York, NY, USA, 3Columbia University, New York, USA, 4Helen Hayes Hospital, West Haverstraw, NY, USA, 5Helen Hayes Hospital, West Haverstraw, USA, 6Columbia Presbyterian Medical Center, New York, NY, USA
Purpose: Osteoporotic fractures aect millions of postmenopausal women worldwide. While measurement of areal BMD (aBMD) by DXA is a powerful clinical tool for identifying patients at increased risk of fracture, half of all postmenopausal fractures occur in women with BMD values above the WHO threshold for osteoporosis. We evaluated the ability of a novel technology, high-resolution peripheral quantitative computed tomography (HRpQCT) to predict fracture risk in postmenopausal women.
Methods: In this cross-sectional study, women with and without a history of postmenopausal fragility fracture had aBMD of central (lumbar spine [LS], total hip [TH], femoral neck [FN]) and peripheral (1/3 radius [1/3R]) sites measured by DXA. Trabecular and cortical volumetric BMD (vBMD) and trabecular microarchitecture were measured by HRpQCT (Xtreme CT, resolution ∼80 μm) of the radius and tibia.
Results: Of 130 postmenopausal women enrolled (85% Caucasian, mean age 69 ± 7 years), 51 (39%) had a history of fragility fracture. The most common sites of fracture were forearm (n=17), vertebrae (n=15) and ankle (n=12). There was no difference in age, race, BMI or GFR between subjects with and without a history of fracture. T-scores of women with a history of fracture were well above the osteoporotic range at all sites (LS: −1.4 ± 1.3; TH: −1.4 ± 0.9; FN: −1.9 ± 0.7; 1/3R: −1.6 ± 1.3. Fracture and non-fracture subjects had similar aBMD, T and Z-scores at the LS and 1/3R. However, fracture subjects had lower BMD at the TH (0.782 ± 0.106 vs. 0.832 ± 0.126 g/cm2; p<0.03) and FN (0.657 ± 0.086 vs. 0.703 ± 0.011 g/cm2; p<0.01). T and Z-scores at the TH and FN were also lower in fracture subjects. Marked differences in vBMD, cortical and trabecular microarchitecture of the radius and tibia were observed by HRpQCT (see Table below). Women with a history of fracture had lower cortical thickness, cortical density, and trabecular density. Trabeculae were fewer, thinner, more widely and unevenly spaced in these women.
Conclusions: In postmenopausal women, HRpQCT measurements of vBMD and microarchitecture differ significantly according to fracture history. Differences were observed in women with both central and peripheral fractures, and were more pronounced than those observed using DXA. HRpQCT can accurately discriminate between postmenopausal women with and without a history of fragility fracture.
Disclosures: Elizabeth Shane, None.
Once-Yearly Zoledronic Acid Increases The Proximal Femur Strength As Assessed By Finite Element Analysis Of QCT Scans.Lang Yang*1, Anya Sycheva2, Lisa Palermo3, Dennis Black4, Richard Eastell5. 1Univesity of Sheeld, Sheeld, United Kingdom, 2University of Sheeld, Sheeld, United Kingdom, 3University of California San Francisco, San Francisco, USA, 4University of California, San Francisco, San Francisco, CA, USA, 5University of Sheeld, Sheeld, United Kingdom
Finite element (FE) analysis of QCT hip scans has shown great promise in non-invasive assessment of whole bone strength. The goal of this study was to apply this FE technique to evaluate the bone's response to drug therapy.
This study involved 179 women from a substudy of the HORIZON_PFT Trial, a randomized controlled trial of once-yearly zoledronic acid 5mg (ZOL 5mg) , who had evaluable hip QCT scans at both baseline and 3-year follow-up. FE models of the right proximal femur were generated (blinded to treatment) from the QCT scans to simulate falls on the greater trochanter . It was assumed that tissue failure in an element occurs if a stress ratio (defined as the stress in it divided by the yield stress) Rs>1 or the strain E>2%. The mean Rs and the percentage elements with Rs>1 over the total hip (femoral neck plus trochanter) were calculated. Similarly, the mean E and the percentage elements with E>2% over the total hip were calculated. The greater these values are, the lower the bone strength and the higher risk of hip fracture. Group means were calculated for percentage changes from baseline and between-treatment differences were evaluated by ANOVA.
Relative to baseline, the ZOL 5mg group (n=94) had significant decreases in the mean Rs (−4.3%, SD 10.1%, p<0.0001) and mean E (−3.2%, SD 12.1%, p=0.011) as well as the percentage elements with Rs>1 (−4.9%, SD 11.7%, p<0.0001). The percentage elements with E>2% decreased non-significantly (−3.8%, SD 28.1%, p=0.19). These imply an increase in the bone strength over time. In contrast, the placebo group (n=85) had significant increases in the percentage elements with Rs>1 (2.7%, SD 10.5%, p=0.0178) and E>2% (9.8%, SD 30.5%, p=0.0041) as well as the mean E (4.1%, SD 15.2%, p=0.0157). These indicate deterioration in the bone strength over time. The placebo mean Rs almost remained the same (0.5%, SD 12.8%, p=0.74). Relative to placebo, the ZOL 5mg group was highly significant in having lower mean Rs (p=0.0057) and E (p=0.0005) as well as the percentage elements with Rs>l (p<0.0001) and with E>2% (p=0.0023). These infer a highly significant effect of ZOL 5mg in improving the bone strength over the placebo group.
In conclusion, based on FE analysis, once-yearly ZOL therapy for 3 years was associated with beneficial effects on proximal femur strength of postmenopausal women.
Black DM et al 2007. N Engl J Med 356:1809.
Keyak JH et al 2005. Clin Orthop Relat Res 437: 219.
Disclosures: Lang Yang, Novartis, Roche, Merck, 2
Secondary Mineralization and the Microhardness of Bone Measured across Menopause in Women.Yohann Bala*1, Susan Bare2, Georges Boivin3, Robert Recker4. 1INSERM Unit 831, University of Lyon, Lyon, France, 2Creighton University Osteoporosis Research Center, Omaha, USA, 3INSERM Unit 831, Lyon, France, 4Creighton University Osteoporosis Research Center, Omaha, NE, USA
The strength of bones depends not only upon bone volume and microarchitecture, but also on bone mineralization. The degree of mineralization of bone (DMB) depends on “age” of the bone structural units (1–3). Our purpose was to measure parameters reflecting DMB and Vickers microhardness of bone in 20 pairs of iliac bone biopsies taken from normal women both before and 12 months after final menses (mean interval between the two biopsies 60 ± 24 months). Embedded bone samples were cut into 100 ± 1 μm-thick sections used for quantitative microradiography (4). DMB (g/cm3) and Heterogeneity Index of the distribution of DMB (HI g/cm3) were measured separately on cortical, cancellous and total bone (= cortical + cancellous). On these sections, Vickers microhardness (Hv kg/mm2) was also measured (5). DMB and HI measured before menopause (1.08 ± 0.09, 0.28 ± 0.08, respectively) were not significantly dierent from those measured 12 months after final menses (1.09 ± 0.08, 0.26 ± 0.06, respectively). Hv was slightly but significantly (p=0.04) decreased 12 months after final menses (45.84 ± 4.04) versus before menopause (47.90 ± 2.85). DMB tended to slightly increase (1.5 %), HI and Hv tended to slightly decrease (1.2 and 4.2 %, respectively) across menopause. These modifications did not depend on the delay between the two biopsies and were similarly observed in cancellous, cortical and total bone. Present results are similar to control values recently reported from human necropsies (5). A previous study, using nanoindentation on 6 pairs of the same biopsies (6) showed an absence of changes in bone hardness contrary to our results at tissue level. Histomorphometry and micro-CT findings in the biopsies taken 12 months after the final menses have demonstrated a doubling of bone remodeling rate, and a deterioration of microarchitecture (7,8). In conclusion, paired iliac samples from women before and 12 months after final menses illustrate that the secondary mineralization of bone was not modified in spite of changes in bone remodeling activity. Microhardness was decreased in postmenopausal women.
Meunier & Boivin 1997, Bone 21:373
Boivin & Meunier 2002, Connective Tissue Res 43:535
Boivin & Meunier 2003, Osteoporos Int 14 Suppl 5:22
Boivin & Meunier 2002, Calcif Tissue Int 70:503
Boivin et al. 2008, Bone 43:532
Polly et al. 2008, J Bone Miner Res 23(suppl.1): S366
Recker et al. 2004, J Bone Miner Res 19:1628
Akhter et al. 2007, Bone 41:111
Disclosures: Yohann Bala, None.
The Effect of Rosiglitazone on Bone Mass and Strength is Reversible and Can Be Attenuated with Alendronate in Ovariectomized Rats.Sanjay Kumar*1, Sandra Homan1, Rana Samadfam2, Peter Mansell3, Susan Y. Smith4, Lorraine Fitzpatrick5. 1GlaxoSmithKline, Collegeville, PA, USA, 2Charles River Laboratories, Senneville, QC, Canada, 3Charles River Laboratories, Montreal, Quebec, Canada, 4Charles River Laboratories Preclinical Services Montreal, Senneville, QC, Canada, 5GlaxoSmithKline, King of Prussia, PA, USA
The objective of this study was to determine the effects of rosiglitazone (RSG) on biomechanical parameters of bone strength, the effect of concomitant treatment with alendronate (ALN) and reversibility of RSG effect. Nine month old rats (N=12 except where noted) were randomized to the following groups: SHAM, OVX (N=24), Metformin (PO, 300mg/kg/d), ALN (SC, 0.03mg/kg/twice weekly), 17β estradiol (E2, SC pellet, 0.01mg), RSG at two doses (PO, 3 or 10 (N=24) mg/kg/d), RSG10+ALN, or RSG10+E2 for 12 weeks. There was an 8-week treatment-free period for animals assigned to the recovery study (N=12; OVX and RSG10 groups). Lumbar spine (LS), calcaneus, femoral neck, femur, metatarsal, and humerus were subjected to biomechanical testing although only LS and femur data are presented due to space limitations. LS compression test showed lower values for OXV and an increase with ALN treatment (up to 40%; statistically significant for peak load, apparent strength, yield load and yield stress). Slight decreases in bone strength parameters were noted for E2 attributed to smaller bone size and in MET treated animals. With RSG treatment, there was a decrease (up to 35%) in bone strength parameters attaining significance for RSG10 for apparent strength and toughness. ALN completely prevented OVX/RSG-mediated reductions in bone strength parameters. The effects of RSG on bone strength showed evidence of reversal after discontinuation of RSG. At femur diaphysis, there were no differences in BMD (DXA, pQCT or uCT) or bone strength parameters for OVX and SHAM, RSG3, RSG10, RSG10+ALN, or RSG10+E2 except for significant increases in cortical thickness for RSG10+ALN. Slightly higher mean values for peak load and ultimate stress (up to 13%) were noted for OVX compared to SHAM. ALN slightly increased bone strength (up to 14%) consistent with slight increases in cortical thickness and BMC. Peak load, ultimate stress, stiffness and modulus were slightly lower for RSG10 (7–21%) and RSG3 (6–8%) relative to OVX with greater effects noted on AUC and toughness. At the end of recovery, values for RSG10 were generally similar to OVX. We conclude that treatment with RSG decreases parameters of bone mass and strength but these effects are prevented by concomitant treatment with ALN. Additionally, the effect of RSG is generally reversible.
Disclosures: Sanjay Kumar, GlaxoSmithKline, 3
This study received funding from: GlaxoSmithKline
Weekly treatment with human parathyroid hormone (1–34) for 18 months increases bone strength via the amelioration of microarchitecture, degree of mineralization, enzymatic and non-enzymatic cross-link formation in ovariectomized cynomolgus monkeys.Tatsuhiko Kuroda1, Keishi Mammo2, Chikara Ushiku2, Ryoko Takao-Kawabata1, Mitsuru Saito*3. 1Asahi Kasei Pharma Corporation, Tokyo, Japan, 2Jikei University School of Medicine, Tokyo, Japan, 3Jikei University School of Medicine, Tokyo, Japan
[Aims] Intermittent administration of PTH stimulates bone formation and increases BMD and mechanical strength. The aim of this study was to investigate dierences in microarchitecture, degree of mineralization, collagen quantity, collagen enzymatic immature and mature cross-links and non-enzymatic cross-links, and mechanical strength with or without weekly treatment of human human PTH(1–34)(hPTH1–34) for 18 months in ovariectomized (ovx) monkeys. The contribution of structural and material properties to mechanical strength was determined.
[Methods] Eighty adult female cynomolgus monkeys were divided into four groups (n=20 each) as follows: sham-vehicle (SHAM), ovx-vehicle (OVX), and ovx groups given once weekly subcutaneous injections of hPTH-(1–34) at 1.2 μg/kg (Low-PTH) or 6.0 μg/kg (High-PTH) for 18 months. BMD, microarchitecture of cancellous bone, calcium and phosphorus content, collagen content, collagen enzymatic immature cross-links (DHLNL, HLNL, and LNL) and mature cross-links (PYD and DPD), and non-enzymatic cross-links (advanced glycation end products; pentosidine) were measured in the third lumbar (L3) vertebrae. Mechanical strength was evaluated using a compression test of cancellous bone. Ultimate load, stiness and breaking energy were obtained as mechanical properties.
[Results] Weekly hPTH-(1–34) treatment increased BMD, cancellous bone volume (BV/TV), trabecular thickness (Tb.Th), calcium/phosphorous content, collagen content, the actual amount of the immature cross-links, and mechanical strength compared with the OVX group. hPTH-(1–34) treatment also improved trabecular bone pattern factor (TBPf) and structure model index (SMI), but decreased pentosidine content in cancellous bone. BMD, BV/TV, Tb.Th, calcium content, phosphorous content, collagen content and enzymatic cross-link formation showed significant positive correlation to mechanical properties. TBPf, SMI, pentosidine and the ratio of pentosidine to total enzymatic cross-links indicated a negative correlation to mechanical properties. Stepwise logistic regression analysis was used to estimate the association between selected parameters and mechanical properties. Ultimate load and breaking energy were significantly aected by cancellous BV/TV, Tb.Th, calcium content and the ratio of pentosidine to enzymatic cross-links, independently. Conversely, stiness was only aected by the ratio of pentosidine to enzymatic crosslinks.
[Conclusion] Weekly intermittent treatment with hPTH-(1–34) increased bone mechanical strength in vertebrae by increasing the actual amount of enzymatic crosslinks, BMD, BV/TV, Tb.Th, calcium content, collagen content, and improving microarchitecture of cancellous bone, whereas non-enzymatic glycation cross-links, pentosidine, were significantly reduced.
Disclosures: Mitsuru Saito, None.
AKT and MAPK Phosphorylation Regulates the Opening/closing of Connexin 43 Hemichannels in Osteocytes in Response to Shear Stress.Sirisha Burra1, Nidhi Batra*2, Xuechun Xia3, Lynda Bonewald4, Eugene Sprague5, Paul Lampe6, Jean Jiang7, 1University of Texas, San Antonio, TX, 2University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, 3Department of Biochemistry, U of Texas Health Science Center, San Antonio, USA, 4University of Missouri, Kansas City, MO, USA, 5Dept. of Radiology, University of Texas Health Science Center, San Antonio, USA, 6Molecular Diagnostics Program, Seattle, USA, 7University of Texas Health Science Center at San Antonio, San Antonio, TX,
Mechanical stimulation in the form of fluid flow shear stress (FFSS) induces opening of Cx43 hemichannels (HC), unopposed halves of gap junctions, resulting in the release of PGE2, an important mechanical signaling molecule. This opening of HC in response to FFSS (30 min, 16 dynes/cm2) is inhibited by a PI3K inhibitor. Long periods of continuous FFSS (up to 24 hrs at 16 dynes/cm2) results in gradual closure of Cx43 HCs. To identify the molecular mechanisms responsible for the opening/closing of hemichannels in response to long term application of FFSS, MLO-Y4 cell lysates from various FFSS time points were probed with antibodies against pERK and pAKT. We observed that pAKT levels increased after 30 min of FFSS when there is maximal opening of Cx43 HCs. Continuous FFSS for longer periods of time (up to 24 hrs) resulted in a gradual decrease of pAKT levels, which correlated with reduced opening of HCs. In contrast, pERK levels increased significantly after 24 hrs of continuous FFSS when there is minimal opening or greater closure of HCs. In addition, we observed phosphorylation of Cx43 at S279/S282, a MAPK-dependent site, that increased by 24 hrs of FFSS. In order to identify the factor(s) responsible for increasing Cx43 phosphorylation, conditioned media (CM) collected after 24 hrs of continuous FFSS was applied to MLO-Y4 cells. CM increased Cx43 phosphorylation at the S279/S282 site within 30 min, whereas continued incubation of MLO-Y4 cells with CM decreased Cx43 phosphorylation at this site. Consistent with these observations, incubation of MLO-Y4 cells with CM for 30 min prior to applying FFSS resulted in decreased opening of HCs. This reduction in Cx43 HC opening induced by preincubation with CM before FFSS was reversed when the cells were also preincubated with PD98059, a p42/44 MAPK pathway inhibitor. Together, these results suggest that Cx43 HC function is intricately regulated by both AKT and MAPK signaling pathways. PI3K-Akt signaling positively regulates the opening of Cx43 HC whereas MAPK signaling is involved in the closing of HC after long term FFSS. This tight regulation of Cx43 HC function is most likely crucial in the regulation of the extracellular anabolic/catabolic signaling factors, such as prostaglandins and other small molecules in response to mechanical loading.
Disclosures: Nidhi Batra, None.
Bone loading modulates neuronal cross-talk between thoracic limbs.Susannah Sample*1, Qiang Wu2, Theresa Baker3, Cathy Thomas3, Mary Behan3, Peter Muir4. 1UW-Madison School of Veterinary Medicine, Madison, WI, 2University of West Virginia, Morgantown, USA, 3University of Wisconsin - Madison, Madison, USA, 4University of Wisconsin, Madison, Madison, WI, USA
Adaptation to loading a single bone involves neuronally regulated responses in multiple bones in different limbs. The periosteum is innervated with a dense net-like meshwork of nerves, suggesting the existence of a sophisticated and specialized neuronal regulatory mechanism optimized for detection of mechanical distortion. The purpose of the present study was to determine whether neuroanatomic connections exist between the peripheral innervation of different limbs and whether such connections are modulated by bone loading.
10 male Sprague-Dawley rats were used for the study. The right ulna of 4 rats was loaded for 1,500 cycles at −18N and 4Hz. 4 rats were sham-loaded, and 2 were baseline controls. To trace and quantify neural connectivity between limbs, attenuated Bartha pseudorabies virus (PRV) was used as a trans synaptic tracer. PRV was injected onto the periosteum of the right ulnar mid-diaphysis 10 days after loading or sham loading. Euthanasia was performed 4–6 days after PRV inoculation, at which time the rats were perfused and the left and right brachial and lumbosacral intumescence dorsal root ganglia (DRG) were removed. The number density of PRV+ neurons was determined for left and right brachial and lumbosacral intumescences.
PRV+ neurons were found in the intumescences of all four limbs. The number density of PRV+ neurons was increased in the left brachial intumescence DRG and the right lumbosacral DRG of rats which had undergone right ulnar loading, when compared to the left brachial DRG of the sham control rats. Although not significant, higher numbers of PRV+ neurons were also seen in the right brachial DRG and the left lumbosacral DRG after loading.
Our results indicate the existence of a complex and dynamic neural cross-talk mechanism between limbs that is responsive to skeletal loading. Additionally, neuroanatomic connections between sensory innervation in right and left thoracic limbs and between thoracic and pelvic limbs must exist, and these connections are enhanced by mechanical loading of the skeleton.
Fig. 1. Pseudorabies virus (PRV) in brachial intumescence dorsal root ganglion (DRG) sensory neurons, after inoculation of the right ulna periosteum. After inoculation small numbers of labeled neurons were detected in both the left (A) and right (B) DRG. When PRV inoculation was performed 10 days after right ulna loading, larger numbers of labeled neurons were found in the left (C) and right (D) DRG. Scale bar=50μm.
Disclosures: Susannah Sample, None.
Gene regulation following mechanically induced woven and lamellar bone formation in the rat ulna.Matthew Silva1, Jennifer McKenzie*2. 1Washington University in St. Louis School of Medicine, St. Louis, MO, USA, 2Washington University, St. Louis, MO, USA
Mechanical loading can induce woven or lamellar bone formation. Our objective was to compare and contrast the gene expression patterns related to loading-induced woven and lamellar bone formation. We hypothesized that genes associated with angiogenesis, cell proliferation and bone formation would be differentially regulated when comparing woven to lamellar bone formation. In prior studies using the rat ulnar loading model, we demonstrated that a single bout of damaging fatigue loading stimulates abundant periosteal woven bone formation as well as lamellar bone at the margins of the woven bone. In contrast, a single bout of non-damaging loading stimulates only formation of lamellar bone. To address our hypothesis, microarray (Illumina) and real time qPCR were performed at 0 (1 hr), 1 and 3 days for both loading conditions. Ulnae loaded using a damaging (woven) protocol (2 Hz hsine; 18 N peak force) were compared to ulnae loaded using a non-damaging (lamellar) protocol (rest-inserted trapezoid; 100 cycles; 15 N peak force) (loading approved by IACUC). Analysis of microarray data (Partek) showed differential regulation of 400 genes for woven vs. lamellar bone at day 0, 5984 at day 1 and 6085 at day 3 (Figure). Analysis of the top ten canonical pathways (GeneGo) activated by woven but not lamellar loading indicates an immune response at day 0 (6/10 pathways). The immune response persists at day 1 (5/10 pathways) with the addition of cytoskeletal remodeling activation (2/10 pathways). By day 3, the immune response has subsided (0/10 pathways), cytoskeletal remodeling still plays a major role (3/10 pathways) and development pathways are now evident (2/10 pathways). Real-time PCR analysis for select genes including angiogenic markers Pecam and VEGF, cell proliferation marker histone H4 (Hist4) and matrix formation marker bone sialoprotein (BSP) showed significant upregulation of all genes for woven versus lamellar bone on days 1 and 3 (Table). Taken together, these data demonstrate the differential regulation of genes associated with woven and lamellar bone formation. In agreement with our hypothesis, woven bone formation significantly upregulates angiogenesis, cell proliferation and bone formation markers. Additionally, we saw a considerable immune response associated with woven bone formation. As a result of this study, we now have a large set of pathway targets that can be analyzed to better understand the complex process of woven bone formation.
Disclosures: Jennifer McKenzie, None.
Does Childhood Forearm Muscle Strength Development Predict Bone Strength in Midlife? Evidence from a 40-year Prospective Study.Amanda Lorbergs*1, J. D. Johnston2, Adam D. G. Baxter-Jones2, Saija Kontulainen1. 1University of Saskatchewan, Saskatoon, SK, Canada, 2University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Strains from muscle contractions produce the largest physiological loads on bones. Maximizing childhood muscle strength is thought to protect against bone fragility later in life. We examined the relationship between forearm muscle strength development in childhood and forearm muscle size, force production and radius bone strength indices in midlife. We measured 24 participants (men and women, mean age 49.5 ± SD2.2 yrs) who were participants of the Saskatchewan Growth and Development Study (1964–2009) (SGDS). Muscle strength development of wrist flexion (FLEX) and extension (EXT) were collected during childhood and adolescence (1964–1973). Peak Strength Velocity (PSV) was determined for each individual by fitting a cubic spline to this serial strength data. Adult muscle strength of the forearm was measured as grip force, peak wrist FLEX and EXT torques and muscle cross-sectional area (MCSA). Non-dominant radius bone strength index in compression (BSI) and polar strength strain index (SSI) were used as estimates of bone strength at the 4% and 65% sites, respectively, using pQCT (Stratec XCT2000). Muscle variables were used in univariate linear regression analyses to determine their ability to predict BSI and SSI. Linear regression analyses identified adolescent wrist FLEX PSV as a significant predictor of adult SSI (R2=0.52, p<0.001). After entering adult wrist FLEX the model improved (R2=0.74, p<0.001) and adolescent wrist flexion maintained significance in the model. Childhood wrist FLEX PSV was also a significant predictor of BSI (R2=0.23, p<0.05) and this association improved with the addition of adult concentric wrist FLEX (R2=0.39, p<0.01). Adult muscle size was also a significant predictor of radius SSI (R2=0.82, p<0.001) and BSI (R2=0.59, p<0.001). Our results indicate that in addition to adult muscle size and strength childhood muscle strength development is an important determinant of estimated radius strength in midlife.
Disclosures: Amanda Lorbergs, None.
Low Sensitivity to Muscle Paralysis-Induced Cortical Bone Loss and Delayed Recovery from Trabecular Bone Loss Upon Reloading in Mice with Connexin43 Gene (Gja1) Ablation in Osteoblasts.Susan Grimston*1, Danial Goldberg2, Marcus Watkins3, Michael Brodt2, Matthew Silva3, Roberto Civitelli3. 1Washington University School of Medicine, St. Louis, MO, USA, 2Washington University School of Medicine, St. Louis, USA, 3Washington University in St. Louis School of Medicine, St. Louis, MO, USA
We have previously reported an attenuated anabolic response to in vivo mechanical loading in mice with a conditional deletion of Gja1, the gene encoding for the gap junction protein connexin43 (Cx43), driven by a 2.3kb fragment of the α1 (I) collagen promoter (cKO). In the present study we determined whether lack of Cx43 in osteoblasts also aects bone response to unloading and re-loading using a transient muscle paralysis model induced by Botulinum toxin. Injection of the toxin (2U/100 g) in the right quadriceps and triceps surae muscles results in a rapid and profound trabecular bone loss — assessed as bone volume/tissue volume (BV/TV) by in vivo μCT — reaching a nadir at around 3 weeks post-injection, equally in cKO and wild type (WT) littermates (−37.37 ± 6.58% vs −42.89 ± 9.81%, respectively, p>0.10, Fig. 1). This bone loss was followed by a slow return towards baseline upon recovery from muscle paralysis and re-loading. At 20 weeks post-injection, mice had recovered about 80% of trabecular bone mass. However, the rate of bone gain (calculated over 8 weeks after nadir) was significantly lower in cKO relative to WT (−0.31 ± 1.54 %/wk vs 3.59 ± 1.03 %/wk; p<0.05, Fig. 1); consistent with attenuated response to anabolic mechanical loading in Cx43 deficient animals. Notably at baseline, cKO mice had larger Ma.Ar (144.2 ± 3.5 mm2 vs 69.3 ± 4.8 mm2) and thinner cortices (1.95 ± 0.11 mm vs 2.11 ± 0.09 mm) relative to WT (p<0.05). Muscle paralysis also induced cortical bone loss, leading to an increased marrow area (Ma.Ar), most likely by activation of endocortical bone resorption. Intriguingly, this eect was barely detectable in cKO compared to WT mice (2.28 ± 0.07% vs 13.76 ± 3.73% Ma.Ar increase from baseline, respectively; p<0.05; Fig. 2). Furthermore, serum markers of bone turnover were marginally higher in cKO relative to WT mice: type I collagen C-telopeptides, 17.36 ± 1.81ng/ml vs 13.22 ± 1.71ng/ml; and osteocalcin 11.45 ± 0.76 ng/ml vs 8.46 ± 1.29 ng/ml, respectively. Thus, Cx43 deficiency reduces the sensitivity to mechanical unloading selectively in the cortical bone, perhaps because of failure to activate osteoclasts in the low remodeling cortical envelope. It also retards recovery of trabecular bone loss after re-loading, which could be related to either an osteoblast defect, as previously shown, and/or to increased osteoclast activity. Hence, Cx43 in osteoblasts and/or osteocytes is a key regulator of skeletal responses to mechanical stimuli.
Disclosures: Susan Grimston, None.
Microfluidic Enhancement of Intramedullary Pressure Increases Interstitial Fluid Flow and Protects Against Hindlimb Unloading-Induced Bone Loss in Mice.Ronald Kwon*1, Diana Meays2, John Frangos2. 1Apple Valley, CA, USA, 2La Jolla Bioen gineering Institute, La Jolla, USA
The capacity for cultured bone cells to sense fluid flow has been well documented. However, investigating flow-induced alterations in bone metabolism in vivo has been dicult, due in large part to the diculties associated with decoupling interstitial fluid flow (IFF) from matrix strain within the small skeletal fluid spaces of mice (the organism of choice for most skeletal research in animals). In this study, our goals were to develop a microfluidic system to enhance femoral intramedullary pressure (ImP) within mice, and determine the capacity of dynamic ImP to enhance IFF and protect against bone loss induced by hindlimb suspension (HLS). 16wk female C57BL/6J mice were used for all studies (all studies were IACUC-approved). ImP was enhanced via a fluid-filled catheter surgically implanted into the femoral intramedullary canal and coupled externally to a custom microfluidic syringe pump. Oscillatory pressure loading (5Hz, peak pump flow rate: 15.7uL/s) substantially increased mean (no load: 12.8 ± 1.7mmHg, load: 32.9 ± 9.8mmHg, n=2) and peak-to-peak (no load: 3.6 ± 0.4mmHg, load: 50.7 ± 18.0mmHg) ImP as quantified by a telemetric pressure transducer. To determine whether IFF was enhanced, we injected catheterized mice with fluorescein, harvested their femurs, and quantified fluorescence recovery after photobleaching (FRAP) of the tracer within periosteal lacunae. Characteristic recovery times (one over the slope of the best fit line of the logarithmic intensity curve) were significantly decreased during pressure loading (no load: 160.3 ± 30.1s, load: 96.9 ± 15.1s, n=8, p=0.02), suggesting that IFF was enhanced through the cortex. Pressure loading for 3min/day significantly eliminated HLS-induced decreases in trabecular BMD after 7 days (no load: −16.5 ± 8.0%, load: 6.5 ± 11.4%, p=0.02) as quantified by in vivo pQCT, and significantly increased trabecular bone volume fraction (no load: 26.9 ± 1.0%, load: 31.4 ± 1.2%, p=0.03) and cortical thickness (no load: 130.7 ± 4.9?m, load: 147.0 ± 4.6um, p=0.01) as quantified by uCT. Analyses of fluorochrome labels revealed loading significantly increased BFR (no load: 15.7 ± 6.2um3/um2/yr, load: 55.4 ± 10.2um3/um2/yr, p=0.03). In conclusion, we have developed a microfluidic system for enhancing ImP in the alert mouse. Our results suggest that dynamic ImP enhances IFF and potently inhibits HLS-induced bone loss in mice. This system may serve as a valuable tool in future investigations examining flow-mediated regulation of bone metabolism.
Disclosures: Ronald Kwon, None.
Whole Body Vibration May Not Alter Bone Volume, Architecture, and Density nor the Effects of Teriparatide in Mature Male ICR (CD-1) Mice.Daniel Perrien*1, Elizabeth C O'Quinn2, Gloria Gutierrez3, Gregory Mundy4, Jery Nyman5. 1Vanderbilt University Medical Center, Nashville, TN, 2Vanderbilt University Medical Center, Nashville, USA, 3Vanderbilit University & Medical Center, Nashville, TN, 4Oates Institute of Experimental Therapeutics, Nashville, TN, USA, 5Vanderbilt University Medical Center, Nashville, TN, USA
High frequency, low amplitude, whole body vibration (WBV) has been proposed as both a stand alone treatment and an adjunct to other anabolic therapies for osteoporotic patients. WBV has been reported to increase trabecular bone mass in young rapidly growing rodents. However, the effects of WBV in more mature rodents are not well characterized. Using previously published WBV protocol shown to be anabolic, this study tested the hypothesis that WBV increases bone volume, architecture, and density independently and/or in conjunction with intermittent hPTH (1–34) (Teriparatide) treatment in mature male mice. Starting at twelve weeks of age, male ICR (CD-1) mice (n=12 per group) were subjected to WBV using a low amplitude plate at 45 Hz, 90 Hz or 0 Hz (Con), where Con mice occupied the housing with the plate turned off. This was done for 20 min/day, 5 days/wk for 6 weeks. Thirty minutes prior to WBV, each mouse received s.c. injection of 40 μg/kg PTH (PTH) or vehicle alone (Veh). All mice were sacrificed following the 6 wk treatment period and bones collected for microCT and histological analyses. Data were analyzed by two-way ANOVA with Tukey's post-hoc test. MicroCT analysis of trabecular bone in the proximal tibial metaphysis revealed that BV/TV, trabecular architecture, vBMD and mBMD in Con+Veh mice were not significantly different from either 45Hz+Veh or 90Hz+Veh (BV/TV; 0.177 +/-0.072; 0.157 +/-0.077; 0.147 +/-0.052, respectively), suggesting that WBV alone did not affect these parameters. BV/TV, vBMD, and mBMD were also unaffected in Con+PTH (BV/TV; 0.150 +/-0.101) compared to Con+Veh mice. However, PTH significantly decreased Tb.N and increased Tb.Th, Tb.Sp, and SMI (all p<0.05). Additionally, PTH effects on trabecular architecture appeared to be unaltered by WBV as these parameters in the 45Hz+PTH and 90Hz+PTH groups were not significantly different from Con+Veh (p<0.05) but not Con+PTH. Although other studies have shown anabolic effects of WBV in young growing animals, this study demonstrates that 45 Hz and 90 Hz WBV, 5 days per wk for 6 wk, alone or in conjunction with intermittent PTH may not significantly affect trabecular volume, architecture, or density in intact, young adult, male ICR mice.
Disclosures: Daniel Perrien, None.
Image Processing for Three Dimensional Visualization of Hemiosteonal Bone Remodeling.Craig Slyfield*1, Evgeniy Tkachenko2, John Siemon2, David Wilson2, Christopher Hernandez1. 1Case Western Reserve University, Cleveland, OH, USA, 2Case Western Reserve University, Cleveland, USA
Introduction: Using a technique known as serial block face imaging, 3D images of cancellous bone and fluorescent bone formation markers can be acquired at a resolution of 0.7×0.7×5 microns/voxel. Images collected with this technique allow direct automated measurements of local bone remodeling activity in 3D. The diffuse nature of bone formation markers makes segmenting signal from background challenging and requires specialized image processing techniques.
Methods: Adult Sprague-Dawley rats (n=5) were ovariectomy sham operated. At 10 months of age, the rats were subjected to two fluorochrome labels (xylenol orange followed 7 days later by calcein). Each L4 lumbar vertebra was excised, embedded in opaque methacrylate, and imaged using an automated serial milling device with attached fluorescence microscope. An automated optical filter changer provided grayscale images of only xylenol orange and only calcein. Bone and fluorescent label surface areas were first measured in 2D using stereology. Images of cancellous bone, 1mm in each direction, were processed by 1) removing fluorescence signal originating below the optical plane 2) grayscale morphological processing to equalize intensities within labels 3) global thresholding 4) 3D morphological closing (dilation followed by erosion) of the segmented image to collate nearby disconnected formation specks and 5) noise removal. Noise was removed based on a minimum volume criterion user determined by evaluating the final 3D image. Global threshold values were automatically determined to match post-processing label surface area with stereology results. Images were processed and visualized using Matlab (MathWorks) and Amira (Visage Imaging).
Results: Complete basic multicellular units (BMU) were observed. Figure 1 shows a complete BMU: a resorption cavity (arrows) and two bone formation labels (left of cavity). A transverse slice illustrates classic double labeling as seen in histological slides. On average, 3.2 ± 1.5 complete BMUs were visible in each 1mm cube.
Conclusions: We demonstrate image processing techniques for identifying bone formation markers in high resolution 3D images acquired through serial block face imaging. The current study also provides the first 3D images of BMUs in cancellous bone and will enable direct dynamic bone histomorphometry measurements such as BMU birthrate, local bone formation rate, and the spatial relationship between bone formation, resorption, and morphology.
Disclosures: Craig Slyfield, NIH/NIAMS, 2
Restriction of Bisphosphonate-associated osteonecrosis (ONJ) to the jaw-Developmental biology considerations and jaw bone specific cytokine expression.Falk Wehrhan*1, Kerstin Amann2, Peter Hyckel3, Phillip Stockmann4, Schlegel Andreas Karl4, Friedrich W. Neukam5, Nkenke Emeka5. 1University of Erlangen-Nuremberg, Erlangen, Germany, 2Institute of Pathology, Erlangen, Germany, 3Dept. of Plastic Surgery/University of Jena, Jena, Germany, 4Dept. of Oral & Maxillofacial Surgery University of Erlangen, Erlangen, Germany, 5Dept. of Oral & Maxillofacial Surgery/University of Erlangen, Erlangen, Germany
Background and purpose of the study:
ONJ is restricted to the jaw. The ONJ-affected sites are associated with hypercalcified bone and prolonged wound healing. The formal pathology of ONJ is unknown. Restriction of ONJ to the jaw bone might be caused by developmental biology related differential interaction of aminobisphosphonates with cranial neural crest derived jaw bone compared to mesenchymal stem cell derived extracranial bone. Sustained expression of the osteoproliferative transcription factor Msx-1 can be found in jaw bone only. Description of bone homeostasis and wound healing related mediators Msx-1, BMP-2/4, TGFβ1 and its downstream effectors Smad 2/3 and Smad-7 in ONJ-sites are missing at present. The aim of the study was to evaluate the expression of Msx-1, BMP-2/4, TGFβ1, Smad 2/3, Smad-7 and Galectin-3 in ONJ-affected sites compared to healthy jaw bone.
Material and Methods:
20 ONJ samples and 20 control specimen of healthy jaw bone were processed for immunohistochemistry of paraffin embedded tissue. Targeting BMP-2/4, Msx-1, TGFβ1, Sox-9, Rank(L), Smad 2/3, Smad 7 and Galectin-3 an autostaining-based APAPP-staining kit was used. Semiquantitative assessment was performed measuring the ratio of positively stained cells/ total amount of cells (labelling index, ANOVA-Test) in ONJ samples (bone and soft tissue) compared to control jaw bone.
A significant suppression (p<0.05) of Msx-1 and Rank(L) and a significantly increased (p<0.05) expression of BMP-2/4 was seen in bone samples of the ONJ-sites compared to normal jaw bone. In the ONJ-related soft tissue a significant suppression (p<0.03) of TGFβ1, Smad 2/3 was detected, whereas Smad-7, Galectin-3 and Sox-9 were significantly (p<0.05) increased in ONJ compared to healthy mucosa.
Suppression of Msx-1 by aminobisphosphonates might explain the development of local ostepetrosis and restriction of ONJ to the jaws since Msx-1 has been shown to be expressed in cranial neural crest derived bone only. Results indicate an interaction of TGFβ1 and BMP-2/4 pathway during bone and oral mucosa regeneration, which is impaired by aminobisphosphonates. Comparative analysis of developmental biology related differences of osteoblast-osteoclast homeostasis in cranial neural crest derived bone compared to mesenchymal stem cell derived bone could explain a variety of bone disorders, restricted to the maxillofacial bone structures.
Disclosures: Falk Wehrhan, None.
Atorvastatin Attenuates Lrp5/Sox9 mediates Degenerative Joint Disease in the Hypercholesterolemic ApoE null mice.Bryan Krueger1, Amy Flores1, Je Park*1, Nalini Rajamannan2. 1Northwestern University, Chicago, USA, 2Pathology, Chicago, IL, USA
Background: Degenerative joint disease is a common clinical problem in the aging population which is responsible for significant morbidity and suering in patients who have this disease. The cellular mechanisms of osteoarthritis in joints are under intense investigation. We have previously shown that Lrp5 mediates abnormal bone formation in the cardiovascular calcification via an osteoblast dierentiation pathway. We hypothesize that lipids may play a role in the development of this degenerative joint disease in parallel to the bone formation in the aortic valves. Methods: In this study we tested the eect on lipids with and with atorvastatin in the ApoE mouse model to determine the gene and protein expression of the Lrp5/Sox9 pathway in the joints and in the valves. ApoE−/− mice (n=60). Group I (n=20) normal diet, Group II (n=20) 0.25% chol diet (w/w), and Group III (n=20) 0.25% (w/w) chol diet+atorv for the development of abnormal matrix synthesis in the joints over 24 weeks. The aortic valve (AVA) was examined for calcification, Lrp5 and sox9. Bone formation was assessed by micro Computed Tomography (microCT). Matrix synthesis in the joint was assessed by Masson Trichrome Staining. Aortic Valve Disease was assessed by Visual Sonics Mouse echocardiography. Results: The cholesterol diet induced complex bone formations in the calcified AV and thickened knee joints with an increase in Sox9, Lrp5 receptor expression and Cyclin as compared to the Control(>3-fold(p<05). Atorvastatin attenuated all markers in the valves and in the joints. Echocardiography demonstrated a mild increase in the peak jet velocity across the aortic valve but not statistically significant. Masson Trichrome visual assessment demonstrated a marked increase in the matrix synthesis as compared to the controls and atorvastatin treated joints. Conclusion: These results demonstrate that Lrp5 mediated bone formation in the hypercholesterolemic aortic valves and abnormal matrix synthesis in the knee joints is associated with aortic valve disease and is attenuated with atorvastatin. Providing further evidence in an atherosclerotic mouse model of joint disease and valvular heart disease, that statins play a role in the treatment of this disease.
Disclosures: Jeff Park, None.
MicroRNAs regulate chondrocyte proliferation and differentiation.Tatsuya Kobayashi*1, Robert Blelloch2. 1Massachusetts General Hospital, Boston, MA, USA, 2UCSF, San Francisco, USA
microRNAs (miRNAs) regulate gene expression mainly at the posttranscriptional level by binding to target RNAs. This miRNA-mediated gene regulatory mechanism plays important roles in various physiological and pathological processes such as tumor suppression and stem cell differentiation. miRNA biogenesis requires sequential cleavage of primary miRNA transcripts first by the microprocessor complex containing Drosha and DGCR8, and then by Dicer. Drosha, DGCR8 and Dicer are individually essential for miRNA generation. In chondrocytes, Dicer-deficiency causes a dramatic reduction in cell proliferation and acceleration of hypertrophic differentiation. Although pre-miRNAs appear to be the dominant substrates of Dicer, Dicer is known to process other RNA species as well. Therefore, the loss of possible miRNA-independent function of Dicer might contribute to the abnormalities caused by Dicer-deficiency. In the present study, to confirm the role of miRNAs in chondrocytes, DGCR8 was conditionally deleted to eliminate miRNAs through actions upstream of Dicer in chondrocytes. DGCR8 conditional knockout (cKO) mice showed growth plate phenotypes similar to Dicer cKO including reduced proliferating chondrocytes, suggesting that the growth plate abnormalities in Dicer cKO are mostly due to miRNA deficiency. Next, in order to understand the mechanism by which miRNA deficiency causes alteration in chondrocyte proliferation and differentiation, we attempted to identify RNAs regulated by miRNAs in chondrocytes. To this end, RNA profiling was performed using Argonaute 2-immunoprecipittants (Ago2-IP). Argonaute proteins are the essential component of the RNA-Induced Silencing Complex (RISC), and therefore Argonaute-associated RNAs are likely regulated by miRNAs. The profile of Ago2-IP RNA prepared from primary rib chondrocytes was significantly different from that of total RNA prepared from the same sample. Gene Ontology analysis of Ago2-IP enriched RNA revealed that Ago2-associated RNAs were enriched in those that function in the nucleus, a result consistent with the previous notion that miRNAs preferably target transcription factors. In summary, the present study suggests that miRNAs are essential for normal chondrocyte proliferation and differentiation. Profiling of Ago2-IP RNA can be used to identify possible miRNA-regulated genes, which provides the basis for understanding roles of miRNAs regulating chondrocyte functions.
Disclosures: Tatsuya Kobayashi, None.
Monocarboxylate transporter-1 is involved in the activation of NF-κB and expression of phagocyte-type NADPH-oxidase in mouse chondrocytes exposed to interleukin-1β.Kentaro Yoshimura*1, Yoichi Miyamoto2, Toshifumi Maruyama2, Rika Yasuhara2, Atsushi Yamada2, Masamichi Takami2, Tetsuo Suzawa2, Kazuyoshi Baba2, Ryutaro Kamijo2. 1Showa University School of Dentistry, Tokyo, Japan, 2Showa University, Tokyo, Japan
We reported previously that interleukin-1β (IL-1β) induced death of primary chondrocytes from mice and rats as well as in mouse chondrogenic ATDC5 cells through mitochondrial dysfunction and energy depletion in a reactive oxygen and nitrogen species-dependent manner. In this process, the expressions of inducible type of nitric oxide synthase (NOS2) and phagocyte-type NADPH-oxidase (NOX2) were induced in these cells, both of which were involved in the cell death. Since we also found that exposure to IL-1β caused intra- and extracellular acidification of the cells, we examined the effects of the inhibitors against various pH modulators on IL-1β-induced cell death. Among the compounds tested, α-cyano-4-hydroxycinnamate, an inhibitor for monocaroboxylate transporters (MCTs), significantly inhibited the chondrocyte death. MCTs are members of the proton-linked membrane transport proteins transport monocarboxylates such as lactate and pyruvate. The most abundant isoform of MCTs in ATDC5 cells was MCT-1 which is known to be distributed in plasma and mitochondrial inner membranes. Therefore we investigated the role of MCT-1 in IL-1β-induced chondrocyte death by knocking down MCT-1 by its siRNA. Expression of MCT-1 was almost completely suppressed in mouse primary chondrocytes and ATDC5 cells, and IL-1β-induced death of ATDC5 cells was significantly inhibited by using the MCT-1 siRNA. In these cells, the expression of NOX2 and production of reactive oxygen induced by IL-1β were significantly suppressed. On the other hand, the effect of MCT-1 knockdown was small on the IL-1β-induced expression of NOS2 and NO production. Since the IL-1β-induced expression of NOX2 in ATDC5 cells was augmented by overexpression of TRAF6 and suppressed by inihibition of NF-κB, we examined phosphorylation of I-κBα and nuclear translocation of RelA in ATDC5 cells. Phosphorylation and degradation of I-κBα was observed from 5 to 60 min after addition of IL-1β. MCT-1 knockdown marginally affected this process. Interestingly, IL-1β induced nuclear translocation of RelA at 48 h after addition of IL-1β in the control ATDC5 cells. On the contrary, this late-phase nuclear translocation of RelA was not observed in the MCT-1-knocked-down cells. While the induction of NOS2 in ATDC5 cells occurs within 3 h, that of NOX2 was detected around 48 h after IL-1β stimulation. These observations indicate that MCT-1 contributes to the late-phase activation of NF-κB in IL-1β-exposed ATDC5 cells.
Disclosures: Kentaro Yoshimura, None.
Neogenin regulates chondrogenesis by modulating BMP receptor association with lipid rafts.Zheng Zhou*1, Daehoon Lee2, Jianxin Xie2, Lijuan Zhou2, Lin Mei2, Wencheng Xiong2. 1Medical College of Georgia, Augusta, GA, USA, 2Medical College of Georgia, Augusta, USA
Neogenin, an immunoglobin family transmembrane receptor, is implicated in axon guidance, neuronal dierentiation, morphogenesis, and apoptosis. It is widely expressed in dierent tissues, including developing cartilage and osteoblast. However, its role in bone development remains largely unknown. Here we provided evidences that neogenin deficient mice showed defect in limb development, reduced endochondral ossification, impaired angiogenesis, and aberrant chondrocyte terminal dierentiation. To investigate mechanisms underlying these events, we found that neogenin is required for BMP induction of Smad1/5/8 phosphorylation, but not p38 signaling in vitro and in vivo. In addition, neogenin is also required for BMP-induced BMP receptors association with lipid rafts, where phosphorylation of Smad1/5/8, but not p38, is induced by BMP. Taken together, our results suggest that neogenin appears to be involved in the regulation of chondrogenesis and bone development through modulating BMP induced Smad1/5/8 signaling pathway.
Disclosures: Zheng Zhou, None.
Noggin Sustained Release Partially Rescues the Tgfbr2Prx1KO Joint Phenotype.Tieshi Li*1, Lara Longobardi2, Froilan Granero-Molto3, Timothy Myers4, Anna Spagnoli3. 1Chapel Hill, NC, USA, 2University North Carolina in Chapel Hill, Chapel Hill, USA, 3University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 4University of North Carolina in Chapel Hill, Chapel Hill, USA
Introduction. Noggin is one of the few genes that have been shown to be necessary in joint development. The regulatory factor(s) that determine Noggin expression in developing joints are undefined.
Purpose. In our previous studies, we have found that the Tgfbr2PRX-1KO mouse, in which the TGF-beta type 2 receptor (Tgfbr2) is conditionally inactivated in the early course of limb development, lacks the interphalangeal joints and Noggin joint expression (Spagnoli A et al, J Cell Biol 177:1105–1117, 2007). To determine the TGF-beta signaling eects mediated by Noggin in developing joints, we performed a Noggin rescue experiment in Tgfbr2PRX-1KO mice.
Methods. E14.5 autopods were dissected from Tgfbr2PRX-1KO embryos and Tgfbr2Flox/Flox control littermate mice. To achieve a sustained release of proteins into dissected autopods, we encapsulated either recombinant Noggin or BSA into hydrophobically modified glycol chitosan (HGC) nanoparticles. In vitro studies had shown that more than 80% of proteins were gradually released from HGC over 48 hours. Control and mutant autopods were injected (∼3ml volume) with Noggin-HGC (0.5mg/ml) or HGC-BSA or HGC or left un-injected. HGC was mixed with Methylene Blue to mark the injection site inside the digits. Dissected autopods were cultured for 2 days in BGJb medium. Histological sections were obtained and subjected to In situ hybridization analyses for Collagen 2, Wnt9a, Gdf5 and TUNEL staining.
Results. We found that HGC-Noggin digital implants partially rescued the joint phenotype of Tgfbr2Prx1KO. In Tgfbr2Prx1KO autopods, Noggin release led to the formation of a zone that morphologically resembled the joint interzone characterized by the segmentation of the growth plate Collagen 2 expressing cells and cell apoptosis. However, Noggin release did not restore the Wnt9a joint specific marker expression. BSA-HGC and HGC implants had not eects on Tgfbr2Prx1KO autopods and did not interfere with the skeletal development, including joints, of controls.
Conclusions. Our findings indicate that Noggin can partially rescue the Tgfbr2Prx1KO joint phenotype and support the hypothesis that Noggin inhibits chondrocyte interzone dierentiation leading to their apoptosis. Whereas Noggin has no eects on the emergence of joint marker expressing cells.
Disclosures: Tieshi Li, None.
Postnatal Deletion of the G-protein Gsα in Epiphyseal Chondrocytes inhibits Longitudinal Bone Growth.Andrei Chagin*1, Takao Hirai2, Lee Weinstein3, Min Chen4, Henry Kronenberg5. 1Massachusetts General Hospital & Harvard Medical School, Boston, MA, USA, 2Massachusetts General Hospital, Endocrine Unit, Boston, MA, 3National Institute of Diabetes & Digestive & Kidney Diseases, Potomac, MD, USA, 4National Institute of Diabetes, Digestive & Kidney Diseases, National Institutes of Health, Bethesda, USA, 5Massachusetts General Hospital, Boston, MA, USA
Patients with Albright hereditary osteodystrophy (AHO) often display brachydactyly and premature closure of the epiphyseal growth plate. AHO results from loss-of-function mutations in one copy of the GNAS gene, which encodes the α-subunit of the stimulatory G-protein (Gsα). Gsα is thought to be a primary downstream target of the PTH/PTHrP receptor (PPR). Recently we have shown that ablation of PPR from epiphyseal chondrocytes of postnatal mice induces abrupt fusion of the growth plate. In the current work we explore the role of Gsα in the growth plate of postnatal mice. For this purpose we crossed mice with collagen-2 (Col2) driven tamoxifen-inducible Cre (Col2-CreERt) with mice harboring floxed exon-1 of the Gsα gene. Ablation of Gsα exon-1 was induced by injection of tamoxifen (tarn) at 3 and 7 days of age. All mice employed in the study were harboring floxed exon-1 of the Gsα gene and injected with tarn. Col2-CreERt negative mice were used as a control. We found that ablation of Gsα in Col2-expressing chondrocytes at 3–4 days of age dramatically delayed growth of long bones. Histological analysis of long bones revealed permanent disruption of bone epiphyses and altered morphology of the growth plate. Fourteen days after tarn administration epiphyseal cartilage was narrowed in Col2-CreERt positive mice with decreased numbers of cells and extent of both the proliferative and hypertrophic layers. Only remnants of epiphyseal cartilage were observed thirty days after tarn injection. Nine days after Gsα ablation we observed decreased chondrocyte proliferation and increased dierentiation. Interestingly, we observed an increase of TUNEL-positive cells at the chondro-osseous junction while the number of TRAP-positive cells (osteo/chondroclasts) was not changed. Metatarsal bones dissected 2 days after tarn administration and cultured ex vivo displayed decreased longitudinal growth, which was prevented by administration of forskolin, an activator of adenylyl cyclase. In conclusion, we have shown that Gsα is essential for normal linear growth of young postnatal mice. Dierences in growth plate phenotype between our Gsα - inactivated mice and mice in which PPR was inactivated in a similar manner suggest that other mediators of PPR signaling may play essential roles in the biology of the epiphyseal growth plate.
Disclosures: Andrei Chagin, None.
PTH/PTHrP Receptor is Required for Maintenance of the Growth Plate in Postnatal Life.Takao Hirai*1, Andrei Chagin2, Susan Mackem3, Tatsuya Kobayashi2, Henry Kronenberg4. 1Massachusetts General Hospital, Endocrine Unit, Boston, MA, 2Endocrine Unit, Massachusetts General Hospital & Harvard Medical School, Boston, USA, 3National Institutes of Health, Bethesda, USA, 4Massachusetts General Hospital, Boston, MA, USA
PTH-related protein (PTHrP), regulated by Indian hedgehog and acting through the PTH/PTHrP receptor (PPR), is crucial for normal cartilage development. These observations suggest a possible role of PPR signaling in the postnatal growth plate; however, the role of PPR signaling in postnatal chondrocytes is unknown, because the embryonic lethality of PPR null mice does not allow examination of the physiological role of PPR signaling in postnatal chondrocytes. To overcome this issue, we have generated tamoxifen-inducible and tissue-specific PPR knockout mice [Coll2-CreERT:floxed PPR (PPRfl/fl)].
To generate Coll2-CreERT:PPRfl/fl mice, we crossed Coll2-CreEPT:PPRfl/fl mice to pppfl/fl mice. Coll2-CreERT:PPRfl/fl and PPRfl/fl control mice were treated with a single dose of 0.5 mg tamoxifen per body at postnatal day (P)3, and the eects on bone development were analyzed at P6, P7 and P10. Three days after treatment of tamoxifen, the columns of chondrocytes in the growth plate were disrupted, and ectopic hypertrophic chondrocytes expressing collagen X were found in the middle of the growth plate. Furthermore, histological analysis of the tibia of P10 mice showed that premature closure of the growth plate had already occurred in Coll2-CreERT:PPRfl/fl mice. To test the possibility that the deletion of PPR signaling could aect cell death of chondrocytes, we performed the TUNEL assay and immunohistochemical detection for cleaved caspase-3 in the growth plate. We observed TUNEL and activated caspase 3-positive cells in the columnar region of the growth plate, along with an increase in apoptotic cells at the end of the hypertrophic layer of Coll2-CreERT:PPRfl/fl at 4 days after tamoxifen administration. Simultaneous administration of a low phosphate diet, that prevents apoptosis of hypertrophic chondrocytes, prevented the disappearance of the growth plate after tamoxifen administration.
These observations suggest that PPR is required for continued survival of chondrocytes in the postnatal growth plate. Postnatal ablation of the PPR gene in chondrocytes results in premature closure of the growth plate and permanent deformity of the epiphysis. Moreover, chondrocyte apoptosis through the activation of a caspase 3 pathway may be involved in the process of growth plate closure by postnatal deletion of PPR in chondrocytes.
Disclosures: Takao Hirai, None.
Putative Mechanism of Runx2 Regulation of Col10a1 Expression and Chondrocyte Maturation In Vivo.Yaojuan Lu1, Ming Ding1, Jun Li1, Yu Song1, Valérie Valérie2, Brendan Lee3, Anna Plaas1, Qiping Zheng*4. 1Rush University Medical Center, Chicago, USA, 2University Paris 7, Paris, France, 3Baylor College of Medicine, Houston, TX, USA, 4Rush University Medical Center, Chicago, IL, USA
Until recently, the molecular mechanisms that regulate cell-specific Coll10a1 expression during chondroyte maturation remain unidentified. We have previously shown that a 4-kb proximal Col10a1 promoter containing Runx2 binding sites can only direct weak reporter activity in lower hypertrophic chondrocytes in transgenic mice (Zheng et al., 2003). More recently, we reported identification of a 90-bp cis-enhancer element within Col10a1 distal promoter that is able to direct its high-level hypertrophic chondrocyte-specific expression in vivo (Zheng et al., 2009). Interestingly, comparative sequence analysis identified two tandem repeat putative Runx2 binding sites within this enhancer region. Specific DNA/protein complex formed when DNA elements containing the putative Runx2 binding sites were used for electrophoretic mobility shift assays (EMSA) with hypertrophic MCT cell nuclear extracts. Up-regulated reporter activity was observed in in vitro transfection studies that use reporter constructs derived from these elements. We have also generated transgenic mice that over express Runx2 in hypertrophic chondrocytes by using a cell-specific Col10a1 regulatory element. Immuno-staining and histological analysis of long bone sections of one transgenic line at P1 stage showed enhanced Col10a1 expression and longer hypertrophic zone compared to wild-type littermate controls. Histological analysis of 4-week old wild type and Runx2 transgenic mice showed a decreased cartilage resorption at the frontal margins of the femoral condyles in transgenic mouse. This raises the possibility that structural abnormalities in joint anatomy can predispose it to accelerated osteoarthritis (OA) in later life. Our preliminary data suggest that Runx2 might be one of the major transcription factors that interact with the Col10a1 cis-enhancer and regulate Col10a1 expression and chondrocyte maturation in vivo. The Runx2 transgenic mouse that have enhanced Runx2 and Col10a1 expression may serve as a useful mouse model to study the biological significance of chondrocyte maturation during endochondral bone formation and the pathogenesis of osteoarthritis.
Disclosures: Qiping Zheng, None.
Reactivation of Indian Hedgehog Signaling Rescues Chondrogenesis Defects in Atf4-deficient Growth Plate Chondrocytes.Weiguang Wang*1, Na Lian2, Lingzhen li2, Florent Elefteriou3, Xiangli Yang3. 1Nashville, TN, USA, 2Vanderbilt University, Bone Research Center, Nashville, USA, 3Vanderbilt University, Nashville, TN, USA
Indian Hedgehog (Ihh) is a major regulator of chondrocyte proliferation and differentiation. Hh signaling pathways are activated through the binding of Ihh to Patched (Ptc), resulting in the inhibition of Ptc-mediated repression on Smoothened (Smo), the signaling transducer. The action of Ihh can be mimicked by the synthetic purine derivative purmorphamine, a known Smo agonist. We have previously identified that ATF4, a leucine zipper-containing transcription factor, acts as a regulator of chondrocyte proliferation and differentiation by targeting Ihh transcription. Atf4−/− mice display dwarfism and growth plate abnormalities such as decreased chondrocyte proliferation, delayed hypertrophic mineralization and reduced expression of Ihh. Quantitative RT-PCR demonstrated that expression Gli1, a transcription target of Smo, was markedly decreased in Atf4−/− mice, thus revealing a defect in Ihh signaling. Based on these observations, we hypothesized that impaired Ihh signaling in Atf4−/− mice leads to their growth defects and that reactivation of Hh signaling should rescue these defects. To test this hypothesis, we isolated limb explants from both E14 wildtype (WT) and Atf4−/− embryos and treated them for 4 days with purmorphamine (10μM) in an organ culture system. The size of purmorphamine-treated Atf4−/− limbs was significantly increased in comparison to control-treated Atf4−/− limbs over the 4 days period, to an extend that made these treated mutant limbs almost similar to the untreated-WT limbs. Histological analysis showed that the increase in size found in purmorphamine-treated Atf4−/− limbs was associated with elongated zones of proliferative chondrocytes. BrdU labeling revealed that the rate of chondrocyte proliferation increased in Atf4−/− limbs upon purmorphamine treatment. H&E staining also showed that purmorphamine treatment partially rescued the hypertrophic chondrocyte phenotypes in Atf4−/− growth plates. These results indicate that reactivation of the Hh pathway in Atf4−/− limbs can rescue the defects in chondrocyte proliferation and hypertrophy of Atf4 mutants, further reinforcing the notion that ATF4 and Ihh act in the same pathways to control limb development and long bone growth.
Disclosures: Weiguang Wang, None.
The roles of ER stress transducer BBF2H7 in chondrogenesis.Shinichiro Hino*1, Atsushi Saito2, Riko Nishimura3, Toshiyuki Yoneda3, Shiro Ikegawa4, Kazunori Imaizumi2. 1University of Miyazaki, Miyazaki, Japan, 2University of Miyazaki, Miyazaki, Japan, 3Osaka University Graduate School of Dentistry, Suita, Japan, 4Laboratory for Bone & Joint Diseases, Center for Genomic Medicine, RIKEN, Tokyo, Japan
The endoplasmic reticulum (ER) is a critical cellular compartment in which protein folding occurs prior to transport to the extracellular surface or different intracellular organelles. A number of cellular stress conditions lead to the accumulation of unfolded or misfolded proteins in the ER lumen. Eukaryotic cells can adapt to deal with unfolded proteins to avoid cellular damage. This system is termed the unfolded protein response (UPR). In mammalian cells, the three major transducers of the UPR are IRE1 (inositol-requiring 1), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6).
Recently, we identified a transmembrane transcription factor, BBF2H7, as a novel ER stress transducer. Its structure is very similar to that of ATF6. BBF2H7 is cleaved at the membrane by regulated intramembrane proteolysis (RIP) in response to ER stress. The cleaved cytoplasmic domain, which contains the basic leucine zipper (bZIP) domain, translocates into the nucleus; and then activates transcription of target genes. However, its physiological functions are still unknown.
To assess the BBF2H7 function in vivo, we generated BBF2H7 deficient mice. The mice exhibited severe chondrodysplasia and died of respiratory failure shortly after birth. The cartilage showed lack of typical columnar structure in the proliferating zone and decrease of the hypertrophic zone involving significant reduction of extracellular matrix proteins. Interestingly, the proliferating chondrocytes showed abnormal expansion of the ER containing aggregated type II collagen and cartilage oligomeric matrix protein. We identified Sec23a, a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7. When Sec23a was introduced to BBF2H7 deficient chondrocytes, the impaired transport and secretion of cartilage matrix proteins were totally restored, indicating that the BBF2H7-Sec23a pathway plays crucial roles in chondrogenesis by the activation of protein secretion. Our findings provide a novel link by which ER stress is converted to signaling for activation of ER-Golgi tracking.
Disclosures: Shinichiro Hino, None.
This study received funding from: Japan Society for the Promotion of Science KAKENHI (#19659082)
Wdr5 is Required for Endochondral Bone Formation in the Chicken Limb.Eric Zhu1, Shimei Zhu2, Sylvain Provot3, Francesca Gori*4. 1Massachusetts General Hospital/Harvard Medical School, Boston, USA, 2Massachusetts General Hospital, Boston, MA, USA, 3University of California San Francisco, San Francisco, CA, USA, 4Massachusetts General Hospital, Boston, MA, USA
In mouse and chicken Wdr5 is developmentally expressed in chondrocytes and osteoblasts. While stable expression of Wdr5 accelerates chondrocyte and osteoblast dierentiation, silencing of Wdr5, using plasmid-based RNA mediated interference, markedly inhibits osteoblast dierentiation in vitro. To address whether Wdr5 is required for endochondral bone formation in vivo, an avian Replication-Competent, ASLV LTR (RCAS) system delivering Wdr5 short hairpin RNA (shRNA) was utilized to silence Wdr5 expression throughout the developing chicken limb. High-titer retroviruses encoding four dierent shWdr5 or a scrambled sequence were injected into the right limb bud of chick embryos at E3 when mesenchymal condensation begins.
Embryos were harvested between E8.5 and E12 to evaluate chondrocyte and osteoblast dierentiation. Uninfected left limbs served as controls. Reduction of endogenous Wdr5 levels resulted in shortening of the cartilage elements and in a decrease in mineralized bone as indicated by Alizarin Red staining. Shortening of the skeletal elements was due to delayed chondrocyte maturation and osteoblast dierentiation as shown by a significant decrease in the expression of chondrocyte and osteoblast specific genes, including type X collagen, osteopontin (OP), Runx-2 and type I collagen (Col I), as assessed by in situ analyses and QRT-PCR. Because Wdr5 associates with the Runx-2 promoter and Wdr5 silencing decreases Runx-2 expression in vitro and in vivo, we investigated whether overexpression of Runx-2 would rescue the delay in chondrocyte and osteoblast maturation seen with Wdr5 silencing. To this purpose, high-titer retroviruses were used to co-express either Runx-2 with shWdr5 or GFP with shWdr5 in chicken limbs. In spite of Wdr5 silencing, by E10 the delay in osteoblast dierentiation was completely rescued in limbs where Runx-2 was co-expressed with shWdr5, as indicated by normal expression pattern of Col I and OP and by mineral deposition. By E12 the delay in chondrocyte dierentiation and in vascular invasion were also rescued in limbs where Runx-2 was co-expressed with shWdr5. Thus, these findings suggest that Wdr5 is required for proper endochondral bone formation in vivo and that Wdr5 influences this process at least in part by regulating Runx-2 expression. In addition, these results illustrate that RCAS-mediated delivery of shRNA in embryonic limbs is a powerful tool to achieve loss-of-function during endochondral bone formation.
Disclosures: Francesca Gori, None.
Ift88/polaris is required for perichondrial architecture.Courtney Haycraft*1, Mandy J Croyle2, Bradley K Yoder2, P. Darwin Bell3. 1Medical University of South Carolina, Charleston, SC, USA, 2University of Alabama at Birmingham, Birmingham, USA, 3Medical University of South Carolina, Charleston, USA
Cilia are expressed on most cells in the mammalian body including mesenchymal cells destined to differentiate into chondrocytes and osteoblasts. They are formed and maintained by a highly conserved process termed Intraflagellar Transport. Ift88/polaris is one of the core proteins of the IFT particle and its loss results in loss of cilia structure and function. Ift88/polaris null mice die midgestation and exhibit a severe pathology including defects in neural tube closure and patterning, randomized left-right axis determination and Polydactyly. Recent work has demonstrated that primary cilia play important roles in mammalian development and tissue homeostasis including roles in mechanotransduction, chemoreception and regulation of signal transduction pathways including hedgehog and Wnt. Loss of Ift88/polaris in limb mesenchyme using cre recombinase expression under the control of the prx1 limb enhancer results in severe defects in endochondral bone formation including loss of the bone collar and development of ectopic chondrocytes along the diaphysis. In the developing long bones, Ptch1 and Glil expression are lost suggesting that primary cilia are essential for Ihh signaling In agreement with their role in Shh signaling during limb and neural tube patterning. These data suggest that primary cilia play important roles in both chondrocyte and osteoblast precursors during endochondral bone formation. To begin to dissect the roles of primary cilia in the osteoblast lineage, we have generated conditional Ift88/polaris mutant mice using the col1a1 3.6kb promoter to drive cre recombinase expression (col3.6cre mutants). This cre strain was chosen to allow disruption of primary cilia in osteoblast precursors prior to differentiation without affecting expression in chodrocytes. Histological analysis of polaris/col3.6cre mutant embryos shows alterations in perichondrial architecture including disorganized cellular arrangements and variations in perichondrial width along the diaphysis. Additionally, ectopic chondrocyte-like cells are visible along the diaphysis similar to that seen in polaris/prx1cre conditional mutant mice. More detailed studies of the perichondrial architecture are ongoing including analysis of osteoblast differentiation and bone formation.
Disclosures: Courtney Haycraft, None.
Iron overload induced bone changes.Jaime Tsay1, Rhima Coleman2, Zhiwei Yang1, Robert Grady1, Patricia Giardina1, Adele Boskey2, Maria Vogiatzi*3. 1New York Presbyterian Hospital, Weill Cornell Medical College, New York, USA, 2Hospital for Special Surgery, New York, USA, 3New York Presbyterian Hospital, Weill Cornell Medical College, New York, NY, USA
Background: Iron overload is a complication of hereditary hemochromatosis and any acquired or congenital disease, such as thalassemias, that requires chronic transfusions. Osteoporosis is a frequent common problem in all these disorders. The exact role of iron in the development of osteoporosis in disorders of iron overload has not been clearly defined. To better understand this, we determined the bone abnormalities, if any, of iron overloaded (IO) wild type (WT) mice.
Methods: Two groups of WT B6/C57 mice (n=6/ group; age 2 mo) were treated with iron dextran (1mg/kg IP X weekly) vs. placebo for 2 mo. Bone mass was studied by micro-CT. Changes in bone material properties were determined by Fourier Transform Infrared Spectroscopic Imaging (FTIRI). Bone turnover was assessed by histology (staining for TRAP and procollagen I) and RT-PCR from bone extracts for genes involved in bone formation (osteocalcin, osteopontin) and resorption (RANKL, OPG, cathepsin K). Oxidative stress (ROS) was determined on bone marrow protein extract (OxiBlot Detection Kit, Chemicon Int'l, CA). Serum TNFa concentrations were measured.
Results: Compared to the placebo group i) the IO mice had increased tissue iron content (×100 and × 11 increase in liver and spleen iron content respectively) ii) trabecular and cortical bone changes by micro-CT, characterized by decreased bone volume fraction and number of trabeculae, thinner cortices and increased marrow area (figure 1) iii) Changes in material properties by FTIRI, consistent with changes in mineralization and protein composition. iv) increased resorption by histology and RT-PCR from bone extracts v) increased bone marrow protein carbonyl content, indicating increased ROS. vi) increased serum TNFa concentrations (77 + 11 vs. 168 + 22 pg/ml, IO vs. placebo p<0.001).
Conclusions: This is the first study to demonstrate that severe iron overload in WT mice results in changes in bone micro-architecture and material properties. These abnormalities are associated with an increase in bone resorption, oxidative stress and serum TNFa concentrations. The mechanisms that regulate bone remodeling in iron overload most likely involve inflammatory/ immune changes and will be the focus of further studies. Studies of iron overload in thalassemia, a disease characterized by transfusional iron overload, are underway using the th3/+ mouse model. A dose effect of iron on the bone needs to be determined.
Disclosures: Maria Vogiatzi, None.
Nephroblastoma Overexpressed Stabilizes Gremlin Transcripts in Osteoblasts.Anna Smerdel-Ramoya1, Stefano Zanotti*2, Ernesto Canalis1. 1St. Francis Hospital & Medical Center, Hartford, CT, USA, 2Saint Francis Hospital & Medical Center, Hartford, CT, USA
Nephroblastoma overexpressed (Nov) is a member of the CCN (Cyr61, Connective tissue growth factor, Nov) family of proteins. Nov inhibits osteoblastogenesis and causes osteopenia in vivo. A central mechanism of Nov action is its physical interaction with bone morphogenetic protein (BMP)-2. However, an important secondary event is the induction of gremlin, a BMP antagonist, which inhibits osteoblastogenesis and bone formation causing osteopenia in vivo. The levels of gremlin mRNA are increased in ST-2 stromal cells transduced with a retroviral vector to overexpress Nov (pLPCX-Nov) and are decreased following the down-regulation of Nov by RNA interference (RNAi) in ST-2 cells, and the inactivation of nov in osteoblasts. Nov does not regulate the transcription of gremlin and induces gremlin expression by post-transcriptional mechanisms, extending the half-life of gremlin transcripts from ∼12 h to ∼48 h. To explore possible mechanisms involved, interactions of cytosolic proteins from Nov overexpressing and control cells with specific segments of the gremlin mRNA were examined by RNA electrophoretic mobility shift assay. 200 base pair radiolabeled RNA sequences spanning the entire gremlin RNA were incubated with cytosolic extracts prepared from ST-2 stromal cells transduced with the retroviral vector pLPCX-Nov and compared to pLPCX (control) transduced cells. RNA sequences from the 5′-untranslated region (UTR) and sequences from the coding region failed to form RNA-protein complexes. Cytosolic extracts from Nov overexpressing cells enhanced the intensity of complexes formed with two RNA segments present in the gremlin 3′-UTR at +1158 to 1357 and at +1358 to 1447. Mutations of ATTA/ATA sequences present in these two regions resulted in a decrease in the intensity of the cytosolic protein-RNA complex, indicating that these elements are required for the formation of the complex. Functional assays using transient transfections of a CMV-driven c-fos gremlin 3′UTR chimeric construct demonstrated that the 3′UTR of gremlin stabilizes c-fos mRNA in transcriptionally arrested ST-2 cells extending the c-fos mRNA half-life from 5 min to ∼2 h. This was prolonged to 4 h in Nov overexpressing cells. In conclusion, Nov induces the formation of cytosolic protein-gremlin 3′UTR complexes, which play a role in the stabilization of gremlin mRNA. This explains in part the inhibitory actions of Nov on osteoblastogenesis and bone formation.
Disclosures: Stefano Zanotti, None.
Pacemaker channel HCN1 affects osteoclast function in vitro and bone remodeling in vivo.Takuya Notomi*1, Shinya Tanaka2, Hitoshi Amano3, Toshitaka Nakamura4, Masaki Noda5, Timothy Skerry6, Miyuki Kuno7. 1Tokyo Medical & Dental University, Chiyoda Tokyo, Japan, 2Saitama Medical Center, Saitama Medical University, Kawagoe, Japan, 3Showa University School of Denistry, Tokyo, Japan, 4University of Occupational & Environmental Health, Kitakyushu, Japan, 5Tokyo Medical & Dental University, Tokyo, Japan, 6University of Sheeld Medical School, Sheeld, United Kingdom, 7Osaka City University, Osaka, Japan
Hyperpolarization-activated cyclic nucleotide (HCN) modulated channels play fundamental roles in excitable cells both in the central nervous system and heart. HCNs control rhythmic activity of cardiac myocytes and neuronal firing patterns of neurons, so-called “Pacemaker channel”. Recently we found that all four HCN subtypes (HCN1–4) were expressed in bone: One HCN subtype, HCN1, was localized at the ruffled border and sealing zone of osteoclasts that is responsible for acid secretion. This raised questions as to the role of HCN1 in osteoclast function and bone remodeling. Here we investigated the physiological roles of HCN1 in bone remodeling, using HCN1 knockout mice (HCN1-KO).
Trabecular bone mass and the structural parameter of Trabecular Number were significantly lower, whereas Trabecular Separation was significantly higher, in HCN1-KO than in wild type (WT) by 26%, 21% and 36% respectively. Histomorphometiric indices of bone formation (MAR and BFR/BS) and resorption (Oc.S/BS and Oc.N/BS) were increased by 13%, 20%, 123% and 96% respectively, suggesting that bone remodeling might be impaired by high bone turnover in the absence of HCN1. The number of TRAP-positive multinucleated cells developed from bone marrow cells was greater in HCN1-KO as compared to WT. Patch clamp recordings showed that HCN1-deficiency decreased the activation rate of the hyperpolarization-activated current although the amplitude was same as WT, probably because other HCN subtypes remained intact. Following acute acidosis of osteoclasts, proton extrusion in the presence of a blocker for sodium-proton transporter, amiloride, was decreased in HCN1-KO showing HCN1-deficiency inhibited proton extrusion through Na+-independent mechanisms. In KO-osteoblasts, proliferation and alkaline phosphatase activity were increased, even though osteoblasts were not found to express HCN1. However in HCN1-KO mice, HCN3 expression was increased in osteoblasts. Overexpression of HCN3 by transfecting the gene enhanced proliferation of WT-osteoblasts as measured by total cell number.
Our findings suggested that HCN1 has important functions in bone physiology. The high bone turnover in HCN1-KO was likely to be based on cellular dysfunctions. HCN1 could modulate acid secretion and differentiation in osteoclasts. In addition, lack of HCN1 upregulated expression of HCN3 in osteoblasts. HCN3 may contribute to osteoblastic bone formation.
Disclosures: Takuya Notomi, None.
Regulation of Osteoblast Activity By Keratocan - A Member of The Small Leucine-rich Proteoglycan Family.John C Igwe1, Qi Gao2, Tiziana Franceschetti1, Winston Kao3, Ivo Kalajzic*4. 1University of Connecticut Health Center, Farmington, USA, 2University of Illinois, Chicago, USA, 3University of Cincinnati, Cincinnati, USA, 4University of Connecticut Health Center, Farmington, CT, USA
Keratocan is an extracellular matrix protein that belongs to the small leucine-rich proteoglycan family, which also includes the lumican, biglycan, decorin, and fibromodulin. Members of this family are known to play a vital role in regulating cellular processes such as proliferation, differentiation and cellular migration. The aims of this study are to evaluate the expression pattern of the keratocan within the osteoprogenitor lineage and its role in regulation of osteoblast activity and function. To elucidate differential gene expression between cells at different stages of the osteoblast lineage we completed a comprehensive analysis of their gene profiles. Selective identification of these populations was achieved by utilization of visual markers of bone lineage cells. We have developed dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by a DMP-1 promoter, while osteoblasts are identified by expression of CFP (cyan) driven by 2.3kb of the Col1a1 promoter. Results from our micro-array analysis indicated that keratocan is differentially expressed in osteoblasts with little or no expression in osteocytes. To evaluate the potential role of keratocan in osteoblast differentiation we have evaluated the bone formation in keratocan deficient mice. Histomorphometric analysis showed that homozygous knockout mice have a significant decrease in bone formation rate as well as mineral apposition rate. To assess the effects of keratocan deficiency on osteoprogenitor cell differentiation we evaluated primary calvarial clutures from keratocan deficient mice. Alkaline phosphatase and von Kossa assay of calvarial osteoblast cultures derived from keratocan−/− mice demonstrated a decrease in alkaline phosphatase activity and mineral deposition. Furthermore, Northern blot analysis of RNA derived from these cultures showed a reduction in the osteoblast differentiation markers; bone sialoprotein and osteocalcin as compared to wild type cultures. Taken together our results demonstrate a novel regulation of osteoblast function by keratocan.
Disclosures: Ivo Kalajzic, None.
Fluid Shear Initiates Early Signaling Events to Activate ERK Phosphorylation in Primary Bovine Articular Chondrocytes.William Yang1, Albert Chen2, Steven Trippel2, Randall Duncan3, Manisha Malik*3. 1University of delaware, newark, USA, 2Indiana University School of Medicine. Indianoplis, USA, 3University of Delaware, Newark, DE, USA
Osteoarthritis, a leading cause of disability in the United States, results from the degeneration of the articular cartilage that acts as the gliding and load bearing surface of joints. Physiologic levels of mechanical loading of articular chondrocytes increases the biosynthetic activity of these cells, yet the mechanical signals that regulate these anabolic actions remain to be elucidated. Here, we postulate that the anabolic response of chondrocytes to mechanical loading utilizes the calcium (Ca2+) influx - purinergic signaling pathway that results in the activation and phosphorylation of ERK. Using primary articular chondrocytes (pBACs) isolated from adult bovine carpal joints, we found that pBACs responded to 12 dynes/cm2 fluid shear with a rapid increase in [Ca2+]i and that this increase in [Ca2+]i that could be restimulated upon reapplication of fluid shear. As we have shown in osteoblasts, the influx of Ca2+ was mediated through mechanosensitive and voltage sensitive Ca2+ channels and that this Ca2+ influx induced a rapid release of ATP from pBACs. While ATP release occurred within 1 min of the onset of shear and peaked at 5 min, the release of ATP remained elevated even after 30 min of shear. As with osteoblasts, inhibition of the mechanosensitive channel with gadolinium or the L-type voltage sensitive Ca2+ channel with nifedipine completely blocked the shear-induced release of ATP. ATP binding to purinergic receptors activates numerous downstream signaling pathways, but ERK1/2 phosphorylation is known to increase the synthesis of a number of anabolic proteins in chondrocytes. Both fluid shear and addition of exogenous ATP to pBACs increased the phosphorylation of ERK1/2 in a time dependent manner. Further, hydrolysis of extracellular ATP during shear significantly abrogated this phosphorylation response. ATP binds to numerous isoforms of purinergic receptors. Using fluorescent antibodies against P2Y2 and P2X7, we found that the P2X7 receptor was highly expressed in pBACs, where as there was slight, but significant staining for P2Y2. These data suggest that one of the mechanotransduction pathways in pBACs involves the both Ca2+ influx and purinergic signaling. Elucidating these early responses, and defining those specific to chondrocytes, may improve our understanding of the process of osteoarthritis and enable the development of markers for osteoarthritis and its treatment.
Disclosures: Manisha Malik, None.
Identification and Characterization of MAPK Phosphorylation Sites in the Runx2 Transcription Factor.Chunxi Ge*1, Guozhi Xiao2, Di Jiang1, Yan Li1, Hernan Roca3, Nan Hatch4, Renny Franceschi5. 1University of Michigan School of Dentistry, Ann Arbor, USA, 2University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 3University of Michigan School of Medicine, Ann Arbor, USA, 4University of Michigan, Ann Arbor, MI, 5University of Michigan, Ann Arbor, MI, USA
Runx2 is an essential transcription factor for bone formation and osteoblast differentiation. Runx2 activity can be regulated by transcriptional and posttranscriptional mechanisms including selective proteolysis and phosphorylation. We previously reported that Runx2 is phosphorylated and activated by the ERK/MAPK pathway (Xiao et al., J Biol Chem 275:4453, 2000). To further understand how the ERK/MAPK pathway regulates Runx2 transcriptional activity, in this study we show that activated ERK can directly bind the Runt domain of Runx2 and phosphorylate S301 and S319 within the proline/serine/threonine domain. These sites are phosphorylated by activated ERK1 in vitro and in cell culture and were confirmed by LC/MS,/MS analysis using a LTQ Orbitrap XL mass spectrometer. Furthermore, we find that these sites are necessary for osteoblast-sped fie gene expression and differentiation. Introduction of S to A mutations rendered Runx2 resistant to MAPK-dependent activation and reduced its ability to stimulate osteoblast-specific gene expression after transfection into Runx2-null calvarial cells and mesenchymal cells. In contrast, S301,319E Runx2 mutants had enhanced transcriptional activity that was minimally dependent on MAPK signaling, consistent with the addition of a negative charge mimicking serine phosphorylation. To examine the function of those sites in osteoblast differentiation, wild type and S301,319A adenovirus were constructed and transduced into C3H10T1/2 mesenchymal cells. After growth for 12 d in differentiation medium, cells transduced with the S301,319A mutant Runx2 were deficient in ability to induce osteoblast differetiation markers such as OCN, BSP and ALP. In addition, the previously reported differentiation-dependent increase in Runx2 binding to the OSE2 enhancer sequence in vitro was shown to require MAPK signaling. Consistent with this result, the S301,319E Runx2 mutant had increased apparent affinity for OSE2 DNA relative to unphosphorylated Runx2 or the S301,319A mutant. These results emphasize the important role played by Runx2 phosphorylation in the control of osteoblast gene expression and provide a mechanism to explain how physiological signals acting on bone through the ERK/MAPK pathway can stimulate osteoblast-specific gene expression.
Disclosures: Chunxi Ge, None.
Osteoblast-Specific Inactivation of Stat3 Decreases Mechanical Responsiveness.Joseph Martin*1, Mark Nelson1, Xiaoguang Yu1, Ian Ferries1, Kaarthik Chandrasekhar1, Jiliang Li2. 1Indiana University-Purdue Universtiy Indianapolis, Indianapolis, USA, 2Indiana University-Purdue University at Indianapolis, Indianapolis, IN, USA
Signal Transducers and Activators of Transcription 3 (Stat3), a transcription factor expressed in many cell types, including osteoblasts and osteoclasts, is emerging as a key regulator of bone mass and strength. Stat3 mutations cause a rare human immunodeficiency disease characterized by extremely elevated levels of IgE in serum that have associated craniofacial and skeletal features, such as reduced bone mineral density and recurrent pathological fractures. Our microarray data and immunohistochemical staining using a normal rat model have shown that Stat3 mRNA and protein levels markedly increase in response to mechanical loading. In addition, as indicated by Stat3 phosphorylation in MC3T3-E1 osteoblastic cells, Stat3 activity significantly increases in response to 30 to 90 minutes fluid shear stress. In order to further study the role that Stat3 plays in bone responsiveness to loading, tissue-selective Stat3 knockout (KO) mice, in which inactivation of Stat3 occurs in osteoblasts, were generated by breeding the transgenic mice in which Cre recombinase cDNA was cloned downstream of a 3.6 or 2.3 kb fragment of the rat Col1a1 promoter (Col3.6-Cre and Col2.3-Cre, respectively) with a strain of floxed mice in which the two loxP sites flank exons 18–20 of the Stat3 gene were used. Mice engineered with bone selective inactivation of Stat3 in osteoblasts exhibited significantly lower bone mineral density (7–12%, p< 0.05) and reduced ultimate force (21–34%, p<0.01) compared to their age-matched littermate controls. The right ulnae of 16-week-old bone specific Stat3 KO mice and the age-matched control mice were loaded with peak forces of 2.5 N and 2.75 N for female and male mice, respectively, at 2 Hz, 120 cycles/day for 3 consecutive days. The bone formation response was measured using histomorphometry. Mice with inactivation of Stat3 specific in bone were significantly less responsive to mechanical loading than the control mice as indicated by decreased relative mineralizing surface (rMS/BS, 47–59%, p<0.05) and relative bone formation rate (rBFR/BS, 64–75%, p<0.001). Bone responsiveness was equally decreased in mice in which Stat3 is inactivated either in early osteoblasts (Col3.6-Cre) or in mature osteoblasts (Col2.3-Cre). We conclude that loss-of-function mutations of Stat3 decrease the osteogenic response following mechanical loading. The mechanism by which Stat3 mediates skeletal response to mechanical stimulation remains to be elucidated.
Disclosures: Joseph Martin, None.
In vivo targeted deletion of Capn4 in cells of the chondrocyte lineage impairs chondrocyte proliferative function.Aki Kashiwagi*1, Mikaela Fein1, Ernestina Schipani2, Peter Greer3, Masako Shimada4. 1Massachusetts General Hospital, Boston, USA, 2Massachusetts General Hospital & Harvard Medical School, Boston, MA, USA, 3Queen's University Cancer Research Institute, Kingston, Ontario, Canada, 4Massachusetts General Hospital, Boston, MA, USA
Calpains are Ca-dependent intracellular cysteine proteases, which include widely expressed μ- and m-calpains. Both calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit. The calpain small subunit encoded by the gene Capn4 directly binds to the intracellular C-terminal tail of the receptor for parathyroid hormone (PTH) and PTH-related peptide, and modulates cellular functions in cells of the osteoblast lineage in vitro and in vivo. To investigate a physiological role of the calpain small subunit in cells of the chondrocyte lineage in vivo, we generated chondrocyte-specific Capn4 knockout mice by mating transgenic mice expressing Cre-recombinase under the control of the collagen IIα1 promoter (Col2-Cre) with Capn4flox/flox mice. Eciency of Cre activity assessed by real-time qPCR of RNA isolated from E15.5 mutant metatarsals was about 80%. Mutant (Col.2-Cre+- Capn4flox/flox) embryos had slightly smaller skeletons. The mean total length of the mutant proliferating chondrocyte zone was shorter than that of controls (Col.2-Cre+/- Capn4flox/+) due to the significantly reduced length of the columnar proliferating chondrocyte layer (Control, 5.5 ± 0.1 vs. Mutant, 4.8 ± 0.1μm, *p<0.005), while the lengths of the periarticular and hypertrophic chondrocyte layers were indistinguishable between two groups. The shortened columnar proliferating chondrocyte layer was also observed at birth. The reduced length of the proliferating chondrocyte layer in mutant embryos could be a consequence of either decreased proliferation or increased apoptosis. Thus, proliferation of cells of the chondrocyte lineage was examined by BrdU incorporation in E15.5 and E18.5 embryos. Dierently from the periarticular region, the number of BrdU-positive cells detected in the columnar proliferating region of the proximal tibial growth plate was significantly reduced in mutants compared with controls. No significant dierence in the number of apoptotic cells determined by TUNEL staining was detected in E18.5 mutants vs. controls. In vitro analysis further revealed that deletion of Capn4 in cells of the chondrocyte lineage showed impaired cell cycle progression at G1/S transition, reduced Cyclin D1 transcription and accumulation of cell cycle proteins including cyclin D and E, and p27Kip1. Collectively, our findings provide the in vivo evidence that the calpain small subunit is essential for proper chondrocyte proliferation in embryonic growth plate.
Disclosures: Aki Kashiwagi, None.
Deletion of 25-Hydroxyvitamin D-24-hydroxylase Gene Restores Normal Skeletal Growth in Hyp Mice.Sophia Biyao Xiao*1, Xiuying Bai2, Dinghong Qiu1, David Goltzman3, Andrew Karaplis3. 1LDI, Jewish General Hospital, McGill University, Montreal, Quebec, Canada, 2Lady Davis Institute, Montreal, QC, Canada, 3McGill University, Montreal, QC, Canada
X-linked hypophosphatemic rickets (XLH) is characterized by hypophosphatemia arising from a defect in the renal reabsorption of filtered inorganic phosphorus (Pi), inappropriately low-normal serum concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels for the degree of prevailing hypophosphatemia, and defective bone mineralization. Much of our understanding of XLH has been facilitated by the availability of the murine homolog, the Hyp mouse. The 25-hydroxyvitamin D-24-hydroxylase enzyme (CYP24) is responsible for the catabolic breakdown of 1,25(OH)2D3, the active form of vitamin D. In this study, we sought to investigate the contribution of CYP24 to the deranged vitamin D metabolism observed in Hyp mice. Toward this end, we bred male Hyp mice with female Cyp24+/- mice to generate obligate heterozygotes at the Phex locus in female mice, and then crossed female Cyp24 +/-/ Hyp mice (Phex+/-) with male Cyp24+/- mice to obtain male Cyp24−/−/ Hyp mice. Mice were sacrificed around 50 days post partum and serum and tissues were procured for analysis and comparison to wild type (WT), Cyp24−/− and Hyp controls.
Genotyping of live animals around the time of weaning demonstrated that 45% of the double mutant animals died before 3 weeks of age, and another 7% died by 7 weeks of age. From 5 weeks of age onward, surviving Cyp24−/−/Hyp mice had a gross appearance similar to their Cyp24−/− littermates while being easily distinguishable from their Hyp littermates by having longer limbs and tails. Serum calcium level in these animals was similar to that of the three control groups while serum inorganic phosphorous was as low as that in Hyp littermates. Serum parathyroid hormone and 1,25(OH)2D3 levels were decreased similar to those in the Cyp24−/− littermates while on the other hand, circulating serum FGF23 levels and serum alkaline phosphatase activity were higher than in Hyp littermates. Expression of Cyp27b1 in the kidney was profoundly decreased in these animals. Importantly, ablation of Cyp24 restored the skeletal abnormalities associated with Hyp, leading to near normalization of skeletal growth and endochondral bone formation, proper mineralization of cartilage and bone, and normalization of trabecular volume.
These findings support the contention that the enzymatic activity of CYP24 partakes in the pathogenetic mechanism of Hyp resulting in the profound rachitic alterations observed in these mice.
Disclosures: Sophia Biyao Xiao, None.
Cyclic AMP-mediated processing of FGF23: Regulation by O-glycosylation.Nisan Bhattacharyya*1, Panagiota Andreopoulou2, Claudia Dumitrescu2, Hazuki Miwa3, Rachel I. Gafni2, Michael T. Collins2. 1NIDCR, NIH, Bethesda, MD, USA, 2NIDCR, NIH, Bethesda, USA, 3NIDDK, NIH, Bethesda, USA
The phosphate and vitamin D regulating hormone, FGF23, circulates in both an active intact (IFGF23) and inactive C-terminal (cFGF23) form. Processing of FGF23 is believed to involve the convertase furin and the glycosyl transferase, GALNT3. Loss of GALNT3, which is seen in the disease familial tumoral calcinosis, results in only cFGF23 in the circulation, and indicates that O-glycosylation may be important in preventing FGF23 degradation by furin. In investigating the relative levels of intact and cFGF23 in various disease states, we found that patients with fibrous dysplasia (FD) had relatively elevated levels of c- vs. iFGF23 compared to normal subjects, or subjects with tumor-induced osteomalacia, and X-linked hypophosphatemia. Since FD is caused by activating mutations in Gsα, which leads to elevated cAMP, we tested the hypothesis that cAMP regulates FGF23 processing via effects on GALNT3 and/or furin. Two different human cell-culture models, embryonic kidney HEK-293 cells and HEKF cells (a clonal HEK-293 cell-line stably overexpressing human FGF23), and a mouse osteocyte cell line, MLO-Y4, were used for this study. Cells were treated with the cAMP analogue, dibutyryl cAMP (db-cAMP), or forskolin, which stimulates adenylate cyclase to produce cAMP. Relative levels of i-vs cFGF23 was measured by Western blotting and ELISA. The relative level of glycosylation of both i- and cFGF23 fragments was assessed by Western blotting after enzymatic deglcosylation. Direct interactions of FGF23 and GALNT3 proteins were observed by co-immunoprecipitation. Furin activity was assessed by examining the effect treatments on the furin-dependent processing of TGF-β. By Western blot and ELISA analyses, both db-cAMP and forskolin resulted in an increase in the relative levels of c- vs. iFGF23, in a dose-dependent fashion. The deglycosylation experiments indicate that both iFGF23 and its C-terminus fragment(s) are glycosylated, and that this process is inhibited by db-cAMP and forskolin. Co-immunoprecipitation demonstrated a decreased level of GALNT3 is associated with FGF-23 after forskolin treatment. cAMP treatment had no effect on TGF-β processing, suggesting that cAMP-mediated FGF-23 cleavage depends on its altered O-glycosylation status. These data demonstrate FGF23 processing by glycosylation can be regulated by cAMP and suggest that glycosylation activity may be a focus of the regulation of the relative levels of active, iFGF23.
Disclosures: Nisan Bhattacharyya, None.
Deletion of PTH Rescues Phenotype of Fgf23 Null Mice.Quan Yuan*1, Despina Sitara2, Konstantina Rowald3, Michael Densmore3, Reinhold Erben4, Beate Lanske5. 1Boston, MA, USA, 2Harvard School of Public Health, Boston, MA, USA, 3Harvard School of Dental Medicine, Boston, USA, 4University of Veterinary Medicine, Vienna, Austria, 5Harvard School of Dental Medicine, Boston, MA, USA
FGF23 has been identified as the circulating phosphaturic factor associated with renal phosphate wasting leading to rickets and osteomalacia. Fgf23−/− mice exhibit a phenotype that includes growth retardation, severe hyperphosphatemia, hyerpcalcemia, and high serum 1,25(OH)2D3 levels, abnormal bone mineralization, and decreased serum PTH levels. Recent studies suggest that PTH plays an important role in the regulation of serum FGF23 levels. To investigate the role of PTH in Fgf23−/− mice, we generated Fgf23−/−/PTH−/− double mutants and compared their phenotype to the one of wild-type, Fgf23−/− and PTH−/− single knockout mice. Our initial observations were quite unexpected. Despite already low serum PTH levels in Fgf23−/− mice, complete ablation of PTH in these mice resulted in larger, heavier, and more active double mutants when compared to Fgf23−/− littermates. Biochemical analyses showed that serum phosphate levels in double mutants were even higher (20.4 ± 1.08 mg/dl) when compared to the already high serum phosphate levels in Fgf23−/− mice (14.1 ± 0.89 mg/dl). In contrast, the hypercalcemia in Fgf23−/− mice (11.6 ± 0.56 mg/dl) was normalized in double mutants (9.15 ± 0.59) and serum Ca levels resembled those found in wild-types (10.4 ± 0.38). These data suggest that extremely high serum phosphate levels alone, in absence of both PTH and high serum Ca are not as detrimental to the health of the animals, as expected. Furthermore, Alizarin staining, histological, and histomorphometric analyses of long bones of all four genotypes was performed. Our data suggest that Fgf23−/−/PTH−/− double mutants do not present with the skeletal abnormalities found in Fgf23−/− single mutants, but rather resemble wild-type or PTH−/− mice. Histological examination of soft tissues including lung, intestine, and spleen showed that emphysema and atrophy of intestine and spleen seen in Fgf23−/− mice were not observed in the double mutants. We are in the process of evaluating the expression pattern of NaPi2a and Napi2c, which might be further up-regulated due to the ablation of both Fgf23 and PTH genes, leading to extremely high serum phosphate levels in Fgf23−/−/PTH−/− double mutants at 6 weeks of age. Further characterization of these mice such as serum 1,25(OH)2D3 concentration and expression of skeletal marker genes will be performed to better understand the contribution of PTH to the Fgf23−/− phenotype.
Disclosures: Quan Yuan, None.
Regulator of G Protein Signaling (RGS)-2 Enhances Bone Anabolic Action of PTH through Inhibition of Gαq/PKC Pathway in Osteoblasts.Naoshi Ogata*1, Fumiko Yano2, Daichi Chikazu3, Ung-Il Chung4, Kozo Nakamura3, Hiroshi Kawaguchi5. 1University of Tokyo School of Medicine, Tokyo, Tokyo, Japan, 2The University of Tokyo, Tokyo, Japan, 3University of Tokyo, Tokyo, Japan, 4University of Tokyo Schools of Engineering & Medicine, Tokyo, Japan, 5University of Tokyo, Faculty of Medicine, Tokyo, Japan
Bone anabolic action of PTH is known to be regulated by the balance between the stimulatory Gαs/cAMP signal and the inhibitory Gαq/Ca2+/PKC signal in osteoblasts. The present study examined the involvement of regulator of G protein signaling (RGS)-2 which is known to bind directly to Gαq protein in the PTH action. The RGS-2 expression was shown by real-time RT-PCR to be induced in response to rhPTH (1–34) (10 nM) in cultured osteoblastic MC3T3-E1 cells. Immunoprecipitation/immunoblotting confirmed the physical association of RGS-2 with Gαq protein, but not with Gαs protein, after the rhPTH treatment. In MC3T3-E1 cells with adenoviral overexpression of RGS-2, cytosolic Ca2+ elevation and PKC activation caused by the rhPTH treatment were significantly suppressed as compared to those in MC3T3-E1 cells with the control empty vector transfection. Among the PKC isoforms, the PKCδ protein level and its subcellular translocation to cell membrane that were enhanced by the rhPTH treatment were markedly inhibited by the RGS-2 overexpression. Contrarily, the rhPTH-induced Ca2+ elevation and PKC activation were further enhanced by the knockdown of RGS-2 by the siRNA. Neither overexpression nor knockdown of RGS-2 in MC3T3-E1 cells altered the PTH-induced cAMP accumulation, indicating no involvement of RGS-2 in the Gαs/cAMP signal. Intermittent treatment of rhPTH (10 nM) in MC3T3-E1 cells stimulated osteoblastic dierentiation and bone matrix synthesis determined by ALP staining and mRNA levels of osteogenesis markers such as COL1A1, ALP, and osteocalcin. These eects were significantly enhanced in the MC3T3-E1 cells overexpressing RGS-2, which were restored by addition of PMA, an activator of PKC. Finally, radiological and histological analyses of RGS-2 deficient (-−/−-) mice showed no significant dierence in bone volume or turnover as compared to the wild-type littermates under physiological conditions. However, when rhPTH (1–34) (0.08 mg/kg × 5 times × 4 weeks) was subcutaneously injected to 8-week-old mice of the two genotypes, the increase in bone densities of the tibia and vertebrae after 4 weeks was much less in the RGS-2-−/−- mice than in the wild-type littermates, suggesting a crucial role of RGS-2 in the PTH action. These lines of results demonstrate a stimulatory act of RGS-2 in the bone anabolic action of PTH through inhibition of the Gαq/PKC signal, indicating a possible application of RGS-2 as an enhancer of PTH for a novel treatment of osteoporosis.
Disclosures: Naoshi Ogata, None.
Sympathetic nervous tone modulates constitutively active parathyroid hormone receptor signaling in vivo.Yoichi Ezura1, Hiroaki Hemmi2, Masashi Nagao3, Ryo Hanyu*4, Shu Takeda5, Takuya Notomi6, Masaki Noda7, Tadayoshi Hayata8. 1Tokyo Medical & Dental University, Medical Research Insititute, Tokyo, Japan, 2Tokyo Medical & Dental University, Chiyoda-ku, Tokyo, Japan, 3Tokyo Medical & Dental University, Chiyoda-ku, Japan, 4Tokyo, Japan, 5KEIO University, Dept. of Nephrology, Endocrinology & Metabolism, Tokyo, Japan, 6Tokyo Medical & Dental University, Chiyoda Tokyo, Japan, 7Tokyo Medical & Dental University, Tokyo, Japan, 8Medical Reserach Institute, Tokyo Medical & Dental University, Tokyo, Japan
Parathyroid hormone, PTH, has been known to be a potent anabolic agent. Sympathetic nervous tone through beta adrenergic receptor in bone cells suppresses bone formation and enhances bone resorption. Parathyroid hormone dependentanabolic action requires bone resorption activity. A crosstalk between parathyroid hormone dependent signaling in bone and beta adrenergic signaling may coexist. Therefore, we tested this hypothesis by crossing Adrb2 heterozygous knock out mice (Adrb2+/-) with PPR-tg mice where constitutively active PTH/PTHrP receptor is expressed under the control of osteoblastic specific promoter, 2.3kb fragment of type I collagen, Wild type and PPR-tg alone or Adrb2+/- alone was similar in terms of the body size. However in contrast to these three groups, double mutant mice (i.e. PPR-tg and Adrb2+/-) were small in their body size. Bone mineral density (BMD) was examined and PPR-tg mice showed higher BMD levels compared to WT. Adrb2+/-did not significantly different from the WT. However, Adrb2+/- suppressed the effects of caPPR signaling on the levels of the BMD in femora. Similar trends were observed in tibial BMD. With respect to the cortical bone volume, PPR-tg alone increased the levels compared to WT. Adrb2+/- exhibited similar cortical bone volume levels compared to WT. Although there was an increase due to PPR signaling in double mutant, the magnitude of the increase was suppressed compared to that in single PPR-tg mice. Regarding histomorphometric parameters, mineral apposition rate(MAR) was increased in PPR-tg mice compared to WT. Adrb2+/- mice showed modest increase in MAR. However, the levels of MAR in double mutant mice tended to decrease though there was no statistically significant difference compared to PPR-tg alone. Similar trends were observed for mineralizing surface as well as bone formation rate. These data indicated that b2 adrenergic receptor signaling is required for fully effective increase of the bone mass and BMD due to caPPR-tg signaling. Basal remodeling events could be activated by PTH signaling to lead to an increase in bone mass. Such basal remodeling activity may in part depend on the enhancement of bone resorption under the control of Adrb2 receptor signaling.
Disclosures: Ryo Hanyu, None.
The Constitutively Active Jansen-Type PTH/PTHrP Receptor Reduces Oxidative Stress and Inhibits Canonical Wnt Signaling In Aortic Vascular Smooth Muscle Cells.Su-Li Cheng*1, Jian Su Shao1, Richard Cohen2, Dwight Towler3. 1Washington University in St. Louis School of Medicine, St. Louis, MO, USA, 2Washington University in St. Louis, St. Louis, USA, 3Washington University in St. Louis, St. Louis, MO, USA
In type II diabetes, arteriosclerotic medial calcification accrues via mechanisms overlapping yet distinct from those of bone formation. In the male LDLR−/− mouse, high fat diets (HFD) induce “diabesity” & vascular calcification, the latter mediated in part via activation of osteogenic Msx2-Wnt signaling in arteriosclerotic vessels. While stimulating trabecular bone formation, pulsatile PTH(1–34) administration reduces aortic calcification & arterial Msx2 expression in diabetic LDLR−/− mice. To assess whether these beneficial vascular actions of PTH were tissue autonomous, we examined the effects of constitutively active Jansen's PTH/PTHrp receptor in aortic vascular smooth muscle cells (VSMC). The construct SM22-PTH1R(H223R) expresses the Jansen's receptor from the VSMC-specific 0.44 kb SM22 promoter. In transfected A7r5 rat aortic VSMCs, SM22-PTH1R(H223R) augmented transcription driven by the cAMP-response element of pCRE-LUC (luciferase reporter). By contrast, as first observed with PTH(1–34) treatment, SM22-PTH1R(H223R) suppressed Wnt3a activation of TOPFLASH, the canonical Wnt/β-catenin reporter. Suppression was dependent upon protein kinase A (PKA), since the PKA inhibitors H8 & KT5720 reversed PTH1R(H223R) inhibition of Wnt signaling. We next generated SM22-PTH1R(H223R) transgenic (Tg) mice, and confirmed human PTH1R receptor expression in Tg aorta by RT-qPCR, western blot, and immunohistochemistry. No differences in BMD, BMC, serum calcium, osteopontin, TRAP, or CTX levels were detected between wild type (WT) and Tg mice indicating that bone metabolism was not affected by this transgene. However, aortic β-Catenin protein levels, an index of canonical Wnt signaling, were decreased by 35% in Tg vs. WT sibs. Moreover, using TOPGAL reporter mice, aortic wall (−43%) & aortic valve (−79%) β-galactosidase staining was reduced in SM22-PTH1R(H223R);TOPGAL vs. non-Tg TOPGAL sibs fed HFD (mixed C57BL/6:CD1 background). Since vascular osteogenic signaling is activated by inflammation & reactive oxygen species, we examined the effects of the SM22-PTH1R(H223R) on VSMC oxidative stress. SM22-PTH1R(H223R) VSMCs produced less superoxide than WT VSMCs as quantified by dihydroethidium staining. Moreover, expression of Nox1, an important source of regulated VSMC superoxide production, was reduced by 40% in Tg vs. WT aortas. Thus, PTH/PTHrP receptor activation exerts vascular benefits in part via suppression of osteogenic Wnt signaling in aortic VSMCs.
Disclosures: Su-Li Cheng, Barnes-Jewish Hospital Foundation, 2; Merck, 5
This study received funding from: National Institutes of Health
A Humanized Model of Breast Cancer Metastasis Revealing Unique Roles for the Primary Tumor and Microenvironment.Robert Goldstein*1, Kristen Anderson2, Heenam Kwon2, Michael Rosenblatt3. 1Tufts University, Boston, MA, USA, 2Tufts University, Boston, USA, 3Tufts University School of Medicine, Boston, MA, USA
The skeletal complications of malignancy represent some of the most serious complications associated with cancer, signaling the entry of the disease into an incurable phase. Despite many recent advances in cancer research and therapy, there remains a need for a better understanding of the metastatic spread of cancer from its primary location to distant “soils” within the body. Animal models are critical to the understanding of the complexities of tumor metastasis. Through creation of an animal model that is representative of human pathophysiology, it should be possible to elucidate the genetic alterations that are required for cancer migration from orthotopic locations to distant sites. Further, clinical observations indicate that organ and stromal environments greatly influence the response of tumors to chemotherapy, suggesting that orthotopic implantation of tumor cells in animal models and human targets for metastasis can better recapitulate the process of cancer metastasis to bone observed in patients. Work in our lab has developed a humanized orthotopic injection model of breast cancer metastasis. We use SUM1315 breast cancer cells injected into the murine mammary fat pad. Subsequently, metastases to subcutaneously implanted human bone cores develop. When compared to results obtained from an intracardiac injection model of cancer metastasis to murine bone, results from our gene array studies and genetic profiling have illuminated a novel metastatic gene signature. Comparison of the expression levels of the specific genes in metastatic samples from different breast cancer cell lines (SUM1315, MDA MB 231), used in an orthotopic injection model of breast cancer metastasis using qRT-PCR, demonstrated four genes that are differentially expressed in mouse and human skeletal metastases (MMP1, HUNK, IL17BR and OPN). These findings suggest that the humanized animal model of breast cancer metastasis provides a novel, human-species specific, metastatic gene signature. Additionally, the model provides a platform to study the human metastatic microenvironment in the context of orthotopic tumor growth and dissemination. Preliminary results suggest that the human bone microenvironment can increase proliferation of breast cancer cells in vitro and in vivo and may induce genetic changes in the primary tumor. Further studies are directed at understanding the influence of human bone cells on migration and metastasis of breast cancer.
Disclosures: Robert Goldstein, None.
Fibronectin Modulates Breast Cancer Metastasis.Norman Hackl*1, Anja von Au1, Matthaeus Vasel1, Marco Cecchini2, Inaam Nakchbandi3. 1Max-Planck Institut for Biochemistry & University of Heidelberg, Heidelberg, Germany, 2University of Bern, Bern, Switzerland, 3Max-Planck Institute for Biochemistry & University of Heidelberg, Heidelberg, Germany
The extracellular matrix has been gaining in importance as it defines tumor cell behavior. Fibronectin is a matrix component that is upregulated in the pre-metastatic niche. Our aim was to delineate the role of fibronectin in the bone marrow on the establishment of breast cancer metastasis.
Deletion of fibronectin in utero results in early embryonic death. We therefore took advantage of mice carrying floxed fibronectin. Using the Mx promoter driving cre expression we deleted fibronectin in the vascular niche. The amount of fibronectin remaining in the bone marrow was diminished by >95% (p<0.001). Crossings to obtain these mice on a nude background were performed and mice were injected intracardially with 100,000 tumor cells of a bone seeking, luciferase expressing MDA line (MDA-231-B/luc+). There was a significant increase in median survival from 47 to 61 days in the conditional knockout mice (Mx-cKO) (p<0.01). There was no decrease in the number of tumor foci per mouse (CT: 7.8 vs. Mx-cKO: 4.7 foci/mouse at 3 weeks, p=0.18) suggesting that homing does not play a relevant role in improved survival. In contrast, we found a significant decrease in the size of tumor foci (9.5×106 vs. 5.0×106 photons/s at 6 weeks, p<0.05). Because fibronectin plays a key role in blood vessel formation we examined CD-31 stained tumor sections and found a significant decrease in the number of blood vessels in tumors developing in Mx-cKO mice (29% decrease, p<0.05).
Deletion of fibronectin in the osteoblastic niche using the collagen alphal(I) promoter driving cre expression resulted in a marginal increase in survival from 47 to 51 days (p=0.06, n=5 col-cKO). There was a decrease in the number of tumor foci per mouse at 3 weeks (CT: 7.8 vs. 4.0, p<0.05) that was no longer present at 4 weeks. In addition, tumor foci were markedly smaller in the col-cKO mice at 3 weeks (CT: 6.0×105 vs. col-cKO: 0.7×105 photons/s, p<0.005), but not at 6 weeks. Thus, there seemed to be an early eect on tumor development that disappeared once the tumors reached a critical size.
In conclusion, fibronectin deletion in the vascular niche results in improved survival and decreased tumor growth resulting from diminished tumor angiogenesis, while deletion of fibronectin in the osteoblastic niche appears to affect early growth only. Thus, the presence of fibronectin in the bone marrow plays a significant role in the growth of metastasic lesions in a mouse model of human breast cancer.
Disclosures: Norman Hackl, None.
Inhibition of BMP Pathway and Osteoclast Activity in Mixed Osteolytic/Osteoblastic Prostate Cancer Lesion in Bone.Mandeep Virk*1, Frank Petrigliano2, Nancy Liu2, Osamu Sugiyama3, Alaee Farhang4, David Stout2, Arion Chatziioannou2, William Dougall5, Jay Lieberman4. 1University of Connecticut Health Center, Farmington, CT, USA. 2University of California at Los Angeles, Los Angeles, USA, 3University of Connecticut, Farmington, USA, 4University of Connecticut Health Center, Farmington, USA, 5Amgen, Inc., Seattle, WA, USA
The purpose of this study was to investigate the influence of combined inhibition of osteoclasts and bone morphogenetic proteins (BMPs) on the progression of a mixed osteolytic/osteoblastic prostate cancer lesion in bone.
A total of 45 male SCID animals were injected with 5×105 C42b prostate cancer cells using a tibial injection model. C42b (Control; n=10); C42b + RANK: Fc (Osteoclasts inhibited using RANK: Fc which is a RANKL antagonist; n=10); C42b +RN (BMP inhibition achieved using ex vivo transduction of C42b cells with retrovirus expressing noggin [RN], which is a natural BMP antagonist; n=10); C42b +RANK: Fc+ RN (combined treatment; n=10); C42b+EV (empty vector control for retroviral transduction; n=5).
Radiographs, hind limb measurements, micro PET-CT, histological and histomorphometric analyses were performed to monitor the tumor growth and bony lesions.
Radiographs demonstrated mixed lytic/blastic lesions in control group tibias (C42B & C42B+EV) at 8–12 weeks. The bony lesions in C42B+RN tibias were smaller and had an osteolytic phenotype. C42b+ RANK: Fc tibias had osteoblastic lesion but without any osteolytic component. The combined treatment group tibias demonstrated smaller bony lesion in two animals and there was no osteolytic component (Fig. 1 & Table 1). Micro CT analysis of bony lesions demonstrated an overall percentage decrease in the bone volume (BV) in the implanted tibias of the control groups and C42b+RN animals at 12 weeks. However, there was net increase in BV in C42B+RANK: Fc and the combined treatment group tibias. The hind limb measurements were lowest in the combined treatment group tibias, but were statistically significant when compared to the control group animals only. FDG micro PET which correlates with cellular glucose metabolism demonstrated smallest FDG lesion sizes in the combined treatment group animals at 12 weeks. However, there were no significant differences with respect to lesion sizes among any of the study group animals. The Bone area/Tissue area (BA/TA) in bony lesions was significantly lower (p<0.05) in C42b+RN tibias when compared to control group tibias. BA/TA in C42b+RANK: Fc tibias were significantly higher when compared to the control tibias and also to combined treatment group tibias. There were abundant osteoclasts in the control groups and C42b+RN group tibias while there were virtually no osteoclasts present in RANK: Fc and the combined treatment group tibias.
Treatment with RANK: Fc alone abolished the osteolytic component whereas inhibition of BMPs with noggin suppressed the osteoblastic component in mixed lytic/ blastic lesions. Combined treatment prevented bone loss, limited the osteoblastic component and resulted in smaller tumors. These results suggest that simultaneous blockade of both osteoblastic and osteolytic pathways are necessary in order to effectively suppress the mixed metastatic prostate cancer lesions in bone.
Disclosures: Mandeep Virk, Amgen Inc, 3
This study received funding from: Amgen Inc.
Inhibition of Focal Adhesion Kinase Suppresses Breast Cancer Induced Bone Metastasis.Naito Kurio*1, Tsuyoshi Shimo2, Munenori Takaoka3, Tatsuo Okui4, Norie Yoshioka4, Nur Mohammad Monsur Hassan4, Shinji Hatakeyama5, Yoshio Naomoto3, Akira Sasaki6. 1Department of Oral & Maxillofacial Surgery, Okayama University, Okayama, Japan, 2Okayama University Graduate School of Medicine, Dentistry & Pharmaceutical, Okayama, Japan, 3Department of Gastroenterological Surgery, Transplant, & Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry & Pharmaceutical Sciences, Okayama, Japan, 4Department of Oral & Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry & Pharmaceutical Sciences, Okayama, Japan, 5Novartis Institutes for BioMedical Research, Basel, Switzerland, 6Okayama University, Okayama, Japan
INTRODUCTION: Focal adhesion kinase (FAK) is a 125-kDa non-receptor protein tyrosine kinase that plays a major role in mediating signal transduction by integrins. FAK overexpression is frequently found in invasive and matastatic cancers of breast, colon, thyroid, prostate, and oral, but its role in osteolytic metastasis has not been evaluated. Using both in vivo and in vitro approaches, we investigated whether/how inhibition of FAK autophosphorylation prevented bone metastasis by using novel FAK (FAK397) inhibitor TAE226.
METHODS: A mouse model of bone metastasis was prepared by inoculating mice with tumor cell suspensions of breast cancer cell line MDA-231 cells via the left cardiac ventricle, as described previously. Oral administration of TAE226 was carried out at a dose of 30 mg/kg every day, starting on 0 day of tumor inoculation and continued throughout the experiment. Osteolytic bone metastases were assessed by radiographs and tartrate-resistant acid phosphatase (TRAP) staining. The expression of FAK related signals were confirmed by Western blot analysis. To evaluate the effects of TAE226 on osteoclastogenesis in vitro, [3H] thymidine incorporation assay, migration assay, adhesion assay, and osteoclast formation assay were done. Furthermore, to investigate the effects of TAE226 on the function of osteoclasts, we performed actin ring formation assay and pit formation assay.
RESULTS: Treatment of mice with a TAE226 greatly decreased osteolytic bone metastasis and osteoclasts involved. TAE226 treatment increased the survival rate of mice in bone metastasis model. TAE226 also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of MDA-231 cells in vitro. TAE226 inhibited the osteoclast formation in murine pre-osteoclastic RAW264.7 cells, and actin ring and pit formation in mature osteoclasts.
DISCUSSION: FAK was critically involved in osteolytic metastasis and was activated in tumor and osteoclast formation. Thus, novel FAK inhibitor, TAE226, can be effectively used in anti-osteolytic therapy.
Disclosures: Naito Kurio, None.
Blockade of VEGFR/PDGFR Axes Abrogate Bone Metastatic Homing and Colonization by Altering Tumor-Stroma Interactions.Raäl Catena*1, Pablo Salazar-Colocho1, Diego Luis-Ravelo1, Iker Antón1, Carolina Zandueta1, Leyre Larzábal1, Alfonso Calvo2, Fernando Lecanda3. 1Center for Applied Medical Research, Pamplona, Spain, 2C, Pamplona, Spain, 3Center for Applied Medical Research, Pamplona, Spain
Bone microenvironment and tumor-stromal interactions are critical for the initiation and development of metastasis. Bone homing requires tumor cell arrest and survival, a process mediated by effective tumor-stromal and/or endothelial engagement. Maintenance of bone microenvironment homeostasis is modulated by a variety of factors. The aim of this study was to delineate the role of VEGFR/PDGFR axes in bone metastatic homing and colonization using a multi-tyrosine kinase inhibitor (SU11248). Incubation of lung A549M1 cells with SU in vitro had no effect on cell proliferation. In contrast, SU decreased cell growth of stromal ST2 cells, and bone marrow derived endothelial cells (BMEC). Direct heterotypic adhesion of tumor cells to a ST2 monolayer was significantly decreased by SU, whereas no effects were found in BMEC or HUVEC monolayers. Dierential transcriptomic analysis in cocultures revealed a marked decrease in the CD44 mRNA levels in A549M1 cells after treatment with SU. To evaluate these eects in an in vivo model of bone homing, we pretreated mice with SU two days before the murine intracardiac inoculation (i.c.) of luciferase expressing A549M1 cells. Dramatic inhibition of bioluminescence was observed in SU treated mice 4 days postinoculation. Quantification of metastatic cell number after bone marrow flushing revealed identical results suggesting an eect of SU in bone homing. To test SU eects in bone metastasis colonization, we used SU alone or in combination with the zoledronic acid (ZA) daily, 6 days after i.c. of A549 M1 cells. As expected ZA treatment showed a slight increase in survival, whereas SU or combination led to overt increase in overall survival (log Rank test p<0.0001). Bioluminescence imaging showed a moderate decrease in skeletal tumor burden in single ZA and SU treated mice, whereas marked reduction in osteolytic lesions assessed by X-Ray imaging and microCT scans was only evident in ZA treated mice (p<0.001). However, combination abrogated tumor burden (p<0.0001) and severely prevented the development of osteolytic lesions as compared to SU treated mice (p<0.0001), which correlated with the number of TRAP positive cells at tumor-bone interphase. These data suggest that disruption of VEGFR/PDGFR axes alters heterotypic tumor-stromal interactions during bone homing preventing osseous colonization. We suggest that SU in combination with ZA could be suitable for the treatment of osteolytic bone metastasis.
Disclosures: Raäl Catena, None.
Bortezomib, a proteasome inhibitor with anti-myeloma activity, stimulates osteoblast differentiation by enhancing ATF4 accumulation in osteoblastic cells.Masahiro Abe*1, Asuka Oda2, Shinsuke Kido2, Ayako Nakano2, Masahiro Hiasa2, Hiroe Amou2, Shingen Nakamura2, Kyoko Takeuchi2, Hirokazu Miki2, Kumiko Kagawa2, kenichiro Yata2, Shuji Ozaki2, Itsuro Endo2, Toshio Matsumoto2. 1University of Tokushima, Tokushima, Japan, 2University of Tokushima, Tokushima, Japan
Bortezomib, a proteasome inhibitor with anti-multiple myeloma (MM) activity, has been shown to have anabolic actions to resume bone formation in patients with MM. Bortezomib was found to enhance Runx2/cbfal expression and activity in mesenchymal stem cells to induce early osteoblast (OB) differentiation. Proteasome inhibition enhances ER stress or unfolded protein response, causing an accumulation of a variety of proteins to modulate cellular differentiation and functions. Among ER stress proteins, activating transcription factor 4 (ATF4) plays a critical role in OB differentiation. ATF4 is expressed in osteoprogenitors and pre-OBs following Runx2, and acts in concert with Runx2 to facilitate terminal maturation of OBs. However, it is unknown whether a change in ATF4 protein level plays any role in OB differentiation induced by proteasome inhibition. In the present study, we therefore explored the role of ATF4 in OB differentiation by proteasome inhibition in Runx2-expressing OB lineage cells. Bortezomib dose-dependently increased ATF4 protein levels in primary bone marrow stromal cells, ST-2 cells and MC3T3-E1 cells at concentrations higher than 10 nM. Because Cmax of serum bortezomib after single injection is around 100 nM, bortezomib treatment of MM patients is expected to cause an accumulation of ATF4 protein in OB lineage cells. Similar effects of bortezomib on ATF4 protein accumulation in OB lineage cells were observed in the presence of MM cell conditioned medium (CM). Bortezomib enhanced osteocalcin promoter activity and BMP-2-induced mineralized nodule formation in MC3T3-E1 cells, and these effects of bortezomib were suppressed by ATF4 siRNA. Similar effects were observed using another proteasome inhibitor, MG132. These results demonstrate that bortezomib treatment inhibits the degradation of ATF4 by blocking proteasomal degradation of ATF4, and suggest that the effect of bortezomib on OB differentiation is mediated via an accumulation of ATF4 protein in OB lineage cells. We have previously demonstrated that OB differentiation is suppressed in MM bone lesions, and that differentiated OBs suppress MM cell growth and survival. Thus, resumption of bone formation by bortezomib may further suppress MM cell growth in concert with its direct anti-MM actions.
Disclosures: Masahiro Abe, None.
EphB4 and ephrinB2 are underexpressed in osteoprogenitors from myeloma patients and activation of the EphB4/ephrinB2 axis affects myeloma bone disease, angiogenesis and tumor growth.Angela Pennisi*1, Wen Ling2, Xin Li2, Sharmin Khan2, Shmuel Yaccoby1. 1University of Arkansas for Medical Sciences, Little Rock, AR, USA, 2University of Arkansas for Medical Sciences, Little Rock, USA
Induction of osteolytic bone lesions in myeloma (MM) is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. In addition to role in cell adhesion, repulsion and neovascularization, bidirectional signaling between the cell surface molecules ephrinB2 and EphB4 also mediates the coupling between osteoblasts and osteoclasts. While osteoblasts express the ligand ephrinB2 and its receptor, EphB4, osteoclasts mainly express EphrinB2. Forward signaling in mesenchymal stem cells (MSCs) promotes osteogenic differentiation and reverse signaling in osteoclast precursors inhibits their differentiation. The aims of the study were to investigate whether the ephrinB2/Eph4 axis is dysregulated in MM osteoprogenitors and whether activation of this axis by ephrinB2-Fc or EphB4-Fc affects MM bone disease, angiogenesis and tumor growth. Gene expression was analyzed by qRT-PCR and microarray, and protein levels were determined by immunohistochemistry, ELISA or flow cytometry. Gene and protein expression of EFNB2 (encoding ephrinB2) and EphB4 were significantly lower in patient MSCs (n=13) than healthy donor MSCs (n=5). Wnt3a induced upregulation of EPHB4 but elicited no change in expression of EFNB2 in patient MSCs, suggesting that EPHB4 is a Wnt signaling target gene. MM cells expressed low to undetectable levels of these genes and did not respond to ephrinB2-Fc or EphB4-Fc in vitro. We exploited our SCID-hu system for primary MM to study the consequences of activation of forward signaling by EphrinB2-Fc or reverse signaling by EphB4-Fc on MM. SCID-hu mice engrafted with MM cells from a patient with active MM were locally treated with Fc (control), EphrinB2-Fc or EPHB4-Fc (5 mice/group) for 4 weeks. BMD of the implanted bone was reduced in the Fc group and increased by EphrinB2-Fc and EPhB4-Fc from pretreatment levels. EphB4-Fc inhibited MM growth, osteoclastogenesis, and angiogenesis and stimulated osteoblastogenesis and bone formation whereas ephrinB2-Fc stimulated angiogenesis, osteoblastogenesis, and bone formation but had no effect on osteoclastogenesis and MM growth. Our study suggests that downregulation of EphrinB2 and EhpB4 in MM MSCs contributes to their impaired osteogenesis and that treatment with EphrinB2-Fc or EphB4-Fc helps restore coupling of bone remodeling in myelomatous bones. The results also indicate that EphB4-Fc treatment is an effective approach to inhibit MM bone disease and indirectly suppress tumor growth.
Disclosures: Angela Pennisi, None.
Identification of Receptor Tyrosine Kinase-Like Orphan Receptor 2 (ROR2) / WNT5B Signaling as a Therapeutic Target against Osteosarcoma.Kazuhito Morioka*1, Koichi Matsuda2, Hirotaka Kawano1, Kozo Nakamura1, Hiroshi Kawaguchi1, Yusuke Nakamura2. 1Sensory & Motor System Medicine, University of Tokyo, Tokyo, Japan, 2Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Molecular therapies against breast cancer and leukemia have recently been developed and are now in clinical use. Aiming at establishment of a molecular therapy against osteosarcoma, we sought to identify the therapeutic target molecule by investigating the gene expression profile using cDNA microarray analysis. From the overall expression patterns of 23,040 genes, 94 genes were up-regulated commonly in 27 osteosarcoma samples that were excised for biopsy before treatment, as compared to normal human mesenchymal cells. Among them, we selected 14 genes that were confirmed to be expressed highly in osteosarcoma samples but little in normal tissues by semiquantitative RT-PCR analysis. Further immunohistochemical and Northern blot analyses have identified a membrane protein receptor tyrosine kinase-like orphan receptor 2 (ROR2) as the most specific molecule that showed high expression in osteosarcoma but no expression in normal vital organs like heart, lung, liver and kidney. Knockdown of ROR2 expression with the siRNA in osteosarcoma cell lines U20S and Saos-2 caused suppression of cell proliferation and invasive ability determined by MTT assay and matrigel invasion assay, respectively. In contrast, the ROR2 overexpression in non-neoplastic normal cell lines COS-7 and NIH3T3 caused enhancement of the cell invasive ability. Since the extracellular portion of ROR2 has a cysteine rich domain resembling that of Frizzled, a receptor of WNT, we then examined the involvement of WNT proteins as possible ligands of ROR2. Among the 19 WNT family members examined by semiquantitative RT-PCR analysis, WNT5B showed the most specific expression in the osteosarcoma samples and the pattern was similar to that of ROR2. Co-immunoprecipitation analysis has confirmed WNT5B binding to the extracellular portion of ROR2. The stimulated cell invasive ability of the ROR2-overexpressed COS-7 cells was further enhanced by adding the conditioned medium from WNT5B-transfected HEK293 cells. In addition, ROR2 and WNT5B showed synergistic effects on the canonical WNT signaling in HEK293 cells transfected with TOPFLASH plasmid containing TCF/LEF-sensitive luciferase reporter gene. In conclusion, the present study succeeded in identification of the signal of ROR2 and its functional ligand WNT5B as a promising therapeutic target for osteosarcoma. This might lead not only to establishment of the first molecular therapy against this condition, but also to development of the diagnostic biomarkers.
Disclosures: Kazuhito Morioka, None.
This study received funding from: Japan Society for the Promotion of Science and Ministry of education, culture, sports, science and technology of Japan
Pharmacological Inhibition of Signaling Through the Activin Receptor Type IIa Heals Osteolytic Lesions and Prevents Anemia Following Chemotherapy.Aaron Mulivor*1, Denise Barbosa2, Ravi Kumar2, Amelia Pearsall2, Kathryn Underwood2, Travis Monnell2, Je Ucran2, Jasbir Seehra3, R. Scott Pearsall1. 1Acceleron Pharma, Cambridge, MA, USA, 2Acceleron Pharma, Cambridge, USA, 3Acceleron Pharma Inc, Cambridge, MA, USA
Skeletal metastases and the associated osteolytic lesions plague cancer patients with bone pain, increased risk of adverse skeletal related events, and poor prognosis. Common therapies for metastatic cancer utilize a chemotherapeutic agent to kill these tumors but do not provide a means to help the bone repair and can render the patient anemic. We have previously demonstrated that treatment with a soluble form of the extracellular domain of the activin type IIA receptor (ActRIIA) fused to a murine IgG-Fc fragment (RAP-011), can restore bone mass in ovariectomized mice with established bone loss. Additionally, the use of the human analog ACE-011 has been shown to aect bone biomarkers and erythrocytes (RBCs) in clinical studies.
Here we demonstrate that RAP-011 can restore bone loss incurred in an osteolytic model of skeletal metastases and replenish RBCs after chemotherapy. To investigate the ability of RAP-011 to restore bone in osteolytic lesions, bioluminescent human breast cancer cells (MDA-MB-231-luc, 5×105 cells) were administered by intra-tibial injection into athymic nude mice. Tumors were implanted and allowed to grow for 14 days with tumor burden being measured weekly by in vivo imaging. Mice then received Paclitaxel (20 mg/kg I.P. 3x/week) treatment to limit tumor growth for the duration of the study. On study day 42, following 4 weeks of Paclitaxel treatment, mice were split into 2 groups of equal tumor burden and osteolytic damage. One group received RAP-011 (10 mg/kg, S.C., b.i.w.) and the other vehicle (VEH). MicroCT scanning of the injected tibia on study days 42 and 60 showed RAP-011 treated mice had fewer lesions at day 60 than day 42 (−43%, P<0.001) whereas the VEH treated mice had more (+28%, P<0.05) and larger (+170%, P<0.001) lesions. At day 60 the RAP-011 treated mice had a reduced tumor burden relative to the VEH treated mice (−49%, P<0.001) suggesting an anti-tumor eect in addition to, or as a result of the anabolic bone growth eect. Furthermore, after 7 days of treatment, RAP-011 treated mice demonstrated a significant increase in RBC parameters (hematocrit + 10%, P<0.001) compared to VEH.
These data suggest that use of a soluble ActRIIA receptor could improve osteolytic bone lesions and reduce anemia in patients receiving chemotherapy treatment for metastatic breast cancer. Towards this end, ACE-011, the human analog of RAP-011, is currently in clinical development for the treatment of cancer-related bone loss.
Disclosures: Aaron Mulivor, Acceleron Pharma, 3
The Zn, p38 and TBS Domains Present in p62 are Critical for Marrow Stromal Cells Support Myeloma Cell Growth and Osteoclast Formation.Fumito Ishizuka*1, Noriyoshi Kurihara2, Jolene Windle3, G. David Roodman4. 1Center for Bone Biology, University of Pittsburgh, Pittsburgh, PA, USA, 2Center for Bone Biology, University of Pittsburgh, Pittsburgh, USA, 3Virginia Commonwealth, Richmond, USA, 4Center for Bone Biology, University of Pittsburgh, VA Pittsburgh Healthcare System, Pittsburgh, USA
We have recently reported that sequestosome 1 (p62) plays a critical role in the formation of signaling complexes that result in NF-κB, p38 MAPK, and PI3k activation in the marrow microenvironment of patients with multiple myeloma (MM) and that p62 is a potential therapeutic target for MM. In contrast to treating patients with inhibitors of multiple specific signaling pathways activated in marrow stromal cells by MM cells such as NF-κB or p38 MAPK, blocking the activation of NF-κB and p38 MAPK mediated by p62 should have a more focused eect on the bone marrow microenvironment. Therefore, the goal of this study was to identify the domains of p62 responsible for increased osteoclast (OCL) formation and MM cell growth mediated NF-κB and p38 MAPK signaling in marrow stromal cells when they interact with myeloma cells, and develop inhibitory peptides as potential therapeutic agents that interfere with p62's role in these signaling complexes. To pursue this objective, we transfected p62−/− and wild type (WT) stromal cells with p62 deletion constructs and assessed their eects on NF-κB and p38 MAPK signaling induced by MM cells or TNF-α. We have shown that p62−/− stromal cells do not support MM growth or OCL formation to the same degree as WT stromal cells. We designed p62 constructs that progressively deleted specific functional domains of p62 including: (1) p62SDLPB1 (83–434)(lacks p56lck, SH2 and PKC interacting domain), (2) p62ΔPB1 and ZZ (164–434) (lacks the PB1, aPKC and Zn finger domains that bind RIP-1) and (3) p62ΔPB1, ZZ, and TBS (252–434) (lacks PB1, aPKC, ZZ, p38 MAPK and TBS (the TRAF6 binding) domains. We then co-cultured MM1.S cells or normal CFU-GM as a source of OCL precursors with p62−/− or WT murine stromal cells transfected with the dierent p62 cDNA deletion constructs. The p62−/− stromal cells transfected with p62ΔPB1 and ZZ or p62ΔPB1, ZZ and TBS only supported myeloma growth and OCL formation by 50% compared to cells transfected with full-length p62. These results provide the Zn, p38 MAPK and TRAF-6 domains present in p62 are critical for marrow stromal cell support of MM cell growth and OCL formation.
Disclosures: Fumito Ishizuka, Amgen, 5; Novartis, 5; Acceleron, 5; Celgene, 5; Novartis, 2; Novartis, 8
Ebf1: a Transcription Factor Required for Regulation of Osteoblast Development and Adipose Tissue Allocation.Jackie Fretz*1, Tracy Nelson2, Yougen Xi2, Chord Rosen3, Mark Horowitz4. 1Yale University, New Haven, CT, USA, 2Yale Medical School, New Haven, USA, 3Maine Medical Center, Scarborough, ME, USA, 4Yale University School of Medicine, New Haven, CT, USA
Early B cell factor-1 (Ebf1) is a transcription factor essential to the development of B-lymphocytes. We have previously published that mice deficient for Ebf1 exhibit markedly increased bone density with increased numbers of osteoblasts, bone formation rate, and serum osteocalcin. The bone marrow of Ebf1−/− mice is also striking in its increased marrow adiposity, which is independent of the lack of B-cells. Therefore, the purpose of this work was to characterize the lipodystrophic phenotype that accompanies the altered bone morphology of Ebf1−/− mice. While marrow adiposity was increased in the KOs compared to the WT controls, deposition of white adipose tissue to other regions of the body was severely reduced (subcutaneously 40–50%, abdominally 80–85%) while brown adipose was not aected. Transcriptional profiling of subcutaneous white adipose tissue showed a marked decrease in the expression of PPARγ and C/EBPβ transcription factors in Ebf1−/− tissue compared to WT. Consistent with the observed decrease in the body-wide white adipose tissue, circulating levels of leptin were decreased in KO animals of all ages compared to their litter-mate controls (down 65–95%). Interestingly, however, the levels of a second adipokine, adiponectin, were comparable to controls at all ages after 2 weeks of age. Serum analysis also found the KO animals to be hypoglycemic and hypotriglyceridemic. The KO animals took longer to recover following IP injection of insulin (t½>360 min) than their WT littermates (t½=270 min). Furthermore, following a glucose clallenge the KO animals reached serum glucose levels almost twice that of their WT counterparts. Measurement of circulating levels of pancreatic hormones reveled normal or reduced circulating insulin levels in the KOs, while circulating glucagon was significantly increased (up 1.7–8.5 fold). In conclusion, it appears that the Ebf1 KO animals exhibit lipodystrophy that manifests as global decreases in white adipose tissue with increased marrow adiposity. This was accompanied by persistent hypoglycemia that is not resolved by elevated circulating glucagon. The striking early increase in all bone formation parameters and the unexpected concomitant accumulation of adipocytes in the bones of Ebf1−/− mice, accompanied by the systemic decreases in white adipose accumulation identifies Ebf1 as a hereto fore-unrecognized transcription factor required for the regulation of osteoblast development and adipose tissue allocation.
Disclosures: Jackie Fretz, None.
A Novel Systems Genetic Resource for Bone Biology.Charles Farber*1, Brain B. Bennett2, Luz Orozco2, Hyun M. Kang3, Hongxiu Oi2, Emrah Kostem3, Anatole Ghazalpour4, Pingzi Wen2, Emily A. Farber2, Ani Berberyan2, Jaijam Suwanwela5, Thomas A. Drake6, Eleazar Eskin7, Aldons J. Lusis2. 1University of Virginia, Charlottesville, VA, 2Department of Medicine, David Geen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, USA, 3Department of Computer Science, University of California Los Angeles, Los Angeles, USA, 42Department of Medicine, David Geen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, USA, 5Department of Oral Biology, David Geen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, USA, 6Department of Pathology/Laboratory Medicine, David Geen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, USA, 7Department of Computer Science, University of California Los Angeles, Los Angeles, CA 90095, Los Angeles, USA
Purpose: Systems genetics is an approach that integrates large scale genomic data types (e.g. transcriptomics) with genetics to dissect the molecular basis of complex traits. In the current study we performed the first systems genetic analysis of bone by characterizing BMD and the cortical bone transcriptome in the Hybrid Mouse Diversity Panel (HMDP); a collection of 96 classical laboratory and recombinant inbred strains.
Methods: Total body areal BMD was measured in male HMDP mice (N=12/strain) using DXA at 16 weeks of age and the cortical bone (femoral diaphysis) transcriptome was quantified using Illumina DNA microarrays. We used the Ecient Mixed-Model Association algorithm to perform a genome-wide association (GWA) analysis for BMD and gene expression values using 135,000 single nucleotide polymorphisms (SNPs).
Results: We first identified cortical bone expression SNPs (eSNPs; SNPs regulating a genes expression) on a genome-wide scale. A total of 5652 significant (P≤1×10−6) eSNPs were identified. Of these, 1971 were local (eSNP and array probe were located within a 5 Mbp window) and 3681 were distant (all others). We next used this set of eSNPs to identify novel transcriptional relationships. For example, Sclerostin (Sost), an osteocyte-derived inhibitor of bone formation, was regulated by a distant eSNP on chromosome 1. In the same genomic region, tumor necrosis factor receptor superfamily, member 11a (Tnfrsf11a) and B-cell leukemia/lymphoma 2 (Bcl2), were the only genes regulated by local eSNP. Causality modeling provided strong evidence that Bcl2, and not Tnfrsf11a, was upstream and causal for the dierence in Sost expression. We also used the HMDP to inform human GWA studies. In the HMDP, a 3 Mbp region on chromosome 12 was identified as significantly associated (P≤1×10−6) with BMD. The most significant SNP in this region was located within the additional sex combs like 2 (Asxl2) gene and its human homolog (ASXL2) demonstrated evidence of association (P≤0.001) in two independent human BMD GWA studies.
Conclusions: We used a systems genetic approach to identify cortical bone eSNPs on a genome-wide scale, uncover a transcriptional relationship between Bcl2 and Sost and identify ASXL2 as novel gene aecting BMD. These data highlight the power of systems genetics for investigating the genetics of transcriptional regulation and dissecting genetically complex bone traits.
Disclosures: Charles Farber, None.
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) in rats: A potential role of abnormal innate immunity in pathogenesis of BRONJ.Ichiro Nishimura1, Akishige Hokugo*2. 1University of California, Los Angeles. Los Angeles, CA, USA, 2UCLA, Los Angeles, CA, USA
Although bisphosphonates have initially been considered to be without major side effects, there have been an increasing number of cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) attributed to these drugs. We have recently demonstrated that coincidental vitamin D deficiency plays a key role in increasing the susceptibility to develop BRONJ in rats. The aim of this study was to elucidate the pathophysological mechanism of BRONJ in this rat model. Vitamin D-deficient as well as normal rats received injections through tail vein a bolus of zoledronate [VitD(-)/ABP and ABP groups, respectively]. Maxillary molars were extracted unilaterally. Four weeks after tooth extraction, rats were subjected to micro positron emission tomography (μPET), followed by euthanasia and the maxillary bone collection. Maxillary bone was fixed and decalcified by EDTA for histological and immunohistochemical examinations. The total-RNA from oral mucosa of tooth-extraction site were prepared and assessed by PCR-Array assay. Histological evaluations of frontal sections through the second and third molars revealed partially exposed necrotic bone fragments in the VitD(-)/ABP group, which mirrored human BRONJ lesions. Necrotic bone sequestra in VitD(-)/ABP rats uniquely associated with actinomyces superinfection and pseudoepitheliomatous hyperplasia. μPET images were reconstructed using filtered back-projection and an iterative three-dimensional reconstruction technique. Standardized uptake value (SUV) calculated to allow relative comparisons between the extracted and non-extracted sites demonstrated the increased accumulation of 18F-fluorodeoxyglucose tracer at the sites of BRONJ lesions of the VitD(-)/ABP group, suggesting the sustained inflammatory reaction at the site of tooth extraction. Immunohitology demonstrated the accumulation of gammadelta T cells juxtaposing the BRONJ lesion. The expression of IL6 and TNF-alpha significantly increased in the oral mucosa of the VitD(-)/ABP group compared to the control and ABP groups. We postulate that the pathophysiological mechanism underpinning BRONJ may involve abnormal innate immunity including gammadelta T cells, which can express MHC-unrestricted cytotoxicity. Because vitamin D deficiency appears to play a role in the postulated innate immune dysregulation, and patients with acquired or inherited disorders that compromise vitamin D functions may present an increased susceptibility to develop BRONJ.
Disclosures: Akishige Hokugo, None.
Comparative phenotypic and biochemical analyses of Crtap−/− mice and patients with recessive osteogenesis imperfecta.Roy Morello*1, Dustin Baldridge2, Jennifer Lennington3, Erica Homan2, Terry Bertin2, Elda Munivez2, Ming-Ming Jiang2, Yuqing Chen2, Douglas Keene4, David Rimoin5, Deborah Krakow6, Dan Cohn5, Shawna Pyott7, Peter Byers7, MaryAnn Weis7, David Eyre8, Brendan Lee1. 1Baylor College of Medicine, Houston, TX, USA, 2Baylor College of Medicine, Houston, USA, 3Texas Children's Hospital, Houston, USA, 4Shriners Hospital for Children, Portland, USA, 5Cedars Sinai Hospital, Los Angeles, USA, 6Cedar Sinai Hospital, Los Angeles, USA, 7University of Washington, Seattle, USA, 8University of Washington Orthopaedic Research Labs, Seattle, WA,
We and others have demonstrated that null mutations in either CRTAP (Cartilage-associated protein) or LEPRE1 (coding for prolyl 3-hydroxylase 1 (P3h1), leprecan) cause lack of type I collagen prolyl 3-hydroxylation and recessive forms of osteogenesis imperfecta (OI). Crtap, P3h1, and cyclophilin B (a prolyl cis-trans isomerase) form a molecular complex in the rER that is responsible for collagen post-translational modification at specific proline residues. We have performed a comprehensive analysis of the Crtap−/− mouse phenotype, a model of recessive OI: KO mice have multiple tissue abnormalities, including in the lungs, kidneys and skin. Both the lung and kidney glomeruli showed an early onset (P10) increase in cellular proliferation by BrdU staining, suggesting an altered tissue growth rate perhaps due to abnormal matrix to cell signaling. Glomerulosclerosis with abnormal collagen deposition was also observed in some Crtap−/− glomeruli. To understand the potential collagen defects underlying these tissue abnormalities, we did a mass spectrometry survey of 3-Hyp occupancy at various proline residues in dierent types of collagen, and assessed the impact on these 3-Hyp sites in Crtap null mice. We identified additional sites where a proline is hydroxylated to become a 3-Hyp in type I, II and V collagens, and we observed that the expected 3-Hyp at two prolines in the a2(V) collagen was missing in absence of Crtap. This may explain the tissue abnormalities seen in Crtap−/− mice. Importantly, preliminary data showed an increase phosphosmad 2 staining in adult Crtap−/− skin compared to control. We also analyzed the stability of Crtap/P3H1/cyclophilin B complex in primary human skin fibroblasts from patients with CRTAP or LEPRE1 null mutations. We demonstrate both by immuno-fluorescence and western blot that LEPRE1−/− fibroblasts completely lose expression of the CRTAP protein and similarly, in CRTAP−/− fibroblasts P3H1 expression is undetectable. These data suggest that both proteins are required to form a stable complex in the rER and that lack of either CRTAP or P3H1 cause degradation of the interacting partner. We propose that CRTAP exerts widespread eects on collagen homeostasis including types V and perhaps type IV in kidney. These studies point to the requirement for prolyl 3-hydroxylation in the widespread regulation of collagen structural and signaling function and suggest a broader clinical assessment of patients with CRTAP or LEPRE1 mutations.
Disclosures: Roy Morello, None.
Expression of Increased Levels of TAFII-17 Combined with IL-6 Normal OCL Precursors Induce Formation of Osteoclasts (OCLs) that Express a Pagetic Phenotype.Yuko Hiruma1, Jolene Windle2, G. David Roodman3, Noriyoshi Kurihara*4. 1Center for Bone Biology, University of Pittsburgh, Pittsburgh, USA, 2Virginia Commonwealth University, Richmond, USA, 3VA Pittsburgh Healthcare System (646), Pittsburgh, PA, USA, 4University of Pittsburgh, Pittsburgh, PA, USA
Osteoclasts (OCL) from Paget's disease (PD) patients have a distinctive phenotype. They express measles virus nucleocapsid protein (MVNP), are hyper-responsive to 1,25-(OH)2D3 and RANKL, produce high levels of IL-6 and TAFII-17, a coactivator of VDR, and have an increased bone resorbing capacity per OCL. Importantly, targeted expression of MVNP to the OCL lineage in transgenic mice results in development of pagetic bone lesions and OCLs in these mice. TAFII-17 is induced by MVNP and results in hyper-responsivity of OCL precursors to 1,25-(OH)2D3. However, increased expression of TAFII-17 in normal OCL precursors by itself is not sucient to induce formation of pagetic OCL in vitro. It is our hypothesis that both increased VDR responsivity and high levels of IL-6 are required for PD-OCL formation. Therefore, we developed transgenic mice with TAFII-17 targeted to the OCL lineage using the TRAP promoter and characterized the eects of IL-6 on OCL formation in marrow cultures from TRAP-TAFII-17 transgenic mice and wild-type (WT) littermates. TRAP-IL-6 mice were also generated to determine the eects of increased IL-6 expression in OCL on the 1,25-(OH)2D3 sensitivity of OCL precursors. OCL precursors from TRAP-TAFII-17 mice were hyper-responsive to 1,25-(OH)2D3 and formed OCLs at very low concentrations (10−11 to 10−12M) of 1,25-(OH)2D3. Further, IL-6 enhanced OCL formation in marrow cultures from TRAP-TAFII-17 mice, and the OCLs that formed were hyper-multinucleated (22 + 6 nuclei/OCL compared to 8 + 1 in cultures lacking IL-6). Similarly, OCL precursors from TRAP-IL-6 transgenic mice produced high levels of IL-6 and were hyper-responsive to 1,25-(OH)2D3, and formed increased numbers of hyper-multinucleated OCL when treated with 1,25-(OH)2D3. In contrast, in marrow cultures from WT mice, IL-6 increased OCL formation in response to 1,25-(OH)2D3, but the OCL that formed were normal. These results suggest that increased endogenous levels of IL-6 in combination with hyper-responsivity of OCL precursors to 1,25-(OH)2D3 may be sucient to induce pagetic OCL in vivo.
Disclosures: Noriyoshi Kurihara, Novartis, 8; Novartis, 2; Celgene, 5; Acceleron, 5; Novartis, 5; Amgen, 5
Stabilization of 1,25-(OH)2D3-VDR Complex by VDR Coactivator TAFII-17 in Pagetic Osteoclast Precursor Cells.Seiichi Ishizuka*1, Noriyoshi Kurihara2, G. David Roodman3. 1Center for Bone Biology, University of Pittsburgh, Pittsburgh, PA, 2Center for Bone Biology, University of Pittsburgh, Pittsburgh, USA, 3Center for Bone Biology, University of Pittsburgh & VA Pittsburgh Healthcare System, Pittsburgh, USA
The pathogenesis of Paget's disease of bone is not fully understood. Recently, we demonstrated that transduction of measles virus nucleocapsid protein (MVNP) gene into osteoclast (OCL) precursors results in formation of pagetic OCLs, which are hyper-responsive to 1,25-(OH)2D3. Thus 1,25-(OH)2D3 hyper-responsivity results from increased expression of TAFII-17, a novel coactivator of VDR. In contrast, normal bone marrow cells are neither hyper-responsive to 1,25-(OH)2D3 nor express high levels of TAFII-17. The presence of increased levels of TAFII-17 in pagetic OCLs and the hyper-responsivity of OCL precursors to 1,25-(OH)2D3 suggest that increased levels of VDR-mediated gene transduction may be required for the development of pagetic OCLs. However, the molecular mechanisms responsible for hyper-responsivity to 1,25-(OH)2D3 by OCL precursors expressing TAFII-17 are still unclear.
We determined if stabilization of 1,25-(OH)2D3-VDR complex by TAFII-17 is in part responsible for the eects of TAFII-17 on VDR activity. Using MVNP-NIH3T3 cells and EV-NIH3T3 cells, VDR content was quantified by Western blot analysis. 1,25-(OH)2D3 blocked VDR degradation and increased VDR content in both cells. Physiological concentrations of 1,25-(OH)2D3 (10−10M) dose-dependently and markedly enhanced the stability of VDR in MVNP-NIH3T3 cells, which expressed TAFII-17. In contrast, high concentrations of 1,25-(OH)2D3 (over 10−8M) were required to enhance VDR content in EV-NIH3T3 cells, which did not express TAFII-17. Moreover, hyper-responsivity of MVNP-NIH3T3 cells to 1,25-(OH)2D3 was diminished by transfection of TAFII-17 siRNA, which also resulted in decreased VDR content. Furthermore, similar results were obtained using bone marrow cells from TRAP-MVNP and TRAP-TAFII-17 mice in which MVNP or TAFII-17 were targeted to the OCL lineage. 1,25-(OH)2D3 (10−12M to 10−7M) enhanced the stability of VDR and markedly increased VDR contents in bone marrow cells from TRAP-MVNP and TRAP-TAFII-17 mice. In contrast, high concentrations of 1,25-(OH)2D3 (10−9M to 10−6M) only slightly increased VDR contents in stromal cells, from the same TRAP-MVNP mice and TRAP-TAFII-17 mice, which did not express TAFII-17. Similarly, 10−8M to 10−6M of 1,25-(OH)2D3 slightly increased VDR stability in bone marrow cells from WT mice. These results suggest that hyper-responsivity to 1,25-(OH)2D3 in pagetic OCL precursors may in part be due to enhanced the stabilization of 1,25-(OH)2D3-VDR complex by TAFII-17.
Disclosures: Seiichi Ishizuka, Novartis, 2; Celgene, 6; Amgen, 5; Novartis, 5; Acceleron, 1; Novartis, 8
A potent stimulator of basal cyclic AMP signaling, XLαs may be involved in the pathogenesis of fibrous dysplasia and McCune-Albright syndrome.Virginie Mariot1, Cumhur Aydin2, Giovanna Mantovani3, Agnes Linglart1, Murat Bastepe*4. 1Hôpital St Vincent de Paul & INSERM U561, Paris, France, 2Massachusetts General Hospital, Harvard Medical School, Boston, MA, 3Department of Medical Sciences, University of Milan, Milan, Italy, 4Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA
McCune-Albright syndrome (MAS) is an endocrine disorder characterized primarily by hyperpigmented skin lesions, precocious puberty, and fibrous dyslasia of bone. Patients with MAS carry postzygotic heterozygous mutations of GNAS leading to constitutive cAMP signaling. GNAS encodes the α-subunit of the stimulatory G protein, which mediates receptor activated adenylyl cyclase stimulation. Also encoded by GNAS is the extra-large variant of Gsα, i.e. XLαs, which is derived exclusively from the paternal allele and can mimic Gsα in stimulating cAMP generation. The activating mutations causing MAS aect both GNAS products, but whether XLαs, like Gsα, can be involved in the pathogenesis remains unknown. Here, we investigated 11 patients with MAS and detected activating mutations of GNAS (Arg201 with respect to Gsα) in seven of the cases. In biopsy samples, the mutation was detected within the paternally expressed XLαs transcript in 3 cases, and the maternally expressed NESP55 transcript in 4 cases. Real-time RT-PCR readily detected XLαs mRNA in samples from the investigated MAS patients, although it was markedly lower than Gsα mRNA. In one patient with paternal GNAS mutation, the level of XLαs mRNA in the fibrous dysplastic bone tissue was higher than that in the surrounding healthy bone tissue. Moreover, XLαs mRNA levels were increased in tissues with the paternal activating mutation of GNAS compared to the maternal. RT-PCR also detected XLαs mRNA in murine preosteoblastic MC3T3-E1 cells and bone marrow stromal cells. When transiently expressed in Gsα and XLαs null mouse embryonic fibroblasts, XLαs demonstrated ∼2-fold higher basal and isoproterenol- induced cAMP signaling than Gsα, despite its expression level being ∼50% less than the latter. Moreover, osteoblastic cells transiently expressing a constitutively active XLαs mutant (XLαsR543H) showed markedly higher basal cAMP accumulation than cells expressing the cognate Gsα mutant (GsαR201H). Thus, XLαs is a more potent signaling protein than Gsα regarding cAMP generation, and taken together, our findings show that constitutive XLαs activity can add to the molecular pathology underlying MAS.
Disclosures: Murat Bastepe, None.
Analysis of CXC Receptor2 Knockout (CXCR2−/−) Mice by MicroCT Imaging.David S Bischo, Nalini S Makhijani, Dean T Yamaguchi. VA Greater Los Angeles Healthcare System, Los Angeles, USA
The role of CXC chemokines with the glu-leu-arg (ELR+) motif in bone formation/repair was studied in CXCR2−/− mice. ELR+ CXC chemokines are released by inflammatory cells during the initial stage of bone repair and serve as angiogenic factors. Previously we have shown that osteogenic dierentiation of hMSCs increases expression of the ELR+ CXC chemokine, CXCL8. CXCL8 signals through the CXCR2 receptor in humans. In mice CXCR2 binds the ELR+ CXC chemokines CXCL1, CXCL2, and CXCL3. To investigate the role of CXCR2 in bone, we studied male CXCR2−/− and wild-type (WT) littermates to see if CXCR2 was involved in establishing a bone phenotype. CXCR2−/− mice were significantly smaller (size/weight) with decreased bone parameters (BMD, BMC, BA) as measured by DEXA. We used 3D microtomography imaging (μCT) to characterize the bones (femur, tibia) from CXCR2−/− (n=6) and WT (n=5) mice at 18 weeks of age (Scanco μCT35). The CXCR2−/− long bones were significantly shorter than WT (femur: 14.8 vs 16.3 mm, p<0.0001; tibia: 17.2 vs 18.3 mm, p<0.0001). μCT measurements show reduced trabecular (Tb) bone volume (femur: 7.7 vs 18.3 %, p<0.0001; tibia: 5.9 vs 13.7 %, p=0.0002), reduced Tb number (femur: 4.7 vs 5.9 mm−1, p=0.0008; tibia: 3.1 vs 4.9 mm−1, p=0.0036), reduced Tb thickness (femur: 33.1 vs 42.7 μm, p<0.0001; tibia: 38.6 vs 43.4 μm, p=0.0023), and increased Tb spacing (femur: 221.8 vs 166.3 μm, p=0.0013; tibia: 377.5 vs 201.1 μm, p=0.0064). Cortical analysis also indicated reduced bone volume (femur: 52.4 vs 62.1 %, p<0.0001; tibia: 63.6 vs 72.2 %, p=0.0011), with increased marrow volume (femur: 47.4 vs 37.9 %, p<0.0001; tibia: 36.4 vs 27.8 %, p=0.0011). There was no dierence in the BMD of the trabecular bone (femur: 915 vs 932 mg HA/ccm; tibia: 968 vs 979 mg HA/ccm); although, the cortical BMD was significantly lower in the CXCR2−/− mice (femur: 1216 vs 1258 mg HA/ccm; tibia: 1170 vs 1210 mg HA/ccm). Computation of the polar moment of inertia suggests that CXCR2−/− bones are weaker than WT bones (femur: 0.3 vs 0.4 mm−4, p<0.0001; tibia: 0.086 vs 0.156 mm−4, p<0.0001). Conclusions: CXCR2−/− mice have 1) reduced weight and size; 2) decreased trabecular bone volume, number, and thickness; 3) decreased cortical bone thickness with increased marrow space suggesting an endosteal formation defect; and 4) decreased moment of inertia suggesting weaker bones. Thus CXCR2 is a determinant factor in the establishment of both trabecular and cortical bone.
Disclosures: David S Bischoff, None.
Bone Marrow Ablation Demonstrates Bone Anabolic Actions of Endogenous PTH in 25-Hydroxyvitamin D-1α-Hydroxylase Null Mice.Jun Yan1, Weiwei Sun2, David Goltzman3, Dengshun Miao*4. 1Second Hospital of Suzhou University, Suzhou, Peoples Republic of China, 2Nanjing Medical University, Nanjing, China, 3McGill University, Montreal, QC, Canada, 4Nunjing Medical University, Nanjing, Peoples Republic of China
To determine whether endogenous PTH plays a role in stimulating bone marrow production of osteogenic cells in vivo, marrow ablations were performed in tibiae and femurs of 6-week-old wild-type (WT) and 25-hydroxyvitamin D-1α-hydroxylase null [1α(OH)ase−/−] mice. 1α(OH)ase−/− mice and WT littermates were maintained on a normal diet, and 1α(OH)ase−/− mice had hypocalcemia, hypophosphatemia and high levels of endogenous PTH. Newly formed bone tissue in the diaphyseal regions were analyzed at 1 and 2 weeks postsurgery by histopathology. RNA and proteins were isolated from diaphyseal regions and bone formation related gene and protein expression levels were evaluated by real-time RT-PCR and Western blots, respectively. At 1 week postsurgery, bone formation parameters including trabecular volume, osteoblast numbers, ALP positive areas, and type I collagen positive areas were all increased significantly in 1α(OH)ase−/− mice compared to WT mice (33.1 ± 1.3 versus 22.2 ± 2.0%; 54.3 ± 3.8 versus 31.3 ± 2.6/mm2; 4.31 ± 0.27 versus 1.15 ± 0.13%; and 31.6 ± 2.1 versus 20.3 ± 2.0%, respectively). Consistent with the histomorphometric observations, gene expression levels of Cbfa1, ALP, type I collagen and OCN, and protein expression levels of Cbfa1, PTHR and IGF-1 levels were also increased significantly in 1α(OH)ase−/− mice at 1 week postsurgery. At 2 week postsurgery, although gene expression levels of ALP, type I collagen and OCN had decreased, trabecular volume, osteoblast number, ALP positive area, and type I collagen positive area were all still significantly higher in 1α(OH)ase−/− mice than in WT mice (41.8 ± 2.3 versus 11.9 ± 1.4%; 74.3 ± 5.8 versus 43.3 ± 4.1/mm2; 8.47 ± 0.73 versus 1.47 ± 0.11%; and 40.9 ± 2.2 versus 7.9 ± 0.5%, respectively). At 1 week postsurgery, TRAP positive osteoclast number and surface were reduced (11.9 ± 1.9 versus 39.3 ± 4.4/mm2; 4.0 ± 0.3 versus 7.9 ± 0.5%, respectively), however at 2 weeks postsurgery, TRAP positive osteoclast number was increased but TRAP positive osteoclast surface was still reduced in 1α(OH)ase−/− mice compared to WT mice (39.7 ± 4.2 versus 24.3 ± 3.2/mm2; 4.5 ± 0.3 versus 9.2 ± 0.3%, respectively). Consistent with the histomorphometric observations, the ratio of RANKL/OPG gene expression was reduced at 1 week postsurgery, but were increased at 2 weeks postsurgery in 1α(OH)ase−/− mice compared to WT mice. These results indicate that endogenous high PTH plays a role in bone formation by stimulating marrow osteogenic cells in vivo.
Disclosures: Dengshun Miao, None.
Fracture Healing is Accelerated in the Absence of the Adaptive Immune System.Daniel Toben*1, Ireen Schroeder2, Thaqif El Khassawna2, Manav Mehta2, Jan Erik Homann2, Jan Frisch2, Hanna Schell2, Jasmin Lienau2, Alessandro Serra3, Andreas Radbruch3, Georg Duda2. 1Charité Universitätsmedizin Berlin, Berlin, Germany, 2Julius Wol Institute & Center for Muskuloskletal Surgery, Charité Universitätsmedizin Berlin, Berlin, Germany, 3German Arthritis Research Center Berlin, Berlin, Germany
Purpose: This project was aimed at further elucidating how the host immune system participates in the process of bone regeneration following fracture. The hypothesis of this study was that fracture healing would be delayed in the absence of the adaptive immune system.
Methods: Complete absence of the adaptive immune system was modelled by using recombination activating gene 1 (RAG1) knockout mice lacking mature B and T lymphocytes. A standard closed femoral fracture was created in 8–10 weeks old wildtype (WT) and RAG1 knockout mice. For μCT analysis and biomechanical testing, animals were sacrificed after 14, 21 and 28 days (n=8/time point). For histological analysis, sections of the callus region were stained with Movat Pentachrome and the callus tissue dierentiation was evaluated by histomorphometry after 7, 14 and 21 days (n=6/time point). Statistical comparisons between the groups were performed using the Mann-Whitney U-test.
Results: Biomechanical testing demonstrated a significantly higher torsional moment at day 14 (77 (69/84) % vs. 60 (50/63) %; [median (25/75 percentile)], p=0.005) and a trend to a higher torsional moment at day 21 (105 (95/122) % vs. 87 (77/91) %, p=0.065) in the RAG1 in comparison to the WT group. μCT evaluation of RAG1 specimens showed a decrease in the total volume of the callus (TV) from day 14 to day 21. In contrast, in the WT specimens the decrease of TV was seen one week later from day 21 to day 28. At day 21, TV was significantly lower in the RAG1 compared to the WT group (21 (16/33) mm3 vs. 36 (25/46) mm3, p=0.01). Histologically, the process of endochondral bone formation and the final remodeling of the bony callus appeared to be accelerated or advanced in the RAG1 compared to the WT mice (FIG. 1). Histomorphometrical analysis at day 7 showed a significantly higher fraction of bone (12 (6/17) % vs. 4 (1/6) %, p=0.015) and a significantly lower fraction of cartilage (40 (33/46) % vs. 50 (44/60) %, p=0.041) in the callus of the RAG1 in comparison to the WT mice.
Conclusion: The results of this study have shown a significant and unexpected phenotype of enhanced bone regeneration in RAG1 knockout mice compared to WT mice suggesting that mature lymphocytes may delay the early phase of fracture healing. The morphometric and biomechanical analyses provide strong evidence for this conclusion, but further analyses are necessary to identify the underlying molecular and cellular mechanisms.
Disclosures: Daniel Toben, None.
Functional Analysis of mutant ALK2 receptors found in Fibrodysplasia Ossificans Progressiva.Toru Fukuda*1, Shoichiro Kokabu2, Satoshi Ohte3, Takenobu Katagiri2. 1Saitama Medical University, Research Center for Genomic Medicine, Hidaka-shi, Saitama, Japan, 2Saitama Medical University, Saitama, Japan, 3Division of Pathophysiology, Saitama Medical University, Research Center for Genomic Medicine, Hidaka-shi, Japan
Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disease characterized by progressive heterotopic endochondral bone formation with great-toe malformations. A c.617G>A mutation in Activin-like kinase 2 (ALK2), a BMP type I receptor, has been found in the most of patients with FOP. This mutation causes an R206H amino acid substitution within the GS domain of ALK2. We have reported that this mutation was observed in 19 Japanese patients with FOP and the mutant ALK2 receptor constitutively transduces intercellular signaling of BMP. Recently, other types of mutations were identified in the GS domain and the serine/threonine kinase domain of ALK2. These patients had distinct clinical features from classical FOP patients including severity or onset age of heterotopic ossification and severe variable reduction deficits of digits. In the present study, we examined a relation between the functional changes of ALK2 mutants and the clinical features of FOP. Transient over-expression of the mutant ALK2 in C2C12 cells induced Id1-luc activity with diverse degrees. The kinase domain mutants (R258S, G328E/R/W, G356D and R375P) tended to be weaker than that of the GS domain mutants (P197F198del, R202I, R206H and Q207E). The osteoblastic dierentiation-inducing activity determined by the induction of ALP in C2C12 myoblasts was the strongest in R206H, one of the GS domain mutants, and the weakest in the kinase domain mutants, G356D and R357P. Other mutants, such as P197F198del, R202I, Q207E, R258S and G328E/R/W, induced moderate levels of ALP activity. Interestingly, the phosphorylation levels of Smad1 induced by each ALK2 mutant were parallel with the osteoblastic dierentiation-inducing activity in C2C12 cells. Taken together, these results indicated that all of the ALK2 mutants found in FOP patients are constitutively active BMP receptors and the heterotopic bone formation may be induced by the Smad-dependent signaling. Inhibitors of the Smad phosphorylation and/or the phospho-Smads would be useful to prevent heterotopic bone formation in FOP.
Disclosures: Toru Fukuda, None.
G protein-cAMP Pathway Suppresses Embryonic Stem Cell-derived Osteoblast Differentiation at Early Stage.Shengliang Zhang*1, Meiqi Xu2, Frederick Kaplan3, Robert Pignolo4, Eileen Shore4. 1Department of Othopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, USA, 2Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Phialelphia, USA, 3University of Pennsylvania Hospital, Philadelphia, PA, USA, 4University of Pennsylvania, Philadelphia, PA, USA
Progressive Osseous Heteroplasia (POH) is a rare genetic disease in which ectopic bone is formed in skin and subcutaneous tissue with progression into deep skeletal muscle. POH patients were found to have inactivating mutations in GNAS, an imprinted gene which encodes multiple gene products such as Gsα, XLαs, 1A, and NESP. Gsα and XLαs are G protein subunits that transmit extracellular signals to intracellular signaling effectors such as cAMP and participate in cell growth and differentiation. Low levels of cAMP were detected in POH patients suggesting that the G protein signaling pathway is involved in regulating osteoblast differentiation. However, whether GNAS plays a role at early or late stages of osteogenesis and whether effects on osteogenesis are mediated through cAMP signaling or an alternative pathway remain unclear. In this study, we used mouse ES cells as a model cell differentiation system. We found that, in response to osteogenic medium containing BMP2, Gsα, XLαs and 1A mRNAs, as detected by qRT-PCR, were expressed at low levels during early stages of osteogenesis then increased at later stages of differentiation. To investigate a role for cAMP in osteoblast differentiation, forskolin, an activator of adenylyl cyclase, was used to induce cAMP levels at different time points during osteoblast differentiation. We found that forskolin treatment at early stages following addition of osteogenic medium containing BMP2 significantly suppressed the expression of the early osteogenic markers msx2, osterix, runx2, and collagen type I. Furthermore, we found that BMP signaling, as assayed by immunoblot analysis for Smad phosphorylation, was decreased immediately upon forskolin treatment, supporting that activation of the G protein-cAMP pathway may inhibit osteoblast differentiation by blocking the BMP-Smad pathway at early stages of osteoblastogenesis. These data suggest that the mechanism of GNAS inactivation in POH patients may function at early stages of osteoblast differentiation and that the mouse ES cell system can be used for further investigation of the molecular causes of ectopic osteogenesis and for investigating treatments for POH.
Disclosures: Shengliang Zhang, None.
Increase of Bone Mass in Aging Prostacyclin Deficient Mice.CHALIDA NAKALEKHA*1, CHIEKO YOKOYAMA2, HIROYUKI MIURA3, NEIL ROCHAN ALLES4, KAZUHIRO AOKI4, KEIICHI OHYA4, IKUO MORITA2. 1Tokyo Medical & Dental University, Tokyo, Tokyo, Japan, 2Dept. of Cellular Physiological Chemistry, Tokyo Medical & Dental University, Tokyo, Japan, 3Dept. of Fixed Prosthodontics, Tokyo Medical & Dental University, Tokyo, Japan, 4Dept. of Hard Tissue Engineering, Tokyo Medical & Dental University, Tokyo, Japan
Prostaglandins are one of regulatory factors which aect on bone metabolism. Prostacyclin (PGI2) is one of major products derived from arachidonic acid by cyclooxygenase and PGI2 synthase (PGIS). Resident bone cells, such as osteoblast and osteocyte, can produce prostaglandin E2 (PGE2), as well as PGI2. Several studies have reported about the role of PGE2 as one of activator in bone remodeling. Unlike PGE2, the role of PGI2 on bone metabolism has not yet been identified. Therefore, we investigated the potential eect of PGI2 on bone metabolism. We used mice with disruption of one (PGIS+/-) or both (PGIS−/−) alleles for the PGIS gene littermates to determine bone morphology in distal metaphysis region of long bones (tibia and femur). PGIS
email@example.com (PGIS+/-) animals have no detectable phenotypic changes. We performed longitudinal study from 5-week-old to 34-week-old mice using pQCT under anesthesia. After sacrifice, microCT analysis was performed in the proximal tibia, bone specimens were collected and prepared for histology and histomorphometry analysis. Serum markers of bone formation and resorption were measured by ELISA.
From pQCT study, PGIS−/− mice gradually displayed an increase of bone mass followed by aging. By microCT analysis at 34-week-old, PGIS−/− mice showed an increase in trabecular BV/TV (PGIS+/- = 7.99% vs. PGIS−/− = 17.20%, n = 8, p<0.01). Histomorphometry analysis showed that PGIS−/− mice displayed 2-fold increase in both bone formation (Tb.N, Ob.S/BS, MAR and BFR) and bone resorption parameters (N.Oc/BS and Oc.S/BS), (p<0.05) compared to PGIS+/-. Serum osteocalcin and serum C-Telopeptides (CTx) were increased in senescent PGIS−/− mice (n=6, p<0.01).
In conclusion, senescent PGIS−/− mice displayed overall increase in the levels of parameters in both bone formation and bone resorption, which suggested that PGI2 deficiency accelerates high bone turnover activity with greater increase in bone mass in aging. These data indicates that PGI2 may contribute to maintain normal bone mass and micro-architecture in mice. Our finding demonstrates for the first time that PGI2 is involved in bone metabolism in vivo.
Disclosures: CHALIDA NAKALEKHA, None.
Local and Controlled Release of Low Dose Lovastatin Improves the Bone Healing Defect Caused by Nf1 Loss-of-function in Osteoblasts.Weixi Wang*1, Jery Nyman2, Heather Moss3, Gloria Gutierrez4, Gregory Mundy5, Florent Elefteriou1. 1Vanderbilt University, Nashville, TN, USA, 2Vanderbilt University Medical Center, Nashville, TN, USA, 3Vanderbilt University, Nashville, USA, 4Vanderbilit University & Medical Center, Nashville, TN, USA, 5Oates Institute of Experimental Therapeutics, Nashville, TN, USA
Endochondral bone fracture healing is a regenerative process that restores bone properties through the formation of a cartilaginous callus that is subsequently replaced by a bony callus and remodeled to give fractured bones their original shape and function. Fracture healing can be delayed or not occur at all, leading to high morbidity and in the worst case amputation. Such healing defects (pseudoarthrosis) are observed in patients with neurofibromatosis (NF1), a congenital disorder due to mutations in NF1. We have shown in Nf1ob−/−mice that Nf1 loss-of-function in mature osteoblasts constitutively activates RAS/ERK, which stimulates ATF4 transcriptional activity and collagen synthesis, delays mineralization and increases Rankl expression and osteoclastogenesis. We thus hypothesized that NF1 pseudoarthrosis could be caused, at least in part, by uncontrolled bone turnover due to Nf1 loss-of-function in osteoblasts. To address this hypothesis, we generated stabilized distal tibia fractures in growing WT and Nf1ob−/− mice. Bone healing was quantified weekly for 4 weeks by X-rays, 3D-μCT, 3-point bending and histomorphometry. Nf1ob−/− calluses were characterized by delayed healing, as shown by increased tissue volume, decreased trabecular and cortical BV/TV and BMD, persistence of cartilaginous remnants and most importantly reduced mechanical properties compared to controls. The observation that bone surfaces of Nf1ob−/− callus were extensively covered by non-mineralized collagen and that osteoclasts were clearly excluded from these surfaces led us to the hypothesis that Nf1ob−/− bones do not heal properly because of impaired access of functional osteoclasts to calcified bone surfaces and thus impaired callus remodeling. Because lovastatin inhibited RAS/ERK activation in Nf1−/− osteoblasts through its inhibitory eect on RAS prenylation/function in vitro, we tested whether lovastatin could correct the bone healing delay in Nf1ob−/− mice. Since oral statins are metabolized by the liver causing low systemic bioavailability, Nf1ob−/− mice were treated, at the time of fracture, by a single injection of carrier or slow release lovastatin microparticles at the fracture site. This treatment significantly increased callus trabecular BV/TV and mechanical properties in treated versus non-treated Nf1ob−/− mice. Together, these results suggest that controlled and local lovastatin delivery could improve callus remodeling and healing in NF1 patients.
Disclosures: Weixi Wang, None.
Schnurri-2 (SHN-2) deficiency counteracts against bone loss induced by ovariectomy.Ryo Hanyu1, Takuya Notomi2, Tetsuya Nakamoto3, Yoshitomo Saita4, Yoichi Ezura5, Masashi Nagao*6, Tadayoshi Hayata7, Masaki Noda3. 1Tokyo Medical & Dental University, Tokyo, Japan, 2Tokyo Medical & Dental University, Chiyoda Tokyo, Japan, 3Tokyo Medical & Dental University, Tokyo, Japan, 4Dept. of Ortopedics, Juntendo Univ., Tokyo, Japan, 5Tokyo Medical & Dental University, Medical Research Insititute, Tokyo, Japan, 6Tokyo Medical & Dental University, Chiyoda-ku, Japan, 7Medical Reserach Institute, Tokyo Medical & Dental University, Tokyo, Japan
Bone loss due postmenopausal estrogen depletion increases fracture risks. After certain levels of bone loss, final bone mass levels stay at the low stage steadily. This bottom levelslevel of the low bone mass after bone loss indicates that a certain new set point for the levels of bone mass is established after estrogen deficiency. As bone remodeling determines bone mass, the contribution of the bone formation as well as bone resorption and its balance is important in understanding the etiology of low bone mass. SHN-2 deficiency in mice increases bone mass through its action on BMP signaling in osteoblasts. In addition, SHN-2 supports the action of the osteoclasts via a pathway through BMP. As SHN-2 regulates both osteoblasts and osteoclasts, the nadir levels of bone mass may be under the control of SHN-2. We therefore tested whether SHN-2 deficiency may affect the level of bone mass after ovariectomy (OVX). Both wild type (WT) and SHN-2 deficient mice were subjected to OVX. After 4 weeks, the bones were examined based on micro-CT. OVX decreased bone mass in WT as known previously. In control knockout (KO) mice (Sham operated), basal levels of bone mass were high and even after reduction due to OVX, the bone mass levels were similar to WT sham control. OVX induced estrogen deficiency was verified by similar reduction of the uterine weight in both WT and KO mice. With respect to the effects of SHN-2 on cellular activity, ex vivo mineralized nodule formation assay was conducted. This assay exhibited an increase due to OVX in WT. The basal levels of the mineralized nodule formation were suppressed in KO mice. However the levels of nodule formation in the cells from ovariectomized SHN-2 deficient mice were similar to those in WT control (Sham) mice. With respect bone resorption, osteoclast parameters in histomorphometry and deoxypyridinoline (DPD) levels were increased in WT after OVX. Control SHN-2 KO mice (Sham operated) indicated reduced base line levels and compared to WT control. Although there was an increase in bone resorption in KO mice after OVX, final levels of bone resorption were soimiarsimilar to WT control. These data indicated that despite of high turnover state and an increase in bone resorption, the lack of SHN-2 counter-acted against bone loss due to OVX . OVX. These cellular features explain why the bone mass levels ended up at comparable levels to WT even after OVX. Luciferase assay indicated that SHN2 deficiency may modulate estrogen deficiency effects on bone. In conclusion, the steady state low levels of bone mass after estrogen deficiency is are at least in part due to the presence of SHN-2 which possibly enhances more on osteoclast activity than that of osteoblasts to contribute to the increase in bone resorption. Thus, SHN-2 could be possible a target of the drug development to increase bone mass after estrogen deficiency.
Disclosures: Masashi Nagao, None.
Powerful Bivariate Genome-wide Association Analyses and Follow-up Replication Studies Identified Several Pleitropic Genes for both Obesity and Femoral Neck Geometry.Fengxiao Bu*1, Lanjuan Zhao2, Yufang Pei3, Robert Recker4, Hong-Wen Deng5. 1Creighton University, Omaha, USA, 2Creighton University, Omaha, NE, USA, 3University of Missouri, Kansas City Medical School, Kansas City, USA, 4Creighton University Osteoporosis Research Center, Omaha, NE, USA, 5University of Missouri, Kansas City Medical School, Kansas City, MO, USA
It is well known that bone and fat share some common genetic factors. Obesity phenotypes and femoral neck geometry are highly heritable. Poor femoral neck geometry is an important risk factor for hip fracture and osteoporosis. Although univariate studies suggest some genes important for either obesity or femoral neck geometry, it is unknown whether the two traits share the same pleiotropic genes. Identifying such pleiotropic genes is important for gaining insights into the molecular mechanism underlying the high genetic correlation between bone and fat. To identify such pleiotropic genes and key mechanistic links between the two groups of traits, we conducted the first bivariate genome-wide association (GWA) study. Our screen cohort is 1,000 unrelated homogeneous Caucasians. We scanned ∼500,000 SNPs to search for genes underlying co-variation of obesity phenotypes and femoral neck geometry traits. According to the covariate association p values (P<10–5) and the functional importance of genes for bone and obesity, we selected the 44 most prominent genes for our replication study. The replication analysis is conducted in 3,038 phenotyped Caucasian individuals from 1,114 families of the Framingham Heart Study. All the subjects have been genotyped using Aymetrix genome-wide 550K SNPs. Family-based association test (FBAT) model was performed for bivariate replication analysis for the selected 44 genes. As a result, significant replicated results (bivariate p<0.001) were observed in four genes: CTNNA3 (catenin alpha 3), PDE3A (phosphodiesterase 3A), INPP4B (inositol polyphosphate-4-phosphatase), and PRKCQ (protein kinase C- theta gene). SNP rs11259272, located in the intron 4 of the PRKCQ gene, achieves a biviarate p value of 2.7×10–5 for femoral neck width and body mass index. Significant bivariate p values are also observed for the SNP for other pairs of femoral neck geometry traits and obesity phenotypes (bivariate p<0.001). Interestingly, the activation of PRKCQ induced by fatty acids is associated with obesity. PRKCQ is also found to inhibit the expression of estrogen receptor alpha (ER-α). ER-α is a major receptor of estrogen, which is a well-known factor regulating both obesity and bone simultaneously. In conclusion, our results suggest four candidate genes bivariately correlated with both femoral neck geometry and obesity phenotypes. It provides important preliminary data for future functional studies in bone and obesity area.
Disclosures: Fengxiao Bu, None.
This study received funding from: NIH
Dullard, a novel phosphatase, that dephosphorylates and degrades BMP receptors, suppresses signaling in osteoblasts.Tadayoshi Hayata*1, Yoichi Ezura2, Masaki Noda3. 1Medical Reserach Institute, Tokyo Medical & Dental University, Tokyo, Japan, 2Tokyo Medical & Dental University, Medical Research Insititute, Tokyo, Japan, 3Tokyo Medical & Dental University, Tokyo, Japan
Bone morphogenetic proteins (BMP) are critical regulator of bone formation via its action on osteoblastic differentiation. BMPs also regulate early development, limb patterning, and bone formation. These activities are under the control of multiple BMP inhibitors, while full spectrum of such inhibitors has not yet been understood. Dullard encodes a phosphatase that associates with type I and type II BMP receptors and has been identified as a negative regulator of BMP signaling. Dullard dephosphorylates activated but not inactive type I BMP receptor (ALK3) and downregulates the protein level of type II BMP receptor by its internalization via caveolin pathway and the subsequent degradation by ubiquitination. Dullard is known to be required for neural development in early amphibian development. However, little is known about function of Dullard in bone formation and metabolism. In this study, we investigated involvement of Dullard in bone formation. Quantitative RT-PCR analysis revealed that Dullard is expressed at the same level in bone tissue including bone marrow-flashed out midshaft of femur and the flashed out bone marrow as well as in osteoblastic cell line MC3T3-E1. To investigate whether Dullard is involved in suppression of BMP signaling in osteoblasts, we transfected MC3T3-E1 with Dullard expression vector or siRNA against Dullard together with 7xBRE (BMP responsive elements)-luciferase construct and performed luciferase assay. As a result, overexpression of Dullard inhibited BMP signaling. On the other hand, siRNA-mediated knowckdown of Dullard mRNA augmented BMP signaling. As expression of Smad6, which encodes a BMP inhibitor, is known to be induced by BMP signal, forming a negative feedback loop, we tested whether Dullard expression is also induced by BMP in osteoblasts. Dullard expression was not induced by BMP treatment in MC3T3-E1, suggesting that Dullard is not involved in the negative feed back loop of BMP signaling. These results suggest that Dullard is a novel BMP inhibitor expressed in osteoblasts.
Disclosures: Tadayoshi Hayata, None.
Fat tissue specific expression of Sprouty1 regulates PPARy and leads to reduced body fat and increased bone mass in a transgenic model.Sumithra Urs*1, Robert Friesel2, Xuehui Yang2, Cliord Rosen3, Lucy Liaw2. 1Maine Medical Center, Scarborough, Maine, 2Maine Medical Center Research Institute, Scarborough, USA, 3Maine Medical Center, Scarborough, ME, USA
Introduction: Spry family proteins are inhibitors of tyrosine kinase signaling antagonizing the FGF signal transduction pathway. Sprouty affects craniofacial development and chondrogenesis, suggesting a potential impact on osteogenesis. However, their role in adipogenesis is unknown. FGF is an important growth factor contributing towards promoting adipogenesis. Although the preadipocytes themselves do not produce FGF1, they are reported to possess the FGF receptors emphasizing the significance of FGF signaling in the adipogenic process. FGFR1 activation by FGF1 results in activation of several other molecules including PPARg, the key transcription factor in adipocyte differentiation. In this study we evaluate the affects of Spry1 expression in fat tissue. Method: mSpry1 was conditionally expressed in mice under the FABP4 promoter (aP2) in a Cre-activated transgene. Double transgene and littermate controls were evaluated based on DEXA scanning, uCT for femurs at 8 and 16 and histology analysis. Bone marrow cultures were subject to osteoblastic and adipogenic differentiation and assayed by staining, q-RT-PCR and western blots. Results: mSpry1 activated by aP2-Cre markedly decreased the number of fat cells, and resulted in hypertrophy of adipocytes in abdominal fat. DEXA analysis revealed a corresponding increase in total bone mass in the transgenic mice. Bone marrow and adipose tissue-derived stromal cell differentiation showed enhanced osteogenesis with 50% increase in ALK-Phos and von Kossa colonies and 15% decrease in ORO stained colonies. Osteocalcin, ALK-Phos and Runx2 transcript levels were 2 fold higher after 1 week of differentiation in the Spry1 expressing mice while there was a 2 fold decrease in the PPARg2 and FABP4 transcripts under adipogenic differentiation. uCT and histology analyses for femurs at 8 and 16 weeks indicated a trend with increased cortical thickness. Further, we report Spry1 acts via inhibition of FGF to elevate expression of TAZ (80 fold), a transcriptional modulator of MSC differentiation providing a potential mechanism for understanding the plasticity of MSC between bone and fat. Conclusion: Sproutyl expression inhibits PPARg2 expression to repress adipocyte differentiation and possibly directing the MSCs towards an osteoblastic lineage. Our findings also support the theory of plasticity in lineage commitment and reciprocal relationship of bone and fat cells.
Disclosures: Sumithra Urs, None.
FGF2-activated ERK/MAP kinase pathway enhances Runx2 protein stability.Ok-Jin Park*1, Hyun-Jung Kim2, Hyun-Mo Ryoo3. 1School of Dentistry, Seoul National University, 2Seoul National University, Seoul, South Korea, 3Seoul National University, School of Dentistry, Seoul, Korea
Runx2 is a key transcription factor regulating osteoblast differentiation and skeletal morphogenesis. Runx2 expression is regulated by various osteogenic signals. Among these osteogenic signals, FGF-2 is one of the most important in vivo regulators of skeletal development and growth. Recently, the importance of MEK-ERK signaling activated by FGF/FGFR activation in the skeletal development has been demonstrated; RNA interference of MEK-ERK signaling, or MEK blocker U0126 could prevent the craniosynostosis phenotype caused by constitutive active mutant of FGFR2 knock-in mice. We previously demonstrated that ERK activation could not stimulate Runx2 gene expression but stimulates Runx2 transcriptional activity. Unfortunately, however, the molecular mechanism underlying the Runx2 activation by FGF/FGFR and/or ERK activation is still unclear. In this study, we found that the FGF2 treatment or FGFR activation increased exogenously overexpressed Runx2 protein level. The increase of the protein level is reversed by ERK inhibitors, U0126 or PD98059. On the other hand, overexpression of constitutive active form of MEK strongly increased Runx2 protein level. The increase of Runx2 protein level was quite well corresponded with the increase of Runx2 acetylation level. As the activation of ERK is directly related with Runx2 protein stabilization and Runx2 acetylation, we tried to indicate the exact phosphorylation sites in Runx2 protein. The two different C-terminal deletion constructs of Runx2 protein showed that middle 1/3 of the protein is responsible for the ERK-induced stabilization and activation. The in silico analysis of possible ERK target indicated three serine residues. The site-directed mutagenesis of each residues showed that only one serine moiety is critical for the ERK-mediated Runx2 stabilization and acetylation. In conclusion, FGF/FGFR activation of ERK MAP kinase strongly increased Runx2 protein and its acetylation level, which is clearly related with the serine residues of the central part of Runx2.
Disclosures: Ok-Jin Park, None.
Increased EFG- and PDGF-receptors signaling by activated FGF-receptor 2 contributes to pathological osteoblast function in Apert craniosynostosis.Hichem Miraoui*1, Jochen Ringe2, Thomas Haupl2, Pierre Marie3. 1Inserm U606 & University Paris Diderot, Paris, France, 2Charite University, Berlin, Germany, 3INSERM Unit 606, Paris, France
The pathogenic signaling mechanisms involved in human craniosynostosis are not fully understood. In this study we investigated the functional signaling pathways that are involved in the pathogenesis of Apert craniosynostosis induced by activating FGFR2 genetic mutations. Transcriptomic analysis in human Apert calvaria osteoblasts bearing the S252W FGFR2 mutation revealed increased EGFR and PDGFR alpha gene expression compared to normal age-matched calvaria osteoblasts. Quantitative RT-PCR and western blot analyses confirmed that EGFR and PDGFR alpha mRNA levels and protein levels were increased in Apert osteoblasts compared to control cells, thus validating the microarray analysis. Consistently, phosphorylated EGFR and PDGFR alpha levels were found to be higher than normal in Apert osteoblasts. Immunohistochemical analysis confirmed that EGFR and PDGFR alpha protein levels were abnormally increased in fused sutures from Apert patients with S252W or P253R Apert mutations. The increased PDGFR alpha and EGFR alpha expression in Apert osteoblasts was reduced by the PKC alpha inhibitor Gö6976 and by a dominant-negative PKC alpha. Apert osteoblasts showed a higher than normal basal AP-1 transcriptional activity, indicating that FGFR2 activation enhanced PDGFR alpha and EGFR expression via activation of PKC alpha and AP-1 transcriptional activation. Additionally, Apert osteoblasts exhibited decreased PDGFR alpha ubiquitination which was associated with increased phosphorylated Sprouty2 resulting in increased Sprouty2-Cbl interaction, suggesting posttranscriptional upregulation of PDGFR alpha mediated by increased Cbl-Sprouty2 interaction. Finally, we showed that EGFR and PDGFR inhibitors reduced the pathological upregulation of phenotypic osteoblast genes (Runx2, ALP, COLIAI, OC) in Apert osteoblasts. These results reveal that upregulation of EGFR and PDGFR alpha expression and signaling induced by activated FGFR2 functionally contributes to the pathological osteoblast function in Apert craniosynostosis, which may help to develop novel targeted therapeutic approaches in this severe cranial dysplasia.
Disclosures: Hichem Miraoui, None.
Sprouty2 Regulates Bone Development.Adriane Joo*1, Ashley Sanders2, Roger Long3, Wenhan Chang1, Ophir Klein4. 1University of California, San Francisco, San Francisco, CA, USA, 2University of California, San Francisco, San Francisco, USA, 3University of California, Davis, Department of Medicine, Division of Endocrinology, Sacramento, USA, 4UCSF, San Francisco, CA, USA
Skeletal development is regulated by the cooperative activity of signaling molecules produced locally by cartilage and bone cells as well as by systemic factors. Receptor tyrosine kinase (RTK) superfamily members, including Fibroblast Growth Factors and Insulin-like Growth Factors, play critical roles in the proliferation, survival, and dierentiation of chondrocytes, osteoblasts, osteoclasts, and other cells in the bone during embryonic development and postnatal bone remodeling. Recently, several molecules that inhibit RTK signaling pathways have been identified, including members of the Sprouty (Spry) family (Spry1, Spry2, Spry3, and Spry4). Sprouty proteins function intracellularly to antagonize RTK signaling through diverse mechanisms. By in situ hybridization and quantitative PCR, we found that Spry1, Spry2, and Spry4, but not Spry3, are expressed by pre- and hypertrophic chondrocytes, osteoblasts, and megakaryocytes, and they share similar expression patterns in adult long bones. To determine the role of Sprouty genes in skeletal development in vivo, we analyzed the skeletal phenotypes of Sprouty knockout (KO) mice by whole-mount staining with alcian blue and alizarin red, micro-computed tomography (μCT), and histology. At 6 weeks of age, Spry2 KO mice have grossly shorter tibiae and femurs compared to wild-type (WT) littermates or to Spry1 or Spry4 KO mice. μCT analysis showed that both the trabecular and cortical bones of Spry2 KO mice are smaller and thinner than those of their WT littermates at 4 weeks, 6 weeks, and 12 weeks of age, while Spry1 and Spry4 KO mice showed minor or no changes. In addition, Spry2 KO mice have reduced body weight (by ∼25%), although serum chemistries indicated that Spry2 KO mice have normal nutritional status and kidney function. To further investigate the function of Spry2 in skeletogenesis, we generated (1) cartilage- or (2) bone-specific KOs of Spry2 by crossing floxed Spry2 mice with transgenic mice expressing Cre under (1) type II collagen-alpha1 or (2) 2.3 kb fragment of the collagen 1-alpha1 promoters, respectively. μCT data showed that both cartilage- and bone-specific Spry2 KO mice had smaller and thinner trabecular bone, although their skeletal abnormalities were milder than those of the global Spry2 KO mice. Thus, Spry2 regulates bone development by modulating both chondrogenesis and osteoblastogenesis.
Disclosures: Adriane Joo, None.
Apolipoprotein E-dependent inverse Regulation of Vertebral Bone Mass and Adipose Tissue Development in C57Bl/6 Mice under a High Fat Diet.Alexander Bartelt1, F. Timo Beil1, Thorsten Schinke2, Kerstin Roeser1, Wolfgang Ruether1, Joerg Heeren1, Andreas Niemeier*2. 1University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2University Hospital Hamburg, Eppendorf, Hamburg, Germany
Purpose The long prevailing view that obesity is associated with beneficial eects on the skeleton has recently been challenged by contrasting studies. The complex relationship between fat mass and bone is regulated by multiple factors. Apolipoprotein E (apoE) is known to influence both adipose tissue development and bone formation. The goal of the current study was to examine the impact of apoE on the development of obesity and bone mass in growing mice under a high fat diet (HFD).
Methods Four weeks-old male C57Bl/6 and apoE-deficient (apoE−/−) mice were randomized to receive a HFD (35,5% fat) or a control diet (5% fat) for 16 weeks (n=14 per group). Total body weight gain was measured weekly. At 20 weeks glucose and insulin tolerance tests were performed. White fat depots were analyzed using μCT and histology. Histomorphometry was performed on sections of undecalcified lumbar vertebra of 8 animals per group. Serum lipids, adipocytokines, bone formation markers and urinary DPD/creatinine were determined with standard methods.
Results Under control conditions, apoE−/− animals displayed less total fat mass and significantly higher lumbar BV/TV (p<0.001) than wildtype (WT) controls. When stressed with HFD, apoE−/− mice accumulated less white adipose tissue (383% over controls) than WT animals (463% over control, p<0.001), in particular in the subcutaneous depots (WT 8.8-fold, apoE−/− 5.6-fold increase) with a smaller increase of the mean adipocyte diameter. Furthermore, apoE deficiency led to improved glucose and insulin tolerance with decreased plasma leptin levels. For WT animals, bone analysis yielded no significant dierences in BV/TV, TbTh, TbN and TbSp between the control and HFD groups. ApoE deficiency, in contrast, led to a significant decrease of BV/TV (21,8 % +/- 2,0 on control to 18,0 % +/-3,3 on HFD; p<0.05), decrease of TbN (p<0.01) and increase of TbSp (p<0.01).
Taken together, under normal dietary conditions, growing apoE-deficient C57Bl/6 mice acquire less fat mass and more bone mass than WT littermates. When stressed with a HFD, the expression of apoE protects against HFD-induced bone loss while it promotes fat mass accrual, rendering the difference of fat mass larger and the difference of bone mass smaller between the genotypes.
Conclusions We conclude that apoE is centrally involved in an inverse regulation of bone mass and fat mass development in WT mice and that this eect is modulated by the amount of dietary fat.
Disclosures: Andreas Niemeier, None.
Centrally Administered Leptin Promotes Bone Growth and Enhances Bone Marrow Cell Differentiation in ob/ob Mice.Shoshana Bartell*1, Srujana Rayalam2, Dhanunjaya Gaddam1, Diane Hartzell1, Mark Hamrick3, Jin-Xiong She3, Mary Anne Della-Fera1, Clifton Baile4. 1University of Georgia, Athens, USA, 2University of Georgia, Athans, USA, 3Medical College of Georgia, Augusta, USA, 4University of Georgia, Athens, GA, USA
Leptin injected intracerebroventricularly (ICV) reduces body weight (BW) & body fat. However, there are contradictory findings when assessing central leptin treatment on bone mass in rodents. The objective of this study was to determine the effect of increasing concentrations of central leptin administration on bone formation & bone marrow gene expression in leptin deficient ob/ob mice (15-wk old, init BW=60.6g). Leptin (1.5 μg/d or 0.38 μg/d) or control was continuously delivered ICV via osmotic pumps for 12 days (n=6). RNA was extracted from the bone marrow adherent cells of the left femur. Leptin decreased BW (41.2 vs 53.1 vs 63.7, p<0.01; mean of 1.5 leptin vs 0.38 leptin vs control), body fat (22.3 vs 30.6 vs 35.0g, p<0.01), & insulin (196.3 vs 446.1 vs 961.4pM, p<0.01) concentrations & increased osteocalcin (92.4 vs 26.4 vs 16.5ng, p<0.01), OPG (1447.6 vs 905.2 vs 660.0pg, p<0.01) & RANK-L (148.3 vs
123.2 vs 103.9pg, p<0.01) concentrations, whole body bone mineral density (BMD; .06 vs .06 vs .05g/cm2, p=0.01) & bone mineral content (BMC; .7 vs .5 vs .4g, p=0.02), distal tibial BMD (.10 vs .07 vs .07g/cm2, p<0.01) & BMC (.05 vs .04 vs .04g, p<0.01) & lumbar BMD (.10 vs .07 vs .07g/cm2, p<0.01) & BMC (.02 vs .02 vs .01g, p<0.01). mRNA expression levels of genes associated with osteogenesis (Runx2, Sp7, Ccl27) were increased, while those associated with osteoclastogenesis (RANK, Csf1) were decreased, which is consistent with the observed leptin-stimulated bone growth & inclination of cells to differentiate into osteoblasts. Expression levels of genes involved in adipogenesis, adipocyte lipid storage & cell survival (eg., PPARγ, Bcl-2, Retn, Aebp1) were decreased, demonstrating an enhancement in the sensitivity to leptin-stimulated adipocyte apoptosis in the bone marrow. The reduction in bone marrow adipocytes & increase in the bone marrow osteoblasts, serum bone markers, BMD & BMC indicates a positive effect on bone formation. These results show that increasing concentrations of ICV leptin injections promote expression of pro-osteogenic factors in the bone marrow which enhance bone formation in ob/ob mice. Supported by the GA Research Alliance (GRA) Eminent Scholar endowment (CAB) & GRA Challenge Grant (CAB & JXS).
Disclosures: Shoshana Bartell, None.
Nemo Like Kinase (NLK), a Novel Inhibitor of Osteoblastic Differentiation.Stefano Zanotti*1, Anna Smerdel-Ramoya2, Ernesto Canalis2. 1Saint Francis Hospital & Medical Center, Hartford, CT, USA, 2St. Francis Hospital & Medical Center, Hartford, CT, USA
NLK is an evolutionary conserved serine-threonine kinase related to mitogen activated protein kinases. NLK regulates the canonical Wnt/β-catenin signaling by phosphorylating and targeting T-cell factor (TCF) transcription factor for ubiquitinylation and proteasomal degradation. Nemo, the orthologous of NLK in Drosophila, phosphorylates Mothers against decapentaplegic (Mad), the orthologous of Smad 1, and induces Mad translocation to the cytoplasm and its subsequent degradation. It is not known whether NLK has a function in mammalian cells. However, it is conceivable that NLK could antagonize the Wnt/β-catenin and BMP/Smad signaling pathways during osteoblastic dierentiation. To test this hypothesis, we studied the consequences of NLK downregulation by RNA interference (RNAi) on osteoblastic dierentiation in ST-2 bone marrow stromal cells and in primary calvarial osteoblasts. For this purpose, cells were transfected with NLK small interfering (si)RNA or scrambled control siRNA. NLK mRNA levels were suppressed 70% to 80% in NLK downregulated cells. NLK downregulation increased alkaline phosphatase and osteocalcin expression in ST-2 cells and primary osteoblasts. Accordingly, ST-2 cells and primary osteoblasts transfected with NLK siRNA expressed increased alkaline phosphatase activity. To study the mechanism of NLK action, we tested its action on the Wnt/β-catenin and BMP/Smad signaling pathways. NLK downregulation enhanced the response of ST-2 cells and osteoblasts to the eect of Wnt3 on the transactivation of the Wnt/β-catenin reporter 16xTCF-Luc. There was no eect on free cytosolic β-catenin levels, suggesting that NLK suppresses Wnt signaling by modulating TCF stability and not β-catenin levels. NLK downregulation in ST-2 cells and osteoblasts enhanced the eect of BMP-2 on the transactivation of the BMP/Smad reporter 12xSBE-Oc-pGL3, and on the levels of phosphorylated Smad 1/5/8, demonstrating that NLK opposes Smad signaling in mammalian cells. In conclusion, NLK suppresses osteoblastic dierentiation by opposing the Wnt/β-catenin and BMP/Smad pathways.
Disclosures: Stefano Zanotti, None.
Regulation of T Cells Function By Osteoclasts-Secreted Factors In Vitro.Francesco Grassi*1, Cristina Manferdini2, Elena Gabusi2, Luca Cattini2, Andrea Facchini2, Gina Lisignoli2. 1BOLOGNA, Italy, 2istituto Ortopedico Rizzoli, Bologna, Italy
Bone Marrow (BM) T cells have been recognized as key players in the maintenance of bone homeostasis and in models of pathological bone loss such as rheumatoid arthritis or post-menopausal osteoporosis. Subsets of antigen-experienced, memory T cells are known to nest to the bone marrow. Yet, whether bone cells can retain and locally regulate T cells is still unclear. As osteoclasts (OCs) arise from antigen-presenting (APC) circulating progenitors, we asked whether mature OCs can retain and regulate T cells. CD11b+ OC precursors were isolated from human PBMCs and OCs were obtained in vitro upon stimulation with M-CSF and RANKL. The question of whether OCs are immunocompetent cells was then approached through gene expression profiling by Real Time PCR, immunohystochemical staining and functional assays. HLA-DR molecules are expressed by unstimulated mature osteoclasts, a unique property of APC cells, and are readily upregulated by IFN?. Moreover, osteoclasts significantly upregulate expression of the key costimulatory molecules CD40 (p<0.05) and CD80 (p<0.001) upon differentiation from CD11b+ monocytes. Mesenchymal stromal cells (MSCs) or osteoblasts (OBs) used as a control showed very low or undetectable levels of these markers. In functional adhesion assays, OCs showed a 2.5-fold (p<0.01) increased ability to retain T cells in adhesion assays as compared to MSCs or OBs. Multiplex analysis of T cell attracting chemokines in cell culture supernatants showed significantly higher levels of chemokines CXCL10, CXCL9 and CCL3 in OC supernatants compared to MSCs and OBs. In functional assays, OCs blunted T cell proliferation in antigen-independent, polyclonal T cell stimulation by PHA or anti-CD3 in co-culture experiments. Moreover, T cell production of TNF? and IFN??as well as activation-induced cell death were significantly inhibited in the presence of OCs. Finally co-coltures of OCs and T cells in the presence of transwell to avoid cell-to-cell contact revealed that OC modulation of T cells response was largely conserved in the absence of cell contact, suggesting a role for OC-secreted factors. Thus, OCs are fully immunocompetent cells with the ability of retain and regulate T cell response in the local microenvironment.
Disclosures: Francesco Grassi, None.
IGFBP-2 regulates bone turnover likely through a mechanism which is independent of IGF-1 binding and involves activation of Wnt/β-catenin signaling.Masanobu Kawai*1, Victoria Demambro2, Anne Breggia1, Wesley Beamer3, David R Clemmons4, Cliord Rosen1. 1Maine Medical Center, Scarborough, ME, USA, 2The Jackson Lab, Bar Harbor, ME, USA, 3The Jackson Laboratory, Bar Harbor, Maine, USA, 4University of North Carolina, Chapel Hill, USA
We previously reported that Igfbp2 null mice (BP2−/− mice) have a skeletal phenotype that is characterized by reduced bone turnover and a low bone volume fraction. In addition, we noted that PTEN was increased in BP2−/− mice. In this study we showed that IGFBP2 regulated bone remodeling in an IGF-I binding independent manner involving modulation of Wnt/β-catenin signaling. To test this we cultured marrow stromal cells and neonatal calvarial osteoblasts (COB) from BP2−/− mice in osteogenic media and treated with either recombinant full-length IGFBP2 (FL-BP2) or a peptide fragment of IGFBP2 that does not contain the IGF binding site (BP2-pep). Addition of FL-BP2 does not have any effect on osteogenesis likely due to the presence of 10% fetal calf serum(FCS) which contains >300 ng/ml of IGFBP-2, whereas BP2-pep enhanced mineralization even in the presence of 10% FCS. We next performed ex vivo cultures of neonatal metacarpals from BP2−/− mice. Metacarpals were cultured in osteogenic media without FCS, and then, labeled with calcein to analyze mineralization. FL-BP2 and BP2-pep increased mineralization. IGF-1 stimulated bone mineralization and when added with BP2-pep, showed an additive effect. Next, we administered a pegylated form of the BP2-pep into BP2−/− mice to see whether BP2-pep could rescue the low bone mass phenotype. Trabecular BV/TV by μCT was higher in mice with BP2-pep treatment compared to controls. To explore the underlying mechanisms, PTEN expression was analyzed. Not only FL-BP2, but also BP2-pep significantly reduced PTEN expression in BP2−/− COBs; this was accompanied by increased phosphorylation of Akt. Since several lines of evidence have shown crosstalk between IGF-1 and Wnt/β-catenin signaling, we analyzed the effect of IGFBP2 on Wnt signaling. In BP2−/− COBs, Wnt3a-induced accumulation of cytosolic β-catenin was impaired compared to controls. In addition, pre-treatment with IGFBP2 in MC3T3-E1 cells enhanced Wnt3a-mediated TCF transcription. Interestingly, BP2-pep partially blocked the suppressive effect of DKK-1 on Wnt/β- catenin signaling. These data indicate that IGFBP2 enhances Wnt/β-catenin signaling in part by blocking DKK-1. In addition, Ser552 phosphorylation of β-catenin by IGF-1 was enhanced in cells pre-treated with BP2-pep compared to controls. In conclusion, the low bone mass phenotype of BP2−/− mice is due in part to a mechanism independent of IGF-I binding, possibly through impaired activation of Wnt/β-catenin signaling.
Disclosures: Masanobu Kawai, None.
IGF-I Signaling Is Required for the Communication between Chondrogenesis and Osteogenesis.Yongmei Wang*1, Hashem ElAlieh2, Wenhan Chang2, Daniel Bikle2. 1University of California, San Francisco, VA Medical Center, San Francisco, CA, USA, 2University of California, San Francisco, VA Medical Center, San Francisco, USA
Our previous studies demonstrated that insulin-like growth factor-I (IGF-I) signaling is critical for the communication between osteoblasts (OBs) and osteoclasts (OCLs). This communication is mediated by ephrin/Eph and RANKL/RANK signaling to stimulate OB dierentiation and osteoclastogenesis, thereby, maintaining normal bone remodeling. In the current study, we used various knockout mouse models to investigate the role of IGF-I signaling in regulating the interaction between chondrocytes and bone cells with particular attention to the role of ephrin B2/EphB4 and RANKL/RANK. Immunohistochemistry of embryonic bones demonstrated ephrin B2 in chondrocytes within each zone of the growth plate (GP), especially in the prehypertrophic (PHZ) and hypertrophic zones (HZ). After birth, the expression of ephrin B2 was found in the chondrocytes in PHZ and HZ and OBs and OCLs in the primary and secondary spongiosa. The expression of ephrin B2 was markedly decreased in GP chondrocytes, OBs and OCLs in global IGF-I knockout (IGF-IKO) mice, and in mice lacking the IGF-I receptor (IGF-IR) specifically in chondrocytes (cart IGF-IR KO) generated by breeding type II collagen Cre recombinase mice with floxed IGF-IR mice. Quantitative real-time PCR (Q-PCR) analysis demonstrated that the mRNA levels of ephrin B2, its receptor EphB4, and the OCL regulator RANKL were decreased by 40%, 43% and 88% respectively in the cart. IGF-IR KO compared with their wild-type littermates (WT). In the mice with IGF-IR null mutation in OCLs (OCLIGF-IR KO), (floxed IGF-IR X TRAP5b driven Cre recombinase), immunohistochemistry demonstrated a reduction in ephrin B2 not only in OCLs, but also in chondrocytes in the PHZ and HZ. Q-PCR analysis showed that the mRNA levels of ephrin B2 and EphB4 in these bones (marrow flushed out) decreased by 70% and 62%, respectively, compared with WTs. Our data indicate that in addition to the expression in OBs and OCLs, chondrocytes also expresses ephrin B2, EphB4 and RANKL. IGF-I/IGF-R in chondrocytes is required for the normal expression of these genes in these cells. In addition, IGF-IR in OCLs regulates not only the expression of ephrin B2 in OCLs, but in chondrocytes, suggesting IGF-I/IGF-IR signaling controls the interaction between these cells and most likely contributes to the regulation of endochondral bone formation via its regulation of ephrin B2/EphB4 and/or RANKL expression,
Disclosures: Yongmei Wang, None.
Mice lacking the TGF-b type I receptor ALK5 in sclerotomes develop severe spinal scoliosis with spina bifida.Tomoya Matsunobu*1, Kiyoyuki Torigoe2, Ashok Kulkarni2, Yukihide Iwamoto3, Yoshihiko Yamada2. 1Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 2National Institute of Dental & Craniofacial Research/NIH, Bethesda, USA, 3Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Transforming growth factor-β (TGF-β) initiates its diverse cellular responses during development and pathogenesis by forming a specific complex with TGF-β type I (Alk5) and type II (TβrII) receptors. This activates downstream signaling through Smad-dependent and -independent pathways such as the MAPK pathways. We showed previously that mice lacking ALK5 in chondrocytes using Col2a-Cre (ALK5Col2CKO) are perinatally inviable, and display defects in axial skeleton formation such as severe spinal scoliosis. In ALK5Col2CKO vertebral cartilage, chondrocytes are dierentiated, but the levels of extracellular matrix and GAG (glycosaminoglycan) are substantially reduced in comparison to wild-type cartilage. Since both notochord and chondrocytes express Col2a1, it was not clear whether ALK5 is required in the notochord for axial skeleton formation. In this study, in order to clarify this point and to test the involvement in vertebral formation of TGF-β signaling in osteoblasts, we created Dermo1-Cre- and Col1a1-Cre-mediated conditional ALK5 knockout mice (ALK5Dermo1CKO and ALK5Col1CKO, respectively). Dermo1-Cre is expressed by osteo-chondroprogenitors in sclerotomes, but not in the notochord or neural tissue. On the other hand, Col1a1-Cre is expressed by mature osteoblasts. We found that ALK5Dermo1CKO knockout mice were perinatally inviable, and exhibited severe spinal scoliosis and myelomeningocele, the most severe form of spina bifida in which the neural tube does not close. In contrast, ALK5Col1CKO mice were viable and no obvious bone abnormalities were observed in their spines. These results suggest that TGF-β signaling in osteo-chondroprogenitors, but not in osteoblasts, plays an important role in vertebral formation. Among over 60 mouse models with neural tube closure defects, no TGF-β signaling-related molecules have been reported. This is the first report that ALK5 is a candidate gene in neural tube defects.
Disclosures: Tomoya Matsunobu, None.
Deletion of retinaldehyde dehydrogenase 1 (Raldh 1) dramatically increases trabecular and cortical bone mass.S Nallamshetty*1, Masanobu Kawai2, Sheila Bornstein2, Terry Henderson3, Mark Horowitz4, Chord Rosen2, Jorge Plutzky1. 1Brigham & Women's Hospital, Boston, USA, 2Maine Medical Center, Scarborough, ME, USA, 3Maine Medical Center Research Institute, Scarborough, USA, 4Yale University School of Medicine, New Haven, CT, USA
Retinoic X Receptor (RXR) is a receptor for retinoids, structural derivatives of vitamin A (retinol). The nuclear receptor peroxisome proliferator activated receptor-gamma, PPARγ, a key regulator of stromal cell fate within the marrow, requires heterodimerization with RXR to be transcriptionally active. RXR selective ligands can independently modulate the PPARγ-RXR heterodimer and recapitulate important PPARγ responses in inflammation and metabolism. As such, RXR and retinoids represent critical nodal points in PPARγ networks. But the role of RXR and its ligands in modifying PPARγ responses in the skeleton remains undefined, even though early work suggested that retinoic acid (RA) inhibited osteoblast differentiation and supplementation. Retinaldehyde (Rald), a metabolite of retinol with no previously documented regulatory role outside of the retina, inhibits the PPARγ-RXR complex and represses adipogenesis in vivo . Mice lacking retinaldehyde dehydrogenase-1 (Raldh1), the enzyme that converts Rald to retinoic acid (RA), have high levels of Rald in fat and are protected against diet-induced obesity and insulin resistance. We hypothesized that if RXR was important in determining PPARγ activation in marrow stromal cells, then mice with global Raldh1 deletion (Raldh1−/−) would have high BMD. We phenotyped the femorae of 12 week n=5 female B6 controls and 5 female Raldh1−/− mice by PIXIMus and by μCT. Femoral aBMD in the Raldh1−/− mice was 20% higher than WT B6 controls: 0.0551+0.0003 g/cm2 vs 0.0440+0.0006 g/cm2; p=0.03. By μCT, femoral trabecular BV/TV was more than 2 fold higher in the Raldh1 −/− mice than controls: 0.1105+0.0053 vs 0.0505+0.016 p<0.001. Trabecular thickness did not differ by genotype but there were more trabeculae in the Raldh1 nulls vs WT: 4.6+0.15 vs 3.22+0.01, p<0.01, and a significant increase in connectivity density. Cortical bone surface in the Raldh1−/− mice was markedly greater than WT(13.23 +0.70 vs 8.19 +0.30 p<0.01). In sum, the micro- architectural changes of the Raldh1−/− mice provide preliminary insights into the potential role of RXR in regulating PPARγ activation within the marrow niche. We cannot exclude the possibility that excess retinaldehyde which occurs after deletion of Raldh1, could have skeletal anabolic properties. Further studies are underway to assess the cellular mechanisms of action in Raldh1−/− mice.
Disclosures: S Nallamshetty, None.
Endogenous Gi Signaling in Osteoblasts Participates in Sexually Dimorphic Regulation of Cancellous Bone Structure.Susan Millard*1, Alyssa Louie2, Carlota Manalac3, Weidar Lu2, Nathalie Cotte3, Thomas Wronski4, Bruce Conklin3, Robert Nissenson5. 1UCSF/VA Medical Center/Northern California Institute for Research & Training, San Francisco, CA, 2Endocrine Research Unit, VA Medical Center & Depts of Medicine & Physiology, University of California San Francisco, San Francisco, USA, 3Glastone Institute, University of California San Francisco, San Francisco, USA, 4University of Florida, Gainesville, FL, USA, 5University of California, San Francisco, San Francisco, CA, USA
We have recently shown that expression of an engineered Gi coupled receptor under control of the CollagenIα 2.3 kb promoter results in severe trabecular osteopenia, yet little is known about the physiological role of endogenous Gi signaling in osteoblasts. In this study, we investigated the skeletal eects of blocking Gi signaling in osteoblasts in vivo. This was accomplished by transgenic expression in osteoblasts of pertussis toxin (PTX), which inhibits receptor-mediated activation of Gi signaling. Here we report that inhibition of endogenous Gi signaling in osteoblasts produces sexually dimorphic eects on cancellous bone. Female Col1(2.3):PTX transgenic mice have increased cancellous fractional bone volume, as assessed in the distal femur of 12wk old animals using histomorphometry (BV/TV: 5.4 ± 1.7 vs 11.9 ± 4.7% p<0.001) and by μCT analysis of a region of interest (ROI) beginning 100 slices (1.05mm) below the first slice on which secondary spongiosa may be circumscribed by a single contour (BV/TV: 9.8 ± 1.3 vs 16.4 ± 1.2% p<0.01). Both analyses showed this dramatic increase to primarily result from an increased trabecular number (Histo: 3.7 ± 0.3 vs 7.3 ± 0.6 per mm, p<0.001). μCT analysis of a ROI closer to the primary spongiosa showed BV/TV to be maintained but not significantly increased (20.7 ± 1.5 vs 23.7 ± 1.8% p=0.2). Thus, in female mice, blockade of Gi signaling produced a marked increase in BV/TV of regional specificity within the secondary spongiosa of the distal femoral metaphysis. In contrast male Col1(2.3):PTX mice displayed decreased BV/TV in both ROIs assessed by μCT (first ROI:16.6 ± 0.01 vs 10.9 ± 0.01%, p<0.001; second ROI:8.0 ± 0.01 vs 6.8 ± 0.01%, p<0.05). An increase in serum markers of bone formation was noted in female Col1(2.3):PTX mice (AlkPhos:69 ± 3 vs 84 ± 3 IU/L p<0.05, Osteocalcin:123 ± 10 vs 155 ± 16 ng/mL p=0.1), but not males (Al-kPhos:62 ± 2 vs 66 ± 4 p=0.4, Osteocalcin:107 ± 10 vs 113 ± 10 p=0.7). Serum levels of pyridinolines, a marker of bone resorption, were unchanged. Increased osteoid width was noted in PTX mice of both genders, suggesting a role for endogenous Gi signaling in promoting normal mineralization. The increased cancellous fractional bone volume which is seen only in female Col1(2.3):PTX mice and is more pronounced in a region further from the primary spongiosa, demonstrates that Gi signaling plays a complex role in the control of skeletal homeostasis that is dependent on both gender and the anatomic site within bone.
Disclosures: Susan Millard, None.
Mice deficient in CuZn-superoxide dismutase exhibit low bone mass and delayed fracture healing.Yoshitomo Saita*1, Hidetoshi Nojiri2, Chizuru Tsuda3, Daichi Morikawa3, Hisashi Kurosawa2, Takahiko Shimizu3. 1Juntendo University, Tokyo, Japan, 2Dept. of Ortopedics, Juntendo Univ., Tokyo, Japan, 3Molecular Gerontology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
Osteoporotic fracture is a major problem in aged society. Bone mass gradually decreases as early as 30 years of age both in men and women. Oxidative stress increases in aging and is believed to contribute to etiology of various degenerative diseases in many organs. Although several in vitro studies suggest that oxidative stress inhibits osteoblast dierentiation and proliferation, the in vivo relationships between oxidative stress and bone tissue remains unclear. Therefore we analyzed bone mass in mice lacking a major antioxidant enzyme, CuZn-superoxide dismutase (Sod1).
Bone mineral density (BMD) of long bones was significantly decreased in Sod1−/− mice compared to wild-type mice. In order to evaluate the bone formation activity, we examined the ability of fracture healing in these mice. Right femur was broken at the half of their length and fixed with intramedullary nail. X-ray analysis and measurement of BMD were performed at 4 weeks after operation. The rate of fracture union was not dierent between wild-type and Sod1−/− mice. BMD at the shaft of femur were significantly increased both in wild-type and Sod1−/− mice as reflected by the newly formed bone around the fracture site, while the rate of increase of BMD was significantly lower in Sod1−/− mice. These results suggest that oxidative stress reduces bone mass and suppresses the ability for bone formation in vivo. In order to investigate further insight into these phenomena, osteoblasts were isolated from neonatal calvarial bone. We compared the expression levels of genes involved in osteoblast dierentiation by real-time PCR analysis. We found that the expression level of Col1a1 mRNA were nearly 30% lower in Sod1−/− mice, suggesting that Sod1 could be regulated to collagen synthesis in osteoblasts.
These results indicate that Sod1 is a novel factor to control bone mass and it might play a crucial role in osteoblast differentiation and/or proliferation through at least in part cell autonomous manner.
Disclosures: Yoshitomo Saita, None.
Mice Lacking Lamin A/C Display Osteopenia Caused by Decreased Bone Formation and Aberrant Bone turnover.Gustavo Duque*1, Wei Li2, Liz Li Sze Yeo3, Diane Fatkin3. 1University of Sydney, Penrith, NSW, Australia, 2Nepean Clinical School, University of Sydney, Penrith, Australia, 3Victor Chang Institute, Sydney, Australia
Nuclear lamina alterations occur in premature aging syndromes that include severe osteoporosis. The role of proteins of the nuclear lamina in bone remains to be elucidated. Our previous study demonstrated that knockdown of lamin A/C inhibits osteoblastogenesis and promotes adipocyte differentiation of mesenchymal stem cells in vitro (Akter et al, JBMR, 2008). In addition, lack of lamin A/C favors an osteoclastogenic milieu and contributes to enhanced osteoclastogenesis in vitro. In this study, we used the lamin A/C knock out (LmnA−/−) mice to identify the role of lamin A/C in bone turnover and bone quality in vivo. At four weeks of age, histological and micro computed tomography measurements of femurs in LmnA−/− mice revealed a significant decrease in bone volume (BV/TV), trabecular number (Tb.N) and both cortical and trabecular thickness in LmnA−/− mice (Fig. D) as compared with their wild type littermates (Fig. A) (p <0.01). Osteoblasts and osteocytes numbers are dramatically diminished by ∼75% in the LmnA−/− mice. In contrast, LmnA−/− mice showed significantly higher osteoclast number and eroded surfaces. In addition, we found aberrant forms of osteoblasts and osteoclasts including giant osteoblasts and lining cells (Fig. E, arrows) as well as “cystic” osteoclasts (Fig. F, arrows). These data indicate that the presence of lamin A/C is necessary for normal osteoblast and osteoclast differentiation and activity in vivo.
Disclosures: Gustavo Duque, None.
Neuropeptide Y controls glucocorticoid signalling to modulate osteoblast activity.Frank Driessler*1, Nikki Lee2, Ronaldo Enriquezz1, Shu Lin2, Amanda Sainsbury-Salis2, Herbert Herzog2, John Eisman3, Paul Baldock4. 1Garvan Institute of Medical Research, Osteoporosis & Bone Biology Program, Sydney, Australia, 2Garvan Institute of Medical Research, Neuroscience Program, Sydney, Australia, 3Garvan Institute of Medical Research, Sydney, NSW, Australia, 4Garvan Institute of Medical Research, Sydney, Australia
Glucocorticoid excess reduces bone mass and osteoblast function. Neuropeptide Y (NPY) has anxiolytic actions, in part, by control of glucocorticoid signaling. NPY also modualtes osteoblast function. This study investigated whether the regulation of glucocorticoids by NPY also extends to their effects on bone. Endogenous glucocorticoid production was elevated with a chronic stress protocol with mice under a cold and restraint stress for 6 weeks prior to collection at 16 weeks. The skeletal effects of NPY involve osteoblastic Y1 and hypothalamic Y2 receptors, thus stress responses were examined in the bones of Y1−/−, Y2−/−, NPY−/− and wild type mice. Primary calvarial osteoblastic cultures from wild type mice were treated with a Y1 receptor antagonist, then NPY or the glucocorticoid dexamathosone (Dex), to evaluate local effects on cell signalling. Chronic stress induced mild bone loss in wild type mice (13%,ns), with a significant loss of trabecular number (Tb.N, /mm). NPY−/− mice lost cancellous bone volume (16.7% ± 1.5 vs 11.6 ± 1.7, p<0.05), again with reduced Tb.N. Stress reduced mineral apposition rate (MAR, μm/d) (2.8 ± 0.1 vs 2.2 ± 0.1, p<0.0001), with no change in osteoclast indices. Y2−/− mice also lost cancellous bone (12.5% ± 0.8 vs 8.7 ± 1.7, p<0.05,) with reduced Tb.N and MAR (2.5 ± 0.2 vs 1.9 ± 0.1, p<0.05). In marked contrast, Y1−/− mice displayed an opposing response to stress. Trabecular thickness was greater (30.9 ± 1.5 vs 38.7 ± 2.3, p<0.02) with increased MAR (2.0 ± 0.1 vs 2.7 ± 0.1, p<0.0001) and mineralizing surface (2.8% ± 0.1 vs 2.2 ± 0.1, p<0.0001). Stress increased corticosterone in all genotypes but to different extents. NPY−/− mice had greater corticosterone than wild type at baseline and following stress (315 ng/ml ± 68 vs 103 ± 11, p<0.005) while Y1−/− mice had reduced levels (57 ± 9, p<0.05). Despite a more adverse stress response, Y2−/− mice had similar corticosterone levels to wild type (116 ± 22). In vitro, both NPY and Dex increased P-Erk1/2 and P-JNK activation. However, both responses were blocked by Y1 antagonism, indicating a common signaling pathway in osteoblasts. NPY appears to be critical for maintaining osteoblast activity during chronic stress, via Y2 receptor signaling, with Y1 playing a counter-regulatory role. This protective effect involves both endocrine (suppression of corticosterone production) and direct neural effects (cell signaling). These pathways may protect skeletal tissue from glucocorticoid excess.
Disclosures: Frank Driessler, None.
Postnatally Induced Inactivation of Osterix in Osteoblasts Results in the Reduction of Bone Formation and Maintenance.Wook-Young Baek*1, Eui-Sic Cho2, Benoit De Crombrugghe3, Jung-Eun Kim1. 1Kyungpook National University School of Medicine, Daegu, Korea, 2Chonbuk National University School of Dentistry, Jeonju, South Korea, 3University of Texas M.D. Anderson Cancer Center, Houston, TX, USA
Osterix (Osx) is a zinc-finger-containing transcription factor that is highly specific to osteoblasts in vivo. Because Osx null mutants die in the immediate perinatal period showing a complete absence of bone formation, it is impossible determine the role that Osx plays in bones that have already formed after birth. To determine whether Osx is essential for bone maintenance and homeostasis, we conditionally inactivated the Osx gene in adult bone using the Cre/loxP system. In previous reports, 2.3-kb Col1a1-CreERT2 mice that expressed a Cre recombinase that is transiently inducible by 4-hydroxytamoxifen (4-OHT) were intercrossed with Rosa26R (R26R) reporter mice, which resulted in the production of Cre-expressing osteoblasts that were detected upon X-gal staining. In the present study, inducible Col1a1-CreERT2 mice and conditional Osx mice (Osxflox/+) were used to generate Osxflox/-;Col1a1-CreERT2 mice. The Osx gene in Osxflox/-;Col1a1-CreERT2 was inactivated in the osteoblasts of already formed bones by active Cre recombinase after 4-OHT administration. The bones from 4-OHT-treated Osxflox/-;Col1a1-CreERT2 and oil-treated control mice were analyzed by radiography, histology, and histomorphometry. Even though no significant dierence was observed in the radiographic images of the whole mouse skeletons, the mineralized trabecular bone volume, thickness, and number in lumbar vertebrae were remarkably reduced in 4-OHT-treated Osxflox/-;Col1a1-CreERT2. The rate of bone formation and area of mineralized surface were also significantly reduced in 4-OHT-treated Osxflox/-;Col1a1-CreERT2. Osx inactivation in already formed bones during the postnatal period caused a functional defect in osteoblasts that was followed by a reduction of bone formation, even though there were no apparent dierences in osteoblast proliferation and osteoclast formation. These results indicate that Osx is required to maintain osteoblast function following adult bone maintenance. * KRF grant (KRF-2008–331-E00039), KOSEF grant (R01–2007−000–20005−0) funded by Korea Government, BK21 Project in 2009.
Disclosures: Wook-Young Baek, None.
The expression of CYP27B1 in bone is associated with osteoblastic differentiation and mineralisation.Andrew Turner*1, Juliette Tyson1, Rebecca Sawyer1, Peter O'Loughlin2, Howard Morris3, Paul Anderson4. 1Endocrine Bone Research Laboratory, Hanson Institute, SA Pathology, Adelaide, Australia, 2Chenical Pathology, SA Pathology, Adelaide, Australia, 3Hanson Institute, Adelaide, SA, Australia, 4SA Pathology, Adelaide, SA, Australia
The endocrine hormone, 1?,25-dihydroxyvitamin D3 (1,25D) is an important regulator of calcium and phosphorus homeostasis. In this context, 1,25D is generally recognized as necessary for the maintenance of a healthy skeleton through its actions on the small intestine. However, we have shown that the bone itself is a site of metabolic conversion of 25 hydroxyvitamin D3 (25D) into 1,25D, by virtue of its expression of the 25-hydroxyvitamin D 1alpha-hydroxylase, CYP27B1. We have used a transgenic (Tg) mouse line that expresses the complete promoter of the human CYP27B1 gene fused to the firefly luciferase reporter gene to demonstrate CYP27B1 expression in femoral bone. Following an 2.5mg i.p. dose of D-luciferin, CYP27B1 promoter activity was located to the to the growth plate of the femur and the cancellous bone of the metaphysis using bioluminescent imaging. When bone marrow cells were cultured from long bones of the Tg mouse in media supporting pro-osteogenic conditions, CYP27B1 promoter activity doubled every 3 days in culture until day 9 and peaked by day 12 of culture (P<0.001). The increase in CYP27B1 promoter activity preceded the marked increased in both osteocalcin mRNA expression and mineral deposition, suggesting that the metabolic conversion of 25D to 1,25D participates in autocrine and paracrine actions of cell differentiation which leads to mineralization of the cells. Furthermore, we used RNAi technology to silence CYP27B1 mRNA expression in human osteosarcoma (HOS) cells. Cells were transfected with CYP27B1 or non-silencing scrambled siRNA (5nM), followed by treatment with 400nM 25D alone or with 200pM 1,25D for 48 hours. RNA was harvested for quantitation of CYP27B1, OCN and b-Actin by qRT-PCR. In HOS cells, RNAi reduced CYP27B1 mRNA levels up to 48% when compared to scrambled control RNA (data not shown). In cells treated with 400 nM 25D, OCN expression was markedly reduced by CYP27B1 siRNA knockdown. The reduction in OCN mRNA levels due to knockdown of CYP27B1 was overcome by the addtion of 1,25D. This demonstrates the importance of 1,25D production in HOS cells for the regulation of OCN expression. We conclude that the skeleton is an intracrine organ for vitamin D metabolism important in the role of bone formation and challenges the long-held notion that 1,25D is solely an endocrine hormone.
Disclosures: Andrew Turner, None.
Ascorbic Acid Regulates Osterix (Osx) Expression in Osteoblasts by a Novel Mechanism That Involves Activation of Prolyl Hydroxylase (PHD) and Proteosomal Degradation of Transcriptional Repressor.Weirong Xing*1, Sheila Pourteymoor2, Subburaman Mohan1. 1JL Pettis VA Memorial Medical Center, Loma Linda, USA, 2JL Pettis VA Memorial Medical Center, Loma Linda, USA
We have previously found that: 1) deletion of the gulonolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA), was responsible for impairment of differentiated functions of osteoblast (OB), bone fracture, and premature death in spontaneous fracture mice, and 2) Osx expression during OB differentiation is dependent on AA. In this study, we found that while 24 h AA treatment of serum-free cultures of MC3T3-E1 cells and mouse calvarial primary OBs increased expression of Osx 5–10-fold (P<0.001), it produced only modest effects (<50%) on expression of Runx2, Dlx3, Dlx5, Msx1 and Msx2, thus suggesting Osx is a key downstream target of AA action in OBs. To determine the mechanism by which AA regulates Osx expression, we evaluated the effects of MAPK inhibitors since AA is known to regulate MAPK signaling in other cell types and since p38 MAPK mediates BMP-induced Osx expression. Pretreatment of MC3T3-E1 cells with effective concentrations of p38 MAPK, ERK1/2 and JNK specific inhibitors had no effect on AA-induced Osx expression. In contrast, BMP-2-induced Osx expression was completely blocked by p38 MAPK and JNK inhibitors. Accordingly, noggin pretreatment did not block AA effects on Osx expression. We next determined if AA effects on Osx expression is mediated via activation of collagen mediated integrin signaling based on the well known effect of AA to stimulate collagen production. However, pretreatment of OBs with neither RGD nor echistatin, inhibitors of integrin signaling, had no effect on AA-induced Osx expression. Because AA is required for PHD activity and because PHD-induced prolyl-hydroxylation targets proteins to proteosomal degradation, we next tested if AA effect on Osx expression involves activation of PHD to hydroxylate and induce ubiquitin-proteosome mediated degradation of transcriptional repressor of Osx gene. Treatment with DMOG and EDHB, known inhibitors of PHD, completely blocked AA effect on Osx expression in both cell types (4–7 without inhibitors vs 0.6–1.5 fold with inhibitors, P<0.001). Furthermore, treatment with MG115, inhibitor of proteosomal degradation, completely blocked AA effects on Osx expression in both cell types. Based on these data, we conclude that AA effect on Osx expression is mediated via a novel mechanism that involves PHD and proteosomal degradation of a yet to be identified transcriptional repressor's that is independent of BMP or integrin-mediated signaling in OBs.
Disclosures: Weirong Xing, None.
Dlx3 Is a Transient Negative Regulator of Osteoblast Genes Linked to Runx2 Activation of Osteoblast Differentiation.Jonathan Gordon*1, M.S. Islam2, Mohammad Hassan3, Tripti Gaur3, Andre Van Wijnen3, Janet L. Stein4, Gary Stein3, Alexander Lichtler5, Maria I. Marasso6, Jane Lian3. 1UMass Medical School, Worcester, MA, USA, 2University of Connecticut Health, Framington, CT, USA, 3University of Massachusetts Medical School, Worcester, MA, USA, 4UMass Medical School, Worcester, MA, USA, 5University of Connecticut Health Center, Farmington, CT, USA, 6N.I.H., Bethesda, MD, USA
Dlx3 activates several gene promoters in mesenchymal stromal cell lines or early stage osteoblasts. The enhancer activity of Dlx3 is reflected by increased mRNA expression of bone-related genes including Runx2 and osteocalcin upon exogenous Dlx3 expression. However, Tricho-Dento-Osseous (TDO) patients (a condition resulting from inactivating mutations in Dlx3) and Dlx3 flox/flox Col1a2.3-Cre-conditional mice exhibit modestly increased bone density. Here we addressed the molecular mechanisms contributing to increased bone mass by investigating osteoblast dierentiation and function in the Dlx3 flox/flox conditional mouse. Calvarial osteoblast precursors were isolated from Dlx3 flox/flox mice and the Dlx3 gene resulted by Adenoviral Cre recombinase excision in an 85% reduction of Dlx3 levels in calvarial osteoblasts. Cell growth (measured by 3thymidine incorporation) was not aected. Osteoblast-related gene expression was increased in all stages of osteoblast dierentiation with mature markers (bone sialoprotein (BSP) and osteocalcin (OCN)) reaching 1.5–2 fold higher levels in the mineralization stage. In Dlx3-deficient osteoprogenitors there was a significant (p<0.01) increase in Dlx5 and Msx2 occupancy of the OCN, BSP Runx2 proximal promoters during proliferative and early dierentiation stages, consistent with previous studies in primary calvarial osteoblast revealing a transient association of Dlx3 with the OCN promoter at the onset of transcription, followed by a displacement of Dlx3 by Dlx5 during osteoblast maturation. The most striking finding is the increased occupancy of Runx2 on the OCN, BSP and Runx2 proximal promoters during all stages of osteoblast maturation. Thus, our results suggest that absence of Dlx3 on osteoblast gene promoters does not significantly alter the timing of osteoblast gene expression, but rather allows for increased Runx2 binding precociously enhancing the expression of these genes. The results of this present in vitro study supports a mechanism for increased osteoblast-related gene expression consistent with the phenotypes of increased bone density in the Dlx3-conditional knockout mouse and increased bone mass in TDO patients.
Disclosures: Jonathan Gordon, None.
Wnt-3a Stimulates Matrix extracellular phosphoglycoprotein expression through the Activation of BMP-2 Autocrine loop.Young Dan Cho*1, Won Joon Yoon2, Kyung Mi Woo2, Jeong Hwa Baek2, Hyun Mo Ryoo2. 1Seoul National University, Seoul, South Korea, 2Department of cell & developmental biology, School of dentistry, Seoul National University, Seoul, South Korea
Matrix extracellular phosphoglycoprotein (MEPE) shows bone cells-specific expression pattern and has antagonistically acting two functional domains. One is AC-100, that stimulates proliferation and dierentiation of bone marrow stromal cell, and the other is acidic-serine-asparate-rich-MEPE-associated motif (ASARM), which inhibits bone mineralization. There are sucient data supporting the role of MEPE function in mineralization, however, little is known about the regulation of MEPE gene expression. Our previous results indicated that MEPE expression is stimulated by BMP-2-induced Dlx5 and Runx2 transcriptional action. Wnt-3a is also one of the most important growth factors for bone formation. We found that Wnt-3a treatment strongly stimulated MEPE mRNA expression, moreover, overexpression of β-catenin or stabilization of β-catenin by LiCl stimulated MEPE expression. In contrast, knockdown by siRNA against β-catenin blocked Wnt-3a-stimulated MEPE expression. It is well known that in the canonical Wnt pathway, stabilized β-catenin makes a complex with LEF-1, which turns on Wnt target genes. As the same context, LEF-1 and/or β-catenin overexpression increased MEPE mRNA level. Furthermore, there are conserved LEF-1 binding elements in the MEPE promoter and we localized the critical response elements by EMSA, ChIP assay and site-directed mutagenesis of the LEF-1 response elements. According to the recent reports, we checked whether there are cross-regulation between BMP-2 and Wnt-3a signaling cascades and found that Wnt-3a treatment strongly stimulated BMP-2 mRNA expression and its downstream-transcription factors Dlx5 and Runx2. To prove a possible cross-regulation between Wnt-3a and BMP-2 in MEPE expression, we collected conditioned media of Wnt-3a-treated MC3T3-E1 cells (CM). The CM strongly increased phospho-Smad1/5 protein level and their downstream Dlx5 and Runx2 mRNA level, which is not blocked by the addition of DKK1, a Wnt-3a blocker, while is strongly suppressed by the addition of Noggin, a BMP-2 blocker, to the CM. Collectively, Wnt-3a stimulates MEPE transcription directly by canonical Wnt signal downstream, β-catenin and LEF-1, and indirectly through the activation of BMP-2 expression and its autocrine loop activation.
Disclosures: Young Dan Cho, None.
This study was supported by the Korea Health 21 R&D Project, Ministry of Health and Welfare (Project No. A010252) and by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD)” (KRF-2006-I00680A).
Notch function in osteoblasts is dependent on canonical Rbpj signaling.Jianning Tao*1, Shan Chen2, Gautam Sule2, Tao Yang2, Brian Dawson2, Brendan Lee1. 1Baylor College of Medicine, Houston, TX, USA, 2Baylor College of Medicine, Houston, USA
Evolutionarily conserved Notch signaling involves in many aspects of development and diseases. The initial importance of Notch function in osteoblast came from in vivo studies in which tissue specific gain and loss of function mutants were generated. We previously generated an osteoblast specific over-express ion of the constitutively active Notch 1 Intracellular Domain (NICD) by using collagen type 1 (Col1a1) promoter in committed osteoblasts. These transgenic mice showed a dramatic increase in osteoblast number, proliferation and formation resulting in a severe osteosclerotic phenotype. However, these founder lines exhibited early lethality and variable expression. To gain an insight into potential mechanistic basis for this phenotypic observation, and also study the effect of this osteoblast-specific mutation on the hematopoietic system, we established a transgenic line with consistent NICD expression and longevity by crossing the RosaNotch/+ with the Col1a1 Cre transgenic mouse. The resulting bigenic mice (RosaNotch/+; Col1a1 Cre or GOF) recapitulated the osteoblast-specific NICD transgenic mice showing similar phenotypes. Using quantitative RT-PCR, a canonical target of Notch signaling, Hey1, was upregulated more than ten fold in GOF bigenic osteoblasts as would be expected for a NICD gain of function model. To explore the relative contributions of canonical (NICD/Rbpj-dependent) vs. non-canonical (NICD dependent but Rbpj-independent) Notch signaling, we crossed the osteoblast-specific Notch GOF bigenic mice (RosaNotch/+; Col1a1 Cre) with a floxed allele of Rbpj. Our analyses showed the addition of the Rbpjf/f allele completely rescued growth retardation and osteosclerosis phenotypes. In summary, our data suggest that Notch intracellular domain regulates osteoblast function through the canonical Rbpj-dependent pathway.
Disclosures: Jianning Tao, None.
Notch Inhibits Wnt Signaling in Osteoblasts by Regulating Cyclic GMP Dependent Kinase II (cGKII) and Glycogen Synthase Kinase 3 β (GSK3β).Stefano Zanotti*1, Anna Smerdel-Ramoya2, Ernesto Canalis2. 1Saint Francis Hospital & Medical Center, Hartford, CT, USA, 2St. Francis Hospital & Medical Center, Hartford, CT, USA
Notch are transmembrane receptors that determine cell dierentiation. Notch1 and 2 inhibit osteoblast maturation in vivo and in vitro, and as a consequence cause osteopenia. In the Notch canonical pathway, interactions of Notch with its ligands result in the release of the intracellular domain of Notch (NICD), which translocates to the nucleus to interact with the CSL family of nuclear proteins to activate transcription. Notch inhibits osteoblastic dierentiation by suppressing free cytosolic β-catenin levels and by inhibiting Wnt signaling. However, the mechanisms by which Notch opposes Wnt signaling and decreases β-catenin levels are not known. cGKII is a membrane tethered serine-threonine kinase critical for skeletal growth, and its inactivation causes dwarfism. cGKII phosphorylates GSK3β on Serine-9, and as a consequence decreases the pool of active GSK3β. Since GSK3β phosphorylates β-catenin to initiate its degradation, a decrease in the pool of active GSK3β should lead to the stabilization and accumulation of β-catenin. Consequently, we asked whether Notch suppressed β-catenin levels in osteoblasts by regulating the cGKII/GSK3β axis. To test this hypothesis, we used primary calvarial osteoblasts from Rosanotch mice, where the NICD coding sequence is preceded by a STOP cassette flanked by loxP sites and by the Rosa26 promoter. In this model, the deletion of the STOP cassette by Cre recombination allows for Notch expression under the control of the Rosa26 promoter. A specific GSK3β inhibitor opposed the eects of Notch and restored β-catenin levels in osteoblasts, indicating that Notch acts, at least in part, through GSK3β to inhibit Wnt signaling. Notch suppressed cGKII mRNA levels in osteoblasts. To test the consequence of this eect, lysates from Rosanotch osteoblasts were treated with cyclic GMP, a physiological activator of cGKII. In this context, Notch reduced the levels of phospho GSK3β, demonstrating that Notch inhibits cGKII mRNA levels as well as activity, and explaining the destabilization of β-catenin by Notch. This was confirmed by demonstrating that 8-Bromo-cyclic GMP increased β-catenin accumulation, and this eect was opposed by Notch. In conclusion, Notch inhibits Wnt signaling in osteoblasts by suppressing cGKII mRNA levels and activity, leading to the accumulation of active GSK3β and the subsequent degradation of β-catenin.
Disclosures: Stefano Zanotti, None.
Immunophenotypic Identification of Mouse Mesenchymal Progenitors in the Bone Endosteum and their Distribution During Intermittent Parathyroid Hormone Administration.Sierra Root*1, Christian Jacome-Galarza1, Joseph Lorenzo2, Hector Aguila2. 1University of Connecticut Health Center, Farmington, USA, 2University of Connecticut Health Center, Farmington, CT, USA
The identification and characterization of osteoblast progenitors is crucial to the understanding of bone development in physiological and pathological conditions. Cells with osteogenic potential can be recovered from the bone marrow and from the endosteal fractions obtained after enzymatic digestion of marrow depleted bones. In both places these progenitors are in tight association with other cells including hematopoietic and vascular endothelial cells defining biological systems important for skeletal and blood homeostasis. Immunophenotypic analysis by flow cytometry has shown that the osteogenic activity in the endosteum is contained within cell populations negative for hematopoietic markers (CD45 and Ter119) and vascular endothelium (CD31). Further dissection using antibodies against the progenitor marker Sca-1 and the adhesion molecule CD166/ALCAM revealed three distinct populations: CD166− Sca-1−, CD166+ Sca-1− and CD166− Sca-1+. To define the stages of mesenchymal development represented in these fractions we have isolated them from transgenic mice expressing green fluorescent proteins under the control of developmentally regulated promoters. We have sorted them, analyzed their expression of signature transcripts for mesenchymal lineage progression, and their developmental potential in vitro. Results from these analyses indicated that the CD 166− Sca-1+ fraction contains cells with early mesenchymal progenitor phenotype and activity, while the other two fractions contain cells more committed to the osteoblastic lineage.
Treatment of C57BL/6 mice with intermittent doses of parathyroid hormone (bPTH 1–34, 80 μg/kg/day) for 7 and 14 days showed an increase in hematopoietic stem cells in the bone marrow and endosteal fractions. Interestingly, these changes correlated with a significant increase in the number of early mesenchymal cell populations and a concomitant decrease in the cell fractions containing more mature osteoblasts. This observation indicates that in the experimental conditions, PTH targets very early stages of mesenchymal development and that this may initiate microenvironment changes that translate in alterations in the dynamics of hematopoietic progenitors. Currently we are dissecting the osteoblast progenitor populations with antibodies against additional cell surface markers to identify the stages of mesenchymal development that influence hematopoiesis, including stability of HSC niches and modulation of osteoclastogenesis.
Disclosures: Sierra Root, None.
Increased FoxO3a Transcription in Osteoblast Progenitors Decreases their Proliferation, Differentiation and Bone Mass in Mice.Elena Ambrogini*1, Li Han1, Marta Martin Millan2, Aaron Warren3, Kanan Vyas3, Joseph Goellner1, Robert Weinstein1, Robert Jilka1, Charles O'Brien1, Maria Jose Almeida1, Stavros Manolagas1. 1University of Arkansas for Medical Sciences, Little Rock, AR, USA, 2Center for Osteoporosis & Metabolic Bone Diseases, University of Arkansas, Little Rock, AR, USA, 3UAMS, Little Rock, USA
FoxO transcription factors are crucial mediators of cellular antioxidant responses and β-catenin is essential for oxidative stress-induced FoxO activation. In osteoblastic cells, H2O2 promotes FoxO-mediated transcription at the expense of Wnt/TCF-mediated transcription and osteoblast differentiation. Based on this and evidence that age-related bone loss is associated with increased oxidative stress and defective osteoblastogenesis, we hypothesized that age-related decrease in bone formation is due, at least in part to increased FoxO activity. We have generated transgenic mice in which FoxO3a can be over-expressed in a cell type-specific manner (FoxO3aC mice) by creating a transgene with a transcriptional/translational stop cassette, flanked by loxP sites, inserted between the hybrid CMV-chicken β-actin promoter and a cDNA encoding an HA-tagged human FoxO3a. Calvaria-derived cells from newborn FoxO3aC and wild-type control mice were infected with adenovirus encoding Cre recombinase (Ad-Cre). Ad-Cre FoxO3a cells exhibited a 2.5-fold increase in FoxO transcriptional activity as determined by a FoxO-luc reporter construct, as well as increased expression of the endogenous FoxO-target genes catalase and p21CIP. In agreement with the up-regulation of the cell cycle inhibitor p21CIP, Ad-Cre FoxO3a cells exhibited decreased proliferation. Similarly, activation of FoxOs by H2O2 increased p21CIP expression in C2C12, OB-6 and bone marrow derived osteoblastic cell, and abrogated basal or Wnt3a-induced C2C12 cell proliferation. On the other hand, basal or H2O2-induced apoptosis was indistinguishable between Ad-Cre FoxO3a and control cells. Importantly, Wnt3a was able to induce TCF-mediated transcription and osteoblastogenesis, as determined by alkaline phosphatase activity, in control cells, while it was ineective in Ad-Cre FoxO3a cells. Wnt-3a induced activation of the TCF-target genes Axin2 and OPG was also attenuated in Ad-Cre FoxO3a cells. Consistent with these findings, transgenic mice overexpressing FoxO3a under the control of Osterix (generated from the cross of FoxO3aC with Osterix-Cre mice) exhibited reduced gain in vertebral BMD between 1 and 3 months compared to controls. These findings suggest that a gain of function of FoxO3a in osteoblast progenitor cells, similar to old age, may lead to decreased bone formation in vivo.
Disclosures: Elena Ambrogini, Radius Health Inc, 1
Mechanical Loading Regulates NFATc1 and β-catenin Signaling through a GSK3β Control Node.Buer Sen*1, Maya Styner2, Zhihui Xie3, Natasha Case4, Janet Rubin5. 1UNC-CH, Chapel Hill, NC, USA, 2University of North Carolina Hospitals, Chapel Hill, NC, USA, 3University of North Carolina, Chapel Hill, USA, 4University of North Carolina at Chapel Hill, Chapel Hill, NC, 5University of North Carolina, Chapel Hill, School of Medicine, Chapel Hill, NC,
Repetitive mechanical stimulation prevents adipogenic dierentiation of mesenchymal stem cells (MSCs). Our previous work suggests that mechanical inactivation of GSK3β, which results in preservation of β-catenin levels, is responsible for inhibition of adipogenic dierentiation; pharmacologic inhibition of GSK3β with SB415286 also inhibits adipogenesis. In follow-up we wished to prove that β-catenin was the primary eector for loading eects. Using C3H10T1/2 cells cultured under strong adipogenic conditions, we first showed that DKK-1 did not prevent the ability of mechanical load (3600cycles/day, 2% strain) to reduce adipogenesis by more than 50% as assessed by decreases in PPARγ and adiponectin mRNA and protein, thus ruling out a role for Wnt binding. siRNA knockdown of β-catenin (to 20% of control protein) blocked by at least half both mechanical and pharmacologic inhibition of PPARγ and adiponectin, indicating that mechanical activation of β-catenin was causally involved. Surprisingly however, the eect of both mechanical and pharmacologic inhibition of GSK3β on a putative β-catenin target, COX-2, was enhanced by β-catenin knockdown: mechanical stimulation increased COX2 mRNA and protein by 5-fold and 10-fold in MSC treated with scrambled siRNA and siRNA to β-catenin respectively. We hypothesized that mechanical inhibition of GSK3β might aect other downstream signaling cascades. Pharmacologic inhibition of GSK3β allows nuclear accumulation of NFATc1, a transcription factor thought to be involved in COX-2 regulation. We thus asked whether mechanical load might alter NFATc1 activation. We demonstrated that nuclear NFATc1 levels increase within 60 min after strain application, unaected by β-catenin knockdown. Further, while the calcineurin inhibitor tacrolimus inhibits the rapid activation of NFAT by ionophore, it does not prevent NFATc1 nuclear accumulation after either mechanical or SB415286 inhibition of GSK3β. Finally, siRNA targeting GSK3β inhibits mechanical eects on both adiponectin and COX-2. Our results indicate that mechanical inhibition of GSK3β causes activation of both β-catenin and NFATc1 signaling pathways, resulting in reduced adipogenesis and very possibly promotion of osteogenic dierentiation via NFATc1. Our novel findings suggest that mechanical load regulates mesenchymal stem cell dierentiation through inhibition of GSK3β, thereby aecting not only β-catenin, but multiple downstream signaling pathways.
Disclosures: Buer Sen, None.
Priming integrin alpha5 Triggers human mesenchymal stromal cell osteoblast differentiation and promotes osteogenesis in vitro and in vivo.Zahia Hamidouche1, Olivia Fromigue2, Jochen Ringe3, Thomas Haupl3, Pascal Vaudin4, Jean-Christophe Pages4, Samer Srouji5, Erella Livne5, Pierre Marie*6. 1Inserm U606 & University Paris Diderot, Paris, France, 2INSERM Unit 606, Paris, France, 3Charite University, Berlin, Germany, 4Inserm U966, Tours, France, 5Faculty of Medicine, Haifa, Israel, 6INSERM Unit 606 & University Paris Diderot, Paris, France
Human adult mesenchymal stromal cells (hMSC) have the potential to differentiate into chondrogenic, adipogenic or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptomic analysis, we found that integrin alpha5 (ITGA5) expression is upregulated during dexamethasone-induced hMSCs osteoblast differentiation. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers as well as in vitro osteogenesis in hMSCs. Downregulation of endogenous ITGA5 using shRNA blunted osteoblast marker expression and in vitro osteogenic differentiation. Pharmacological and molecular functional analyses showed that the enhanced hMSCs osteoblast differentiation induced by ITGA5 was mediated by activation of FAK/ERK1/2-MAPKs and PI3K signaling. Remarkably, activation of ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and the osteogenic capacity of hMSCs. Furthermore, we demonstrated that hMSCs engineered to over-express ITGA5 exhibited a marked increase in their osteogenic potential in vivo in a standard subcutaneous assay in mice. These findings not only reveal that ITGA5 is required for osteoblast differentiation of adult human MSCs but also provide a novel targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs, which may be used for tissue regeneration in bone disorders where the recruitment or capacity of MSCs is compromised.
Disclosures: Pierre Marie, None.
c-Cbl and Cbl-b Displace HDAC6 from β-Tubulin and Stabilize Microtubules and Podosomes in Osteoclasts.Enkhtsetseg Purev1, Lynn Ne1, William Horne*2, Roland Baron3. 1Harvard School of Dental Medicine, Boston. USA, 2Harvard School of Dental Medicine, Boston, MA, USA, 3Harvard School of Medicine & of Dental Medicine, Boston, MA, USA
c-Cbl and Cbl-b are adaptor proteins and ubiquitin ligases that share many highly homologous structural features. One or both of the Cbl proteins are involved in several mechanisms that affect osteoclast differentiation, adhesion and motility, bone resorption and survival, incuding M-CSF and RANK signaling and the regulation of podosome organization and function. While deleting both c-Cbl and Cbl-b genes leads to embryonic lethality, indicating the existence of essential redundant functions of the two Cbl proteins, the single deletions have relatively mild and different effects in osteoclasts. To examine the redundant actions of c-Cbl and Cbl-b in osteoclasts, we used a short hairpin RNA (c-Cbl-shRNA) to deplete c-Cbl by ∼75% in Cbl-b−/− osteoclasts. The podosome belt and the microtubule network, which is required for podosome belt formation, were disrupted in the doubly Cbl-depleted OCLs and bone-resorbing activity was decreased, in contrast to the eects of either single deletion. Stabilizing the microtubules with paclitaxel prevented the loss of the podosome belt. Inhibiting histone deacetylase 6 (HDAC6), which destabilizes microtubules by deacetylating tubulin, also prevented the disruption of both the microtubule network and the podosome belt. Examination of the mechanism involved demonstrated that the conserved four-helix bundle of c-Cbl's TKB domain bound to β-tubulin, and both c-Cbl and Cbl-b displaced HDAC6, suggesting that the Cbl proteins promote podosome belt formation at least in part by preventing the destabilizing eect of HDAC6 on microtubules. In addition to, and independent of the eects on microtubules and the podosome belt, depleting both Cbl proteins increased apoptosis of the OCLs. Consistent with the increased apoptosis, the level of the pro-apoptotic protein Bim was moderately increased when either c-Cbl or Cbl-b was deleted and markedly increased in the doubly Cbl-depleted OCLs. Thus, both c-Cbl and Cbl-b promote the formation of the podosome belt in osteoclasts by stabilizing the microtubule network and independently promote osteoclast survival.
Disclosures: William Horne, None.
Paxillin Regulates Osteoclast Function.Wei Zou*1, Carl DeSelm2, Haibo Zhao2, Jikun Zha2, Scott Vande Pol3, Kyunghee Choi2, F Patrick Ross2, Steven L Teitelbaum2. 1Washington University in St. Louis School of Medicine, St. Louis, MO, 2Department of Pathology, Washington University School of Medicine, St. Louis, USA, 3Department of Pathology, University of Virginia, Charlottesville, USA
The osteoclast (OC) cytoskeleton, which is essential for its bone resorption, is characterized by a sealing zone, composed of a ring of F-actin surrounded by integrinassociated proteins such as paxillin. Paxillin is an integrin-binding scaffolding protein essential for cytoskeletal organization. As the paxillin−/− mouse is early embryonic lethal, little is known about paxillin's function in the OC. We therefore studied the function of paxillin in the bone resorptive cell by developing techniques for generating large numbers of authentic OCs from embryonic stem (ES) cell lacking both paxillin alleles. Pax−/− and Pax+/- ES cells were differentiated to the embryonic body stage, then cultured with M-CSF until a macrophage-like cell forms, which when exposed to M-CSF and RANKL generates OCs. Paxillin deficiency does not affect OC differentiation, as confirmed by differentiation markers, including β3 integrin, c-src and cathK, as well as M-CSF and RANKL induced MAP kinase and IκB activation. However, given its critical role in cytoskeletal organization in other cells, we were surprised to observe that pax−/− OCs actually spread and form larger OCs on plastic dish; furthermore, while Pax+/- OC contains numerous small actin ring when cultured on bone, each Pax−/− OC contains only a single ring which is substantially larger and encompasses a greater percentage of the cell's area. Moreover, we found that while Pax+/- OCs generate well-demarcated bone resorptive lacunae, those formed by Pax−/− OCs are larger but have indistinct margins. Transverse sections of bone slices show that the Pax+/- OCs generated deep resorptive pits, while Pax−/− OCs are more spread but generate shallow pits, and measurement of pit depth confirms this result (P<0.01). These phenotypic abnormalities are rescued by lentivirally transducing Paxillin into Pax−/− cells.
Paxilin contains 5 LD motifs which mediate interaction with other proteins and several tyrosines regulating its function. We find RANKL, M-CSF and integrin all induce c-src-dependent paxillin tyrosine phosphorylation. However, double tyrosine to phenylalanine mutant (Y31F, Y118F) completely rescues Pax−/− OC function, suggesting other less well characterized Y residues may be operant in the OC. On other hand, paxillin lacking the LD4 domain (ΔLD4) fails to rescue Pax−/− OC. Our results indicate that paxillin mediates OC cytoskeleton reorganization on bone and its LD4 domain, but not Y31 or Y118, is essential for its function.
Disclosures: Wei Zou, None.
RANKL Induces Osteoclast Differentiation from TRAF6 Knockout Mouse Osteoclast Precursors through Autocrine Induction of TNF and Interaction with Bone Matrix.Zhenqiang Yao*1, Bryant Darnay2, Yanyun Li3, Lana Hur4, Betty Lamothe5, Lianping Xing1, Brendan Boyce6. 1University of Rochester, Rochester, NY, USA, 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA, 3University of Rochester Medical Center, Rochester, USA, 4University of Texas MD Anderson Cancer Center, Houston, USA, 5MD Anderson Cancer Center, Houston, TX, USA, 6University of Rochester Medical Center, Rochester, NY, USA
Expression of TRAF6 is essential for bone resorption. RANKL does not induce osteoclast (OC) formation from TRAF6 KO OC precursors (OCPs) in vitro, but there are OCs in the bones of osteopetrotic TRAF6 KO mice, suggesting that some other factor induces OC formation in vivo in the mice. We found that TNF, but not IL-1 or TGF-b induced TRAF6 KO OCP dierentiation to OCs along with M-CSF (50+/-6 vs 60+/-8/well from WT OCPs), and importantly these cells formed resorption pits. Unexpectedly, RANKL increased TNF-induced OC formation on plastic (376+/-18 vs 98+/-12/well), but it did not mediate OC formation by itself or with IL-1 or TGF-b. Surprisingly, RANKL induced KO OCP dierentiation into OCs on bone slices (186+/- 25/slice) and on the adjacent plastic. We previously reported that WT OCPs expressing c-Fos dierentiate into OCs in response to autocrine secretion of IL-1 and TNF by interacting with bone matrix. To examine if cytokines are involved in KO OCP dierentiation on bone slices, we treated them with RANKL+/-specific cytokine inhibitors. TNFR:Fc almost completely and TGF-b neutralizing antibody partially blocked RANKL-mediated OC formation (15+/-8 and 86+/-19/slice, respectively vs 175+/-29), while IL-1R antagonist had no eect. We measured TNF levels in the culture medium to determine if TRAF6 is involved in the production of TNF by OCPs on bone slices in response to PBS control or RANKL. KO OCPs + PBS on bone slices secreted significantly less TNF than WT cells (50+/-2 vs 169+/-43 pg/ml), and addition of RANKL did not change TNF production in either KO or WT OCPs (39+/-12 vs 170+/-32 pg/ml). We next examined the relationship between TRAF6 and c-Fos. RANKL or TNF induced similar levels of c-Fos mRNA in KO and WT OCPs on plastic. Furthermore, c-Fos retroviral over-expression did not rescue RANKL-mediated OC formation from KO OCPs on plastic. In summary: 1) TNF induces OC formation and activity independent of TRAF6 in vitro; 2) RANKL can stimulate OCP dierentiation independent of TRAF6 by an autocrine TNF-mediated mechanism through OCP interaction with bone matrix. These findings suggest that RANKL could stimulate OC formation through OCP interaction with bone matrix by a TRAF6-independent mechanism and that TNF expressed by OCPs in response to this interaction could induce these cells to resorb bone. This could be one mechanism to explain the increased bone resorption seen in inflammatory arthritis in which TNF levels are increased.
Disclosures: Zhenqiang Yao, None.
ADAM8 Enhances Osteoclast (OCL) Precursor Number and Increases OCL Activity in Vitro.Hisako Ishizuka*1, Noriyoshi Kurihara2, Jolene Windle3, G. David Roodman4. 1Center for Bone Biology, University of Pittsburgh, Pittsburgh, PA, USA, 2Center for Bone Biology, University of Pittsburgh, Pittsburgh, USA, 3Virginia Commonwealth University, Richmond, USA, 4Center for Bone Biology, University of Pittsburgh & VA Pittsburgh Healthcare System, Pittsburgh, USA
ADAM (a disintegrin and metalloprotease) family proteins are type 1 transmembrane glycoproteins involved in cell proliferation, differentiation, migration, adhesion, and morphogenesis. ADAM8 is one of several factors produced by OCL that is involved in osteoclastogenesis in vitro. To characterize the role of ADAM8 in OCL formation in vivo, we generated TRAP-ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage. Overexpress ion of ADAM8 in OCL precursors resulted in increased OCL formation and decreased trabecular bone volume in vivo. Because the mechanisms for ADAM8 effects in OCL formation and activity have not been clearly defined, we characterized OCL formation in mice overexpressing ADAM8. Transgenic mice overexpressing ADAM8 had increased OCL precursor numbers and formed 3-fold more multinucleated TRAP(+) cells than wild type (WT) mice in vitro. The OCLs that formed were hyper-multinucleated compared to OCL derived from CD11b(+) mononuclear cell cultures from WT mice. OCL precursors from TRAP-ADAM8 mice displayed up to a 5-fold increase in p-ERK activation as compared to OCL precursors from WT littermates. The NF-B canonical pathway stimulated by RANKL was not affected by overexpression of ADAM8 in OCL precursors. Interestingly, TRAP-ADAM8 OCLs demonstrated an increased bone resorption capacity, which was associated with increased levels of Pyk2, p-src activation, and expression of cathepsin K and rab7. These data demonstrating that increased expression of ADAM8 targeted to the OCL lineage results in enhanced formation of OCL with an increased bone resorption per OCL and by increased numbers of OCL precursors, and demonstrate an important role for ADAM8 in OCLs.
Disclosures: Hisako Ishizuka, Celgene, 5; Amgen, 5; Novartis, 5; Novartis, 8; Novartis, 2; Acceleron, 5
Chemokine-mediated migration control of osteoclast precursors visualized by dynamic in vivo bone imaging: a novel point of regulation for bone homeostasis.Masaru Ishii. Immunology Frontier Research Center, Osaka University, Osaka, Japan
Osteoclasts are bone-resorbing multinucleated giant cells that dierentiate from mononuclear macrophages/monocyte-lineage hematopoietic precursors. They play critical roles not only in normal bone homeostasis (“remodeling”) but also in the pathogenesis of bone destrctive disorders such as osteoporosis, rheumatoid arthritis, periodontosis, and bone metastatic cancers. Although many molecules, M-CSF and RANKL cheif among them, are known to play crucial contributions in osteoclast dierentiation, a critical process that has been less well documented is the tracking of osteoclast precursors to and from the bone surface, where they undergo cell fusion to form the fully dierentiated multinucleated cells that actually mediate bone resorption. By using an advanced imaging technique with intravital two-photon microscopy I have established a new system for visualizing in situ behavior of osteoclasts and their precursors within intact bone tissues (see an attached figure), and found that sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, controls the movement of osteoclast precursors between the blood and endosteum (their site of final dierentiation). Osteoclast precursor monocytes in bone tissues express functional S1P receptor, and potent S1P receptor agonist stimulated their mobilization in vivo. Because S1P concentration in blood is always higher than that in tissues, S1P-mediated chemotaxis of osteoclast precursors contributes to their recirculation from bone tissues to systemic blood flow. Treatment with FTY720, a clinicaly usable S1P agonist, relieved ovariectomy-induced murine osteoporosis by facilitating recirculation of osteoclast precursors and thus by reducing the number of mature osteoclasts attached to bone surface.
Further examinations are revealing the possible roles of several bone-enriched chemokines on “bone-attraction” of osteoclast precursors. The sum of these results support the concept that fine tuning of osteoclast precursor migration mediated by various chemokines and lipid mediators dynamically modulates bone homeostasis, suggesting a unique point of action on osteoclastogenesis that may be promosing as a future therapeutic target. In this presentation I will show the latest results on this novel concept in the field of bone and mineral research, and will also present the methodology of intravital in vivo bone imaging using two-photon microscopy and discuss its further application in this field.
Disclosures: Masaru Ishii, None.
IL-27 Abrogates RANKL-mediated Osteoclastogenesis of Human Granulocyte-Macrophage CFU Cells through STAT1-dependent Inhibition of c-Fos.Mitsuru Furukawa*1, Hironari Takaishi2, Tomohiro Hikata3, Akihiro Hakozaki3, Shinichi Uchikawa4, Tomoaki Mori5, Masaki Yoda5, Takahide Tohmonda5, Morio Matsumoto5, Kazuhiro Chiba5, Koichi Matsuo3, Hiroki Yoshida6, Yoshiaki Toyama5. 1Tokyo, Japan, 2Keio University, Tokyo, Japan, 3Keio University School of Medicine, Tokyo, Japan, 4Department of Orthopaedic Surgery, School of Medicine, Keio University, Tokyo, Japan, 5Department of Orthopaedic Surgery, Keio University, Tokyo, Japan, Tokyo, Japan, 6Departmrnt of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan, Tokyo, Japan
IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hematopoietic stem cell differentiation, and anti-tumor activities. However, hIL-27's role in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing hGM-CSF, hSCF (stem cell factor), and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, a WSX-1/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kB ligand (hRANKL) induced the differentiation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts cultured on a dentine slice, caused a marked increase in STAT1 phosphorylation, and enhanced the STAT1 protein level 10-fold. It also inhibited the RANKL induced c-Fos and NFATc1 expression, which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and a STAT1 small-interfering RNA partially rescued IL-27's inhibition of osteoclastogenesis. A WSX-1 deficient(KO) mice caused severe inflammatory bone destruction primed by E. coli cell-wall lysate in vivo. The number of inflammatory cells and osteoclasts were invaded into bare area of femur in WSX-1 KO mice. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis by hCFU-GMs via STAT1-dependent downregulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.
Disclosures: Mitsuru Furukawa. None.
The role of splenic IL-34 in CSF-1-independent osteoclastogenesis induced by active vitamin D3.Yuko Nakamichi*1, Nobuyuki Udagawa1, Toshihide Mizoguchi2, Yasuhiro Kobayashi2, Naoyuki Takahashi3. 1Matsumoto Dental University, Shiojiri, Japan, 2Matsumoto Dental University, Shiojiri, Japan, 3Matsumoto Dental University, Nagano, Japan
CSF-1 (colony stimulating factor-1) is essential for proliferation of macrophages and their dierentiation into osteoclasts (OCs). Therefore, CSF-1-deficient CSF-1op/op mice exhibit monocytopenia, and severe osteopetrosis with OC deficiency. We previously reported that 2MD, a highly potent 1α,25-dihydroxyvitamin D3 analog, induced OC formation in 3-week-old CSF-1op/op mice (J Bone Miner Res 22, Suppl 1; S380, 2007) . This finding suggests the existence of the substitute for CSF-1 in CSF-1op/op mice, expression of which may be up-regulated by 2MD. Recently, IL-34 was discovered as the second ligand for CSF-1R, in addition to CSF-1, and was shown to be expressed prominently in spleen among human tissues (Science 320:807–11, 2008). We speculated that IL-34 is involved in 2MD-induced OC formation in CSF-1op/op mice.
The osteoclast-inducing activity of IL-34 was quite similar to that of CSF-1 in cultures of mouse bone marrow macrophages. In consistent with the previous report in human, IL-34 was highly expressed in mouse spleen. We also found that IL-34 was barely expressed in bone. 2MD (30 pmol/head/injection) was administered i.p. twice into 3-week-old CSF-1op/op mice 2 days apart. Many TRAP-positive OCs were observed in both the epiphysis and metaphysis of tibiae at 48 h after the second administration of 2MD. Prior to the appearance of OCs in bone, the expression of IL-34 in spleen was increased in response to 2MD at 24 h after the first administration. Interestingly, TRAP-positive mononuclear cells were also induced in spleen by 2MD administration at 48 h, 24 h after the increase in IL-34 expression was observed. The number of TRAP-positive cells in spleen was decreased at 48 h after the second administration of 2MD, at the time point that TRAP-positive OCs were observed in bone. To investigate the relationship between 2MD-induced splenic TRAP-positive cells and bone OCs, spleen was removed from 3-week-old CSF-lop/op mice, and 2MD was given to those mice. 2MD failed to induce TRAP-positive OCs in bone in splenectomized CSF-1op/op mice. These results suggest that spleen is responsible for 2MD-induced osteoclast generation in CSF-1op/op mice, and that IL-34 is a new target of 1α,25-dihydroxyvitamin D3.
Disclosures: Yuko Nakamichi, None.
Bone Quality in Rac1 and Rac2 knock out Female Mouse Model.Joyce Magalhaes*1, Michael Glogauer2, Marc Grynpas3. 1Faculty of Dentistry. University of Toronto, Toronto, Ontario, Canada, 2Faculty of Dentistry, University of Toronto, Toronto, ON, Canada, 3Samuel Lunenfeld Research Institute, Toronto, ON, Canada
Purpose: Rho small GTPases are important regulators of a variety of cell functions, including osteoclast differentiation. We have investigated the role of Rac1 and Rac2 in bone resorption and remodeling activity, which is a key determinant of bone quality. Our purpose was to determine the role of Rac1 and Rac2 in the bone quality of 4-month old female mice. Methods: 46 C57BL/6J female mice were divided into 4 groups — 15 wild type (WT), 12 Rac1 null (Rac1KO), 15 Rac2 null (Rac2KO) and 4 Rac1 and Rac2 double null mice (DKO). After sacrifice, the bones were harvested for mechanical tests, densitometry and histomorphometric analyses. In addition, back-scattered electron imaging (BSE) was used to evaluate bone mineralization and architecture. The results were compared between groups using ANOVA and LSD post hoc test. Results: Rac1KO cortical bones showed higher bone mineral density (BMD) and mechanical properties. Higher mineral density was also observed in their trabecular bones, and they showed increased trabecular connectivity and structural parameters such as trabecular bone number and volume compared to the control and Rac2KO. In contrast, Rac2KO showed lower BMD in both cortical and trabecular bones. Their bones were less mineralized and lower structural properties were observed in the mechanical tests. Additionally, Rac2KO cortical bones showed significantly smaller bones compared to the others. However, no difference was observed when material properties were analyzed between Rac2KO and the other groups. All knock out groups showed higher proportion of osteoclast surface to bone surface, but there was no difference in osteoclast number between the various groups. Despite the higher BMD, DKO samples did not show difference in most of the analyses. Conclusion: While our results suggest that Rac1KO improves bone quality, they also imply that Rac2 deletion had a significant impact in bone formation and consequently on bone structural properties. Apparently Rac deletion did not affect osteoclast formation. Our results suggest that while Rac1 seems to play a more significant role on osteoclasts activity than on differentiation, Rac2 may be more important in bone formation. Despite the similarity of the two GTPases molecules, no synergism was found in DKO mice.
Disclosures: Joyce Magalhaes, None.
Phosphoinositide 3-Kinase gamma (PI3K gamma) Knockout Mice Have Increased Bone Mass due to Deficient Osteoclast Development.Heeseog Kang*1, Agnes Vignery2, Dianqing Wu1. 1Yale University, New Haven, USA, 2Yale School of Medicine, New Haven, CT, USA
PI3Ks are a family of dual specificity kinases that regulate various biological processes, including cell growth, dierentiation, survival, and migration. PI3Kgamma is a 110-kDa catalytic subunit of class IB PI3K family and activated by G-protein-coupled receptors, unlike the class IA PI3K family members that are activated by receptor-type and non receptor-type tyrosine kinases. PI3Kgamma is mainly expressed by myeloid cells, while other types of PI3Ks are ubiquitously expressed in various tissues. Being one of the key enzymes in leukocyte signaling, PI3Kgamma became a promising target for drug development for the treatment of rheumatoid arthritis and other immune-mediated diseases. Despite the recent development of selective PI3K inhibitors for immune disorders, direct eect of PI3Kgamma inhibition on skeletal health has not been explored. To investigate the role of PI3K gamma in the bone homeostasis, we generated PI3Kgamma knockout (KO) mice and carried out functional characterization of PI3Kgamma in bone. Dual energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) analyses of femurs from two month-old male mice showed 10% increased bone mineral density (BMD) and bone mineral content (BMC) in PI3Kgamma KO compared to control mice with statistical significance (P<0.05). Microcomputed tomotraphy (microCT) analysis of the same bone samples further supported these observations showing that PI3Kgamma KO mice had 35% higher trabecular bone volume fraction (BV/TV) compared with wild-type control mice. Moreover, trabecular bone thickness was 11% higher in PI3K gamma-deficient mice. Histological examination of tibial bone sections revealed that PI3Kgamma KO mice had a decreased number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts on trabecular bone surfaces. To further investigate the underlying mechanism of this bone phenotype, osteoclasts were dierentiated from bone marrow-derived monocytes and recognized as multinucleated cells with TRAP activity. Quantification of TRAP-positive cells with three or more nuclei demonstrated that osteoclast development was significantly delayed in PI3Kgamma-null cells. In conclusion, PI3Kgamma deficiency results in the increased bone mass and it is attributable to the deficient osteoclast development. This study suggests that PI3Kgamma plays a role in osteoclast development and thereby in bone homeostasis.
Disclosures: Heeseog Kang, None.
Constitutive Protein Kinase A Activity in Osteocytes Leads to Increased Trabecular Bone.Richard Kao*1, Alyssa Louie1, Weidar Lu2, Robert Nissenson3. 1UCSF/VAMC, San Francisco, USA, 2UCSF/VAMC, San Francsico, USA, 3University of California, San Francisco, San Francisco, CA, USA
Osteocytes have been implicated in the control of systemic mineral homeostasis and in regulating the rate of bone formation. However, the signal transduction pathways that regulate the biological function of osteocytes are poorly defined. Limited evidence suggests an important role for the Gs/cAMP pathway in osteocyte function. In the present study, we explored the hypothesis that PKA activation in osteocytes plays a key role in controlling skeletal homeostasis. To test this hypothesis, we mated mice harboring a Cre-conditional, mutated PKA catalytic subunit allele that encodes a constitutively-active form of PKA (CA-PKA) with mice expressing Cre under the control of the osteocyte-specific promoter, DMP1. This allowed us to direct the expression of CA-PKA to osteocytes in double transgenic progeny. Cortical and trabecular bone parameters from 12-week old female mice were determined by μCT. Expression of CA-PKA in osteocytes produced a significant increase in the overall size of the tibia-fibular junction (TFJ) as compared to DMP1Cre single transgenic controls. This was exemplified by increases in total volume (TV), bone volume (BV), and medullary volume but not BV/TV. In cancellous bone of the distal femur, BV/TV, connectivity density, trabecular number, and trabecular thickness showed increases while there was a decrease in trabecular spacing. To assess possible causes for the increase in trabecular bone, qRT-PCR was carried out using RNA isolated from whole bones to detect the expression of osteoblast and osteocyte marker genes. No pronounced changes in the expression of osteoblast markers such as osterix, runx2, collagen 1α1, alkaline phosphatase, and osteocalcin were detected. Interestingly, some of the osteocyte-specific products showed dramatic alterations in their gene expression. FGF23 expression was greatly increased by several fold over DMP1Cre controls. On the other hand, expression of SOST was reduced by more than 50% compared to control. In summary, constitutive PKA signaling in osteocytes led to a small expansion of the size of TFI and an increase in trabecular bone in female mice. This was associated with down-regulation of the expression of SOST and up-regulation of FGF23. Activation of the PKA pathway in osteocytes appears to be sufficient for the initiation of an anabolic skeletal response.
Disclosures: Richard Kao, None.
Mice with Targeted Deletion of E11/gp38 in Late Osteoblasts Have Reduced Canaliculi per Osteocyte Which May Be Responsible for The Enhanced Trabecular Bone Volume.Dayong Guo1, Hong Zhao*2, Yuji Mishina3, Jerry Feng4, Stephen Harris5, Lynda Bonewald6. 1Kansas City, MO. USA, 2UMKC, Kansas City, USA, 3UMSD, Ann Arbor, USA, 4Baylor College of Dentistry, Dallas, USA, 5UTHSCSA, San Antonio, USA, 6University of Missouri, Kansas City, MO, USA
E11/gp38/pdpn is a membrane glycoprotein thought to be involved in the formation of cellular processes in cells such as kidney podocytes, type 1 alveolar lung cells, lymphatic endothelial cells and in osteocytes. E11 is highly expressed in osteoid osteocytes morphing from polygonal osteoblasts into dendritic cells during the embedding process. To test the hypothesis that depletion of E11 in osteocytes would reduce the dendrite formation and potentially osteocyte function, targeted deletion was performed by crossing the E11 flx/flx mice with transgenic mice expressing Cre recombinase driven by osteocalcin promoter. It was discovered that the E11 fl/fl mice were hypomorphs with reduced E11 expression in lung, kidney and bone. The majority of animals were unaected by this reduction resulting in normal sized animals with normal survival, while a few animals with defective kidney and lymphatic function had involving the osteocyte network poor survival. Conditional knockout, cKO, animals used for these studies were of normal size and immunohistochemistry confirmed removal of E11 protein in cKO bone. The adult cKO mice showed slightly higher body weight and whole body bone mineral density as examined by PIXIMUS densitometry compared to control littermates with one allele of the wild type E11 gene. By uCT, the cKO had a highly significant two fold increase in the number and area of trabecular bone, but no significant dierences in cortical bone compared to control littermates at 3 months of age. Dierences in the lacuna-canalicular network were observed in the cKO bone compared to control using scanning electron microscopy images of acid etched resin embedded ulnae. Quantitative measurements revealed that the number of canaliculi between the newly embedded osteocytes and bone surface cells was reduced by half, while the number of osteocyte lacunae per area was not changed. These findings suggest that osteocytes in trabecular bone may regulate bone remodeling by a dierent mechanism than osteocytes in cortical bone involving the osteocyte canalicular network and that reduction of canaliculi in trabecular bone may be a way to increase trabecular bone mass.
Disclosures: Hong Zhao, None.
Sclerostin and abnormalities in regulation of cortical bone formation at osteon level: a hip fracture model of osteoporosis.Jonathan Reeve*1, Jon Power2, Michael Doube3, Kenneth Poole4, Rutger v Bezooijen5, Socrates Papapoulos5, Nigel Loveridge2. 1Addenbrookes Hospital, Cambridge, United Kingdom, 2University Dept Medicine, Cambridge, United Kingdom, 3Imperial College, London, United Kingdom, 4MRC Bone Research Group, Cambridge, United Kingdom, 5LUMC, Leiden, Netherlands
Purpose: Osteocytes regulate bone formation by secreting sclerostin and other molecules. Osteoblasts are seeded on the previously resorbed surface during the reversal phase of cortical bone remodeling. It has been postulated that in osteoporosis osteoblasts undergo excessive apoptosis impairing bone formation, so leading to excessive cortical porosity. Osteocytes are derived from osteoblasts and osteocyte formation might be reduced in osteoporosis through a shortage of osteoblasts. We examined osteonal remodelling and its regulation by osteocytic sclerostin.
Methods: We studied cases of femoral neck fracture (FNF) who donated femoral neck bone discarded at surgery and post mortem controls. Using specific antibody staining, we determined the osteocytic expression of sclerostin within osteons of the femoral neck cortex (FNF: 5M, 5F 73–87y; controls C: 5M, 6F 61–90y). Sclerostin expression, distances of each osteocyte to the canal surface and cement line and areal osteocyte density were assessed for all osteonal osteocytes in 636 unremodelled osteons chosen from fields (∼0.5mm diameter) with at least one canal staining for alkaline phosphatase (ALP; a marker of bone formation). In adjacent sections, qualitative ALP staining was used to classify BMUs as quiescent or actively forming (ALP+). 5 quantiles of osteocyte distance from the canal surface were calculated for each subject.
Results: FNF cases had more ALP+ osteons (42 vs 27%, p<0.05). In both C and FNF, BMU-level ALP expression was inversely related to the areal density of sclerostin expressing osteocytes (logistic regression: χ2 = 30.1, p<0.0001). In C but not FNF the probability of an osteon being ALP+ was related inversely to wall thickness ie maturity (C: p=0.0002). Positive associations were seen between wall thickness and osteocytic sclerostin expression in both C and FNF. In FNF, the 10th and 25th quantiles of osteocyte distance from the canal were significantly larger by 6.6 (+41%) and 7.4 (+27%) micrometers (p 0.004 and 0.002) due to fewer newly incorporated osteocytes.
Conclusions: Sclerostin expression was a strong determinant of bone forming activity at BMU level in both C and FNF. FNF was associated as expected with reduced osteocyte density localized adjacent to the canal in maturer osteons. Loss of the normal acceleration in the early phase of bone formation in FNF was unexpected, could not be attributed to impaired osteocyte function and requires further investigation.
Disclosures: Jonathan Reeve, P & G, Novartis, Merck, 5; P & G, 2
A Prospective Study of DXA-Based Structural Engineering Models for the Prediction of Hip Fractures.Kim Naylor*1, Eugene McCloskey2, Richard Eastell2, Lang Yang3. 1The University of Sheeld, Sheeld, United Kingdom, 2University of Sheeld, Sheeld, United Kingdom, 3Univesity of Sheeld, Sheeld, United Kingdom
In a previous cross-sectional study, we demonstrated that structural engineering models (SEM) of the proximal femur were at least as good discriminators of hip fractures compared to the bone mineral density (BMD) alone. The aim of the present study was to determine whether SEM variables could predict hip fracture prospectively in a nested case control study.
We studied 728 women, mean age 82yr, 182 with incident hip fractures during a median of 4 years follow-up, and 546 age-height-weight matched controls. DXA scans of the hip at baseline were reanalyzed using a specially-designed version of Hologic software to produce a pixel-by pixel bone map. A finite element (FE) model was generated to calculate the stress within the bone as a consequence of a sideways fall. A stress ratio (SR) at each pixel was defined as the FE model calculated stress divided by the yield stress; a higher value indicating greater fracture risk. The maximum and mean SRs were determined over the femoral neck (FN), trochanter (TR) and total hip and compared to BMD as hip fracture predictors.
Baseline mean BMDs at the total hip and sub-regions were significantly lower in the hip fracture cases compared to controls (P<0.001). The baseline maximum and mean SRs were all significantly higher in fracture cases than in controls (P<0.0001). The odds ratio (OR) of fracture for a 1 SD decrease in FN BMD was 2.10 (95% CI 1.69 to 2.62). The ORs for 1 SD increase in the stress ratio parameters were all significantly greater than 1 (P<0.05). The maximum compressive SR in the FN had the largest OR (2.25, 95% CI 1.83–2.75), and its area under the ROC curve (0.74, 95% CI 0.70 to 0.78) was significantly greater than FN BMD alone (0.66, 95% CI 0.62 to 0.71). After adjusting for FN BMD, the maximum compressive and tensile SRs in the FN and TR remained significantly greater than 1. Forward conditional logistic regression including FN BMD and stress ratio parameters identified the best regression model to have stress ratio parameters only: maximum compressive and tensile SRs in the FN and tensile SR in the trochanter. The area under the ROC curve for the best model (0.75, 95% CI 0.72–0.79) was significantly greater than FN BMD alone (0.66) and similar to that of maximum compressive SR.
In conclusion, this finite element model can be applied to routine hip DXA scans and is more predictive of hip fracture than femoral neck BMD alone.
Disclosures: Kim Naylor, Hologic Inc, MA USA, 99
Bone DESTINY™ Predicts 10-year Clinical Fracture Risk in Men in the CaMos Cohort.Karen Beattie*1, Maggie Larche2, George Ioannidis3, Alexandra Papaioannou2, Shawn Davison4, David Hanley5, Robert Josse6, Stephanie Kaiser7, Christopher Kovacs8, Nancy Kreiger9, Wojciech Olszynski4, Jerilynn Prior10, Tanveer Towheed11, Jonathan D. Adachi2, William Bensen12. 1McMaster University, Hamilton, ON, Canada, 2McMaster University, Hamilton, Ontario, Canada, 3, Kitchener, ON, Canada, 4University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 5University of Calgary, Calgary, Alberta, Canada, 6St. Michael's Hospital, University of Toronto, Toronto, ON, Canada, 7Dalhousie University, Halifax, NS, Canada, 8Memorial University, St. John's, Newfoundland & Labrador, Canada, 9University of Toronto, Toronto, Ontario, Canada, 10University of British Columbia, Vancouver, British Columbia, Canada, 11Queen's University, Kingston, Ontario, Canada, 12Self-Employed, Hamilton, ON, Canada
Objective: To validate the use of Bone DESTINY as a one-step fracture risk predictor in men using the CaMos cohort.
Methods: Bone DESTINY is a one-step fracture risk predictor that considers bone mineral density, sex, age, previous fragility fracture (any site excluding toes, fingers, face, skull), current unprotected steroid use >3 months and history of falls (past month) to predict overall fracture risk using 5 fracture risk-specific colours where green is associated with very low risk of fracture over 10 years, yellow with low risk, orange with moderate risk, red with high risk, and purple with very high risk. While conducting DEXA scans, technicians input all fracture risk factors into a hand-held computer. A software program produces a diagrammatic DESTINY report (Figure 1). We hypothesized that very low fracture risk corresponded to <5% overall fracture risk, low risk to 5–10%, moderate risk to 11–20%, high risk to 21–25% and very high risk to >25%. To validate the use of this tool in men, bone DESTINY fracture risk colours were determined at baseline for each participant >50 years of age in the CaMos cohort. Ten-year clinical fracture data were used to determine the fracture risk associated with each colour to validate the accuracy of these fracture-risk categories.
Results: Of 1215 men, mean age (SD) 63.3 (8.1) yrs, 90 experienced a clinical fragility fracture (10 clinical vertebral). Table 1 depicts the proportion of men in each fracture risk colour category at baseline who experienced a fracture within 10 years of follow-up. Odds ratios associated with each colour are also shown. Fracture risk associated with green, yellow and orange are consistent with our hypotheses while fracture risk associated with red and purple appear reversed. When grouped into three fracture risk specific groups similar to Osteoporosis Canada, (low fracture risk = green + yellow, moderate fracture risk = orange, high fracture risk = red + purple), the proportion of men who experienced a fracture was also calculated. Results are shown in Table 2.
Conclusions: Results from this study suggest that the colour-specific fracture risk categories appear to be associated with an increasing 10-year risk of fracture. Very low fracture risk (green) was associated with 3.2% risk of fracture while very high risk (purple) was associated with 23.1% risk of osteoporotic fracture over 10 years.
Disclosures: Karen Beattie, None.
DXA Whole Body Reference Data from the 2008 NHANES Data Release.Steven Heymsfield1, Kevin Wilson2, Thomas Kelly*2. 1Merck & Co., Inc., Whitehouse Station, USA, 2Hologic, Inc., Bedford, MA, USA
In 2008 NHANES released a DXA whole body dataset containing bone and body composition results for approximately 20,000 subjects from the survey years 1999 through 2004. We partitioned the NHANES dataset into groups according to gender and ethnicity (White, Black, and Mexican American) and developed reference values for whole body bone and body composition measures and selected derivative values.
The LMS curve fitting procedure  was employed to develop gender and ethnicity specific DXA whole body reference values for %fat, fat mass/height2, lean mass/height2, appendicular lean mass/height2, %fat trunk/%fat legs ratio, trunk/limb fat mass ratio, bone mineral content (BMC) and bone mineral density (BMD). The LMS procedure handles any skewness that may be present in the underlying reference data by dividing the independent measure (e.g. age) into groups and then applying a power transformation which extends one tail of the distribution and contracts the other. The resultant LMS reference values may be used to generate robust z-scores and percentiles even in cases where the reference data are not normally distributed. For adults ages 20 to 85 the DXA whole body reference values were normalized to age. For children ages 8 to 19+ whole body BMC and BMD databases included both total body and sub-total body (head excluded) results and were normalized to age and height. Sub-total BMC was also normalized to lean mass, which may be helpful in the assessment of the muscle-bone unit .
These reference values should be helpful in the evaluation of a wide variety of adult and childhood abnormalities involving fat, lean, and bone, for establishing entry criteria into clinical trials, and for other medical, research, and epidemiological uses.
Cole TJ (1990) The LMS method for constructing normalized growth standards. Eur J Clin Nutr 44: 45–60.
Schoenau E, Frost HM (2002) The “muscle-bone unit” in children and adolescents. Calcif Tissue Int 70: 405–407.
Disclosures: Thomas Kelly, Hologic, Inc., 3
External Validation of Nomograms Predicting Fracture Risk and Hip Fracture Risk: Canadian Men and Women.Lisa Langsetmo*1, Tuan V Nguyen2, David A Hanley3, Christopher S Kovacs4, Nguyen Nguyen5, John Eisman5, Jacqueline Center5, Jonathan D. Adachi6, Jerilynn C Prior7, Robert G Josse8. 1Canandian Multicenter Osteoporosis Study, Montreal, QC, 2Garvan Institute of Medical Research, Sydney, Australia, 3University of Calgary, Calgary, Alberta, Canada, 4Memorial University, St Johns', Newfoundland & Labrador, Canada, 5Garvan Institute of Medical Research, Sydney, NSW, Australia, 6McMaster University, Hamilton, Ontario, Canada, 7University of British Columbia, Vancouver, British Columbia, Canada, 8University of Toronto, Toronto, Ontario, Canada
Background: A set of nomograms has been developed by Nguyen et al to predict the 5-year and 10-year absolute risk of fracture based on age, BMD, prior falls, and low-trauma fracture history. These nomograms were derived from an Australian population, but might be applicable in other populations. For validation purposes, we tested the calibration and discrimination of these nomograms in the Canadian Multicentre Osteoporosis Study (CaMos).
Methods: The study sample included all participants in the adult cohort of CaMos between 55 and 95 years old at baseline with baseline femoral neck BMD measurement, and at least one year of follow-up data. Femoral neck T-scores were based on published reference standards for Canadians. Self-reported incident clinical fractures were identified by yearly postal questionnaire or at the scheduled interviews (Year 3, 5, and 10). All low-trauma fractures prior to year 10 were included. Fractures of the skull, face, hands and feet were excluded. We used a Cox proportional hazards model.
Results: There were 4152 women with mean 8.6 years follow-up and 1606 men with mean 8.3 years of follow-up. During follow-up 518 women and 116 men had at least one clinical low-trauma fracture. Increasing age, lower BMD, prior fracture and prior falls were associated with increased risk of fracture. The model parameters based on the CaMos population were similar, but not identical to the parameters from the derivation cohort for the nomogram. For low-trauma fractures, the concordance between predicted risk and actual fracture outcomes (Harrell's C) was 0.69 in women and 0.70 in men. For hip fractures, the concordance was 0.80 in women and 0.85 in men. The observed fracture risk was lower than predicted by the nomogram, and notably so in the highest risk quintile (see Figure).
Conclusions: The published nomograms provide good fracture risk discrimination in a representative sample of the Canadian population, but may overestimate the risk of fracture for those with high fracture risk. The overestimation of risk in the highest quintile does not affect clinical decisions as treatment would be recommended even at the risk level observed in our study.
Disclosures: Lisa Langsetmo, None.
Use of longitudinal data to derive peak bone mass discloses sex and skeletal site heterogeneity and differences in osteoporosis prevalence.Claudie Berger*1, David Goltzman2, Lisa Langsetmo3, Nancy Kreiger4, Alan Tenenhouse5, Lawrence Joseph5, K. Shawn Davison6, Robert Josse7, Jerilynn Prior8, David Hanley9. 1CaMos, MUHC research Institute, McGill University, Montreal, QC, Canada, 2McGill University, Montreal, QC, Canada, 3Canandian Multicenter Osteoporosis Study, Montreal, QC, 4University of Toronto, Toronto, Ontario, Canada, 5McGill University, Montreal, Quebec, Canada, 6Laval University, Saskatoon, SK, Canada, 7St. Michael's Hospital, University of Toronto, Toronto, ON, Canada, 8University of British Columbia, Vancouver, BC, Canada, 9University of Calgary, Calgary, AB, Canada
We determined peak bone mass (PBM) using longitudinal data from the Canadian Multicentre Osteoporosis Study (CaMos). We studied 328 women and 292 men aged 16–24 (recently recruited youth cohort) together with 287 women and 235 men aged 25–40 from the original CaMos cohort. All had at least 2 BMD measurements at the lumbar spine (L1-L4) and total hip. Individual rate of change was determined by calculating the slope of BMD versus time, and rates were averaged to find the mean rate of change by baseline age. PBM was achieved at the age at which there was no further increase in BMD, i.e. when the rate of change was no longer positive. When no positive rate of change was observed from the earliest age (16), we used the youngest 4 ages to estimate PBM. To do so, we used hierarchical models weighted according to the 2006 Canadian census. We also compared the average percent change over 2 years of bone mineral content (BMC), skeletal area, and BMD within the youth cohort. BMD was measured on Hologic and Lunar densitometers, with the latter measurements converted to Hologic by use of the Genant equation.
At L1-L4, PBM occured at 33–40 years in women with PBM=1.046g/cm2 (SD=0.123) and occurred at 19–33 years in men with PBM=1.066g/cm2 (SD=0.129). The age of PBM for total hip was 19–21 in men with PBM=1.093g/cm2 (SD=0.169). The rate of change of BMD for total hip was zero at age 16; therefore PBM for total hip was defined to occur at 16–19 years with PBM-0.981g/cm2 (SD=0.122). In women who were premenarchal, total hip BMD increased (0.007g/cm2/year with 95%CI: 0.002; 0.013) up until 3 years after menarche and then remained stable. Longitudinal changes in BMD, BMC, and area appeared to occur in parallel. Based on these PBM and using the CaMos cohort, we found the prevalence of osteoporosis (T-score<−2.5) was 11.6% (L1-L4) and 8.5% (total hip) in women aged ≥50 years and 3.0% (L1-L4) and 1.0% (total hip) in men aged ≥50 years. This compares to a prevalence of osteoporosis for total hip of 4.8% in women aged ≥50 years and 1.2% in men aged ≥50 years using NHANES PBM in the CaMos cohort.
In summary, using the longitudinal rate of change of BMD, we found that the age at which PBM is achieved varies by sex and skeletal site, with hip PBM occurring at an earlier age than L1-L4 PBM and with men achieving L1-L4 PBM earlier than women but achieving hip PBM later than women. Our different reference values for PBM lead to different prevalence of osteoporosis and therefore demonstrate the importance in the choice of reference values.
Disclosures: Claudie Berger, None.
Relation of Bone Density and Structure to Vertebral Deformities.Shreyasee Amin*1, B. Lawrence Riggs2, Tony Keaveny3, Sara Achenbach4, David Kopperdahl5, Jon Camp4, Peggy Rouleau4, Elizabeth Atkinson4, Richard Robb4, Terry Therneau4, Sundeep Khosla6, L. Joseph Melton1. 1Mayo Clinic, Rochester, MN, USA, 2Mayo Clinic, Little Rock, AR, USA, 3University of California, Berkeley, Berkeley, CA, USA, 4Mayo Clinic, Rochester, USA, 5O.N. Diagnostics, Berkeley, CA, USA, 6Mayo Clinic College of Medicine, Rochester, MN, USA
Prediction of vertebral fracture risk by skeletal parameters (e.g., areal bone mineral density [aBMD]) has been modest, in part because mild, moderate, and severe vertebral deformities are considered together, and mild vertebral deformities could include false positive cases. To further address this issue, we compared postmenopausal women with no vertebral deformity (n=90) to those with mild (semiquantitative grade 1, n=142) or moderate/severe (grade 2/3) deformities (n=57) on lateral digital radiographs. Lumbar spine (LS) and hip aBMD were measured by DXA, LS volumetric BMD (vBMD) and bone geometry by quantitative computed tomography (QCT), bone microstructure by high resolution peripheral QCT (HRpQCT) as determined at the distal radius, and vertebral failure load (∼strength) by voxel-based finite element analysis (FEA). Compared to those without deformities, women with grade 2/3 deformities were older and had significantly worse values for almost every bone density, structure and strength parameter. Bone strength was also reduced among women with grade 1 deformities, which was intermediate between moderate/severe cases and controls. By age-adjusted logistic regression, the most significant predictors of any vertebral deformity in each of five main variable categories (see Table) were for: bone density (LS trabecular vBMD), geometry (vertebral apparent cortical thickness), microstructure (radius bone volume/total volume); strength (vertebral trabecular compressive strength by FEA), and factor-of-risk (> for 90° forward flexion ÷ overall vertebral compressive strength), all of which were much more strongly associated with grade 2/3 than with grade 1 deformities (Table). In an overall multivariable analysis, the most significant predictor of severe deformities was LS trabecular vBMD (OR, 4.3; 95% CI, 2.4–7.7), with an area under the ROC curve (AUC) of 0.81. While many parameters were interchangeable, LS and hip aBMD were not independent predictors of fracture risk in the multivariable analysis. Although some women with grade 1 deformities may represent false positive cases, the fact that their bone structural and strength parameters were reduced compared to controls suggests that many in this group may actually have early osteoporotic fractures. This has practical implications for clinical decision making and emphasizes the need for a more specific definition of vertebral fracture.
Disclosures: Shreyasee Amin, On Diagnostics, 1
Bone Geometry and the Risk of Non-vertebral Fractures: The MrOS Study.Yahtyng Sheu*1, Joseph Zmuda1, Robert Boudreau2, Moira Petit3, Kristine Ensrud4, Douglas Bauer5, Christopher Gordon6, Eric Orwoll7, Jane Cauley8. 1University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, 2University of Pittsburgh, Pittsburgh, USA, 3University of Minnesota, Minneapolis, MN, USA, 4Minneapolis VA Medical Center / University of Minnesota, Minneapolis, MN, USA, 5University of California, San Francisco, San Francisco, CA, USA, 6Dundas, ON, Canada, 7Oregon Health & Science University, Portland, OR, USA, 8University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA
Although femoral neck bone mineral density (FNBMD) is known to effectively predict overall fracture risk, bone geometry is also thought to be informative to assess skeletal health. Therefore, we used bone geometry parameters at the radius and tibia measured by peripheral quantitative computed tomography (pQCT) to evaluate their relationships with incident non-vertebral fracture risk and fracture prediction. FNBMD measured by dual energy x-ray absorptiometry was also included. The study population consisted of 1,143 Caucasian men aged 65+ years with valid pQCT measures from the Minneapolis and Pittsburgh centers of the Osteoporotic Fractures in Men (MrOS) study. Principal component analysis was used to identify 36 bone density and geometry measures that accounted for most of the observed variability in a total of 58 pQCT parameters. This initial analysis was followed with Cox proportional hazards modeling with backward elimination to identify 9 out of 36 variables with a major contribution to non-vertebral incident fractures. Seven additional parameters with significant associations with incident fractures independent of FNBMD were also included. Sixteen geometry parameters were identified and Cox proportional hazards modeling was used to test their relationships with fracture risk. After a mean 2.9 years of follow up, 28 non-vertebral fractures occurred. Men without incident fractures had significantly greater bone mineral content, area and strength than those with fractures. Every one standard deviation (SD) decrease in bone geometry parameters was significantly associated with increased fracture risk (HRs: 1.56 to 2.12, Table) independent of age, study site, BMI and FNBMD. The combination of single pQCT parameter and FNBMD improved overall fracture prediction (Table: shown in area under the curve (AUC)). Bone geometry measures are associated with fracture risk and may improve our ability to identify men at high risk of fracture.
Disclosures: Yahtyng Sheu, None.
Ethnic/Geographic Differences in Older Men's Bone Mineral Densities.Hae Sung Nam*1, Min-Ho Shin2, Joseph Zmuda3, PC Leung4, Edith Lau5, Elizabeth Barrett-Connor6, Eric Orwoll7, Jane Cauley8. 1University of Pittsburgh, Graduate School of Public Health, Pittsburgh, PA, USA, 2Department of Preventive Medicine, Chonnam National University School of Medicine, Gwangju, South Korea, 3University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, 44Jockey Club Centre for Osteoporosis Care & Control, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, New Territories, Hong Kong, 5Hong Kong Orthopaedic & Osteoporosis Center for Treatment & Research, Hong Kong, Peoples Republic of China, 6University of California, San Diego, La Jolla, CA, USA, 7Oregon Health & Science University, Portland, OR, USA, 8University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA
There is insufficient epidemiologic information about men's bone mineral density (BMD) levels across ethnic groups and geographic locations. In a cross-sectional design, we compared older men's mean BMD in seven ethnic groups and four countries. Femoral neck, total hip and lumbar spine BMD were measured in men (age 65 to 78 yrs) from the Osteoporotic Fractures in Men (MrOS) study (4074 Caucasian, 208 African American, 157 Asian and 116 Hispanic men in U.S.), Tobago Bone Health Study (422 Afro-Caribbean men), MrOS Hong Kong study (1747 Hong Kong Chinese men) and Namwon Study (1116 South Korean men). BMDs were corrected according to the cross-site calibration results for all scanners. Least square means of BMD (LSMs) among ethnic groups were estimated by multivariable general linear models and the percentage differences in LSMs between U.S. Caucasian men and other ethnic groups were calculated. All p-values to test these differences were based on Tukey-Kramer adjustment for multiple comparisons of LSMs.
Compared with U.S. Caucasian men, the percentage differences in the age adjusted mean BMDs at femoral neck, total hip and lumbar spine, respectively, among ethnic groups were 11.6 (p<0.001), 7.9 (p<0.001) and 1.0% (p>0.1) in Tobago Afro-Caribbean men; 11.3, 7.3 and 6.1% (p<0.001 for all) in African American men; 1.8, 0.3 and −2.9% (p>0.05 for all) in U.S. Hispanic men; −3.5 (p=0.055), −4.9 (p<0.001) and −2.9% (p>0.1) in U.S. Asian men; −6.8, −7.5 and −10.1% (p<0.001 for all) in Hong Kong Chinese men; and −15.4, −25.4 and −17.6% (p<0.001 for all) in South Korean men. Additional adjustment for weight and height greatly attenuated the differences between U.S. Caucasian men vs. Asian ethnic groups such as U.S. Asian, Hong Kong Chinese and South Korean men (Figure). Despite this attenuation, some differences, particularly those between South Korean and US Caucasian men, still persist.
In conclusion, men of African origin had substantially higher hip BMD than U.S. Caucasian men. U.S. Hispanic, U.S. Asian and Hong Kong Chinese men had similar hip BMD. South Korean men had the lowest hip and spine BMD even after adjusting for age, height and weight. These South Korean men were all age 8 to 22 yrs at the beginning of Korean War. Malnutrition and other factors may have influenced their peak skeletal mass.
Disclosures: Hae Sung Nam, None.
Fracture Risk Thresholds in Men and Women: Misleading Effects of Sex-Specific Reference Data.William Leslie*1, Wei Zhou2, Lisa Langsetmo3, David Goltzman4, Robert Josse5, Christopher S. Kovacs6, Wojciech P. Olszynski7, K. Shawn Davison7, Jerilynn Prior8, Tassos Anastassiades9, Tanveer Towheed9, David Hanley10, Stephanie Kaiser11, Nancy Kreiger12. 1Winnipeg, MB, Canada, 2McGill University, Montreal, Quebec, Canada, 3Canandian Multicenter Osteoporosis Study, Montreal, QC, 4McGill University, Montreal, QC, Canada, 5St. Michael's Hospital, University of Toronto, Toronto, ON, Canada, 6Memorial University, St. John's, Newfoundland & Labrador, Canada, 7University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 8University of British Columbia, Vancouver, British Columbia, Canada, 9Queen's University, Kingston, Ontario, Canada, 10University of Calgary, Calgary, Alberta, Canada, 11Dalhousie University, Halifax, NS, Canada, 12University of Toronto, Toronto, Ontario, Canada
Background: Although bone mineral density (BMD) predicts fragility fractures in both men and women, there are postulated sex dierences in the level of absolute BMD at which fractures occur (“fracture threshold”). Some studies demonstrate higher mean BMD for men with fractures than for women with farctures, while others note similar risk at the same level of BMD. This question was addressed in the population-based Canadian Multi-centre Osteoporosis Study (CaMos). Methods: The cohort included women and men 50+ years old at the time of baseline measurement of hip BMD. We evaluated 4,752 women (614 with fragility fractures) and 1,908 men (125 with fragility fractures) over a 10 year follow-up period. Age-adjusted Cox proportional hazards models were constructed for time to first fragility fracture. Three methods of BMD normalization were compared: Method 1 used the same reference population mean and SD; Method 2 used sex-specific reference population means but the same SD; Method 3 used sex-specific reference population means and SDs. Results: Mean BMD in men with fractures was higher than in women with fractures. However, men without fractures also had higher BMD than women without fractures. In Model 1 (common reference population), there were strong eects of age (HR 1.37 [95% CI 1.24–1.50] per decade increase) and total hip BMD (HR 1.65 per SD decrease [95% CI 1.49–1.82]) for prediction of future fractures, but no eect of sex (HR 0.91 [95% CI 0.73–1.12] for male vs female). In Model 2, an apparent sex dierence emerged (HR 0.59 [95% CI 0.48–0.72] for men versus women) that approximately balanced the higher calculated risk for men when using sex-specific reference means (normalized BMD scores for men 0.83 SD lower than for women with the same absolute BMD). Method 3 (sex-specific means and SDs) gave similar results to Model 2 with an apparent sex dierence (HR 0.59 [95% CI 0.48–0.72] for men vs women) attributed to the introduction of sex-specific dierences in normalized BMD scores at the same absolute BMD. Similar results were seen when femoral neck BMD was assessed instead of total hip BMD and when fractures were limited to hip, forearm, humerus and clinical spine. Conclusions: Sex is not predictive of fragility fracture when using a common reference mean and SD. The use of sex-specific reference data creates spurious sex dierences in modeled fracture risk. Overall, men and women have a similar risk of fracture at the same level of absolute BMD.
Disclosures: William Leslie, None.
The Ability of QCT and DXA BMD of the Spine to Predict Clinical Vertebral Fracture in Men: The MROS Study.Dennis Black*1, Lynn Marshall2, Kristine Ensrud3, Thomas Lang1, Jane Cauley4, Eric Orwoll2, Lisa Palermo1. 1University of California, San Francisco, San Francisco, CA, USA, 2Oregon Health & Science University, Portland, OR, USA, 3Minneapolis VA Medical Center / University of Minnesota, Minneapolis, MN, USA, 4University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA
The relationship of BMD and vertebral structure to new vertebral fractures (vertFX) among elderly adults has not been studied prospectively. We examined these associations among community dwelling men ages 65–100 years using data from the MROS study. A total of 3489 men had QCT at the spine (L1-L2 vertebrae) at the baseline exam. Parameters calculated from spine QCT scan include trabecular BMD, cross sectional area and vertebral strength index (VSI), an estimate of bone strength derived from BMD and cross sectional area. Lumbar spine BMD from DXA was also obtained for all men. The primary endpoint was new clinical vertFX. These were initially self-reported but verified by comparing the clinically-obtained radiograph at the time of the event to the study-obtained baseline radiograph using semiquantitative (SQ) criteria. Over a mean 7.2 years of follow-up, 72 vertFX were confirmed among men with baseline QCT. Hazard ratios per SD decrease in the skeletal parameter were adjusted for age, clinical center and BMI in Cox PH models.
The risks of new vertFX declined with increasing quartile of DXA BMD, QCT trabecular BMD, and VSI, but not vertebral cross sectional area (Table). For DXA-BMD, the RH/SD was 3.0 (95% CI:2.3, 4.0) which did not differ significantly from that for QCT trabecular BMD (RH/SD=3.7 (2.6,5.2)). The RR/SD for VSI was higher (NS) (RH/SD=5.6 (3.2,10)) primarily due to a large number of fractures at the lowest VSI levels (29 of 72 fractures occurred in men in the lowest VSI decile). There was no significant relationship between cross sectional vertebral area and vertFX risk. In initial multivariate analyses including both QCT and DXA variables, each remained independently predictive but overall prediction (as assessed by ROC analysis) did not improve.
Limitations of this analysis include the fact that the number of vertFX was limited and there was no assessment of baseline prevalent fractures available for inclusion in adjustments.
We conclude that bone mineral density from spine QCT or DXA in men is highly predictive of future clinical vertebral fractures. While QCT parameters were not significantly more predictive than DXA BMD, other measures of bone strength derived from QCT may be more strongly predictive of vertebral fracture and deserve future study.
Disclosures: Dennis Black, Roche, 2; Zosano, 5; Nycomed, 5; Novartis, 2; Merck, 2
Variability in Hispanic Hip Fracture Incidence Statewide in the US.Stuart Silverman*1, David Zingmond2. 1Cedars-Sinai/UCLA, Beverly Hills, CA, USA, 2UCLA David Geen School of Medicine, Los Angeles, USA
We have previously reported Hispanic hip fracture incidence rates in California for individuals over the age of 55 using the California inpatient file to be intermediate (4 fractures per 1000 person-years in year 2000) between Caucasians and African-Americans with a suggestion of increasing secular trends between 1983 and 2000. It was unclear if these data are representative of Hispanic fracture rates across the US.
National Medicare data were used to identify acute hip fractures and estimate hip fracture incidence among Hispanic Fee-For-Service Medicare enrollees 65 years of age and older in the nine states with the largest Hispanic populations: Arizona (AZ), California (CA), Colorado (CO), Florida (FL), Nevada (NV), New Jersey (NJ), New Mexico (NM), New York (NY) and Texas (TX) for the years 1997, 2000, 2003, and 2006. Hispanic Fee-For-Service Medicare enrollees were identified using the Medicare Beneficiary Annual Summary File. Acute hip fractures were identified using the annual Medicare MedPAR file. Estimated poverty and rural residence status was derived from U.S. Census 2000 zip code-level data. Incidence rates with confidence intervals were estimates using Poisson analyses. Poisson regression models were used to estimate state variation after accounting for patient case-mix and secular trends. Because of multiple comparisons, a P-value of < 0.01 was considered significant.
During the four years examined, annual identified Hispanic enrollment increased from 423,939 to 502,061. Unadjusted hip fracture incidence rates varied significantly: higher in FL (6.9 fractures per 1000 person-years), NM (6.5), and CO (6.5); lower in NY (3.1), NJ (3.5), CA (3.9), and NV (3.9); and intermediate in AZ (5.8) and TX (5.9). After accounting for case-mix and secular trends, NY had a significantly lower incidence rate ratio (compared to CA), while AZ, CO, FL, NM, and TX had significantly higher incident rate ratios. No significant secular trend was seen.
Differences in case-mix adjusted hip fracture incidence rates do occur in Hispanic populations in different states across the U.S. Variability across populations may be due to a number of unmeasured differences, including differences in Hispanic ancestry, socioeconomic status, and nutritional history.
Disclosures: Stuart Silverman, Proctor & Gamble, 2
Bone micro-architecture assessed by TBS at the AP spine predicts clinical spine fractures independently of BMD in 22234 women aged 50 and older: the Manitoba prospective study.Didier Hans*1, Andrew L. Goertzen2, Marc-Antoine Krieg3, William Leslie4. 1Lausanne University Hospital. Lausanne, ACT, Switzerland, 2University of Manitoba, Departments of Medicine & Radiology, Winnipeg, Ontario, Canada, 3University Hospital, Lausanne, Switzerland, 4St. Boniface General Hospital, Winnipeg, MB, Canada
BMD as assessed by DXA constitutes the gold standard for osteoporosis diagnosis. However, it does not take into account deterioration in bone microarchitecture. Trabecular Bone Score (TBS), a new grey-level texture measurement that can be extracted from the DXA image, correlates with 3D parameters of bone microarchitecture. Previous cross-sectional studies reported the ability of spine TBS to discriminate fractured women from age- and BMD-matched controls. The aim of our study was to prospectively evaluate the ability of lumbar spine TBS to predict clinical spine fractures in comparison with AP spine BMD.
22,234 women age 50 years and older at the time of baseline hip and spine DXA were identified in a database containing all clinical results for the Province of Manitoba, Canada. Health service records were assessed for the presence of non-trauma osteoporotic fracture codes subsequent to BMD testing. Lumbar spine TBS was derived by the Bone Disease Unit, University of Lausanne, for each spine DXA examination using anonymized files (blinded from clinical parameters and outcomes). We used Cox proportional hazard regression to model the hazard of first spine fracture. Age-adjusted HRs for fracture per SD decrease in TBS and/or BMD are reported. Incremental gain in prediction information when TBS was added to age and BMD was assessed using the log-likelihood ratio test (LLR).
The mean age of the population was 65.0 ± 9.5 y and the numbers of fractures during mean 4.6 y of follow up were clinical spine 297 (1.3%). Significantly lower spine BMD and spine TBS parameters were found in fracture than non fracture women for clinical spine fracture (p<.0001). Correlation between spine BMD and spine TBS was modest (r=.32) and less than correlation between spine and hip BMD (r=.72), consistent with a skeletal parameter largely unrelated to BMD. Spine BMD and TBS predicted fractures equally well and independently.
In conclusion, we have demonstrated that spine TBS predicts clinical spine fracture. Furthermore, TBS provided information that was independent of spine BMD. Combining the TBS micro-architecture index with BMD from conventional DXA incrementally improved fracture prediction in postmenopausal women.
Disclosures: Didier Hans, None.
Prediction of Subsequent Fracture Risk: Contribution of Baseline Risk Profile.Nguyen Nguyen*1, Dana Bliuc1, Steven Frost2, Jacqueline Center1, John Eisman3, Tuan Nguyen4. 1Garvan Institute of Medical Research, Sydney, NSW, Australia, 2University of Western Sydney, Penrith South, NSW, Australia, 3Garvan Institute of Medical Research, Sydney, Australia, 4Garvan Institute of Medical Research, Sydney, Australia
The risk of refracture is increased after an initial fracture in men and women. While, bone mineral density (BMD) and age are robust predictors of fracture risk, we hypothesized that prior (study entry) BMD would be as robust a predictor of subsequent fracture as BMD at the time of an incident fracture. Our aim was to compare absolute refracture risk estimates using these two models.
Women (n=411) and men (n=144) with an initial fracture and aged at least 60 years (1989) from the Dubbo Osteoporosis Epidemiology Study were followed for up to 18 years with femoral neck BMD (DXA, GE-LUNAR) at baseline and every 2 years. As expected, BMD at study entry was higher than at incident fracture, 5.5 years (0.3–18.1) later in women and men; 0.72 vs. 0.70 g/cm2 in women and 0.86 vs. 0.83 g/cm2 in men (p<0.0001). Cox's proportional hazards models were used to develop two models for comparison: Model 1 included BMD at study entry, adjusting for time to first fracture and Model 2, BMD at time of first incident fracture. Both models included age at this initial fracture. Prognostic value for each model was estimated from reclassification with respect to 5-year absolute risk of fracture into low, moderate and high risk tertiles. The proportion of ‘correct’ reclassifications between models was regarded as the net reclassification improvement (NRI) in prediction.
During the follow-up, 166 women (40%) and 32 men (22%) sustained a second fracture. The hazard ratio (HR) for study-entry BMD (per −0.12g/cm2) was 1.48 (1.24–1.76) in women and 1.55 (1.15–2.08) in men. The HR for BMD at fracture was 1.46 (1.23–1.74) in women and 1.52 (1.15–2.02) in men. There was no dierence in terms of predicting the risk of second fractures between models. For women, study entry BMD had similar predictive value to BMD measured at initial fracture. In men, study entry BMD reclassification resulted in a non-significant loss 8% (p=0.35).
Prior BMD measurement and interval since measurement provided comparable information to BMD at initial fracture, with respect to subsequent fracture risk, which could influence clinical decision-making.
Disclosures: Nguyen Nguyen, Amgen, deCode, Eli Lilly and Company, GE-Lunar, Merck Sharp & Dohme Ltd., Novartis, Organon, Roche-GSK, Sanofi-Aventis and Servier, 99
The Factor-of-Risk (Φ) Biomechanical Approach Predicts Hip Fracture and, In Women, Is Independent of Bone Mineral Density (BMD): The Framingham Study.Alyssa Dufour*1, Benjamin Roberts2, Douglas Kiel3, Mary Bouxsein4, Marian Hannan5. 1Hebrew Senior Life, Boston, MA, USA, 2Beth Israel Deaconess Medical Center, Boston, USA, 3Institute for Aging Research, Hebrew SeniorLife, Boston, MA, USA, 4Beth Israel Deaconess Medical Center, Boston, MA, USA, 5HSL Institute for Aging Research, Boston, MA, USA
Hip fractures are disabling events for older persons. Studies have identified low BMD as a major risk factor, yet up to half of those suffering a hip fracture do not have low BMD; thus, alternative methods of assessing fracture risk are needed. One such method is the biomechanical Φ, defined as the ratio of force applied to the hip from a sideways fall to femoral bone strength. We examined the relation between Φ and risk of hip fracture in men and women from the population-based Framingham Study.
Subjects included 1100 Framingham Original Cohort members who had hip BMD scans (LUNAR DPA) in 1988–89 (baseline). Body weight, height (used to calculate BMI (kg/m2)) and age were measured at baseline. Incident hip fractures were ascertained and confirmed by interview and review of medical records through 12/31/05. We calculated Φ in two ways: using the ratio of either the estimated peak force or the attenuated (atten) force applied to the hip in a sideways fall from standing height divided by the femoral strength. Peak force (N) was calculated from height (m) and weight (kg). The attenuated force incorporated the cushioning eect of trochanteric soft tissue thickness (mm), as estimated by BMI, using published sex-specific regressions. Femoral strength (N) was estimated from femoral neck BMD, using a relation derived from published cadaveric femoral strength testing. Sex-specific crude and age-adjusted Cox Proportional Hazards regressions were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) for the relation between hip fracture risk and both Φpeak and Φatten.
In the 425 men and 675 women (mean age 76 yrs) mean BMI was 26.8 kg/m2. Median follow-up was 11.3 yrs and 155 hip fractures occurred (28 in men). Φpeak and Φatten were associated with increased risk of hip fracture, adjusting for age (Table). A 1 SD increase in Φpeak was associated with a HR of 1.88 in men and 1.23 in women. Similarly, for Φatten, the HR for hip fracture was 1.78 in men and 1.41 in women. After adding BMD to the model, Φatten remained predictive of hip fracture in women. In men, we were not able to adjust for BMD due to collinearity with Φ.
In summary, both Φpeak and Φatten predict subsequent hip fracture in men and women. In women, we showed for the first time that Φatten predicts hip fracture independent of hip BMD. These findings provide strong rationale for additional studies testing the ability of this biomechanical approach to predict hip fracture.
Disclosures: Alyssa Dufour, Merck, 5; Amgen, 5; Lilly, 8; Hologic, 2; Philips Lifeline, 5; Amgen, 2; Novartis, 8; GSK, 8; Novartis, 2; Hologic, 5; Merck, 8; Wyeth, 2; Novartis, 5; Merck, 2; Procter and Gamble, 5; Eli Lilly, 5; GSK, 5; Pfizer, 2; Wyeth, 5
Abdominal Aortic Calcification and Risk of Vertebral and Hip Fractures Among Older Women.Kristine Ensrud*1, Lisa Palermo2, Dennis Black3, Douglas Kiel4, Brent C. Taylor5, Jane Cauley6, Eric Vittingho2, Nicolas Rodondi7, John Schousboe8, Marc Hochberg9, Teresa Hillier10, Steven Cummings11. 1Minneapolis VA Medical Center/University of Minnesota, Minneapolis, MN, USA, 2University of California - San Francisco, San Francisco, USA, 3University of California, San Francisco, San Francisco, CA, USA, 4Institute for Aging Research, Hebrew SeniorLife, Boston, MA, USA, 5VA Medical Center / University of Minnesota, Minneapolis, USA, 6University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA, 7University of Lausanne, Lausanne, Switzerland, 8Park Nicollet Clinic, Minneapolis, MN, USA, 9University of Maryland School of Medicine, Baltimore, MD, USA, 10Kaiser Center for Health Research, Portland, OR, USA, 11San Francisco Coordinating Center, San Francisco, CA, USA
Some evidence supports shared biological pathways between vascular calcification and osteoporosis, conditions that increase in prevalence with advancing age. To test the hypothesis that older women with greater severity of abdominal aortic calcification (AAC) are at increased risk of vertebral and hip fracture, we conducted a case-cohort study (within a cohort of 9704 women ≥65 years) of 94 women with incident vertebral fracture identified at 4 years (average 3.7 yrs between baseline and follow-up spine film), 105 women with incident vertebral fracture identified at 15 years (average 15.0 years between baseline and follow-up spine films), 185 women with first hip fracture (identified over an average of 14.3 yrs), and a sample of 951 women randomly selected from the overall cohort. A semi-quantitative method was used to determine severity of AAC (score 0–24) on baseline spine radiographs. Among the women in the random sample, median AAC score was 1; 49% had an AAC score of 0, 26% had an AAC score of 1–4, and 25% had an AAC score ≥5. There was an independent, graded association between AAC and risk of vertebral fracture at 4 years (p trend 0.008) (table). After adjustment for multiple potential confounders, women with an AAC score ≥5 had a 2.3-fold increase in risk of vertebral fracture. However, AAC was not associated with an increase in vertebral fracture at 15 years (p trend 0.92). In addition, higher AAC was associated with an increased risk of hip fracture during the 1st 5 years of follow-up (p trend 0.05), but the association waned with increasing length of follow-up and AAC was not related to risk of hip fracture during the entire follow-up period (mean follow-up 14.3 years) (p trend 0.38). In conclusion, greater severity of AAC in older women is associated with an increase in the short-term risks of vertebral and hip fractures, but not associated with the long-term risk of these fractures. Our results may be at least in part be explained by differential survival in older women with and without AAC.
Disclosures: Kristine Ensrud, Hologic, Inc., 2; Amgen, 5; Amgen, 2; Hologic, Inc., 5
Application of the WHO fracture risk assessment tool FRAXR in a Spanish population.Luis Del Rio*1, Cristian Tebe2, Helena Johansson3, Silvana Di Gregorio4, Dolors Estrada2, Mireia Espallargues2. 1Cetir Centre Medical, Barcelona, Spain, 2Agència d'Avaluació de Tecnologia i Recerca Mèdiques, Barcelona, Spain, 3University of Sheeld, Sheeld, United Kingdom, 4CETIR Centre Medic. Densitometría Ósea, Barcelona, Spain
The FRAX tool has been developed to estimate the 10-year probability of hip and major osteoporotic fracture using country-specific data. This algorithm may combine clinical risk factors with or without the bone mineral density (BMD) measurement in order to identify subjects in high risk of fragility fracture. The aim of this study was to test the Spanish version of the WHO fracture risk assessment tool (FRAX) on a cohort of women with BMD measurement indication. Methods: Clinical and BMD data from a large population cohort taken from public and private health care facilities distributed over the metropolitan area of Barcelona were used for this study. Inclusion criteria were: age range 40–90 yrs, clinical data with fracture risk factors, femoral neck BMD T-score available and follow-up longer than 7 years. Main outcome was: major osteoporotic fracture at least 7 years after the first BMD measurement. The total number of predicted fractures by the FRAX algorithm was compared with the total number of new registered fractures during the follow-up time in the study population and expressed as observed - expected fracture (O/E) ratio. Results were stratified by age group, follow-up time and number of clinical risk factors used in the FRAX algorithm Results: a total of 8,573 women were included, 69% were under 60 years and 14% presented a previous fracture. After follow-up, 23% had a major osteoporotic fracture. Wrist was the most incident fracture site and hip was accounted only for 1.6% of the total. 10% of women with none risk factors had a major osteoporotic fracture, in turn 57% of those women with more than three risk factors had a major osteoporotic fracture. However the fracture ratio (O/E) was 6.2 CI95% [5.5; 7.0]. The O/E ratio was lower as higher was the age of women (for those older than 70 O/E = 4.0 CI95% [2.9; 6.0]), longer the follow-up time (for those with more than 10 years O/E-5.4 CI95% [4.5; 6.8]) or lower the number of risk factors (for those without risk factors O/E-4.2 CI95% [3.5; 5.3]). In conclusion, the Spanish version of the FRAX algorithm for this population underestimates the observed fracture incidence independently of the T-score, number of risk factors and follow-up time. More in-depth analyses are warranted to find a reasonable explanation to this issue.
Disclosures: Luis Del Rio, None.
Can Active Shape Modelling be used to generate an additional risk factor for hip fracture?.Simon Goodyear*1, Jennifer S. Gregory2, Rebecca J. Barr3, Eugene McCloskey4, Salvatore Alesci5, Richard Aspden6, David Reid6, 1University of Aberdeem, Aberdeen, United Kingdom, 2School of Medicine & Dentistry, University of Aberdeen, Aberdeen, United Kingdom, 31School of Medicine & Dentistry, University of Aberdeen, Aberdeen, United Kingdom, 4University of Sheeld, Sheeld, United Kingdom, 5Wyeth Pharmaceuticals, USA, Collegeville, USA, 6University of Aberdeen, Aberdeen, United Kingdom
Fracture-preventive drugs are often abandoned during late stage clinical development due to the cost of the trials. Active Shape Modelling (ASM) has been proposed as a method for predicting risk of fracture. Use of ASM to identify individuals at the highest risk of hip fracture may reduce the size, duration and therefore, cost of clinical trials, bringing such drugs to market sooner.
ASM was used to create a template describing the outline of the hip joint (including the proximal femur, acetabulum and parts of the pelvis) from DXA images. The coordinates of 72 landmark points were found semi-automatically in each image and principal components analysis used to reduce these variables to a small number of new independent variables (modes of variation). Each image was then assigned a score for each mode, designating how many standard deviations it lay from the overall mean for that mode derived from the image dataset.
We built the model from baseline DXA images of 364 women entering the MRC Clodronate trial. 182 subjects sustained a hip fracture and were matched by age, height and weight with 182 subjects who did not fracture.
Mode 1 scores were significantly different between fracture and control baseline images (Odds Ratio (OR) = 0.80, 95% CI = 0.65–0.98, P = 0.034). Hips that fractured typically had a narrower, more upright appearance (Figure 1). After adjusting for BMD using binary logistic regression, the dierence in Mode 1 scores was still significant (OR = 0.79, 95% CI = 0.64–0.98, P = 0.033). The lack of change in the OR suggests the two variables are independent, an observation confirmed by the lack of correlation (R = −0.001, P = 0.99). Stepwise logistic regression, including the first 20 modes, BMD, age, height and weight, confirmed that Mode 1 and BMD were the only significant risk factors associated with hip fracture.
This study shows that ASM of DXA images can identify risk factors for hip fracture that are independent of BMD. Combining these factors will, therefore, provide a more powerful tool for identifying individuals at greatest risk of hip fracture.
Disclosures: Simon Goodyear, TMRI Ltd, 2 This study received funding from: TMRI Ltd
No Link Between C-reactive Protein (CRP) and Bone Mineral Density (BMD) in Men, But Menopause Status Modifies the Relation in Women: The Framingham Osteoporosis Study.Robert McLean*1, Xiaochun Zhang2, Emelia J. Benjamin3, L. Adrienne Cupples4, Douglas Kiel5, Marian Hannan6. 1Hebrew SeniorLife Institute for Aging Research & Harvard Medical School, Boston, MA, USA, 2Hebrew SeniorLife, Boston, USA, 3Framingham Heart Study & Boston University School of Medicine, Framingham, USA, 4Biostatistics Dept, Boston University School of Public Health, Boston, USA, 5Institute for Aging Research, Hebrew SeniorLife, Boston, MA, USA, 6HSL Institute for Aging Research, Boston, MA, USA
Laboratory studies suggest that several pro-inflammatory cytokines influence bone resorption, and this mechanism may partly mediate estrogen deficiency-related bone loss. Epidemiologic studies have been unable to elucidate the interrelation of inflammatory biomarkers, menopause status and BMD, and few have examined inflammation and BMD in men. Thus, we determined the cross-sectional association between elevated systemic inflammation, indicated by increased serum CRP concentration, and BMD among men and women in the Framingham Ospring Study. We hypothesized an inverse relation between CRP and BMD, and that it would be strongest in postmenopausal women not on estrogen. Fasting blood samples were obtained from 1,291 men and 1,614 women (1998–2001) and CRP concentrations (mg/L) were measured using a high-sensitivity assay (interassay CV=5.3%). Using clinical cut points, participants were categorized into CRP groups (<1, 1 to 3, >3 mg/L). Femoral neck BMD (g/cm2) was measured with a Lunar DPX-L. Women were categorized as premenopausal (n=231), postmenopausal on estrogen (n=498), and postmenopausal not on estrogen (n=893). We used analysis of variance to compare crude mean BMD among CRP groups, separately for men and groups of women. Using analysis of covariance, analyses were further adjusted for age (y), height (in), weight (lbs), physical activity (PASE) and current smoking (y/n). Mean age was 61 y (range 29–86). Due to a consistent threshold eect at 1 mg/L the upper 2 categories were combined (≥1 mg/L). In crude analyses, mean BMD was similar between CRP groups for men and premenopausal women, yet was greater in the ≥1 mg/L group in postmenopausal women. After adjustment the lack of association in men remained (Table), yet premenopausal women with CRP ≥1 mg/L tended to have 3.6% lower BMD (P=0.06). In postmenopausal women not on estrogen the ≥1 mg/L group had 2.3% higher BMD (P=0.04), but the dierence was not significant for those on estrogen. Our findings suggest increased systemic inflammation may be a risk factor for lower BMD among premenopausal women, but not for men or postmenopausal women on estrogen. Elevated inflammation was associated with higher BMD among postmenopausal women not on estrogen, contrary to our hypothesis. Future studies should examine the role of estrogen using biomarkers rather than crude estrogen status categories, and explore comorbidities that could explain our unexpected findings among postmenopausal women.
Disclosures: Robert McLean, None.
Reciprocal Relations of Subcutaneous and Visceral Fat to Bone.James Chalfant*1, Ashley Mo1, David Lee1, Steven Mittelman1, Vicente Gilsanz2. 1Childrens Hospital Los Angeles, Los Angeles, USA, 2Children's Hospital of Los Angeles, Los Angeles, CA, USA
The traditional paradigm is that adiposity is not detrimental to bone health, but rather protects against osteoporosis. Low body weight and recent weight loss are well-known risk factors for osteoporosis and fractures, and most studies have revealed a positive relation between body weight and bone mass. The contention for a positive fat-bone association is credited not only to stresses from mechanical loading, but also to the metabolic eects of bone-regulator hormones secreted or regulated by adipocytes. Challenging this widely held view, recent information suggest that fat mass is not associated with, or is negatively related to, bone mass. Given these discrepancies in results, a greater phenotypic characterization, beyond total fat accumulation, is needed to explain the complex relation between bone and adipose tissue.
The relations between subcutaneous and visceral adiposity and the cross-sectional dimensions of the femur in 100 healthy women ages 15 to 25 years were obtained using computed tomography. Positive simple correlations were present between measures of subcutaneous and visceral fat and both bone phenotypes; however, for visceral adiposity, neither of the relations achieved statistical significance. Multiple linear regressions analyses indicated that, after adjusting for leg length and thigh musculature, both subcutaneous and visceral fat had strong and independent associations with femoral cross sectional area and cortical bone area. However, while subcutaneous fat had a positive predictive value with all femoral bone phenotypes, a similar but negative effect was observed between visceral fat and these measures (Table). Overall, the ratios of the standardized coefficients of subcutaneous to visceral fat were around 1.5 in all models. The calculated condition number of 12.85 for the model suggested multicollinearity not to be a concern.
Our findings provide evidence that visceral and subcutaneous fat have strong and opposite effects on the structure of bone in young women. They support the hypothesis that subcutaneous fat is beneficial to peak bone mass, while visceral fat serves as a unique pathogenic fat depot. A greater understanding of the molecular and functional differences of visceral and subcutaneous adipocytes and how they interact with bone could likely lead to novel therapeutic approaches for obesity and osteoporosis.
Disclosures: James Chalfant, None.
Relationship Between Low BMD And Fracture In Mortality Risk: An 18-Year Prospective Study From Dubbo Osteoporosis Epidemiology Study.Dana Bliuc*1, Nguyen Nguyen1, Tuan Nguyen2, John A Eisman3, Jacqueline Center1. 1Garvan Institute of Medical Research, Sydney, NSW, Australia, 2Garvan Institute of Medical Research, Sydney, Australia, 3Osteoporosis & Bone Biology Program, Garvan Institute of Medical Research, Sydney, Australia
Mortality risk is increased both by all major osteoporotic fractures and by low bone mineral density (BMD), a factor that also contributes to fracture risk. It is unclear whether low BMD has a direct effect on mortality or whether its contribution is indirect, through increased fracture risk. The aim of this study was to examine the association of low BMD with all cause and post-fracture mortality risk in elderly women and men participating in the Dubbo Osteoporosis Epidemiology Study over an 18-year period (April 1989-May 2007).
All low trauma fractures and mortality data were recorded from 1164 women and 819 men. Information on co-morbidities as well as measurements of BMD, quadriceps strength and sway were obtained at baseline and up-dated 2-yearly. Cox proportional hazards models were used to determine mortality risk.
There were 359 fractures in women and 129 in men, and 435 deaths in women (153 post-fracture) over 15001 person-years and 386 in men (80 post-fracture) over 9367 person-years. Mortality risk was increased following a fracture by 1.85 (1.48, 2.31) in women and by 2.30 (1.70, 3.11) in men after adjustment for co-morbidities and markers of frailty, including BMD. Low BMD independently predicted all cause mortality in both genders [adjusted HR 1.17 (1.01, 1.37) and 1.22 (1.07, 1.38) in women and men, respectively]. However, when fracture was included as a time dependent variable, there was an interaction between fracture and BMD in women, such that only in those with osteoporosis was BMD significant. On the other hand, in those fewer women with fractures, BMD taken at the time of fracture was associated with mortality [HR 1.67 (1.23, 2.26)], but not in even fewer men [HR 1.07 (0.88, 2.32)].
Thus mortality risk was increased following all osteoporotic fractures, after accounting for co-morbidities, including BMD in both genders. Low BMD was associated with all cause mortality, but its effect was modified by fracture particularly in women. On the other hand a lower bone density at the time of fracture marked increased mortality risk in women but not in men. This suggests that circumstances surrounding the fracture event may play a particularly important role in the associated premature mortality in men and that, although low BMD is associated with increased mortality in both sexes, in men its effect may be primarily through increasing fracture events.
Disclosures: Dana Bliuc, Amgen, Eli Lilly, GE Lunar, Merck Sharp & Dohme, Novartis, Organon, Sanofi Aventis, 99
DLK1/FA1 and Parameters of Bone Metabolism and Body Composition in the Elderly.Ghada El-Hajj Fuleihan*1, Asma Arabi2, Tala Ghalayini3, Rola El-Rassi3, Ghassan Baliki3, Moustapha Kassem4. 1American University of Beirut Medical Center, Beirut, 00000, Lebanon, 2American University of Beirut-Medical Center, Beirut, Lebanon, 3American University of Beirut-Medical Center, Beirut, Lebanon, 4Odense University Hospital, Odense, Denmark
Background: DLK1/FA1 is a novel circulating factor that belongs to the Notch/Delta family of signaling molecules and is a negative regulator of bone and fat mass in mice (Abdallah et al., Endocrinology, 2007). Thus, we studied the relationship between serum levels of DLK1/FA1 and DXA derived parameters of bone mass, body composition, and indices of bone turnover in elderly subjects.
Methods: 129 ambulatory subjects (42 men and 87 women), age 72.3 ± 5.0 years, participated in a prospective population based study assessing bone mass, bone loss and fractures in the elderly Lebanese. The following measurements were obtained at baseline and at a median follow-up of 4.2 ± 0.3 years: bone mass, body composition on a DXA 4500 densitometer (Hologic), and routine biochemistries, PINP, cross-laps, and DLK1/FA1.
Results: DLK1/FA1 at study entry negatively correlated with total hip BMD in the overall group (R = −0.183, P = 0.041), and in females (R = −0.223, P = 0.044). Follow up DLK1/FA1 and % change DLK1/FA1 did not correlate with changes in BMD or BMC at the spine, hip or femoral neck, nor did they correlate with baseline or changes in BMI and body composition parameters. Baseline DLK1/FA1 correlated with 25-OHD level (R=0.235, p=0.009) and % change DLK1/FA1 correlated with follow up serum Cr (R=0.252, p=0.005). Among the bone turnover markers, DLK1/FA1 was significantly and negatively correlated with PINP, as detailed in the overall group, and by gender (table).
Conclusion: Serum level of DLK1/FA1 is influenced by kidney function and is correlated to bone formation markers. Increased level of s-DLK1/FA1 in elderly patients may be a mechanism mediating the negative effects of aging on bone formation. Targeting DLK1/FA1 may be a novel approach to prevent the deleterious effects of aging on the skeleton.
Disclosures: Ghada El-Hajj Fuleihan, None.
Effects of Exercise on Appendicular Bone Marrow Fat and Its Relationship to Changes in Bone Density, Structure and Strength in Older Men.Robin Daly*1, Peter Ebeling2, Sonja Kukuljan3, Sue Harvey4, Caryl Nowson3. 1The University of Melbourne, Western Hospital, Melbourne, Australia, 2University of Melbourne, Footscray, Australia, 3School of Exercise & Nutrition Sciences, Deakin University, Melbourne, Australia, 4Department of Medicine, The University of Melbourne, Western Hospital, Melbourne, Australia
Ageing and reduced loading are associated with bone loss and increased bone marrow adiposity. Since osteoblasts and adipocytes share a common bone marrow progenitor cell, it has been suggested that these changes may be due to a preferential differentiation towards the adipoctye rather than osteoblast lineage. However, a recent murine study revealed that increased loading can bias this differentiation towards osteoblastogenesis (Luu Y et al JBMR 24:50–61, 2009). However, the effects of exercise (Ex) on changes in marrow adiposity and its relationship to changes in bone remain unknown in humans. This study examined: 1) whether an 18-mth targeted bone loading program could improve bone marrow fat density (reduce adiposity) in older men, and 2) whether changes in marrow adiposity were related to changes in bone turnover, density, and structure, and markers of inflammation. Men (n=180) aged 61 ± 7 yrs (± SD) were randomised to an Ex or Non-Ex group with or without additional Ca (1000mg/d)-Vit D3 (800IU/d). Because there were no Ex-Ca-Vit D3 interactions, we report the main eects of Ex which involved resistance training (60–85% 1RM) and weight-bearing activities performed 3 d/wk for 18 mth. Mid-femur marrow fat density (FD), total (TotAr), cortical (CortAr), medullary (MedAr) areas, vBMD, Ipolar and muscle CSA (QCT); total body fat (FM) and lean (LM) mass (DXA), and bone turnover (CTx and P1NP) and inflammatory markers (TNFα, IL-6, CRP) were assessed. 172 men (95%) completed the study; Ex compliance averaged 63%. Ex significantly improved FM (−1.1 kg), LM (0.6 kg) and muscle CSA (1.8%) (p<0.05-<0.001), and also led to an increase in marrow FD (reduced adiposity) relative to No-Ex (0.20% vs 0.05%, p<0.05), independent of age and changes in FM. However, there was no eect of Ex on any mid-femur bone trait [CortAr decreased similarly in both groups (−0.35%, p<0.01) due to an increase in MedAr (1.1%, p<0.001)]. In contrast, Ex led to a net 1.8% and 2.2% gain in FN aBMD (p<0.001) and LS trabecular vBMD (P<0.01), but there was no eect on bone turnover or inflammation. Changes in marrow FD were inversely related to age in the No-Ex group (r=-0.35, p<0.01) and changes in MedAr in the Ex group (r=-0.23, p=0.05), but not with changes in any other bone trait, body composition or biochemical marker in either group. In conclusion, Ex can reduce bone marrow adiposity in older men, and these changes were associated with reduced endocortical bone loss.
Disclosures: Robin Daly, None.
Effects of self-administered alcohol on cortical bone mass, architecture, and remodeling in cynomolgus monkeys.Dawn Olson*1, Kathleen Grant2, Kristine Wiren2, Andrew Rau2, Misa Odagiri2, Kathleen Howe3, Gianni F. Maddalozzo3, Russell T. Turner3, Urszula Iwaniec3. 1Oregon State University, Corvallis, OR, USA, 2Oregon Health Sciences University, Portland, USA, 3Oregon State University, Corvallis, USA
Alcohol abuse is an established risk factor for osteoporotic as well as non-osteoporotic fractures. Alcohol suppresses biochemical markers of bone turnover. However, the precise effect of alcohol on cancellous versus cortical bone compartments in humans is unknown. The rat has been the predominant animal model for investigating the effects of alcohol on cancellous bone growth and turnover. In the rat, chronic alcohol abuse results in progressive cancellous bone loss due to reduced bone formation. Alcohol also suppresses acquisition of cortical bone in growing rats. However, because of limited intracortical remodeling, the rat is less suitable for evaluation of alcohol effects on cortical bone turnover. Therefore, the purpose of this study was to assess the effects of alcohol on intracortical bone remodeling in a non-human primate model for alcohol abuse. Young adult (8-year-old) female cynomolgus monkeys (n=10) were trained to drink 4% (w/v) ethanol in water and allowed to self-administer ethanol for 22 hrs/day over a period of 52 weeks. Monkeys (n=4) who refused alcohol during the training interval were subsequently given an isocalorically-matched maltose-dextrose solution. The monkeys self-administered an average of 3.2 ± 0.2 g ethanol/kg/d (mean ± SE) resulting in blood alcohol levels of 96 ± 11 mg/dl (mean ± SE). Intracortical bone formation was evaluated in ground sections obtained from tibia midshaft. Alcohol consumption resulted in drastically lower numbers of labeled osteons/bone area (Figure A) and drastically lower intracortical bone formation rate (Figure B). These findings suggest that a reduction in intracortical bone remodeling contributes to the decreased bone quality observed in chronic alcohol abusers.
Disclosures: Dawn Olson, None.
Does Proton Pump Inhibitor Therapy Decrease Calcium Absorption?.Karen Hansen*1, Andrea Jones2, Mary Lindstrom3, Lisa Davis3, Kristina Penniston3, Martin Shafer4. 1University of Wisconsin, Madison, WI, USA, 2University of Wisconsin School of Medicine & Public Health, Madison, WI, USA, 3University of Wisconsin, Madison, USA, 4WI State Lab of Hygiene, Madison, USA
Epidemiologic studies link proton pump inhibitor (PPI) therapy to higher risk of osteoporotic fracture, presumably by decreasing calcium absorption. Such studies raise significant public concern regarding the safety of PPI therapy, now available without a prescription. However, existing literature does not clarify whether PPI therapy truly decreases calcium absorption. We hypothesized that PPI use might simply mark individuals who, due to poor general health, have a higher risk of fracture.
We used dual stable isotopes to clarify the impact of short-term omeprazole therapy on calcium absorption and bone resorption in healthy postmenopausal women. We excluded women <5 years past menopause and those with conditions interfering with nutrient absorption. Women underwent three inpatient calcium absorption studies, the first two before and the third following at least 30 days of omeprazole 40 mg daily. During each study, women ingested their typical diet and received 44Ca orally and 42Ca intravenously. We collected urine for 24 hours and measured its calcium isotope content by mass spectrometry. We measured bone resorption by collecting fasting urine c-telopeptide the morning of each calcium absorption study visit.
Twenty-one women completed the study. The monthly variation in calcium absorption without omeprazole was <1%, demonstrating the high precision of our method to estimate calcium absorption. Omeprazole therapy had no effect on calcium absorption (Figure) nor bone resorption (Table).
We conclude that short-term omeprazole therapy does not decrease calcium absorption nor increase bone resorption. We plan future studies to evaluate whether long-term omeprazole has untoward effects on calcium homeostasis.
Disclosures: Karen Hansen, None.
Prevalence and Costs of Subsequent Fractures Among Osteoporotic Non-vertebral Fracture Patients in the United States.Crystal Pike1, Howard Birnbaum2, Matt Schiller2, Hari Sharma2, Anna Gu2, Russel Burge*3, Eric Edgell3. 1Boston, MA, USA, 2Analysis Group, Inc., Boston, USA, 3Eli Lilly & Company, Indianapolis, USA
The objective was to estimate the prevalence and costs of osteoporotic patients with subsequent fractures that occur in the year after an incident non-vertebral (NV) fracture in the U.S.
Using a privately-insured employer claims database (age 18–64) and the Medicare Standard Analytic Files 5% sample (age 65+), osteoporotic patients with NV fractures (1999–2006) were identified and matched to osteoporotic controls without fractures. Patients were followed for one year after a new osteoporotic NV fracture (the “index” fracture). To avoid categorizing misdiagnoses/miscodings/follow-up as subsequent fractures, only claims for NV fractures (at any site) occurring more than three months after the index date were identified as subsequent fractures (more than six months for claims for fractures occurring at the same site as the index fracture). Subsequent fracture rates were calculated by dividing the number of patients with each type of subsequent osteoporotic NV fracture by the total number of patients with each type of osteoporotic NV fracture. Costs were reported in 2006 dollars (from a third-party payer/Medicare perspective).
In the privately-insured population (n=4,764), 14.1% of patients with an NV index fracture had a subsequent NV fracture. Patients with index fractures of the hip, upper arm, and multiple sites had the highest prevalence of subsequent NV fractures (20.0%, 19.5% and 18.9%, respectively). Among all NV index fracture patients, 1.6% had subsequent hip and 13.0% had subsequent non-vertebral, non-hip (NVNH) fractures. The mean annual excess medical and drug costs per subsequent fracture patient were $13,579 ($19,243 v. $5,664; p<0.01). In Medicare (n=48,742), 22.6% of patients with an NV index fracture had a subsequent NV fracture. Patients with index fractures of multiple sites, hip, and femur had the highest prevalence of subsequent NV fractures (35.2%, 32.0% and 27.8%, respectively). Among all NV index fracture patients, 9.4% had subsequent hip and 15.5% had subsequent NVNH fractures. The mean annual excess medical costs per subsequent fracture patient were $28,928 ($36,485 v. $7,557; p<0.01).
NV fracture patients are at substantial risk for a subsequent NV fracture within one year. Patients with subsequent fractures are significantly more costly than controls, and both the prevalence and cost of subsequent fracture patients were significantly higher in the Medicare population.
Disclosures: Russel Burge, Eli Lilly and Company, 5 This study received funding from: Eli Lilly and Company
Idiopathic Osteoporosis in Premenopausal Women: Is the Osteoblast to Blame?.Adi Cohen*1, Mishaela Rubin2, David Dempster3, Serge Cremers4, Cliord Rosen5, John Manavalan6, Emily Stein7, Hua Zhou8, Thomas Nickolas1, Joan Lappe9, Halley Rogers6, Robert Recker9, Elizabeth Shane7. 1Columbia University Medical Center, New York, NY, USA, 2Columbia University, New York, NY, 3Columbia University, West Haverstraw, NY, USA, 4Columbia University, New York, NY, USA, 5Maine Medical Center, Scarborough, ME, USA, 6Columbia University, New York, USA, 7Columbia University College of Physicians & Surgeons, New York, NY, USA, 8Helen Hayes Hospital, West Haverstraw, NY, USA, 9Creighton University Osteoporosis Research Center, Omaha, NE, USA
Purpose: Idiopathic osteoporosis (IOP) in premenopausal women is a poorly understood entity in which young, otherwise healthy women have low trauma fracture(s) or very low BMD.
Methods: To investigate the etiology of IOP, we measured calciotropic hormones and bone turnover markers, and performed transiliac bone biopsies in 40 women with IOP and 52 healthy controls (C) with normal BMD and no fractures. Secondary causes of osteoporosis were excluded.
Results: IOP and C did not differ by age (37 ± 8 yrs) or BMI (24.7 ± 5.8 kg/m2). BMD was significantly lower at all sites in IOP. Serum PTH, 25-OHD, bone alkaline phosphatase (BAP) and CTx were similar in IOP and C, as was IGF1 (175 ± 58 vs 184 ± 55 ng/mL). By histomorphometry, IOPs had thinner cortices and fewer, thinner, more widely spaced trabeculae than C (p=0.06-<0.0001). Wall thickness (W.Th; the thickness of completed osteons), an index of osteoblast (OB) function, was approximately 10% lower in IOP on cancellous (Cn), endocortical (Ec) and intracortical (Ic) surfaces (Table); Cn osteoid width was 15% (p=0.005) lower. Remodeling parameters, including mineralized perimeter (Md.Pm), bone formation rate (BFR) and activation frequency (Act.F) did not differ between groups and correlated directly with CTx (r=0.602–0.665, p<0.0001) and BAP (r=0.382–0.561, p=0.02–0.0002) at the Cn surface in both IOP and C In C, IGF1 correlated directly with Act.F, Md.Pm, BFR and bone resorption rate (BRs.R) on Cn and Ec surfaces (Table). After controlling for age, these relationships remained significant on the Ec surface (p=0.04–0.02). In IOP, IGF1 did not correlate with any remodeling parameter. Hypothesizing that an OB defect could underlie the lower mean W.Th, we used flow cytometry and OB-specific ligands to isolate circulating OB precursors from peripheral blood mononuclear cells in a subset of IOP (n=7) and C (n=5) subjects. Osteocalcin (OCN)+ OB precursors were 62% lower; immature OCN+CD146+ cells were 2.6 fold and OCN+CD34+ cells 2.1 fold higher in IOP than C.
Conclusions: In IOP, mean W.Th was lower and there was no relationship between serum IGF1 and histomorphometric parameters of bone remodeling. A subset of IOP had significantly fewer circulating OB precursors, and a preponderance of more immature forms. These preliminary results suggest that OB synthesize less bone matrix per remodeling site in IOP and may be less responsive to IGF1. Abnormal OB number or function may contribute to the pathogenesis of IOP.
Disclosures: Adi Cohen, None.
Relationship Between Serum Levels of Osteocalcin and Atherosclerotic Disease in Patients with Type 2 Diabetes Mellitus.Manuel Munoz-Torres*1, Pedro Rozas-Moreno2, Mariela Varsavsky3, Antonia Garcia Martín4, María José Lara Villoslada5, JA Garcia salcedo5, A. Rocio Gonzalez Ramirez5. 1University Hospital, Granada, Spain, 2Endocrinology Department. Hopsital General de Ciudad Real, Ciudad Real, Spain, 3Endocrinology, Bone Metabolic Unit. Hospital Universitario San Cecilio, Granada, Spain, 4Endocrinology, Bone Metabolic Unit. Hospital Universitario San Cecilio, Granada, Spain, 5Hospital Universitario San Cecilio, Granada, Spain
Osteocalcin, an osteoblast-specific protein, has several hormonal features and its actions are related not only with bone metabolism but also glucose metabolism and fat mass (Kanazawa et al JCEM 2009). If osteocalcin is a marker of atherosclerotic disease is not well established. Aims: To analyze the relationship between serum levels of osteocalcin and atherosclerotic disease in patients with type 2 diabetes mellitus (DM2). Patients & Methods: We studied 78 DM2 patients (55% males) with and without prevalent cardiovascular disease: coronary heart disease (CHD), cerebrovascular disease (CVD) and peripheral vascular disease (PVD). Mean duration of diabetes was 13.4 years. Mean hemoglobin A1c levels was 8.01%. Intima-media thickness (IMT), atherosclerotic plaque and aortic calcifications were evaluated. Serum osteocalcin levels were determined by RIA. Results: A total of 57.7% of patients had clinical cardiovascular disease: 37.2%, CHD, 20.5% CVD and 12.8% PVD. 25% of the patients showed abnormal IMT, 26.9% carotid plaque and 32.1% aortic calcifications. Mean serum levels of osteocalcin were 1.62 ± 1.33 ng/ml in females 1.35 ± 1.19 ng/ ml in males. CHD was associated with higher levels of osteocalcin in males (1.95 ± 1.36 vs. 0.93 ± 0.86 ng/ml, p= 0.006). Also, we found higher concentrations of osteocalcin in females with abnormal IMT (2.17 ± 1.84 vs. 1.25 ± 0.67 ng/ml, p= 0.042), carotid plaque (2.86 ± 2.10 vs. 1.43 ± 1.09 ng/ml, p= 0.03) and aortic calcifications (2.85 ± 1.97 vs. 1.26 ± 0.83 ng/ml, p= 0.002). Conclusions: In type 2 diabetic patients serum osteocalcin levels are associated with atherosclerotic parameters, suggesting that osteocalcin is important not only for bone metabolism but also for atherosclerotic disease.
Disclosures: Manuel Munoz-Torres, None.
Eighteen months of Teriparatide treatment results in cortical bone, mass, density, area and thickness increase in tibia. A pQCT study.Eleftheria Metania*1, Kostantinos Stathopoulos2, Pelagia Katsibri3, Aristidis Zoubos2, Grigoris Skarantavos4. 1Chaidari, Greece, 2Metabolic Bone Diseases Unit, 1st Department Of Orthopaedic Surgery, Medical School, University of Athens, ‘ATTIKON’ University Hospital., Athens, Greece, 3Metabolic Bone Diseases Unit, 1st Department Of Orthopaedics Surgery, Medical School, University of Athens, ‘ATTIKON’ University Hospital., Athens, Greece, 4Metabolic Bone Diseases Unit, 1st Department Of Orthopaedics Surgery, Medical School, University of Athens, ‘ATTIKON’ University Hospital., Athens, Greece
PURPOSE: Peripheral Quantitative Computed Tomography (pQCT) is a highly sensitive method used to monitor changes in long bones density and geometric properties following therapeutic intervention. Teriparatide [TPTD- Recombinant human parathyroid hormone (1–34)], has been shown to stimulate new bone formation, resulting in rapid gains in bone mass with improved skeletal architecture in osteoporotic patients. The purpose of this study was to investigate the eects of TPTD treatment in bone after 6, 12 and 18 months of treatment, using pQCT. MATERIAL AND METHODS: The study was performed in a subgroup of 36 postmenopausal women that received once-daily, self-administered subcutaneous injection of TPTD 40 μg, and completed successfully the required pQCT measurments at 6,12 and 18 months after baseline. All patients received 1000 mg calcium and 400 IU vitamin D daily. Measurements were performed at the nondominant tibia, at baseline and after 6, 12 and 18 months exposure to treatment, using a Stratec XCT 2000 pQCT (Stratec Medizintechnik GmbH, Pforzheim, Germany) at the 4%, 14% and 38% of tibia length sites. Statistical analyses were performed using pair samples t-test. RESULTS: There was no statistically significant dierence in cancellous bone from baseline to 6, 12 and 18 months of treatment with TPTD. After 6 months, at the 14% site there was an increase in the Total Density (360,36 ± 70,9 vs 347,15 ± 69,07, p: 0,049), Subcortical Density (609,08 ± 123,06 vs 587,77 ± 121,90, p: 0,046), Cortical Content (108,67 ± 37,45 vs 104,55 ± 38,64, p: 0,038), Cortical Area (107,15 ± 31,13 vs 103,25 ± 32,26, p: 0,033), and Cortical Thickness (1,50 ± 0,44 vs 1,43 ± 0,45, p: 0,025). Cortical Thickness was increased at the 14% site after 18 months of treatment (1,49 ± 0,34 vs 1,46 ± 0,36, p: 0,050). Total Density and Subcortical Density were also increased, though with no statistical significance. (TOT.D: 343,78 ± 73,85 vs 339,16 ± 75,5, p: 0,065, SUB.D: 588,71 ± 111,82 vs 580,08 ± 115,35, p:0,073, CONCLUSIONS: According to our results, after 18 months of treatment, TPTD increases bone mass, volumetric density, cortical thickness and cortical area at the 14% of tibia length.
Disclosures: Eleftheria Metania, None.
Bone loss after Discontinuation of Teriparatide can be Prevented with a Single Infusion of Zoledronic Acid.Katherine Tuthill1, Chad Deal*2, Audrey Kriegman3. 1Cleveland Clinic, Cleveland, USA, 2Cleveland Clinic Foundation, Cleveland, OH, USA, 3Novartis Pharmaceuticals, East Hanover, NJ, USA
Teriparatide (TPTD) is an anabolic agent approved for patients at high risk for fracture. Two years of TPTD results in a significant increase in bone mass but after discontinuation significant bone loss occurs. Treatment with alendronate has been shown to prevent bone loss and increase bone mass after TPTD discontinuation. Zoledronic acid 5mg (ZOL) is an IV bisphosphonate approved as a one-yearly infusion for the treatment of osteoporosis. We tested the ability of ZOL to maintain bone mass after treatment with TPTD. We treated 35 postmenopausal women with ZOL immediately after finishing a course of TPTD. The primary endpoint was change in bone density (grams/cm2) in the lumbar spine at 12 months. Secondary endpoints were change in bone density at the total hip (TH), femoral neck (FN), and total body (TB). Markers of bone turnover (serum c-telopeptide type I collagen and serum n-propeptide type I collagen) were measured in all patients throughout the 12 months of the study. We report on the bone density results on 33 patients who have finished the trial (35 enrolled). The complete bone density dataset and changes in bone turnover markers will be reported.
The study cohort had a mean age of 68.8 +/- 11.2, a mean T-score of −2.1 for the LS and TH, −2.3 for FN, and −1.8 for TB. Table 1 summarized the number of patients exhibiting stable, increase or decrease BMD at the LS, FN, TH and TB (LSC 0.03g/cm2). In 33 patients at 132 skeletal sites, 123 sites (93.2%) had stable or increased bone mass, 9 had a significant decline in bone mass.
A single infusion of zolendronic acid 5mg preserves or increases bone mass in patients after TPTD treatment in the LS, FN, TH and TB in 90%, 90%, 94% and 97% respectively.
Disclosures: Chad Deal, Novarties Pharmaceuticals, 3 This study received funding from: Novartis Pharmaceuticals
Characterization of and Risk Factors for Acute Phase Reactions Following Zoledronic Acid.Ian Reid*1, Gregory Gamble2, Peter Mesenbrink3, Dennis Black4. 1University of Auckland, Auckland, New zealand, 2University of Auckland, Auckland, New zealand, 3Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA, 4University of California, San Francisco, San Francisco, CA, USA
The occurrence of flu-like symptoms in patients receiving high-dose oral or intravenous amino-bisphosphonates (the acute phase reaction, APR) has been recognized for >20 y but has not been systematically analyzed. It has been suggested that previous exposure to oral bisphosphonates, glucocorticoids, non-steroidal antiinflammatory drugs (NSAIDs) and statins might reduce the severity/frequency of the APR. In this analysis, we examine the symptoms seen after zoledronic acid and risk factors for the APR in the HORIZON-PFT study. This is an international, randomized, controlled trial comparing annual injections of zoledronic acid 5mg to placebo over 3 years in 7765 women with postmenopausal osteoporosis.
Adverse events (AEs) were collected using the MEDRA dictionary, in which AEs are categorized using ∼2000 individual terms. To identify the components of the APR, all AEs occurring within 3 days of the first zoledronic acid infusion were compared between treatment groups.
We found >30 AE terms that were significantly more common in the zoledronic acid group, and these could be clustered into 5 groups, as shown in the Table. The musculoskeletal group included pain and joint swelling; gastrointestinal included abdominal pain, vomiting and diarrhea; “other” included non-specific systemic symptoms such as fatigue and malaise, but also a significant increase in the frequency of nasopharyngitis and peripheral edema in the zoledronic acid group. Overall, 42.5% of subjects treated with zoledronic acid showed evidence of an APR after the first infusion, compared with 11.7% of the placebo group. All APR components had their peak onset within 1 day of infusion, median duration of the APR was 3 days (interquartile range 2–5 days), each component having a similar time-course except for eye symptoms which had a median duration of 5 days. Severity was rated as mild or moderate in 90%, and the APR resulted in drug discontinuation in 0.8%. Most putative risk factors had only a small impact on incidence of APR. Stepwise regression showed that APR was more common in Chinese, younger subjects and those using NSAIDs, and was less common in smokers, diabetics, previous bisphosphonate users and Latin Americans (P<0.05 for all).
Disclosures: Ian Reid, Novartis Pharmaceuticals, 2; Novartis Pharmaceuticals, 5 This study received funding from: Novartis
Esophageal Cancer Reports Coincident With Risedronate Use.John Bilezikian*1, Andrea Klemes2, Ethel Siris1. 1Columbia University College of Physicians & Surgeons, New York, NY, USA, 2, Mason, OH, USA
In the January 1, 2009 issue of the New England Journal of Medicine a letter to the editor cited reports of esophageal cancer subsequent to alendronate use as well as alendronate combined with other oral bisphosphonates.
We examined risedronate clinical and postmarketing data. Risedronate has been studied in placebo-controlled Phase III osteoporosis trials of up to three years duration in over 15,000 patients and in active control studies with 4000 additional patients. There were no exclusions for patients having any GI diseases including GERD in these trials.
Patients in the placebo controlled studies were randomized to receive risedronate 2.5 mg daily (n = 4998), 5 mg daily (n = 5395), or placebo daily (n = 5363). The incidence for esophageal cancer in these combined risedronate studies, was 0.04% (2 pts) in the 2.5 mg group, 0.02% (1 pt) in the 5 mg group, and 0.04% (2 pts) in the placebo group. The incidence rate for esophageal cancer was 19 per 100,000 patient-years of observation in placebo patients, 22 per 100,000 in 2.5 mg daily patients and 9 per 100,000 in the 5 mg daily patients
The only case in the active control studies with multiple dosing regimens was in the 5 mg daily (n=1731) control group that was compared to the 35 mg weekly (n=485), 50 mg weekly (n=491), 75 mg two consecutive days per month (n=620) and 150 mg once a month (n=650). There were no cases in prevention, flexible dosing, male osteoporosis or open label extension studies.
In post-approval surveillance from May 1998 to January 9, 2009, with more than 18 million patient years of exposure, the rate of reporting for esophageal cancer coincident with risedronate use was very rare (per CIOMS) or less than 0.1 per 100,000 patient-years exposure, without attribution of causation. Most of the reports lack clinical details (e.g., histopathology, radiography, or full patient history) for definitive assessment.
Esophageal cancer incidence in U.S. men and women ≥65 years of age is 23.3 per 100,000 per year according to National Cancer Institute Surveillance Epidemiology and End Results (SEER), cancer statistics review (2002–2005). The incidence is 11.2 per 100,000 per year for Caucasian women ≥65 years of age. The incidence of esophageal cancer events observed among risedronate or placebo treated patients was consistent with the background rates in the population.
In summary, these data do not support a causal relationship or an association between the use of risedronate and esophageal cancer.
Disclosures: John Bilezikian, Procter & Gamble Pharmaceuticals, 5 This study received funding from: Procter & Gamble Pharmaceuticals
Osteoporosis Medication And Mortality Risk In Elderly Women And Men: An 18-Year Prospective Study From Dubbo Osteoporosis Epidemiology Study.Jacqueline Center*1, Dana Bliuc1, Nguyen Nguyen1, Tuan Nguyen2, John Eisman1. 1Garvan Institute of Medical Research, Sydney, NSW, Australia, 2Garvan Institute of Medical Research, Sydney, Australia
Osteoporotic fractures are associated with premature mortality and with increased risk of subsequent fracture, which again raises mortality risk. While it is clear that osteoporosis treatment reduces the incidence of fracture, some studies suggest that it reduces mortality risk. The aims of this study were to examine the effect of osteoporosis treatment on 1) all cause mortality in women and men over 60+ and 2) post-fracture mortality in women.
There were 359 and 129 low trauma fractures (women and men, respectively) and 435 deaths over 15001 person-years in women and 386 deaths over 9367 in men amongst 1164 women and 819 men participating in the Dubbo Osteoporosis Epidemiology Study (April 1989- May 2007). Bone mineral density and information on co-morbidities and medications were obtained. Osteoporosis medication was classified as: bisphosphonates (BP), current hormone replacement therapy (HRT) and calcium ± vitamin D only (CaD). Propensity matching was used to adjust for baseline differences.
There were 317 women (BP, n=81; HRT, n=94, CaD, n=142) and 36 men (BP, n=14; CaD, n=22) who received osteoporosis medication. The majority of those on BP had had a fracture (women, n=74; men, n=10).
In men, mortality rates were lower for those treated with BP, but not with CaD compared with no treatment [BP 1.8/100 person-years (0.5, 7.2) and Ca D 3.8/100 person-years (2.5, 5.2) vs no treatment 4.6/100 person-years (4.2, 5.1)]. The association between BP and mortality after adjusting for propensity scores, was similar but not statistically significant [HR: 0.52 (0.1, 2.1)].
In women, mortality rates were lower for those treated with BP and HRT, and as for men, not with CaD [BP 1.2/100 person-years (0.6, 2.2), HRT 1.3/100 person-years (0.7, 2.3), and CaD 3.0/100 person-years (2.5, 6.3); vs. no treatment 4.0/100 person-years (3.6, 4.4)]. After adjusting for propensity scores mortality risk remained lower for women treated with BP [HR: 0.5 (0.3, 0.9)] but was not significant for HRT [adjusted HR: 0.65 (0.35, 1.23)]. When limiting the analysis to the 359 women with fracture, mortality risk was still reduced in the group treated with BP [adjusted HR: 0.4 (0.2, 0.9)].
These data suggest that bisphosphonates may reduce the risk of all cause and post-fracture mortality in women, and perhaps in men. This finding warrants further exploration in randomised trials.
Disclosures: Jacqueline Center, Amgen, Novartis, GE Lunar, Organon, Sanofi Aventis, 99
The Rapid Onset and Sustained Efficacy (ROSE) Study: Zoledronic Acid vs. Alendronate - First Results.Peyman Hadji*1, Dieter Gamerdinger2, Wolfgang Spieler3, Peter Kann4, Helena Löer5, Konstantin Articus5, Volker Ziller6. 1Philipps-University of Marburg, Marburg, Germany, 2Orthopaedische Praxis, Bautzen, Germany, 3Reseach Center for Osteoporosis & Rheumatology, Zerbst, Germany, 4Philipps University Marburg, Marburg, Germany, 5Novartis Pharma GmbH Nürnberg, Nuremberg, Germany, 6Philipps-University-Marburg, Marburg, Germany
Purpose: Annual i.v. infusion of Zoledronic acid (ZOL) 5 mg is an eective and overall well-tolerated therapy for osteoporotic patients and also in patients with a recent low impact hip fracture. An annual dosing regimen of ZOL 5 mg showed improved compliance as compared with weekly or daily oral bisphosphonates used in the treatment of osteoporosis (1). Besides compliance, comparing the ecacy and safety as well as bone metabolic eects of once-yearly i.v. ZOL 5 mg and once-weekly oral Alendronate (ALN) 70 mg is important. The primary objective of this study was to assess serum N-telopeptide (NTx) area under curve (AUC), while the secondary objectives were evaluation of serum N-terminal propeptide of type I collagen (P1NP) AUC, serum NTx and P1NP levels at dierent time points (0, 3, 6, 9 month and 1-year) compliance, quality of life, patient's preference assessment and safety.
Methods: The Rapid Onset and Sustained Ecacy (ROSE) study was a 1-year, open-label, multi-center, randomized, controlled trial in postmenopausal women (n=594) aged 55–99 years, with documented osteopenia/osteoporosis (T-score ≤ −2.0 at total hip or spine measured by DXA). Exclusion criteria were prior bisphosphonate therapy, calculated creatinine clearance <35 mL/min, secondary osteoporosis, primary hyperparathyroidism, and presence of contraindications to study drugs, calcium or vitamin D. Patients were randomized (2:1) to receive either once-yearly i.v. ZOL 5 mg or once-weekly oral ALN 70 mg. All patients received daily doses of 1200 mg calcium and 800 I.U. vitamin D.
Results: At study start there were no dierences in age and anthropometric data of both groups (table 1). An analysis of the preliminary data available till date showed that ZOL has a greater and more rapid reduction of serum NTx compared to ALN. The pattern of NTx changes over time was dierent in treatment groups as NTx level were reached at 3 months with ZOL but only after 9 months with ALN treatment. At month 12, NTx levels were still slightly but significantly higher in the ZOL group as compared with the ALN group (P-value=0.013) (Figure 1). Conclusion: The results of the ROSE study showed greater and more rapid reduction of NTx with once-yearly i.v. ZOL 5 mg as compared to once-weekly oral ALN 70 mg.
Disclosures: Peyman Hadji, Novartis Pharma GmbH, 5; Novartis Pharma GmbH, 2 This study received funding from: Novartis Pharma GmbH
Time to Onset of Anti-Fracture Efficacy and Persistence of Effect of Zoledronic Acid 5 mg in Women with Osteoporosis or Recent Hip Fracture.Richard Eastell1, Dennis Black*2, Steven Boonen3, Peter Mesenbrink4, Chrisitna Bucci-Rechtweg5. 1Department of Human Metabolism & Clinical Biochemistry, University of Sheeld, Sheeld, United Kingdom, 2University of California, San Francisco, San Francisco, CA, USA, 3Center for Metabolic Bone Disease, Leuven, Belgium, 4Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA, 5Pediatric Advisory Group, Novartis Pharmaceuticals, Basel, Switzerland
Purpose: Fragility fractures, especially those of the hip and spine are associated with increased morbidity and mortality, thus, necessitating a highly ecient therapy that achieves a rapid onset of action. Bisphosphonates (Bp), such as zoledronic acid (ZOL), are known to prevent bone loss and preserve trabecular architecture in postmenopausal women with osteoporosis. ZOL 5 mg is the only Bp given as a once-yearly i.v. treatment because its high binding anity and 100% bioavailability allows rapid distribution of the drug in bone tissue. ZOL 5 mg i.v. was shown to reduce the risk of new clinical fractures in men and women with a recent low trauma hip fracture. Method: This is a pooled post-hoc analysis of two HORIZON trials to observe the time-to-onset of anti-fracture ecacy with ZOL 5 mg. A total of 9375 women from two HORIZON trials were included in this analysis. HORIZON-Pivotal Fracture Trial (PFT) [3-year] included women with postmenopausal osteoporosis and HORIZON-Recurrent Fracture Trial (RFT) [median follow-up 1.9 years] included women with recent low-trauma hip fracture. The reduction in clinical vertebral and non-vertebral fractures were observed at 6, 12, 18, 24, and 36 months adjusting for the dierences across studies using Cox regression Results: ZOL 5 mg showed a reduction in fracture risk for all fracture types as early as 6 months. Furthermore, ZOL 5 mg significantly reduced the risk of clinical vertebral fractures (Figure 1) by Month 12 (risk reduction [95% CI]: 57% [25–76%]) and of non-vertebral fractures (Figure 2) by Month 18 (23% [8–36%]) [both p<0.005 vs. placebo]. Conclusion: ZOL 5 mg (once-yearly i.v.) demonstrated its anti-fracture ecacy as early as 6 months which persists for at least 3 years. However, ongoing studies would examine the long term eects of ZOL 5 mg in postmenopausal women with osteoporosis.
Disclosures: Dennis Black, Yes, 5
This study received funding from: Novartis
Implementation of guidelines by a fracture nurse in patients presenting with non-vertebral fractures: effect on subsequent fracture incidence and survival.Piet Geusens*1, Kirsten Huntjens2, Sven van Helden2. 1University Hasselt, Diepenbeek, Belgium, 2Maastricht University Medical Center, Maastricht, Netherlands
A history of fracture increases the risk of subsequent fractures and some fractures are associated with increased mortality. We evaluated the ecacy of implementation of guidelines for osteoporosis by a dedicated fracture nurse on incidence of subsequent non-spine fractures and mortality within 2 years in patients presenting with a non-spine fracture.
Two cohorts of patients were compared: before (January 1999 - January 2001) and after intervention (September 2004 - September 2006). Patients were older than 50 years and living in the postal area of the hospital. Pathological fractures and vertebral fractures were excluded. All baseline and subsequent fractures were confirmed by radiography. Mortality was searched in the national obituary database. The intervention consisted of evaluation of bone and fall related fracture risks by a fracture nurse and accordingly treatment advice was given. Multivariate Cox regression analysis was used to calculate hazard ratio's (HR) for subsequent fracture incidence and mortality corrected for age, sex and baseline fracture location (major fractures: hip, pelvis, proximal tibia or humerus, multiple ribs and distal femur and minor fractures: all other fractures).
In total, 3334 patients (2467 (74%) women and 867 (26%) men)) were included, 1921 before and 1413 after intervention. Groups differed at baseline for fracture location (before vs. after intervention: major fractures 44.9% vs. 40.6% and minor fractures 55.1% vs. 59.4%, p<0.05). The absolute risk of a subsequent fracture decreased from 9.9% to 7.3% (p=0.008). Cox regression analysis showed a risk reduction of 30% for a subsequent fracture (HR 0.70; 95% Conficence Interval (CI): 0.55 – 0.89). The mortality decreased from 17.9% to 11.4% (p< 0.001). Cox regression analysis indicated a risk reduction of 37% in mortality (HR 0.63; 95%CI: 0.52 – 0.76). After major fractures mortality decreased by 44% (HR 0.56; 95%CI: 0.45.−0.69) (32.1% vs. 19.9%; p<0.001) Mortality after minor fractures was lower and remained unchanged after intervention (6.2% vs. 5.7%).
Conclusions. Fracture nurse risk assessment and intervention in a fracture population are associated with a decrease in subsequent fracture incidence and mortality.
Due to the multifactorial nature of the assessment and intervention by the fracture nurse, it is not clear which part of this intervention is associated with the decrease in new fractures and mortality. Further research to clarify this is needed.
Disclosures: Piet Geusens, None.
Is More Really Better? The Complex Relation Between Adherence and Fracture Reduction.Daniel Solomon*1, Suzanne Cadarette2, Amanda Patrick3, Elena Losina3, John Schousboe4, M Brookhart3. 1Harvard Medical School, Boston, MA, USA, 2Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada, 3Brigham & Women's Hospital, Boston, USA, 4Park Nicollet Clinic, Minneapolis, MN, USA
Purpose: Consistent data support a relation between medication adherence and fracture reduction. Prior work suggests that the link between adherence and fracture reduction is substantially affected by how one measures adherence. We further examined this relation by varying adherence assessment.
Methods: The study cohort consisted of Medicare beneficiaries initiating an oral bisphosphonate for osteoporosis. Adherence was assessed in sequential 60-day periods from the time of initiation until censoring by fracture, death, loss of eligibility or end of follow-up. In Cox regression models incorporating time-varying measures of adherence, the adjusted relation between adherence and an osteoporotic fracture (hip, wrist, humerus, and pelvis) was examined using several methods for calculating the medication possession ratio (MPR), a common adherence metric derived from pharmacy dispensing records. Method 1 (“cumulative”) examined all 60-day periods from initiation until the index period; Method 2 (“instantaneous”) assessed only the 60-day period immediately prior to the index period; and Method 3 (“short-term cumulative”) only focused on the three 60-day periods immediately prior to the index period. All models were adjusted for variables known to be associated with osteoporosis or fracture.
Results: 23,579 persons fit the eligibility criteria for this study. 22,813 (97%) were women and the mean age was 79 years. Prior fractures were noted in the one-year baseline period for 6% of subjects and 40% had a BMD test in the prior year. All three methods showed a reduced risk of hip or any fracture when comparing an MPR of > 66% with < 33% (see Table). This reduction was slightly more pronounced for hip fractures compared with any fractures. There was evidence for a significant trend toward a reduction in relative risk of fractures and improving MPR (p-values for all trend tests < 0.05). This was most consistent for the cumulative method for measuring MPR.
Conclusions: We found a consistent relation between adherence with osteoporosis treatment and fracture reduction, regardless of method for measuring MPR. Since randomized controlled trials are not feasible in this area, observational data will need to be used to describe this relation, however it is not clear which method of measuring MPR is correct. A cumulative method produces the most gradual trend toward a reduced relative risk of fracture with incremental improvement in adherence.
Disclosures: Daniel Solomon, None.
Most non-persistent patients with osteoporosis do not switch to other drug treatments: a 3½ year market survey of 240,000 patients in the Netherlands.Coen Netelenbos*1, Piet Geusens2, Servaas Buijs3. 1VU Medical Center, Amsterdam, The Netherlands, 2University Hasselt, Diepenbeek, Belgium, 3IMS, Capelle aan den IJssel, Netherlands
Compliance in drug treatment of osteoporosis with oral medication such as oral bisphosphonates (BP) is low. We evaluated the compliance after one year of osteoporotic treatment and analysed the percentage of non-compliant patients who did not switch to other antiosteoporotic medication, based on longitudinal data of drug dispensing.
Methods: The study was carried out in the routine practice setting in the Netherlands, and involved an analysis of prescriptions dispensed by pharmacies and dispensing GPs. In the Netherlands, ambulant patients visiting a specialist also receive their medication via the retail channel, and so this dispensing is also covered by the database. Data for each patient were anonymized and provided detailed information of the therapy used, with no risk of sampling bias or hidden influences. Data was collected by IMS Health in a representative sample with a coverage of 72%. Adherence was evaluated by measuring the MPR (medication possession ratio), persistence (period of use for the drug), non-adherence (no refill) and switch between drugs used in the treatment of osteoporosis.
Results: Of the 240,000 patients (projection based on a sample coverage of over 50%) who were on medication for osteoporosis, 82% were on bisphosphonates. Of the 1.4M prescriptions, 89% were refill, 11% new and 1% switch between drugs. Compliance - measured by the MPR- was 97% for daily BPs, 98% for weekly BPs and 94% for monthly BPs. On average 91% of the chronic users had a MPR of ≥ 80%, which is considered the minimal useful dosage for BPs and which is much more then the 25–45% that is found in other reports. After 1 year, persistence was 40–54% and 45–60% of the starters had stopped treatment with the specific drug. During a follow up of 18 months after stopping a drug for osteoporosis, only 20% (95% CI: 17%, 25%) switched to other drugs for osteoporosis. Thus 40% of all patients in whom treatment was started for osteoporosis were non-adherent and had drug treatment for less than one year and did not switch to other osteoporosis drug regimens. Conclusion: About half of the patients with osteoporosis taking oral BP discontinued this treatment after one year, of which 80% did not switch to other treatment regimens during the first 18 months after stopping. These data indicate a major failure to adequately treat patients at high risk for fractures in real clinical practice which represents also a huge economic loss.
Disclosures: Coen Netelenbos, IMS, 3
The OPTIMUS intervention: Primary Care Practitioners treating osteoporosis in up to 64% of patients with fragility fracture.Gilles Boire*1, Michele Beaulieu2, Dominique Lambert3, François Cabana4. 1Université de Sherbrooke, Sherbrooke, Quebec, Canada, 2Merck Frosst Canada Inc., Montréal, Quebec, Canada, 3Procter&Gamble Pharma Canada Inc., Québec City, Quebec, Canada, 4Centre hospitalier universitaire de Sherbrooke, Sherbrooke, Quebec, Canada
Objective. To compare 2 strategies of osteoporosis (OP) treatment with current care in patients with fragility fractures.
Methods. Outpatients ≥50 years seen by orthopedists at the Centre Hospitalier Universitaire de Sherbrooke were screened for incident fragility fracture. Consenting patients with fragility fracture were randomized to Standard Care (SC) or to either Minimal (MI) or Intensive Interventions (II). Current use of OP treatments was collected by phone at each follow up and confirmed with patients' pharmacists. Appropriate OP treatment was defined as Ca and vitamin D supplements with an eective OP treatment (usually an aminobisphosphonate). Patients untreated at 12 months were oered a rescue. In SC, OP treatments were obtained at 6 and 12 months; if untreated at 12 months, an II was oered. In MI, a research assistant gave oral and written information about OP, informed through a standardized letter the Primary Care Practitioner (PCP) of the patient's fragility fracture; the importance to treat OP after a fragility fracture and the appropriate investigation and treatment were outlined. At 6 months, the PCP was again urged to treat OP in still untreated patients; at 12 months, an II was oered. Intensive Intervention (II) included all components of a MI plus an immediate lab investigation for primary causes and for conditions aecting treatment of OP. The PCP received the results of the investigation and a personalized counseling letter for treatment of the patient. When patients were untreated at 4, 8 and 12 months, PCP were again urged to treat their patient's OP. Results were compared with the rates of OP treatment at 12 months in hip fracture patients (HIP) evaluated as inpatients by rheumatologists.
Results. From January 15 2007 to March 31 2009, 734 patients were recruited: 200 in SC, 183 in MI, 170 in II and 181 in HIP. Of these, 70, 101, 80 and 93 respectively had completed a 12 months evaluation. Appropriate OP treatment at Inclusion and at 12months was present in 22% and 37% (+15%) in SC, in 31% and 58% (+27%) in ML in 28% and 64% (+36%) in II, and in 25% and 77% (+52%) in HIP, respectively.
Conclusions. The OPTIMUS intervention aims to improve self management in patients and PCP. OPTIMUS increased rates of appropriate treatment of OP by PCP to up to 64% of patients with fragility fractures, with low costs and minimal involvement of specialized human resources. In addition, the long term approach of PCP to OP may be improved.
Disclosures: Gilles Boire, Pfizer Canada, 5; Alliance for Bone Health (Aventis Canada and Procter & Gamble Pharma), 2; Abbott Canada, 2; Bristol Myers Squibb, 5; Wyeth Canada, 5; UCB Canada, 5; Homan LaRoche Canada, 5; Abbott Canada, 5; Amgen Canada, 5; Merck Frosst Canada, 2
This study received funding from: Merck Frosst Canada and Bone Health Alliance (Aventis Canada and Procter Gamble Pharma)
Assessment of the Effect of Bazedoxifene on Non-vertebral Fracture Risk.Eugene McCloskey*1, Helena Johansson2, Anders Oden2, Arkadi Chines3, John Kanis4. 1University of Sheeld, Sheeld, United Kingdom, 2University of Sheeld, Sheeld, United Kingdom, 3Wyeth Research, Collegeville, PA, USA, 4University of Sheeld, Brussels, Belgium
In a phase III study, bazedoxifene significantly decreased the risk of vertebral fractures in postmenopausal women. No significant effect was noted on non-vertebral fractures, but fracture risk reduction was reported in a post hoc subgroup analysis in a high risk group categorised on the basis of BMD and prior fracture. We recently demonstrated a significant reduction in all clinical fractures with bazedoxifene in higher risk patients assessed by FRAX®. We have now re-evaluated the efficacy of bazedoxifene on non-vertebral fracture outcomes in the pivotal study.
The present analysis compared the effects of two doses of bazedoxifene (20 and 40 mg daily combined) with placebo on the risk of all non-vertebral clinical fractures. The risk of a major osteoporotic fracture was assessed using the FRAX® algorithms, and the relationship between pre hoc 10 year fracture probabilities and efficacy examined by Poisson regression.
This independent re-analysis confirmed that hazard ratios for the effect of bazedoxifene on all non-vertebral fractures decreased with increasing fracture probability, and that at low probability values, the 95% confidence estimates crossed unity. At the higher probabilities, the effect became significant (Table). In the more complete FRAX® model (i.e., with BMD), treatment with bazedoxifene reduced non-vertebral fractures in women with a ten year probability at or exceeding 20%, representing 11.9% of the population studied.
Bazedoxifene (20 and 40 mg doses combined) significantly decreases the risk of non-vertebral clinical fractures in women at or above a FRAX® based fracture probability threshold. The efficacy increases with increasing fracture probability. These results, consistent with the previous clinical fracture analysis, suggest that bazedoxifene should be targeted preferentially to women at increased fracture risk.
Disclosures: Eugene McCloskey, Wyeth, 2 This study received funding from: Wyeth Pharmaceuticals
Supplementation with Alfacalcidol Demonstrates a Greater Bone Sparing Effect in Raloxifene Therapy than when Raloxifene is Used Alone in Postmenopausal Japanese Women with Osteoporosis or Osteopenia.Yasuhisa Iwaoki1, Itsuo Gorai*2, Shin Hattori3. 1JA Yoshida General Hospital, Yoshida, Japan, 1International University of Health & Welfare, Atami, Japan, 3International University of Health & Welfare Atami Hospital, Atami, Japan
Purpose: Vitamin D insufficiency is prevalent in osteopenic and osteoporotic postmenopausal women. The persistence of increased PTH secretion caused by vitamin D insufficiency reduces bone density response to anti-resorptive agents in these postmenopausal women. Alendronate has been reported to strongly suppress bone resorption with elevation of endogenous PTH secretion. It is not well known whether administration of raloxifene might induce secondary PTH secretion in postmenopausal women with osteopenia or osteoporosis. We aimed to assess whether raloxifene might induce secondary PTH secretion and whether the addition of alfacalcidol to raloxifene could have favorable effects on bone mineral density (BMD) and bone turnover as compared to raloxifene alone therapy in postmenopausal Japanese women with osteoporosis or osteopenia (<−2.0 SD). Methods: A total of 153 subjects were randomly assigned to groups receiving 60 mg raloxifene (R), or 1 μg alfacalcidol (D), or a combination of both (R+D) for 2 years. Lumbar (L)-BMD, biochemical indices, and i-PTH were monitored for 2 years. The FAS was used for statistical analysis. Results: There were no significant differences in background characteristics, including age, YSM, BMI and bone density among the three groups at baseline. Baseline 25(OH)D and i-PTH were 23.8 ng/ml and 38.7 pg/ml, respectively. At 6 month, i-PTH demonstrated a significant decrease (?15.6%) in the D-group and combination-group (?9.2%) whereas a significant increase in i-PTH (+22.4%) was observed in the R-group. In the combination-group there was a significant increase in L-BMD (+4.6% at 1 year and +5.8% at 2 years, P<0.0001), and the increase was significant compared with that in the D-group (vs. +0.4% at 1 year and ?0.3% at 2 years, P<0.05) and in the R-group (vs. +2.6% at 2 years, P<0.05). The decrease in BAP at 3 month was significantly greater in the combination group than in the R-group whereas the decreases in BAP, urinary CTX-I and NTX-I after 6 month were significantly greater in the combination- and the R-groups than in the D-group. There were no significant differences in the number of patients who stopped taking their medications because of adverse events among three groups. Conclusion: Supplementation with alfacalcidol demonstrates a greater bone sparing effect by suppressing the secondary increment of PTH induced by raloxifene and lowering bone turnover more than when raloxifene is used alone in this population.
Disclosures: Itsuo Gorai, None.
Absolute Ten Year Fracture Risk Reporting Changes Osteoporosis Treatment Initiation in Routine Clinical Practice: A Before and After Study.William Leslie*1, Suzanne Morin2. 1Winnipeg, MB, Canada, 2McGill University Health Center, Montreal, QC, Canada
Background: BMD testing is valuable for fracture risk assessment and guiding the need for osteoporosis therapy (OTX), but clinical risk factors (CRF) can further refine farcture risk estimates. How absolute ten year fracture risk reporting impacts on physician behaviour in terms of OTX initiation is unknown.
Methods: Clinical BMD testing for the Province of Manitoba, Canada, uses an integrated program structure with a common report format. A BMD database can be linked with administrative health databases to identify reported fractures and all prescription dispensations. Prior to Jan 1, 2006, fracture risk reporting was based upon T-scores only; after this date ten year fracture risk was reported using a FRAX-like model (http://apps.sbgh.mb.ca/bmd-web-calculator/calculator.action) using age, hip T score and multiple clinical risk factors. Risk was categorized as low (<10%), moderate (10–20%) or high (>20%). OTX initiation was studied before (PRE = Apr 1-Dec 31, 2005) and after (POST = Jan 1-Dec 31, 2006) introduction of ten year fracture risk reporting. The population consisted of adults age 50+ without OTX dispensation, major corticosteroid use, or major osteoporotic fracture in the preceding year.
Results: The PRE and POST groups were similar in terms of age, BMD and risk factors. OTX initiation was 584/2,002 (29.2%) in PRE and 873/3,820 (22.8%) in POST (21.7% reduction, P<.0001) with similar fracture outcomes. The eect of the BMD reporting change on OTX initiation was greatest in the categories of low risk (48.1% reduction, P<.0001), medium risk (25.0% reduction, P < .0001) and low bone mass-osteopenia (53.5% reduction, P<.0001). Osteoporotic women in the POST period had less OTX initiation (13.6% reduction, P<.0001) due to avoidance of OTX in a lower risk group (younger, fewer CRFs and non-osteoporotic hip). Logistic regression was used to assess factors associated with OTX initiation. During PRE only T-score (P<.0001) and parental hip fracture (P=.027) were associated with OTX initiation. During POST T-score (P<.0001), parental hip fracture (P=.027), older age (P=.017), personal fracture (P=.0001), and failed chair test (P<.0001) were all associated with OTX initiation.
Conclusions: These findings are consistent with a change in physician behaviour following introduction of an absolute risk based reporting system. There was an overall reduction in intervention rates that was most evident in low risk, moderate risk and low bone mass-osteopenic women.
Disclosures: William Leslie, Novartis; Genzyme Canada, 99; Merck Frosst Canada Ltd; The Alliance for Better Bone Health: Sanofi-Aventis and Procter & Gamble Pharmaceuticals Canada, Inc.; Amgen Pharmaceuticals Canada, Inc.; innovus 3M, 2; Merck Frosst Canada Ltd, 8
A UK Denosumab Cost-Effectiveness Model Incorporating FRAX® and Adherence.Oskar Strom*1, David Macarios2, Enkhe Badamgarav2, Fredrik Borgstrom3, Anna Tosteson4, John Kanis5. 1I3 Innovus, Stockholm, Sweden, 2Amgen, Thousand Oaks, USA, 3Karolinska Institute, Stockholm, Sweden, 4Dartmouth Medical School, Lebanon, NH, 5University of Sheeld, Brussels, Belgium
Denosumab is an investigational osteoporosis therapy given as a SC injection every 6 months. In clinical studies, it has been shown to reduce the risk of vertebral and non-vertebral fractures in postmenopausal women. A major problem in osteoporosis treatment is lack of adherence, and a 6-monthly regimen for denosumab would provide 6 months of treatment adherence. In traditional cost-effectiveness modeling of osteoporosis, the fracture risk has been estimated based on only 3 factors: age, BMD, and prior fractures. However, the FRAX®-tool provides methods for incorporating other clinical risk factors (CRFs) that contribute to risk including parental hip fracture, alcohol consumption, BMI, rheumatoid arthritis, secondary osteoporosis, smoking and glucocorticoid use. The objective of this study was to construct a cost-effectiveness model that incorporated both FRAX® and adherence in order to evaluate cost-effectiveness and intervention thresholds for denosumab treatment compared to placebo or risedronate from a health care-perspective in the UK. A Markov-cohort-model was adapted to incorporate FRAX®. The model calculates cost-effectiveness in patient populations with any combination of the CRFs and is populated with relevant cost, adherence and epidemiological data. The cost of risedronate was assumed to be £301 and since no cost is available for denosumab (not yet commercially available) the average cost of available branded bisphosphonates was assumed to be £305. Patients on denosumab were assumed to have a 60% lower risk of treatment drop-out than patients on oral risedronate for 5 year maximum treatment duration. In case of treatment-drop-out, denosumab was assumed to have a declining residual effect for a maximum of 1 year while risedronate was assumed to have a declining residual effect equal to the time on treatment, up to a maximum of 5 years after discontinuation. In 70 year old women with a T-score at −2.5 (osteoporosis threshold) without other CRFs, the incremental cost-effectiveness ratio for denosumab was £14,300 /QALY and £10,700/QALY compared with placebo and risedronate, respectively. Using a willingness-to-pay of £20,000, an intervention threshold could be found at a 10-year risk of major osteoporotic fracture of ∼8–14% (denosumab vs. risedronate) and ∼10–20% (denosumab vs. placebo). Thus, given the model's assumptions, denosumab has the potential to be a cost-effective alternative to both “no-treatment” and risedronate in a UK setting.
Disclosures: Oskar Strom, Amgen, 5 This study received funding from: Amgen, Inc.
Positive Changes of Markers of Bone Turnover in Postmenopausal Women, Following therapy with Nitroglcerin.Carola Springer1, Anoja Wamsawithana2, Sunil Wimalawansa*3. 1U RWJMS, New Brunswick, USA, 2RWJMS, New Brunswickl, USA, 3Robert Wood Johnson Medical School, New Brunswick, NJ, USA
Purpose: A variety of therapeutic options are studied for osteoporosis. Emerging therapies are effective, but prohibitively expensive; hence cost-effective therapies are necessary. At appropriate doses, nitroglycerin (NG), a nitric oxide (NO) donor has favorable effects on osteoblast and osteoclast. Since effects of estrogen on bone in part mediated via NO, NG is an alternative to estrogen for postmenopausal women.
Method: We analyzed biomarkers, serum C-Telopeptide (CTx) and osteocalcin (OC) levels from IRB-approved, randomized, double-blind, controlled clinical trial (n=186) that used NG for preventing bone loss. Postmenopausal women were randomized to receive NG or placebo ointment over a 3-year period. Intent to treat analysis demonstrated no difference in the primary endpoint, lumbar spine BMD. Taking compliance (∼75%), the dose of NG used was ∼50% of that originally intended to use (sub-therapeutic). To understand the relationship of biomarkers with BMD, we analyzed changes of serum CTx and OC in those who responded vs. non-responders in both active and calcium + vitamin D treated groups.
Results: Those subjects who had ↑ BMD (responders) following NG-therapy had a significant ↓ of serum CTx (−0.43 ± 0.09 ng/ml, ↓ of 42%; p< 0.0002), and ↑ OC levels (1.32 ± 1.43, 7.7%; p=0.27). In NG-group, non-responders had change of CTx of −0.12 ± 0.05; ↓ of 15% p< 0.025), and OC −4.99 ± 1.2 ng/ml; ↓ by 22.5%; p<0.0008. In the NG-treated group, responders vs. non-responders for CTx was p<0.0005; and for OC, p<0.001. We cannot state whether this is due to adherence to therapy, or individuals needed different doses of NG to protect their skeleton.
In comparison, those who had ↑ BMD in the placebo-treated group had no major change in serum CTx (−0.16 ± 0.06 ng/ml; 18%, p<0.36), or OC levels (−4.11 ± 1.23 ng/ml, 23%; p<0.054). Furthermore, in the NG-treated group, BMD changes observed were correlated with the baseline serum CTx (p<0.001) and change of OC (p<0.001) (levels from the baseline). But no such correlation was seen with the change of BMD with the baseline CTx or OC levels.
Conclusion: Those who had ↑ BMD in response to this novel therapy had a significant ↓ in serum CTx & stable OC levels. Correlations were restricted to NG-treated group only, suggesting that changes of these two biochemical markers are specific to NG-treated group. Hence, these markers could be used to identify responders to NG, adjusting its dose, and/or assess the adherence to therapy.
Disclosures: Sunil Wimalawansa, Novartis, 8
The Effect of Denosumab on Risk of Fractures in Subgroups of Women with Osteoporosis.Steven Cummings*1, Michael McClung2, Henry Bone3, Jonathan Adachi4, Steven Boonen5, Claus Christiansen6, Richard Eastell7, Jordi Farrerons8, Nathalie Franchimont9, Kurt Lippuner10, Zulema Man11, Salvatore Minisola12, Ian Reid13, Rene Rizzoli14, Javier San Martin15, Ethel Siris16, Ove Torring17, Andrea Wang18. 1San Francisco Coordinating Center, San Francisco, CA, USA, 2Oregon Osteoporosis Center, Portland, OR, USA, 3Michigan Bone & Mineral Clinic, Detroit, MI, USA, 4St. Joseph's Hospital, Hamilton, ON, Canada, 5Center for Metabolic Bone Disease, Leuven, Belgium, 6Nordic Bioscience A/S, Herlev, Denmark, 7University of Sheeld, Sheeld, United Kingdom, 8Hospital de la Santa Creu I Sant Pau, Barcelona, Spain, 9Amgen (Europe) GmbH, Zug, Switzerland, 10Bern University Hospital Andd University of Bern, Berne, Switzerland, 11Centro Médico TIEMPO, Buenos Aires, Argentina, 12University of Rome, Rome, Italy, 13University of Auckland, Auckland, New zealand, 14University Hospital, Geneva, Switzerland, 15Amgen, Inc., Thousand Oaks, CA, USA, 16Columbia University College of Physicians & Surgeons, New York, NY, USA, 17Karolinska Institutet, Stockholm, Sweden, 18Amgen Inc., Thousand Oaks, USA
Denosumab reduces the risk of new vertebral fracture and nonvertebral fracture. A few previous trials suggest that the efficacy of antiresorptives on nonvertebral fractures might differ by patients' bone density, age, presence of vertebral fracture or other characteristics.
In FREEDOM 7,868 women aged 60–90 years with spine or total hip BMD T-scores <−2.5 and not < −4.0 at either site were randomly assigned to denosumab 60 mg every 6 months or placebo; all received daily calcium and vitamin D. Nonvertebral and clinical vertebral fractures were radiologically confirmed. We excluded nonvertebral fracture from severe trauma and appendicular and head fractures that are not associated with low BMD. We prospectively planned 9 subgroup analyses of the effect of denosumab on new vertebral and nonvertebral fractures. Statistical significance was based on tests for quantitative interactions.
Denosumab reduced all nonvertebral fractures by 20% (95% CI, 5 to 33%). The effect was significantly greater in women with low BMI, femoral neck BMD T-score ≤-2.5, and those without prevalent vertebral fracture (Table) but did not differ by age (< 75 vs. ≥ 75 years; p = 0.64) or prior nonvertebral fracture (p = 0.61). Compared with placebo, denosumab decreased the risk of new vertebral fracture by 68% (95% CI, 59 to 74%). This effect did not significantly differ by age, prevalent vertebral fracture, prior nonvertebral fracture and BMI (P>0.29 for all potential interactions).
Denosumab 60 mg for 3 years in women with osteoporosis reduced the risk of new vertebral fractures to a similar degree in all subgroups tested. However, as observed in several other trials, treatment reduced the risk of nonvertebral fractures significantly more in women with femoral neck BMD T-score ≤-2.5 than in those with higher BMD. It was also more effective in thin than heavier women and those without existing vertebral fractures.
Disclosures: Steven Cummings, Amgen, Eli Lilly, GlaxoSmithKline, Organon, Pfizer, Novartis, 5
This study received funding from: Amgen
The Relationship Between Baseline Bone Resorption and Fracture Risk Reduction with Denosumab.Richard Eastell*1, Douglas Bauer2, Claus Christiansen3, Peter Ebeling4, Andreas Grauer5, Peter Lakatos6, Willem Lems7, Cesar Libanati8, Michael McClung9, Ian Reid10, Christian Roux11, Philip Sambrook12, Andrea Wang13, Steven Cummings14. 1University of Sheeld, Sheeld, United Kingdom, 2University of California, San Francisco, San Francisco, CA, USA, 3Nordic Bioscience A/S, Herlev, Denmark, 4University of Melbourne, Footscray, Australia, 5Amgen, Thousand Oaks, CA, USA, 6Semmelweis University Medical School, Budapest, Hungary, 7Free University Medical Center, Amsterdam, The Netherlands, 8Amgen, Inc., Thousand Oaks, CA, USA, 9Oregon Osteoporosis Center, Portland, OR, USA, 10University of Auckland, Auckland, New zealand, 11Hospital Cochin, Paris, France, 12Royal North Shore Hospital, Sydney, NSW, Australia, 13Amgen Inc., Thousand Oaks, USA, 14San Francisco Coordinating Center, San Francisco, CA, USA
Denosumab is an investigational human monoclonal antibody to RANKL and it reduced the risk of new vertebral, non-vertebral and hip fracture in the FREEDOM Trial. However, it is not known whether the reduction in fracture risk with denosumab is related to the level of bone resorption at baseline. FREEDOM was a randomised, placebo-controlled trial of denosumab 60 mg sc or placebo every 6 months in women ages 60 to 90 years with postmenopausal osteoporosis defined as a T-score less than −2.5 at the spine or hip and not less than — 4 at either site. All received daily calcium (1000 mg) and vitamin D supplement (400 to 800 IU). We measured 2 bone resorption markers, serum CTX (ELISA, Osteometer) and TRACP5b (immunocapture activity assay, Suomen Bioanalytiikka Oy, Oulu, Finland) in baseline fasting serum samples from 3906 in the placebo and 3902 in the denosumab group. New vertebral fractures (n=350) were identified as an increase of ≥ 1 semiquantitative grade from a baseline grade of 0 on spinal radiographs taken annually, and non-vertebral fractures (n=531) were confirmed radiologically. Overall, denosumab decreased new vertebral fracture rates 68% (RR=0.32, p<0.0001) and non-vertebral fracture rates by 20% (HR=0.80, p=0.01). We divided the study population by bone turnover marker levels at baseline into quartiles and examined the denosumab vs. placebo risk/hazard ratios for each fracture type and then tested for a trend across quartiles of bone turnover among subjects treated with denosumab (Table). The eect of denosumab on risk of new vertebral fracture was greater among patients with higher baseline serum levels of CTX (p=0.01). Denosumab's nonvertebral fracture ecacy did not relate to baseline bone resorption. Denosumab reduced the risk of new vertebral fracture regardless of the level of baseline bone resorption. Patients with higher levels of CTX have a greater reduction in new vertebral fracture risk.
Disclosures: Richard Eastell, Medical Research Council, the National Institutes of Health Research (UK Department of Health), AstraZeneca, Procter & Gamble, Novartis, 2; Amgen, Novartis, Procter & Gamble, Servier, Ono, GlaxoSmithKline, Eli Lilly, 5
This study received funding from: Amgen Inc.
Effects of a combined treatment with alfacalcidol and alendronate versus treatment with alendronate alone on BMD (DXA) at lumbar spine and total hip, and on peripheral fractures in postmenopausal women with osteoporosis or osteopenia, 3 year results.Oliver Bock*1, Hendrikje Börst1, Martin Runge2, Erich Schacht3, Marlies Kalbow1, Ze'ev Mazor4, Junko Hashimoto5, Peter Martus6, Dieter Felsenberg1. 1Charité - University Medicine Berlin, Campus Benjamin Franklin, Centre for Muscle & Bone Research, Berlin, Germany, 2Aerpah Kliniken Esslingen-Kennenburg, Esslingen, Germany, 3ZORG - Zurich Osteoporosis Research Group, Zurich, Switzerland, 4Bone Metabolism Unit, Teva Pharmaceutical Industries, Jerusalem, Israel, 5Bone Disease Area Department, Chugai Pharmaceutical Co. Ltd., Tokyo, Japan, 6Charité - University Medicine Berlin, Campus Benjamin Franklin, Institute for Biometrics & Clinical Epidemiology, Berlin, Germany
Objectives: To examine the possible additive eect of alfacalcidol on bone mineral density (DXA-BMD) and non-vertebral fracture incidence in postmenopausal, alendronate treated women with reduced bone mass. Comparison of eects in patients with osteoporosis and osteopenia, respectively.
Patients and Methods: 279 postmenopausal women (mean age 73.7 ± 4.7 years), randomized into a bi-centric, placebo-controlled, double-blind study of 3 years (ALFA study) with two treatment arms: alfa-calcidol 1μg daily or placebo. All patients received alendronate 70mg once weekly and calcium 500mg daily. DXA-BMD at lumbar spine (LS, L1–4) and total hip was examined in yearly intervals using HOLOGIC QDR-1000+ and Delphi W (Bedford, MA, USA). The data of apost-hoc, sub-group analysis of the ITT population comparing patients with either osteoporosis or osteopenia will be presented.
Results: In the entire ITT population, the mean change of DXA-BMD at the LS was significantly greater in alendronate treated patients receiving additionally alfacalcidol (7.12% vs. 4.46%, p=0.003)). DXA-BMD at the total hip showed also slightly higher, but statistically non-significant, increases in these patients (2.79% vs. 2.05%). The additive effect of alfacalcidol on DXA-BMD at the LS is comparably verifiable in osteoporotic (7.92% vs. 4.77%, p=0.033) as well as in osteopenic patients (6.50% vs. 4.21%, p=0,036). In general, when comparing patients with osteoporosis and patients with osteopenia, the effects of additional alfacalcidol treatment on DXA-BMD increase at the LS and total hip were very similar in both sub-groups (table). A total number of only 45 non-vertebral fractures occurred during the 3 years' course of the study. The incidence of non-vertebral fractures was lower by 38% (8 vs. 13) in osteoporotic patients treated with alendronate + alfacalcidol as compared to those with alendronate alone.
Conclusions: Alfacalcidol improves the effects of alendronate therapy on DXA-BMD at the LS in patients with osteoporosis as well as in patients with osteopenia, and results in a similar additional DXA-BMD increase in both sub-groups. The low incidence of non-vertebral fractures in osteoporotic patients treated with alendronate is further reduced by combining it with alfacalcidol.
Disclosures: Oliver Bock, Teva Deutschland, 5
This study received funding from: Chugai Pharmaceutical Co. Ltd., GRY-Pharma GmbH, Teva Pharmaceutical Industries Ltd.
Normalization of Secondary Hyperparathyroidism and Hypovitaminosis D in Ambulatory Elderly Women: a Calcitriol-Controlled Randomized Trial with Cholecalciferol.Giulio Pioli1, Antonella Barone*2, Andrea Giusti3, Giuseppe Girasole4, Monica Pizzonia2, Monica Razzano2, Mario Pedrazzoni5, Ernesto Palummeri2, Gerolamo Bianchi6. 1Parma, Italy, 2Galliera, Genoa, Italy, 3Galliera Hospital, Genoa, Italy, 4Ospeoale, Genova, Italy, 5University of Parma, Parma, Italy, 6Lelicomar Onlus, Arenzano, Italy
Objective: The optimal treatment capable of normalizing secondary hyperparathyroidism (sHPTH) due to hypovitaminosis D in older adults is still uncertain. The aim of the study was to compare the eects on PTH, 25-OH vitamin D (25OHD) and bone turnover markers of 2 dosing regimens of cholecalciferol (D3) versus calcitriol in elderly women with sHPTH.
Methods: 65 women with sHPTH due to hypovitaminosis D were randomized to receive: D3 300,000 IU every 3 months (21 patients, 3MD3 group), D3 1,000 IU/day (23 patients, DD3 group) or calcitriol 0.5 ug/day (21 patients, 1,25D group). All subjects received calcium supplements (daily intake of 1,500 mg). The percent and absolute changes of serum PTH and 25OHD from baseline at 6-month were the primary outcomes. Serum calcium, bone alkaline phosphatase (BAP), carboxyterminal collagen crosslinks (CTX), phosphate and 24-hour urinary calcium excretion were measured.
Results: The 3 groups were similar with regard to baseline characteristics. At 6 months, PTH significantly decreased from baseline (p<.001) in all groups (3MD3, 73 ± 30 vs 113 ± 38; DD3, 66 ± 20 vs 93 ± 27; 1,25D, 56 ± 12 vs 98 ± 23). Mean percent decrease of PTH in the 1,25D group (−38.4%) was significantly (p<.001) greater compared to the other two groups (3MD3, −27.5%; DD3 −26.4%). At 3 months the proportion of subjects with normalized PTH was similar in the 3 groups (1,25D, 39%; DD3, 44%; 3MD3, 29%; p=NS). At 6 months a high proportion of patients in the DD3 (50%) and 3MD3 (44%) groups reached normal PTH values. However, these percentages were significantly lower compared to patients treated with calcitriol (72%, p<.01). 25OHD levels did not significantly change from baseline after 6 months of treatment with calcitriol, while showed a significant increase from baseline in the other 2 groups (3MD3, from 8.8 ± 4.9 to 32.2 ± 11.9, p<.001; DD3, from 9.4 ± 5.1 to 23.1 ± 7.6, p<.001). Both CTX and BAP showed a significant decrease from baseline in the 3 groups. In the subgroups of patients treated with D3, BAP absolute reduction at 6 months demonstrated a positive correlation with PTH decrease (r=.377, p=.01).
Conclusion: The two dosing regimens of D3 demonstrated to reduce PTH level and increase 25OHD value similarly in elderly women with sHPTH and hypovitaminosis D, even if a high proportion of patients still presented sHPTH at 6 months. Therefore, this results support the use of long-term high doses of D3 for the treatment of sHPTH in older adults.
Disclosures: Antonella Barone, None.
Oral supplementation with 25-hydroxyvitamin D3 versus vitamin D3: effects on 25-hydroxyvitamin D status, lower extremity function, and blood pressure.Heike Bischo-Ferrari*1, Bess Dawson-Hughes2, Jana Henschkowski3, Hannes B Staehelin4, Elisabeth Stoecklin5, Swen Wolfram5, Stephen M Ferrari3, Andreas Egli6. 1University of Zurich, Zurich, Switzerland, 2Tufts University Center for Aging, Boston, MA, USA, 3University of Zurich, Zurich, Switzerland, 4University of Basel, Basel, Switzerland, 5DSM Nutritional Products Ltd, Basel, Switzerland, 6Centre on Ageing & Mobility, Zurich, Switzerland
Background: Raising 25-hydroxyvitamin D levels to at least 75 nmol/l has been identified as a desirable range for anti-fracture ecacy, optimal lower extremity function, and the prevention of incident hypertension.
Aim: To assess the difference in efficacy of 20 μg of oral 25-hydroxyvitamin D (25(OH)D) per day compared to 20 μg of oral vitamin D3 in increasing 25-hydroxyvitamin D levels over a 16 week trial. Secondary endpoints were the odds of maintaining or improving lower extremity function, as well as difference in mean blood pressure by treatment.
Methods: We enrolled 20 healthy postmenopausal women with 25(OH)D levels below 60 nmol/l. Mean age was 61.5 years (SD+/- 7.2). Women were randomized to either 20 μg of oral 25(OH)D per day or 20 μg of vitaminD3 per day (800 IU / day) in a double blind randomized controlled trial. 25-Hydroxyvitamin D levels were measured with LC-MS/MS on 13 visits. The odds of maintained or improved lower extremity function at 16 weeks follow-up was compared between the 25(OH)D and vitaminD3 treatment groups across 4 tests including knee extensor and flexor strength, the timed up and go test, and the repeated sit-to-stand test. Standardized blood pressure measurements were taken in a sitting position after a 5 minute rest on 13 visits.
Results: Over 16 weeks, mean 25-hydroxyvitamin D levels increased from 30.7 to 162.2 nmol/l in the 25(OH)D group and from 35.5 to 76.2 nmol/l in the vitamin D3 group. With 25(OH)D treatment, the odds of maintained or improved function across the lower extremity test battery was 2.79-fold higher than with vitamin D3 (95%CI: 1.18–6.58). For each of the 4 tests and controlling for age and body mass index, as well as baseline strength/function, the 25(OH)D- treated group appeared to be better than the vitamin D3 group at 16 weeks with a significant difference for knee extensor strength (p = 0.03). Across time, and controlling for baseline blood pressure, 25(OH)D treatment resulted in a 6.1 mmHG decrease in systolic blood pressure compared to vitamin D3 (p = 0.0002). There was also a small and non-significant decrease in diastolic blood pressure with 25(OH)D compared to vitamin D 3 (1.4 mmHG; p = 0.24). No hypercalcemia was observed for 25(OH)D or vitamin D3 treatment at any time point.
Conclusions: 25(OH)D treatment at a dose of 20 μg per day resulted in a safe, immediate and sustained mean increase in 25(OH)D levels to 162 nmol/l compared to 76 nmol/l with the same dose of vitamin D3. In addition, there appeared be a significant benefit of 25(OH)D treatment on lower extremity function and systolic blood pressure if compared to vitamin D3 at the same dose.
Disclosures: Heike Bischoff-Ferrari, DSM Nutritional Products Ltd, 5 This study received funding from: DSM Nutritional Products Ltd
Splitting the Daily Dose of Parathyroid Hormone PTH(1–34) Results in a Faster Bone Anabolic Response in Rats.Juerg A. Gasser*1, Peter Ingold1, Andrea Venturiere1, Markus John2. 1Novartis Institutes for BioMedical Research, Basel, Switzerland, 2Novartis Pharma AG, Basel, Switzerland
Purpose: The only available bone-forming therapy involves a daily subcutaneous (s.c.) injection of parathyroid hormone (PTH) 1–84 or the PTH fragment 1–34. Anabolic agents that could be administered less frequently or by oral route would mark a substantial improvement. Calcilytics induce PTH secretion by antagonizing the calcium-sensing receptor which controls the release of the hormone from stores in the parathyroid glands. Widler et al (JBMR 2008;23:Abstract 1173) reported that oral administration of the calcilytic ATF936 resulted in peak plasma immunoreactivity of 270 pg/mL hPTH(1–84), which is roughly 50% of the peak plasma level achieved after s.c.-administration of 5μg/kg hPTH(1–34) (620 pg/mL). Since there may be a limit to the total amount of PTH that can be released from the parathyroid gland, the aim of our study was to evaluate whether splitting the total daily dose of hPTH(1–34) would achieve a similar bone anabolic response as single dosing in skeletally mature rats.
Methods: Ten month-old female virgin Wistar rats were injected s.c. for 6 months with PTH(1–34) 2.5, 5 or 10μg/kg once daily, or bid with 2.5, 5 or 10μg/kg per dose. Bone mineral density (BMD), cortical and trabecular architecture plasma osteocalcin and TRAP5b were measured monthly by pQCT/μCT in the proximal tibia metaphysis for 6 months.
Results: Daily and bid injections resulted in a rapid dose-dependent increase in total BMD, trabecular BMD, cortical thickness, trabecular bone volume and trabecular thickness. The total bone gain at the end of the 6 month treatment was also dose-dependent. The PTH-dose of 2×2.5μg/kg/day resulted in a faster onset of the bone anabolic response than the once-daily dose of 5μg/kg, while at 6 months, no difference in the total amount of bone gained was detectable between animals receiving the same total daily dose.
Conclusions: Data indicates that splitting the total daily dose of PTH(1–34) into two injections does not compromise the bone anabolic effect and even leads to a more rapid onset of the anabolic response. If these animal data translate into the clinical situation with oral calcilytics, multiple daily dosing may be an interesting option to achieve full bone anabolic action more quickly than with once-daily dosing, and at the same time may improve the adverse effect profile of calcilytics or PTH, should those be Cmax associated. The study argues against peak PTH-plasma levels being a predictor of the bone anabolic response to PTH(1–34).
Disclosures: Juerg A. Gasser, Novartis Pharma AG, 3 This study received funding from: Novartis Pharma AG
Teriparatide (rhPTH(1–34)), but not strontium ranelate, demonstrates bone anabolic properties in osteopenic, ovariectomized rat.Yanfei Ma*1, Qingqiang Zeng2, Leah Porras2, Allen Schmidt2, Mary Dee Adrian3, Anita Harvey3, Terry Moore3, Andrew Geiser1, Timothy Shelbourn3, Thomas Wronski4, Stephen Iturria3, Henry Bryant3, Masahiko Sato5. 1Eli Lilly & Company, Indianapolis, IN, 2Eli Lilly & Company, Indianapolis, USA, 3Eli Lilly Company, Indianapolis, USA, 4University of Florida, Gainesville, FL, USA, 5Lilly Research Labs, Indianapolis, IN, USA
We compared the effect of strontium ranelate (SR) and teriparatide (TPTD) on gene expression, BMD, biomechanical properties and histomorphometry in a rat model of estrogen-deficiency osteoporosis. Eight months old rats were ovariectomized (Ovx) at age 6 months and permitted to lose bone for 2 months before treatment for 3 or 12 weeks (n= 8–11) with TPTD (5 or 15 μg/kg/d sc) or SR (150 or 450 mg/kg/d po). Dunnett's test (p<0.05) was used for all comparisons. After 3 weeks of treatment, RT-PCR analyses of the distal femur showed TPTD elevation of collagen 1a2 (Col 1a2), osteocalcin (OCN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and Runx2 gene expression at both doses, relative to Ovx controls (Table). SR had no effect on these genes at either dose. Neither compound affected osteoprotegerin expression. Compared to OVX controls, 12 weeks of TPTD5 and TPTD15 treatment increased lumbar vertebral (LV) BMC and BMD while SR150 and SR450 increased only BMC. Further, TPTD5 and TPTD15 increased LV strength (121%, 180%), and stiffness (56%, 68%), while neither SR150 nor SR450 had significant effects on vertebral strength, and only SR450 had a 27% effect on stiffness. There were dose-dependent effects of TPTD5 and 15 on strength (36%, 57%) at the proximal femur, but not for SR at either dose. At the femoral midshaft, there were dose-dependent effects of TPTD5 and 15 on BMD and BMC, and TPTD15 increased strength (22%). SR150 increased midshaft BMC; SR450 increased BMD and BMC, but neither dose of SR significantly affected strength. Histomorphometry at the proximal tibial metaphysis (PTM) showed dose-dependent effects of TPTD5 and 15 on trabecular thickness and number, and relative bone volume and formation rates. There were no significant effects of SR on histomorphometric parameters. SR BMD and BMC efficacy could be explained by incorporation of SR into bone, but neither dose stimulated an anabolic bone response and did not significantly improve the bone biomechanical properties of Ovx rats. These findings in osteopenic Ovx rats demonstrated that the skeletal efficacy of the low dose of TPTD exceeded efficacy at either dose of SR, especially in inducing bone formation activity.
Disclosures: Yanfei Ma, Eli Lilly Company, 3
Analyzing The Skeletal Distribution And Cellular Uptake Of Fluorescently-Labeled Bisphosphonate Analogues in vivo.Frank Ebetino*1, A Roelofs2, Alan Boyde3, Mark Lundy4, C McKenna5, K Blazewska5, S Sun5, B Kashemirov5, Z Henneman6, G Nancollas6, R Graham Russell7, Michael Rogers2, F Coxon2. 1Procter & Gamble Pharmaceuticals, Inc., Cincinnati, OH, 2University of Aberdeen, Aberdeen, United Kingdom, 3Queen Mary University of London, London, United Kingdom, 4MDS Pharma Services, Cincinnati, OH, 5University of Southern California, Los Angeles, USA, 6SUNY at Bualo, Bualo, USA, 7University of Oxford, Oxford, United Kingdom
To determine whether differences in the mineral affinity of bisphosphonates (BPs) leads to differential skeletal distribution, we synthesized fluorescently-conjugated compounds; risedronate (higher affinity) and its phosphonocarboxylate (PC) analogue 3-PEHPC (lower affinity), coupled to the fluorophores carboxyfluorescein or Alexa Fluor 647. Mineral affinity differences were confirmed by HAP crystal growth studies. These compounds were also utilized to determine whether cells other than osteoclasts can internalize BPs in vivo.
Lower and higher affinity compounds conjugated to different fluorophores were administered simultaneously to rats or mice, in some cases together with xylenol orange (XO), a fluorophore with even lower affinity than the PC. 24h or one week later, the proximal tibia and mandible were removed, fixed, and embedded in PMMA. Bones were examined directly in the blocks by confocal microscopy in optical section planes a few microns beneath the block surfaces. In some cases, morphological analysis was assisted by overlaying subsequent backscattered electron SEM images of surface fields.
Both BP and PC compounds were detected at sites of resorption on remodeling metaphyseal cancellous surfaces and modeling periosteal surfaces, and in the walls of vascular channels. However, as well as some areas of co-localization, there were differences in sites of labeling on both forming and resorbing surfaces. At both formation and resorption sites, the PC penetrated more deeply into mineralized bone and incisor dentine than the BP, while XO penetrated even further. Finally, the extent of labeling of osteocyte lacunae was inversely proportional to bone affinity, with lower affinity compounds appearing to penetrate further into the osteocyte network.
To assess intracellular uptake of BP in vivo, neonatal rabbits were subcutaneously injected with Alexa Fluor 647-RIS. After 24h, uptake was detectable in multinucleated osteoclasts and CD14+ bone marrow mononuclear cells in vivo, whereas CD14- cells showed very little uptake, as assessed by flow cytometry and confocal microscopy.
These observations support the hypothesis that mineral affinity influences the distribution of BPs on bone surfaces, and possibly influences the extent to which they can penetrate the osteocyte canalicular network. These findings also confirm osteoclasts as the main cell type targeted by BPs in vivo, but suggest that bone marrow monocytes may also be affected by these drugs.
Disclosures: Frank Ebetino, Procter & Gamble Pharmaceuticals, 3 This study received funding from: Procter & Gamble Pharmaceuticals
Bisphosphonate Treatment of Postmenopausal Women Results in a Reduction in Circulating Endothelial Progenitor Cells Co-expressing Osteoblastic Cell Surface Markers.Ulrike Moedder*1, Pilar Peris2, Mario Gössl3, Louise McCready3, Amir Lerman3, Sundeep Khosla4. 1Mayo Clinic, Rochester, MN, USA, 2Hospital Clinic of Barcelona, Barcelona, Spain, 3Mayo Clinic, Rochester, USA, 4Mayo Clinic College of Medicine, Rochester, MN, USA
Postmenopausal osteoporosis and increased bone turnover have been associated with vascular calcification and increased cardiovascular mortality, but the underlying mechanism(s) for these associations are unclear. Circulating endothelial progenitor cells (EPCs) have been shown to coexpress the osteoblastic marker, osteocalcin (OCN), and EPCs expressing OCN are increased in patients with severe coronary atherosclerosis (J Am Coll Cardiol 52:1314, 2008). These data suggest that EPCs expressing osteogenic proteins may contribute to vascular calcification as opposed to initiating normal vascular repair. Clinical studies in postmenopausal women have suggested that in addition to reducing bone turnover, bisphosphonates may also reduce arterial inflammation and calcification. Thus, in this double blinded, randomized pilot study, we tested whether the bisphosphonate, risedronate, would lead not only to a decrease in bone turnover but also a decrease in EPCs coexpressing osteoblastic cell surface markers. Healthy early postmenopausal women (N=10 per group, 1 to 5 years following menopause) were treated either with placebo or with risedronate (35 mg/week) for 4 months. Peripheral blood was collected at the beginning and at the end of the study for determination of serum bone turnover markers and post-Ficoll mononuclear cells were obtained for flow cytometry analysis. The peripheral blood mononuclear cells were stained for EPC markers (CD133, CD34, and the VEGF receptor [KDR]) as well as osteoblast markers (OCN, Stro-I, and alkaline phosphatase [AP]). Risedronate treatment resulted in a significant reduction in bone turnover (by 21% for TRAP 5b [P < 0.05] and by 38% for PINP [P < 0.01]). While there was only a small change (−1.6%) of the total number of EPCs and the number of EPCs expressing osteoblastic markers (−6%) in the placebo group, there was an ∼35% reduction in the total number of circulating EPCs in the risedronate group. Moreover, as shown in the Figure, risedronate treatment resulted in 30–50% reductions in EPCs coexpressing OCN, Stro-1, or AP. These data thus suggest that bisphosphonate treatment may modulate vascular calcification by reducing the number of “osteogenic” EPCs and provide a strong rationale for further studies examining the possible role of bisphosphonates in inhibiting vascular calcification.
Disclosures: Ulrike Moedder, None.
This study received funding from: Research Grant from Procter&Gamble
Cell therapy of human adipose-derived mesenchymal stem cells prevent bone loss in ovariectomized nude mice.Hwa Young Cho*1, Sun Wook Cho2, Hyung Jin Choi3, Hyun Jin Sun3, Ju Yeon Jung3, Jae-Yeon Yang3, Jee Hyun Ahn3, Sang Wan Kim4, Seong Yeon Kim3, Kyu-Joo Park5, Chan Soo Shin6. 1Seoul National University hospital, Seoul, South Korea, 2Seoul National University Hospital, Seoul, Korea, 3Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea, 4Massachusettes General Hospital, Boston, MA, 5Department of Surgery, Seoul National University College of Medicine, Seoul, South Korea, 6Seoul National University, Seoul, Korea
We studied the effects of cell therapy with human adipose-derived mesenchymal stem cell (ADMSC) on ovariectomy (OVX)-induced bone loss in nude mice. ADMSCs were isolated from fat tissue from patients who underwent abdominal surgery due to benign surgical condition and maintained in vitro. Ten-week-old female nude mice were divided into 4 groups and underwent intravenous injection of ADMSCs or PBS as follows: Sham-operated mice treated with PBS (Sham op + PBS), OVX mice treated with ADMSC (OVX + ADMSC), Estradiol (OVX + E2) or treated with PBS only (OVX + PBS). Measurement of BMD by dual energy x-ray absorptiometry (PIXImus) revealed that OVX + ADMSC group exhibited significantly greater protection against OVX-induced bone loss compared to OVX + PBS group during 8weeks after cell therapy (0.0453 ± 0.0009 vs. 0.0436 ± 0.0013 at 4 weeks, p<0.05, 0.0473 ± 0.0011 vs. 0.0455 ± 0.0030 at 8 weeks, p<0.05). Notably, the effect in OVX + ADMSC group was comparable to that in OVX + E2 group. In accordance with BMD, we found that OVX + ADMSC group showed significant increase in bone volume (17.93 ± 0.34 vs. 12.48 ± 0.28%, p<0.05) and trabecular number (3.08 ± 0.24 vs. 2.22 ± 0.19 /mm, p<0.05). Serum osteocalcin was increased at 4 weeks after OVX and the level was significantly higher in OVX + ADMSC group compared with OVX + E2 or OVX + PBS groups. In addition, cell homing assay using PKH26-labeled cells showed that appreciable amount of ADMSCs were indentified in bone marrow of OVX + ADMSC group. Moreover, PCR analysis of human specific β-globin using genomic DNA isolated from recipient mice bone also confirmed homing of infused cells to bone marrow. Collectively, these results suggest that cell therapy with ADMSCs could be a promising option for ameliorating bone loss.
Disclosures: Hwa Young Cho, None.
Effects of Odanacatib on Bone Mass, Turnover and Strength in the Femoral Neck of Estrogen Deficient Adult Rhesus Monkeys.Tara Cusick*1, Brenda Pennypacker2, Kevin Scott1, Le Thi Duong2, Donald Kimmel1. 1Merck & Co., Inc., West Point, PA, USA, 2Merck Research Laboratories, West Point, PA, USA
Cathepsin K (CatK) is a cysteine protease highly expressed in bone resorbing osteoclasts and its inhibition represents a novel and promising mechanism for the treatment of osteoporosis. Odanacatib (ODN) is a selective, reversible CatK inhibitor and appears to be a bone formation-sparing antiresorptive. We previously demonstrated that ODN, fully prevented bone mineral density (BMD) loss at the spine and hip and maintained normal bone strength at the central femur in 21-mo. old ovariectomized (OVX) rhesus monkeys. Here we characterize ODN effects on cortical and trabecular bone in the femoral neck (FN). Rhesus monkeys (13–19 yrs) were assigned to four groups (n=8–11): intact and OVX + vehicle or ODN (6 and 30mg/kg, q.d., p.o.). For histomorphometry, two 15-d interval labels were given with calcein at 10-mo. and tetracycline just prior to necropsy. Bone strength in shearing mode, surface-based mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR) were determined in the FN. Short term and long-term (LT)BFR represented the distance between 15-d tetracycline labels and between tetracycline and calcein labels, respectively. ODN treatment led to BMD gains of 11% and 15% (p<0.01, vs. vehicle), and peak-load increases of 17% and 19% in the 6 and 30mg/kg ODN groups, respectively. In trabecular FN, MS/BS and BFR were unaltered by the 6mg/kg dose, but trended lower with the 30mg/kg dose vs. vehicle. MAR, TbTh, TbN and TbSp. were unaffected by ODN. Periosteal FN MS/BS, MAR and BFR were also not affected by low dose treatment, but trended higher with 30mg/kg ODN vs. vehicle. Dose-dependent effects of ODN on periosteal FN BFR were clearly demonstrated by a LTMAR increase of 1.5-fold and a 3.5-fold LTBFR increase with the 30mg/kg dose vs. vehicle. In summary, we show that in OVX monkeys, both doses of ODN treatment protected against BMD loss and preserved normal bone quality in the FN. Similar to previous findings in the spine and proximal femur of these monkeys, ODN appeared to have differential effects on FN trabecular and cortical bone at doses which fully prevented OVX-induced bone loss. While 6mg/kg ODN left bone formation unaffected, at 30mg/kg it partially stimulated periosteal BFR. Our findings in the femoral neck suggest that ODN, unlike conventional antiresorptives, can effectively inhibit trabecular bone remodeling while simultaneously building cortical bone, at least in part via stimulating periosteal bone formation.
Disclosures: Tara Cusick, None.
This study received funding from: Merck & Co.
Orally Active Cathepsin K Inhibitor, ONO-5334, Potently Improved Bone Mineral Density not only in Trabecular Bone but also in Cortical Bone in Ovariectomized Cynomolgus Monkeys.Hiroyuki Yamada*1, Hiroshi Mori2, Yasutomo Nakanishi2, Akiko Kunishige2, Satoshi Nishikawa2, Makoto Tanaka2, Tsutomu Shiroya2. 1Mishima-gun Osaka, Japan, 2ONO Pharmaceutical Co Ltd, Osaka, Japan
Cathepsin K, a cysteine protease highly expressed in osteoclasts, is capable of degrading bone type I collagen. Recently, cathepsin K inhibitors have drawn much attention as novel therapeutic agents for treatment of elevated bone turnover including osteoporosis. We evaluated cathepsin K inhibitor, ONO-5334, in ovariectomized cynomolgus monkeys.
One hundred twenty female cynomolgus monkeys, aged between 9 and 12 years old, were assigned into the following 6 groups (n=20): sham operated, ovariectomized (OVX) control treated with vehicle, ONO-5334 1.2, 6 or 30 mg/kg, or alendronate group. ONO-5334 was given orally once daily from the next day of ovariectomy for 16 months. Alendronate (0.05 mg/kg) was administered intravenously once every two weeks. Ovariectomy resulted in significant increases in both bone resorption and formation markers and decreases in bone mineral density (BMD) at spine, femur, radius and tibia. ONO-5334 potently and rapidly suppressed serum C-terminal telopeptide of type I collagen (CTX), urinary CTX, serum N-telopeptide of type I collagen (NTX), urinary NTX, but weakly suppressed serum osteocalcin and serum bone-specific alkaline phosphatase (BAP). Interestingly, ONO-5334 increased serum intact-parathyroid hormone (PTH). In DXA analysis, alendronate completely improved the OVX-induced lumbar spine BMD loss up to sham level. However ONO-5334 strongly increased in lumbar spine BMD over sham level. Surprisingly, ONO-5334 showed the potent efficacy at femur, radius and tibia over sham group, which were not observed in alendronate-treated group. As the results of pQCT analysis, ONO-5334 increased in BMD at both trabecular and cortical bone. Although the efficacy of alendronate could be mainly observed at trabecular bone. There were no remarkable differences in clinical signs and body weights of animals treated with ONO-5334 at any dose levels and alendronate compared with the OVX control group.
These results suggest that an orally active cathepsin K inhibitor, ONO-5334 preferentially suppressed bone resorption and strongly improve BMD in ovariectomized cynomolgus monkeys. Especially, ONO-5334 has a greater effect on cortical bone than bisphosphonates. We conclude that ONO-5334 has the remarkable therapeutic potential not only in vertebral bone, but also in non-vertebral bone. ONO-5334 would be a new ideal drug for osteoporosis therapy.
Disclosures: Hiroyuki Yamada, None.
A Novel Active Vitamin D Analogue, ED-71, Increases Bone Mass and Bone Strength more Efficiently than Alfacalcidol in CYP27B1 Knockout Mice.Toshio Okano*1, Kimie Nakagawa2, Keigo Isomoto3, Naoko Okuda3, Natsumi Sawada3, Toshiyuki Sakaki4, Shigeaki Kato5. 1Kobe Pharmaceutical University, Kobe, Japan,2 Kobe, Japan, 3Kobe Pharmaceutical University, Kobe, Japan, 4Toyama Prefectural University, Toyama, Japan, 5University of Tokyo Institute of Molecular & Cellular Biosciences, Tokyo, Japan
Calcitriol, 1α,25(OH)2D3, appears to have a superior ecacy in reducing incidence of fractures in osteoporotic patients compared to its ecacy in maintaining/increasing bone mass. It would be probable that the incidence of fractures may be further reduced if an active vitamin D analogue possessing potent bone mass-increasing activity were to become available. Based on this idea, ED-71, a unique analogue of 1α,25(OH)2D3 of bearing a hydroxypropoxy substituent at the 2β-position of A-ring has been developed in Japan. ED-71 is proven in both animals and humans to suppress a decrease of bone mass and an incidence rate of bone fracture to a greater extent than alfacalcidol, an 1α,25(OH)2D3 prodrug, which is used worldwide for the treatment of osteoporosis. The question as to whether or not the above bone eects of ED-71 are independent of the so-called vitamin D endocrine system, however, remains to be answered. In this study, the ecacy of ED-71 and alfacalcidol in increasing bone mineralization and bone strength was evaluated in CYP27B1 knockout (1αOHase−/−) mice. 1αOHase−/− mice and wild-type (WT, controls) mice, after weaning, were orally administered either MCT-vehicle, alfacalcidol (0.25 μg/kg) or ED-71 (0.25 μg/kg) three times per week for 6 weeks. During the experimental period, the animals were fed a diet containing 0.6% calcium, 0.3% phosphorus and 240 IU /100g vitamin D3. At 3 and 6 weeks after the initiation of administration, the animals were sacrificed and lumbar spines, femoral bones, kidneys, and intestine were excised for immnostaining, taking soft X-ray photographs, microcomputed tomography, mechanical testing, contact microradiography, and histomorphometrical measurements.
1αOHase−/− mice treated with MCT-vehicle had no serum 1α,25(OH)2D3 and moderate levels of serum 25-hydroxyvitamin D3. They were growth retarded, hypocalcemic, and had poor bone mineralization and strength compared to WT mice treated with MCT-vehicle during the experimental period. Treatment with alfacalcidol and ED-71 improved these impairments remarkably at 3 weeks and restored completely at 6 weeks after the initiation of administration compared to WT mice treated with alfacalcidol or ED-71. Furthermore, ED-71 increased bone mass and bone strength in 1αOHase−/− mice more eciently than alfacalcidol at 3 and 6 weeks after the initiation of administration. These results suggest that ED-71 is more superior than alfacalcidol in increasing bone mass and strength, and acts on bone independent of endogenous 1α,25(OH)2D3.
Disclosures: Toshio Okano, None.
PTH Administration Reverses Abnormal Bone Remodeling Dynamics and Structure in Hypoparathyroidism.Mishaela Rubin*1, David Dempster2, James Sliney3, Hua Zhou4, Thomas Nickolas3, Emily Stein5, Maryann Dellabadia3, Elizabeth Shane5, Shonni Silverberg6, John Bilezikian5. 1Columbia University, New York, NY, USA, 2Columbia University, West Haverstraw, NY, USA, 3Columbia University, New York, USA, 4Helen Hayes Hospital, West Haverstraw, NY, USA, 5Columbia University College of Physicians & Surgeons, New York, NY, USA, 6Columbia University, New York, NY, USA
In hypoparathyroidism (HypoPT), a disease of low PTH, there is increased BMD and suppressed bone turnover. We studied histomorphometric properties in HypoPT and investigated how PTH treatment would alter them. HypoPT subjects (N=64; 48 women; age 46 ± 14 yr) were treated with 100 μg of hPTH(1–84)[PTH] every other day for up to 2 yrs. Tetracycline double-labeled iliac crest bone biopsies (bx) were performed prior to PTH treatment (n=48) and compared to matched controls (C), with repeat bx at 1 yr (n=14) or 2 yrs (n=12) of PTH treatment. The 16 other HypoPT underwent bx after 3 months of PTH with a quadruple-label protocol (double label at baseline and 3 mo).
Bone remodeling variables at baseline were profoundly suppressed as compared to C, including mineralized perimeter (Md.Pm: 1.00 ± 2 vs 4.33 ± 3%, p<0.0001), mineral apposition rate (MAR: 0.49 ± 0.3 vs 0.73 ± 0.2 μm/d, p<0.0001) and bone formation rate (BFR: 0.008 ± 0.02 vs 0.032 ± 0.02 μm3 / μm2/d, p<0.0001). Remodeling was increased after 3 mo of PTH (MdPm 5.46 ± 6%, p= 0.004; MAR 0.67 ± 0.3 μm/d, p<0.0001; BFR 0.042 ± 0.05, p=0.004), peaking at 1 yr (MdPm 7.05 ± 6%, p= 0.001; MAR 0.82 ± 0.2 μm/d, p=0.006; BFR 0.067 ± 0.06, p=0.002) and remaining above baseline at 2 yrs (MdPm 2.77 ± 4%, p= 0.01; MAR 0.70 ± 0.2 μm/d, p=0.02 BFR 0.022 ± 0.03, p=0.02). The 2-yr values approximated normal levels. Bone structural variables were higher than C at baseline, including trabecular width (Tb.Wi: 129.2 ± 36 vs 114.8 ± 20 μm; p= 0.02) and cancellous bone volume (BV/TV: 23.3 ± 7 vs 19.8 ± 4; p=0.007). Tb.Wi decreased at 1 yr (127.5 ± 34 μm, p=0.03) and 2 yrs (122.5 ± 26 μm, p=0.03). Trabecular number increased at 1 yr (1.7 ± 0.4 to 2.1 ± 0.6 #/mm, p=0.02). BV/TV did not change. Cortical porosity (%Ct.PoAr), which was normal at baseline (HypoPT: 6.97 ± 3 vs. C: 7.41 ± 4%), increased, peaking at 1yr (10.22 ± 4%, p=0.01) and remaining above baseline at 2 yrs (8.84 ± 2%, p=0.04).
These data show that PTH treatment of HypoPT restores abnormal dynamic and structural skeletal properties towards normal. The dynamic changes peak within the 1st year of therapy, with most bone remodeling parameters approximating normal levels by 2 yrs. Structural changes that are consistent with less dense bone emerge at 1 yr. These changes might have a beneficial impact on bone strength. Furthermore, in a model of PTH insufficiency, these results help to demonstrate the importance of PTH in the establishment and maintenance of skeletal health.
Disclosures: Mishaela Rubin, None.
Analysis of jck Mouse: A Disease Model of Progressive Renal Failure with CKD-MBD.Stephen O'Brien*1, Yves Sabbagh1, Wen Tang2, Shiguang Liu1, Susan Schiavi1. 1Genzyme Corporation, Framingham, MA, USA, 2Genzyme Corporation, Framingham, USA
Mortality in chronic kidney disease is correlated with hyperphosphatemia, cardiovascular disease, and elevations in mineral metabolism hormones such as FGF23 and PTH. Evidence suggests mineral metabolism dysregulation occurs early in chronic kidney disease (CKD) prior to the rise in serum phosphate levels. Understanding these initial mechanisms has been limited by lack of appropriate animal models. Recently a new classification of chronic kidney disease — mineral bone disorder (CKD-MBD) has been proposed based on the presence of specific laboratory abnormalities, bone disease and soft tissue calcification. The jck mouse is a slowly progressing model caused by a mutation in the Nek8 [NIMA (never in mitosis A)-related kinase] gene that develop phenotypic autosomal dominant polycystic kidney disease (ADPKD) and die of complications associated with CKD by 20 weeks of age. The progressive nature of this renal disease in the absence of surgical or chemical procedures provides an opportunity to assess early changes associated with CKD-MBD. Jck mice but not age-matched wild type mice fed a grain based chow diet containing 0.6% Pi and 0.8% Ca develop progressive but moderate hyperphosphatemia (9.2 ± 0.98 vs. 8.0 ± 0.17 mg/dl) and severe hypercalcemia (11.0 ± 0.31 vs 8.2 ± 0.15 mg/dl). Elevated levels of FGF23 (>1100 pg/ml at 15 weeks) and PTH (> 600 pg/ml at 15 weeks) in jck correspond to increasing levels of uremia (80 mg/dl) and mimic human CKD conditions. Immunohistological analysis confirmed elevated FGF23 production in bone. Static and dynamic bone histomorphometric analysis at 15 weeks reveal that jck relative to wild type mice have elevated bone volume (9.9 ± 0.6 vs. 6.5 ± 0.5%), increased trabecular numbers (3.4 ± 0.2 vs. 2.3 ± 0.2/mm), decreased trabecular spacing (270 ± 15 vs. 425 ± 36 μm), suppressed mineral apposition rate (1.6 ± 0.13 vs. 2.2 ± 0.07 μm/day), and mildly suppressed bone formation rates (0.45 ± 0.09 vs. 0.64 ± 0.05 μm3/μm2/day). As hypercalcemia is atypical in CKD, jck mice were fed a low calcium synthetic diet containing 0.4% calcium with 1.0% Pi. These animals remain normocalcemic but achieve significant hyperphosphatemia (18.0 ± 1.9 vs. 8.0 ± 0.27 mg/dl). Importantly, the induced levels of the phosphaturic hormones FGF23 and PTH are similar to those observed in mice fed a 0.6% phosphate diet. Based on the mineral bone abnormalities induced by renal failure, we conclude that jck mice may represent a new model for studying CKD-MBD associated with PKD.
Disclosures: Stephen O'Brien, Genzyme Corporation, 3; Genzyme Corporation, 1 This study received funding from: Genzyme Corporation
Dual Role of Type III Sodium-Dependent Phosphate Transporter in the Pathogenesis of Arteriosclerosis Caused by Hyperphosphatemia.Yutaka Taketani*1, Emi Shuto2, Asuka Shiota2, Ayako Tanimura2, Eriko Watari2, Tomoyo Kitamura2, Hironori Yamamoto3, Eiji Takeda4. 1University of Tokushima, Tokushima, Japan, 2Department of Clinical Nutrition, University of Tokushima, Tokushima, Japan, 3University Tokushima School of Medicine, Tokushima, Japan, 4University of Tokushima School of Medicine, Tokushima, Japan
Hyperphosphatemia is now recognized as a risk factor for cardiovascular disease in healthy people as well as chronic kidney disease patients. Hyperphosphatemia can be involved in vascular calcification by stimulating the differentiation of vascular smooth muscle cells (VSMC) to osteoblast-like cells with increase phosphate (P) influx. It has been known that type III sodium-dependent P transporter (NaPi-III) plays an important role in the P-influx and following processes to promote calcification. In addition, we recently reported that increase in extracellular P can deteriorate endothelial function through enhancement of ROS production and decreased NO availability. These data suggest that NaPi-III may also play another role in the pathogenesis of arteriosclerosis moreover calcification. In this study, we investigate the role of NaPi-III in the P-mediated endothelial dysfunction using bovine aortic endothelial cells (BAECs). RT-PCR analysis demonstrated that BAECs express both PiT-1 and PiT-2 (subtype of NaPi-III) mRNA, but not express NaPi-IIa and NaPi-IIb. Time-lapse confocal microscopy analysis of ROS production with aminophenyl fluorescein, specific reactive oxygen species (ROS) indicator, demonstrated that P-mediated ROS production in BAECs was completely inhibited by phosphonoformic acid (PFA), a specific inihibitor of sodium-dependent P transporter. We also found that elevation of extracellular P increased conventional PKC activity in BAECs. The activation of PKC would be related to activation of NADPH oxidase that is a major supplier for ROS. Interestingly, the activation of PKC was also inhibited by PFA. These results indicate that P-influx via NaPi-III can induce endothelial dysfunction. Thus, NaPi-III has dual role in the pathogenesis of arteriosclerosis mediated by hyperphosphatemia; one is in the calcification of VSMC, the other is in the endothelial dysfunction.
Disclosures: Yutaka Taketani, None.
Mild Renal Dysfunction is a Risk Factor for Bone loss and Vertebral Fractures in Postmenopausal Women.Mika Yamauchi*1, Toru Yamaguchi1, Hiroshi Kaji2, Toshitsugu Sugimoto3. 1Shimane University Faculty of Medicine, Shimane, Japan, 2Kobe University, Kobe, Japan, 3Shimane University School of Medicine, Shimane, Japan
Although it is well known that patients with end-stage renal disease are at increased risk for hip fracture, the eect of less severe renal dysfunction on bone loss and fracture risk is uncertain. We therefore evaluated whether or not mild renal dysfunction would aect bone mineral density (BMD) and the risk of vertebral fractures (VFs) in postmenopausal women.
We enrolled 659 postmenopausal healthy women who had examination of osteoporosis (mean age 65years). Serum creatinine level was measured and creatinine clearance (CCr) was calculated using the Cockcroft-Gault formula (mean levels of CCr 89ml/min/1.73m2). Estimated glomerular filtration rate (eGFR) was calculated using the formula presented by the Modification of Diet in Renal Disease (MDRD) study (mean levels of eGFR 76ml/min/1.73m2). BMD values of the lumbar (L), femoral neck (FN), and radius (R) were measured by DXA. Renal function was categorized by the criteria from the Kidney Disease Outcomes Quality Initiative (K/DOQI) committee.
At baseline, 22.3% of participants had chronic kidney disease (CKD) stage1 (eGFR 90?), 64.8% had CKD stage2 (60–89), and 12.9% had stage3 or more (<60). Based on CCr, these percentages were 46.3%, 46.9% and 6.8%, respectively. One hundred eighty women had one or more VFs. Comparison of fracture prevalence by CKD stages revealed that the stage3 or more group by eGFR had a significantly higher rate of VFs (45.3%) than the stage1 (23.8%) and 2 (25.3%) groups (p<0.01), suggesting that lower eGFR was associated with increased risk for VFs. In the stage2 group, there were significant positive correlations between eGFR and BMD values at FN and R, as well as between CCr and BMD values at all sites. Moreover, postmenopausal women with VFs had lower eGFR (p<0.05) and CCr (p<0.01) values than those without VFs in stage2. When multivariable logistic regression analysis was performed with the presence of VFs as a dependent variable and CCr levels adjusted for years after menopause, smoking habit, alcohol intake, and BMD as an independent variable, CCr levels were identified as a factor associated with the presence of VFs in postmenopausal women (odds ratio 0.36, 95% confidential interval 0.17–0.77 per SD increase, p< 0.01).
In conclusion, the present results suggest that postmenopausal women with mild renal dysfunction are at increased risk for bone loss and VFs. Thus, assessment of renal function could partly predict the risk of bone loss and vertebral fragility.
Disclosures: Mika Yamauchi, None.
Calcium Sensing Receptor (CaR) regulates the secretion of ACTH and PTHrP in Mouse Pituitary Tumor (AtT20) cells.Ramanaiah Mamillapalli. Section of Endocrinology, Internal Medicine, Yale School of Medicine, Yale University, New Haven, USA
The calcium-sensing receptor (CaR) regulates many cellular functions in a variety of cells in response to changes in the extracellular concentration of calcium and other cations. The CaR is a typical 7-pass transmembrane receptor that signals by coupling to different G proteins. Classically, the CaR has been described to utilize G alpha i, G alpha q and G alpha 11/12 for signaling. In normal and malignant breast epithelial cells the CaR has been shown to regulate the secretion of parathyroid hormone-related protein by regulating cAMP production. In normal breast cells, the CaR couples to G alpha i and inhibits cAMP and PTHrP production. However, in malignant breast cells, the CaR instead couples to G alpha s and stimulates both cAMP and PTHrP production. In order to determine if the CaR also couples to G alpha s in any other cells, we studied mouse pituitary tumor (AtT20) cells, which have previously been described to stimulate cAMP production in response to increases in extracellular calcium. We found that AtT20 cells express the CaR and that stimulation of the CaR with calcium and calcimimetics increases cAMP levels and PTHrP and adrenocorticotropic hormone (ACTH) secretion. PTHrP and ACTH secretion from these cells is also stimulated by forskolin and the effects of high calcium are blocked by treatment with the PKA inhibitor, H89. The effects of high calcium on PTHrP and ACTH secretion were mediated by the CaR, since siRNA-mediated knockdown of CaR gene expression blocked the effects of calcium and calcimimetics. Finally, using agonist stimulated 35S-GTPrS G-protein binding assays, we demonstrated that the CaR coupled to G alpha s in AtT20 cells. These data demonstrate that the CaR regulates the production of PTHrP and ACTH in pituitary cells by coupling to G alpha s and stimulating cAMP production. Thus, depending on the cell type the CaR can couple either to G alpha i or to G alpha s.
Disclosures: Ramanaiah Mamillapalli, None.
A Novel Persistently Active PTH-PTH Receptor Complex.Beena Thomas*1, Thomas Gardella2, Edi Goihberg3, John Potts, Jr.2, Michael Rosenblatt1. 1Tufts University School of Medicine, Boston, MA, USA, Massachusetts General Hospital, Boston, MA, USA, 3Department of Physiology, Tufts University School of Medicine, Boston, USA
The interaction of parathyroid hormone (PTH) with the PTH1-receptor (PTHR) is usually brief in normal physiology. Hormone-binding, followed by receptor activation, followed by dissociation of the PTH—-PTHR complex occur on a time scale of fractions of a second to seconds. Although constitutively active forms of the receptor are known, they arise from receptor mutations that alter the interaction of PTHR with G proteins. The prolonged signaling that results is ligand-independent. Recently, we designed and created a series of PTHR mutants, each of which contains a pair of cysteine substitutions within transmembrane (TM) helices 5 and 6 of the receptor. Each set of mutant receptors has the potential to form a new intra-receptor disulfide bridge across TM5 and TM 6. These helices are of particular interest in receptor activation because they are connected by intracellular loop (ICL) 3, the receptor domain that interfaces with the G protein-coupled signal transduction apparatus. We were interested in determining if the formation of a new disulfide bridge between extracellular ends of TM5 and TM6 could “lock” the PTHR into an active conformation. It was observed that some of the double cysteine-containing PTHR mutants, especially the L368C/M425C (LM) mutant PTHR, produced prolonged increases in cAMP levels upon addition of a biologically active PTH analog, MPTH(1–15). In fact, elevated cAMP levels persisted for at least 1h after “wash-out” of ligand. Further examination of the mechanism reveals that despite “wash-out,” MPTH (1–15) remains bound to PTHR in a productive bimolecular complex. This finding stands in sharp contrast to the documented rapid dissociation of MPTH (1–15) from the native sequence receptor. Interestingly, addition of GTPγS, which uncouples the receptor from G protein complexes, has no adverse effect on the prolonged binding pattern observed with the LM mutant. Using cells expressing a GαS dominant negative (GSDN) form of the G protein which is known to prolong ligand binding, the expected prolongation in binding was observed with the native PTHR, but not much difference was observed for the binding properties of the LM mutant.
We conclude that certain double cysteine-containing mutant forms of the PTHR are able to adopt a conformation of the PTHR which traps the ligand, resulting in a ligand-receptor complex that is persistently active.
Disclosures: Beena Thomas, None.
AF-2 In Estrogen Receptor-α (ERα) Is Not Required For the Effect of ERα On Pubertal Bone Growth In Female Mice.Anna Borjesson*1, Sara H Windahl2, Marie K Lagerquist2, Cristina M Antal3, Andrée Krust3, Pierre Chambon3, Claes Ohlsson4. 1Gothenburg University, Göteborg, Sweden, 2Center for Bone Research, Institute of Medicine, Sahlgrenska Academy, Göteborg University of Gothenburg, Gothenburg, Sweden, 3Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France, 4Sahlgrenska University Hospital, Goteborg, Sweden
Estrogen affects skeletal growth mainly via the estrogen receptor-α (ERα). The DNA binding domain of ERα is surrounded by transcription activation function 1 (AF-1, N-terminal, ligand independent) and AF-2 (C-terminal, ligand dependent). We have previously seen that specific inactivation of AF-1 in ERα results in growth plate closure while total inactivation of ERα results in increased bone growth in elderly female mice, suggesting that a specific inactivation of AF-1 results in hyperactive ERα function, resulting in growth plate closure in elderly female mice.
The aim of the present study was to determine the importance of AF-2 for the pubertal growth in female mice. The skeletal growth in female mice with a specific inactivation of AF-2 in ERα (ERαAF-20) and mice with total inactivation of ERα (ERα−/−) were compared with wild type mice. The axial skeleton (crown rump length) and appendicular skeleton (femur length) were evaluated before sexual maturation (4 weeks of age) and after sexual maturation (12 and 17 weeks of age) using X-ray.
Before sexual maturation, the size of both the axial and the appendicaular skeleton was normal in both ERα−/− and ERαAF-20 female mice. During puberty the ERα−/− mice demonstrated a more pronounced increase in length of both femur and crown rump compared to wild type mice (Fig 1, p<0.01). In contrast, ERαAF-20 displayed a normal pubertal growth both regarding the appendicular and the axial skeleton (Fig 1, non signficant). The uterus weight was clearly reduced in both ERα−/− and ERαAF-20 compared to wild type mice (Fig 1, p<0.001).
In conclusion, ERα inhibits pubertal bone growth in female mice and the ligand dependent AF-2 is not required for this inhibition. In contrast the ERα-mediated regulation of uterus weight is dependent on AF-2, suggesting that the role of AF-2 is tissue/function dependent.
Disclosures: Anna Borjesson, None.
Estrogen Deficiency Augments TZD-induced Bone Loss and Fat Accumulation in Bone in Vivo.Shilong Huang*1, Farhan Syed2, Larry Suva3, Beata Lecka-Czernik4. 1University of Toledo Health Sciences Campus, Toledo, OH, USA, 2Mayo Clinic, Rochester, MN, USA, 3University of Arkansas for Medical Sciences, Little Rock, AR, USA, 4University of Toledo College of Medicine, Toledo, USA
Antidiabetic drugs thiazolidinediones (TZDs) are high affinity ligands for the PPARγ nuclear receptor, which is a key regulator of insulin sensitivity and glucose metabolism. PPARγ also regulates bone cell development; following activation with TZDs it acts as a negative regulator of osteoblastogenesis and a positive regulator of osteoclastogenesis and adipogenesis. Recent evidence showing that older diabetic women on TZDs therapy are more susceptible to bone loss and have an increased fracture risk prompted us to develop an animal model to study the mechanism by which PPARγ and estrogen may control bone mass. C57BL/6 female mice, at 4 mo of age, were either ovariectomized (OVX) or sham-operated and fed a diet supplemented with rosiglitazone (Rosi) (20 mg/kg/day) or non-supplemented for 4 weeks following surgery. MicroCT evaluation of trabecular bone in the L5 vertebra showed that treatment with Rosi of OVX animals decreased bone mass by 58%, compared to 36% and 22% in Rosi- and OVX-only animals, respectively. Accordingly, the decrease in BV/TV was the result of a decrease in both Tb.Th and ConnD. Histomorphometric analysis revealed a significant decrease in osteoblast number and activity, as well as an increase in osteoclast number per bone perimeter, in all three groups (OVX, Rosi, OVX+Rosi). Furthermore, analysis of tibial gene expression demonstrated that the increase in RANK and RANKL mRNA expression in the OVX and Rosi groups was further increased in the OVX+Rosi group, suggesting a potential additive effect on bone resorption. Estrogen has been shown to antagonize the pro-adipocytic activity of PPARγ. In OVX animals body weight was increased by 30%, primarily due to an increase in visceral fat (WAT) and interscapular (BAT) fat content. Interestingly, treatment with Rosi prevented WAT accumulation, but enhanced BAT accumulation, suggesting that Rosi-activated PPARγ interferes with the energy metabolism profile induced by estrogen deficiency. Moreover, while the number of fat cells in the vertebra was slightly increased in the OVX group and increased by 15-fold in Rosi group, OVX+Rosi had an apparent synergistic effect and increased the number of fat cells by 25-fold. Collectively, these data demonstrate that estrogen deficiency exacerbates the pathologic bone loss observed in TZD-treated animals, thereby providing an explanation for the increased risk of bone loss and fracture observed in older women on TZD therapy.
Disclosures: Shilong Huang, None.
A Characterization of the Interactions Between the Unliganded VDR and LEF1.Marie Demay1, Hilary Luderer*2. 1Massachusetts General Hospital & Harvard Medical School, Boston, MA, USA, 2Massachusetts General Hospital, Boston, MA, USA
The Vitamin D Receptor (VDR) is a nuclear receptor that plays an important role in cutaneous homeostasis. Humans and mice lacking a functional VDR develop Hereditary Vitamin D Resistant Rickets, which is often accompanied by alopecia totalis. Ligand-independent actions of the VDR in keratinocyte stem cells (KSC) are essential for normal hair follicle regeneration in mice. Therefore, elucidating the mechanism by which the VDR maintains normal KSC function is expected to elucidate novel molecular interactions of this receptor.
Previous studies demonstrated that the VDR is necessary for synergistic activation of Wnt reporter genes by LEF1 and β-catenin in primary keratinocytes. The VDR is also present in a complex with LEF1 and β-catenin in COS-7 cells. GST pull-down assays were performed to characterize the interactions of the VDR with these effectors of the canonical Wnt signaling pathway. These studies identified a novel interaction between the unliganded VDR and LEF1 that is independent of β-catenin.
The purpose of this study was to determine which specific domains of the VDR are necessary and sufficient for interaction with LEF1. Several deletion mutants of the VDR were engineered as N-terminal GST-fusions and expressed in bacteria. GST-VDRs were bound to glutathione sepharose beads in the absence of ligand and then incubated with nuclear extracts isolated from COS-7 cells expressing LEF1-HA. Proteins were eluted using reduced glutathione and subjected to Western analysis for detection of LEF1-HA and the VDR. Deletion of the Activation Function 1 (AF1) domain or the Activation Function 2 domain (AF2) did not affect the VDR-LEF1 interaction. However, deletion of both the AF1 and the DNA binding domain (DBD) abrogated this interaction, suggesting that sequences within the N-terminal region of the VDR are required for interaction with LEF1. To determine if the AF1 and DBD regions of the VDR are sufficient to mediate interactions with LEF1, a C-terminal truncation mutant VDR containing only these two domains was engineered. This mutant retained the ability to interact with LEF1 demonstrating that the N-terminal region of the VDR is not only necessary, but also sufficient to mediate interactions with LEF1.
In conclusion, these data demonstrate novel ligand-independent interactions of the VDR with LEF1. We hypothesize that these interactions are required for maintenance of KSCs, which are critical for cyclic hair follicle regeneration.
Disclosures: Hilary Luderer, None.
Elevated Vitamin D Receptor (VDR) Regulates Multiple Target Gene Expressions Through Histone Modification in Genetic Hypercalciuric Stone-Forming (GHS) Rats.Hongwei Wang*1, Randal Zhou1, Ayodele Adesanya1, Jikun Shen1, Hongang Ye1, Qiang Wang1, David Bushinsky2, Murray Favus3. 1University of Chicago, Chicago, USA, 2University of Rochester, Rochester, NY, USA, 3University of Chicago, Chicago, IL, USA
GHS rats are an excellent model for studies of human idiopathic hypercalciuria (IH), as both share increased intestinal Ca absorption and bone resorption, decreased renal tubule Ca reabsorption, low bone mass and Ca kidney stone formation. In GHS rats, vitamin D receptor (VDR) over-expression and elevated tissue VDR levels in intestine, kidney, and bone can mediate the tissue Ca transport changes that cause hypercalciuria. The specificity of increased VDR in Ca transporting tissues is not known. Therefore, the present study was undertaken to determine: whether VDR is over-expressed in non-Ca transporting tissues that normally express VDR; whether the VDR repressor Snail is also expressed in all VDR-containing tissues; and to explore the molecular mechanisms of VDR up-regulation of key VDR-dependent genes. Tissues were harvested from GHS and control (Sprague Dawley, SD) rats that were fed a normal Ca (0.6%Ca) diet. Using Western blot, GHS rats had higher VDR expression levels in Ca transporting tissues (intestine, kidney, and bone marrow stromal cells) and thymus and prostate. GHS and SD rats had comparable levels of VDR in lung, brain, heart, and liver. Snail was down-regulated in kidney, intestine, and thymus in GHS rats. To explore the molecular mechanisms whereby high VDR levels modulate gene expression, GHS rats were injected ip with either 1,25(OH)2D3 (200 ng/100g body weight) or ethanol control and sacrificed 12 hr after injection. Using real-time PCR analysis, 1,25 (OH) 2D3 up-regulated intestinal Ca transporters TrpV6, and renal Ca Sensing receptor (CaSR), vitamin D 24-hydroxylase (CYP24), and VDR; and down regulated renal 25-hydroxyvitamin D3–1α-hydroxylase (CYP27) gene expression. ChIP assay revealed VDR specific binding to multiple regions of the proximal promoter of the several target genes followed by hyperacetylation or hypermethylation of the histone H3 protein around the target gene promoters. In conclusion: in GHS rats, Snail and VDR expressions were inversely correlated in all tissues examined; 1,25(OH)2D3 levels were the result of VDR-mediated suppression of synthesis through down-regulation of CYP27 and stimulation of 24-hydroxylation through up-regulation of CYP24; VDR regulated Ca transport by TrpV6 and CaSR by modifying histone and chromatin structures; and non-Ca transporting tissues may over-express VDR. The molecular basis of the tissue specificity for VDR over-expression in GHS rats requires further investigation.
Disclosures: Hongwei Wang, None.
Meningioma 1 (MN1) is Required for Appropriate Osteoblast Proliferation, Motility, Differentiation and Function.Xiaoxue Zhang1, Diane R. Dowd1, Meika C. Moore1, Tanya A. Kranenburg2, Magda A. Meester-Smoor3, Ellen C. Zwartho3, PAUL MACDONALD*4. 1Case Western Reserve University, Cleveland, USA, 2St. Jude Children's Research Hospital, Memphis, USA, 3Erasmus Medical Center, Rotterdam, Netherlands, 4CASE WESTERN RESERVE UNIVERSITY, CLEVELAND, OH, USA
The vitamin D endocrine system is essential for calcium and phosphate homeostasis and skeletal mineralization. The 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone binds to the vitamin D receptor (VDR) to regulate gene expression. These gene products, in turn, mediate the actions of 1,25(OH)2D3 in mineral-regulating target cells such as the osteoblast. We showed previously that meningioma 1 (MN1) is a novel target of 1,25(OH)2D3 in MG-63 osteoblastic cells and that it is a coactivator for VDR-mediated transcription (Sutton AL, et al. Mol Endocrinol. 2005; 19:2234–2244). This, combined with the undermineralized cranial bone phenotype of the MN1 null mice, suggests a potential role for MN1 in osteoblast function. However, the functional significance of MN1 in osteoblastic cell biology is largely unknown. Using calvarial osteoblasts derived from wild-type and MN1 knockout mice, we provide data supporting an essential role of MN1 in maintaining appropriate osteoblast proliferation, dierentiation, and function. Here, we demonstrate that MN1 expression is increased dramatically during dierentiation of wild-type primary osteoblastic cells. MN1 knockout osteoblasts displayed altered morphology, decreased growth rate, impaired motility, and attenuated 1,25(OH)2D3/VDR-mediated transcription. Expression of osteoblastic genes, including alkaline phosphatase, Runx2, osterix, BSP2, DMP1, and osteocalcin, were impaired in dierentiating MN1 null calvarial osteoblasts and/or in intact calvaria obtained from the MN1 null mice compared to wild-type controls. Indeed, in vitro osteoblast dierentiation studies demonstrated that MN1 knockout cells had reduced alkaline phosphatase activity and mineralized nodule formation compared to wild-type cultures. MN1 null osteoblasts were also impaired in supporting osteoclastogenesis in co-culture studies presumably due to marked reduction in the RANKL:OPG ratio in the MN1 null cells. Mechanistic studies supported a transcriptional role for MN1 in controlling RANKL gene expression through activation of the RANKL promoter. Cumulatively, these studies indicate an important role for MN1 in maintaining the appropriate maturation and function of calvarial osteoblasts. Due to early postnatal lethality of the MN1 knockout model, conditional strategies are needed in the future to address the significance of MN1 in osteoblastic cells of the postnatal axial and appendicular skeletons.
Disclosures: PAUL MACDONALD, None.
Novel Mechanisms Involved in the Regulation of Immune Function by 1,25(OH)2D3: Inhibition of Cytokine Activation by Runx1 and Induction of Foxp3.Sneha Joshi*1, Kenji Ichiyama2, Masahiro Ono3, Yu Wakabayashi4, Akihiko Yoshimura5, Shimon Sakaguchi6, Sylvia Christakos7. 1UMDNJ-NJMS, Newark, NJ, USA, 2Division of Molecular & Cellular Immunology, Medical Institute of Bioregulation, Kyushu University 3–1−1 Maidashi, Higashi-ku, Fukuoka, Japan, 3Department of Experimental Pathology, Kyoto University, Kyoto, Japan, 4Department of Microbiology & Immunology, Keio University, Tokyo, Japan, 5epartment of Microbiology & Immunology Keio University, Tokyo, Japan, 6CREST, Japan Science & Technology Agency, Kawaguchi, Japan, 7New Jersey Medical School, Newark, NJ, USA
1,25(OH)2D3 has immune suppressive actions and has been shown to prevent induction, in mouse models, of various autoimmune diseases including collagen induced arthritis. However, the molecular mechanisms involved in the eects of 1,25(OH)2D3 on the immune system are still poorly understood. We previously reported that the proinflammatory cytokine IL-17 is inhibited by 1,25(OH)2D3. 1,25(OH)2D3 also suppresses IL-2 expression. Runx1, which is required for hematopoiesis, is also required for activation of IL-2 and IL-17. We report here that Runx1 mediated activation of IL-2 and IL-17 transcription is inhibited by 1,25(OH)2D3 and that this inhibition of Runx1 activation is due, at least in part, to sequestration of Runx1 by 1,25(OH)2D3/vitamin D receptor (VDR), blocking Runx1 binding to its site. Using the mouse 1.6 kb IL-2 promoter or the mouse 2.0 kb IL-17 promoter and transfection in Jurkat cells, there was a 3 to 4 fold enhancement of transcription in the presence of Runx1 that was inhibited after treatment of Jurkat cells with 1,25(OH)2D3 (10−8M, 24h). Co-immunoprecipitation indicated interaction of VDR and Runx1. Chromatin immunoprecipitation (ChIP) showed that activation with PMA and ionomycin results in the recruitment of Runx1 to the Runx sites in the IL-2 and IL-17 promoters. This recruitment was inhibited in the presence of 1,25(OH)2D3/VDR. However, ChIP analysis indicated that VDR does not bind to the Runx sites. Foxp3 is a transcription factor involved in the function of regulatory T cells that are important in suppressing autoimmune processes. Foxp3 interacts with Runx1 and inhibits IL-17 and IL-2 transcription. 1,25(OH)2D3 enhances Tregs and the expression of Foxp3. Using the mouse Foxp3 promoter/enhancer (−3523/+6668) we found that 1,25(OH)2D3/VDR has a direct eect on the Foxp3 gene (15 fold maximum induction; 10−8M 1,25(OH)2D3, 24h). A putative vitamin D response element (VDRE) was identified within the core enhancer region (+2114/+2350) at +2159. EMSA showed binding of VDR to this site and mutation of this site suppressed the transcriptional response to 1,25(OH)2D3. These findings suggest, for the first time, that the negative eect of 1,25(OH)2D3 on IL-2 and IL-17 transcription involves sequestration of Runx1 by 1,25(OH)2D3/VDR and induction of Foxp3 by 1,25(OH)2D3. Our results describe novel mechanisms and new concepts with regard to vitamin D and the immune system that suggest therapeutic targets for the control of autoimmune diseases.
Disclosures: Sneha Joshi, None.