Above diagonal — Genetic correlations (RhoG) * p < 0.05; ** p < 0.005; *** p < 0.0001
ASBMR 29th Annual Meeting
Article first published online: 4 MAR 2010
Copyright © 2007 ASBMR
Journal of Bone and Mineral Research
Supplement: 30th ASBMR Annual Meeting Advancing The Future
Volume 22, Issue Supplement S1, pages s102–s151, September 2007
How to Cite
(2007), ASBMR 29th Annual Meeting. J Bone Miner Res, 22: s102–s151. doi: 10.1002/jbmr.5650221404
- Issue published online: 4 MAR 2010
- Article first published online: 4 MAR 2010
Biglycan and Fibromodulin Control Bone Mass by Regulating Osteoclast Differentiation Through Bone Marrow Stromal Cells.Y. Bi*, T. M, Kilts*, A. C. Griffin*, M. F. Young. National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.
Members of the Small Leucine-Rich Proteoglycan (SLRP) family populate numerous sites in the musculoskeletal system including tendon, cartilage and bone, but, their precise role in these tissues is still unclear. Previous work showed that mice with combined deficiencies in two SLRPs, biglycan (bgn) and fibromodulin (fmod), acquire osteoarthritis and ectopic tendon ossification. Here we show that the bgn-/0/finod-/- mice have delayed bone formation during development judged by alizerin red/alcian blue staining and severe age-dependent osteopenia determined by X-ray and microCT analyses. In order to understand the molecular basis for this osteopenia, osteogenic bone marrow stromal cells (BMSCs) were isolated and examined. Colony-forming unit fibroblastic (CFU-F) analysis, which estimates the number of osteogenic stem cells, showed no significant differences between WT and bgn-/0/fmod-/- mice. Further, in vitro osteogenesis assays showed that Ca2+ accumulation was increased in the bgn-/0/fmod-/- BMSC cultures compared to WT BMSCs. Western analysis of the BMSC protein extracts showed that the bgn-/0/fmod-/- cells had increased levels of Runx2 compared to WT controls. The bgn-/0/finod-/- BMSCs also had increased BMP-2 and TGF-betal signaling indicated by increased levels of p-smadl and p-smad2/3 in the presence of BMP-2 and TGF-betal compared to WT BMSCs, respectively. Furthermore, in vivo bone formation was not significantly different between WT and bgn-/0/fmod-/- BMSCs tranplanted subcutaneously into immuno-compromised mice. From these data, we concluded that the decreased bone mass in bgn-/0/fmod-/- mice was not primarily due to a defect in osteogenesis. We next determined whether the osteopenia in bgn-/0/fmod-/- mice could be due to increased differentiation and/or activity of osteoclasts. To test this, sections of long bone and BMSC transplants were stained for Tartrate-Resistant Acid Phosphatase (TRAP), an osteoclast marker. The number of TRAP positive cells was higher in bone and in vivo BMSC transplants from bgn-/0/fmod-/-mice. Osteoclastogenesis was assessed by adding bone marrow suspensions to plates in which BMSC had formed individual colonies and showed that greater numbers of colonies in the bgn-/0/fmod-/- cultures supported formation of TRAP-positive multinucleated cells compared to WT. In summary, we have shown that bgn and fmod when depleted in combination have a negative influence on bone mass by increasing osteoclast formation and function and thereby pointing to the possibility that these SRLPs could be novel targets to modulate bone turnover.
Disclosures: Y. Bi, None.
This study received[funding from: IRP-N1DCR, NIH.
Implication of Two Matrix Proteins in Bone Healing: Osteopontin (OPN) and Bone Sialoprotein (BSP).L. Monfoulet*1, J. Fricain*1, L. Malaval2, L Aubin*3, O. Chassande*11INSERM US77 - Univ Victor Segalen Bordeaux2, bordeaux, France, 2INSERM U890 - Univ Jean Monnet, St Etienne, France, 3Medical Genetics and Microbiology, Faculty of Medecine, University of Toronto, Toronto, ON, Canada.
Bone fracture healing involves a well characterized cascade of events that includes: hematoma, inflammation, “soft” callus, neovascularization, callus mineralization and remodelling of the “hard” callus to generate mature lamellar bone. This complex sequence of biological processes is controlled by chemotactic factors, growth factors and bone matrix proteins. Our study is focused on two major non-cotlagenous bone matrix proteins belonging to the Small Integrin-Binding LIgand N-linked Glycoprotein (SIBLING) family: osteopontin and Bone Sialoprotein. OPN promotes angiogenesis, inhibits bone mineralization, and regulates osteoclast function. BSP is involved in osteoclast adhesion and differentiation, in matrix mineralization, and mediates endothelial cell migration and angiogenesis. Together, these data suggest that BSP and OPN can play an important role in bone fracture healing. To investigate the role of these proteins of bone healing, we used mice knockout for the OPN or BSP genes. We developed a new model of bone healing in mice which consists in a 0.9 mm diameter cortical defect in the femoral diaphysis. Micro-computed tomography was used to quantify mineralization at 14 and 21 days. Histology (at 14 and 21 days) and quantitative RT-PCR (at 10 days) were used to evaluate cellular functions related to ECM formation, bone formation and remodelling. We also established primary cultures of osteoblasts from mutant and wild type bone marrow, characterized by quantitative RT-PCR and alkaline phosphatase activity. Using micro-CT, we have demonstrated in BSP-/- mice a significant delay of bone healing as showed by a slower decrease of the Bone Volume / Tissue Volume (BV/TV) volume within the defect. At 10 days, RT-PCR analysis reveals a lower expression of osteocalcin mRNA in BSP-/- mice compared to wild-type mice. These preliminary results suggest that the delay in bone healing observed in BSP-/- mice could be due to a decreased osteoblast activity. In contrast, the OPN-/- mice show an advanced bone healing in comparison with wild-type. We have also developed primary cultures of mouse Bone Marrow Stromal Cells to investigate the role of this bone matrix protein on osteoblast differentiation. The first results tend to show that OPN deficiency accelerates differentiation into mature osteoblasts. This early differentiation could explain the advanced bone healing observed by microCT in OPN-/- mice. In summary, our results reveal antagonist roles for these two major bone matrix proteins during the process of bone healing.
Disclosures: L. Monfoulet, None.
Delayed Cartilage and Bone Remodeling During Fracture Healing in Matrix Metalloproteinase 13 Null Mutant Mice.C. Colnot1, D. J. Behonick*2, S. Lieu*1, Z. Xing*1, R. Marcucio*1, Z. Werb*2, T. Miclau*11Orthopaedic Surgery, University of California San Francisco, San Francisco, CA, USA, 2Anatomy, University of California San Francisco, San Francisco, CA, USA.
Adult bone does not heal through the production of scar tissue rather through a regenerative process, that recapitulates early skeletal development. Matrix metalloproteinase 13 (MMP13, collagenase-3) is an extracellular matrix (ECM) protease whose preferred substrates include the major components of cartilage and bone ECM. MMP13 has been shown to be important for skeletal development via endochondral ossification (1). This observation led us to question the role of MMP13 in skeletal repair. We compared bone healing in Mmpl3−/− and wild type mice during non-stable, stable fracture repair and cortical defect healing. In order to understand which cell types were affected by the Mmpl3−/− mutation, we examined the expression profile of Mmpl3 during bone healing using in situ hybridization. In addition, we transplanted bone marrow from wild type mice into Mmpl3−/− mice and compared healing to Mmpl3−/− mice, which received bone marrow from a Mmpl3−/− donor. Tissues were analyzed by cellular, molecular and histomorphometric analyses on paraffin tissue sections.
We show that MMP13 is required for proper resorption of hypertrophic cartilage and normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification (Fig. 1). Transplant of bone marrow from wild type mice is not sufficient to rescue the Mmpl3−/− healing phenotype. These results indicate that impaired healing is intrinsic to cartilage and/or bone and is not due to defects in osteoclasts. These results are consistent with the expression of Mmpl3 which is restricted to hypertrophic chondrocytes and osteoblasts but absent from osteoclasts. Furthermore, Mmpl3−/− mice also exhibit a delay in bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification, demonstrating that the bone phenotype is independent from the cartilage phenotype. Overall, our findings demonstrate that MMP13 is crucial for normal production and remodeling of cartilage and bone during adult fracture repair.
Disclosures: C. Colnot, None.
This study received funding from: NIH.
Rescue of MT1-MMP Expression in Cartilage Increases Survival, Chondrocyte Proliferation and Bone Formation in MT1-MMP Deficient Mice.L. Szabova*, S. Yamada*, K. Holmbeck*. CSDB/MMPU, NIDCR, Bethesda, MD, USA.
Membrane type-1 matrix metalloproteinase (MT1-MMP) is a potent collagenase essential for proper remodeling of collagen rich tissues. Mice deficient for MT1-MMP (KO) are dwarfs with severe fibrosis of connective tissues, bone loss and retention of embryonic cartilages in the skull due to impaired degradation of unmineralized type I and II collagens. Here we define the contribution of disrupted cartilage remodeling in the development of the MT1-MMP-deficient phenotype. Specifically, we created transgenic mice where MT1-MMP expression was selectively re-introduced in cartilage tissues using a transgene driven by the type II collagen promoter/enhancer. We have bred these mice into the MT1-MMP-deficient background and evaluated me developmental effects of MT1-MMP expression in the cartilage tissue of otherwise MT1-MMP deficient mice. Cartilage specific MT1-MMP expression in KO mice resulted in complete rescue of the pre-weaning death observed in KO mice, increased body weight and prolonged survival. Transgenic KO mice further displayed increased bone formation in the skull compared to KO mice. Strong expression of the MT1-MMP transgene increased chondrocyte proliferation in the epiphyseal growth plate, which resulted in increased growth of the long bones. Increased bone formation quite unexpectedly coincided with expression of the transgene in a subset of bone cells. Expression of type II collagen was documented in bone cells of normal mice using in situ hybridization and immunostaining thus documenting that transgene expression was not ectopic, but mirrored the normal expression pattern of type II collagen. This finding explains the increased bone formation observed in transgenic KO mice since MT1-MMP is necessary for maintaining the bone formation. In conclusion, reintroduction of MT1-MMP in the cartilage of MT1-MMP KO mice results not only in cartilage specific expression, but also directs transgene expression in a subset of bone cells thereby facilitating increased bone formation. These bone cells are most likely the descendants of a common progenitor of bone cells and chondrocytes expressing type II collagen and support our previous observation that some chondrocytes can differentiate into bone cells. Our data suggest that MT1-MMP provides type II expressing chondrocytes and bone cells with the necessary collagenolytic activity required for chondrocyte proliferation and bone formation in vivo.
Disclosures: L. Szabova, None.
Identification of a Novel Stem Cell for Bone and Cartilage of the Myeloid Lineage.Z. W. Lazard*1, C. M. Fouletier-Dilling*1, F. H. Gannon2, M. H. Heggeness*3, E. A. Olmsted-Davis1, A. R. Davis11Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA, 2Orthopedic Surgery and Pathology, Baylor College of Medicine, Houston, TX, USA, 3Orthopedic Surgery, Baylor College of Medicine, Houston, TX, USA.
We have previously shown that osteoblasts can be derived from hematopoietic stem cells (HSC), through mesenchymal progenitors. In this study, we focused on identifying the pathway that leads from a bone marrow HSC to a mesenchymal progenitor for bone and cartilage. To better characterize this process, we used a BMP2 inductive model which recruits chondro-osseous progenitors in the mouse to a heterotopic location. In this bone formation model, cells transduced with an adenovirus expressing BMP2 were implanted into the muscle in the mouse hind limb. Bone formation occurs rapidly, with cartilage appearing within 6 days and mineralized osteoid detected by 7 days after implantation of me transduced cells. Initial characterization of the tissues isolated three days after induction showed a substantial increase in tentative progenitors. These cells possessed the marker of the donor HSCs used to repopulate the bone marrow in ablated recipients, suggesting they were bone marrow derived. Cells isolated from the day 4 tissues were capable of undergoing osteogenic or chondrogenic differentiation in vitro. Immunohistochemical and microarray analysis of these tissues showed specific elevation in genes associated both with mesenchymal stem cells (CD44, CXCR4) as well as myeloid cells (CDIlb, CD68, CD166) in tissues receiving the AdBMP2 transduced cells as compared to those receiving cells transduced with an empty vector. To further delineate the tentative myeloid origin of these progenitors we performed heterotopic bone assays in mice which express myeloid restricted β-galactosidase. In these mice ere recombinase is expressed by the myeloid specific LysM gene promoter which can remove the intervening DNA sequence between the LacZ gene promoter and its transcriptional start site leading to permanent activation. Analysis of the tissues shows specific β-galactosidase activity in the tentative progenitors, as well as the mature chondrocytes, and osteoblasts in the heterotopic bone as well as adjacent skeletal bone. Since these bone marrow derived cells must circulate to reach the heterotopic location, we next attempted to purify these from peripheral blood. Results showed significant elevation in both myeloid and mesenchymal stem cell markers on peripheral blood mononuclear cells by 3 days after induction of bone formation. The data collectively demonstrates a novel myeloid progenitor, easily isolated from peripheral blood, that is recruited for endochondral bone formation.
Disclosures: Z. W. Lazard, None.
This study receivedfunding from: N1B1B: R01EB005173 and DOD PR03H69.
BMP Canonical Signaling Through Receptor Smads 1 and 5 Is Required for Endochondral Bone Formation.K. N. Retting*1, R. R. Behringer*2, K, M. Lyons11Orthopaedic Surgery, UCLA, Los Angeles, CA, USA, 2Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
Transforming growth factor-β (TGFβ) superfamily members including bone morphogenetic proteins (BMP) play multiple roles during endochondral bone formation. It is believed that TGFβ superfamily members signal predominantly through canonical Smad pathways during chondrogenesis. The BMP family activates Smads 1, 5 and 8. We have found nuclear localization of BMP R-Smads in proliferating and differentiating chondrocytes in the growth plate and in primary cultures. These and other studies establish that BMP signaling is required for chondrogenesis. R-Smads share structural homology, yet the extent to which Smad proteins are functionally redundant, regulate distinct targets, and control endochondral bone formation in vivo is unknown. To study the role of BMP R-Smads during chondrogenesis, we generated knockout models of Smads 1. 5 and 8 individually and in combination. It has been reported that Smad8 null mice have no obvious defects. Because mice null for either Smadl or 5 die too early in embryogenesis for chondrogenic analysis, we conditionally deleted Smads 1 and 5 in endochondral elements utilizing the cartilage-specific collagen II-Cre. Cartilage-specific loss of Smadl as well as the combined loss of Smads 1 and 8 results in apparently normal mice. However, Smadl(fx/fx);Smad5(fx/fx);Col2Cre mutant mice exhibit severe chondrodysplasia, resulting in lethality at birth. Smadl/5CKO mutants display little to no mineralization of the axial skeleton. The dramatically shortened limbs lack an organized growth plate and display excessive growth in the bone collar. These data demonstrate that canonical Smad signaling is required for endochondral bone formation, and suggest that Smads I and 5 exhibit functional redundancy. The phenotype is more severe than that seen in Smad4(fV fx);Col2Cre mice, challengeing the dogma, at least in chondrocytes, that co-Smad4 is required to mediate Smad signaling through TGFβ and BMP pathways. Furthermore, skeletal defects in the Smadl/5CKO are not as severe as the cartilage-specific knockout of BMP receptors, indicating that BMP signaling is not completely abolished. We are currently testing these hypotheses. Overall, these data demonstrate the importance of the BMP canonical Smad pathway in chondrogenesis and answer the questions of functional redundancy of Smads 1,5 and 8.
Smad1 (TX/fx): Smad5(TX/fX); Col2Cre mice. Wild type (*,C) and mutant (B,D) littermates at PO; MicroCT scan (A,B); proximal tibia stained with Alcian blue and nuclear fast red (CD).
Disclosures: K.N. Retting, None.
Role of TROY in Tooth Development.N. Yamaguchi1, H. Ichioka*1, SL Tovosawa*2, H. Harada*3, R. Nishimura1, T, Yoneda11Biochemistry, Osaka University Graduate School of Dentistry, Suita, Japan, 2Oral Pathology, Osaka University Graduate School of Dentistry, Suita, Japan, 3Oral Anatomy 2, lwate Medical University School of Dentistry, Morioka, Japan.
Mutations of ectodysplasin (Eda), a member of TNF family, cause hypohydrotic ectodermal dysplasia (HED), characterized by ectodermal abnormalities, including sweat glands defects, impaired hair formation, anodontia/oligodontia, and amelogenesis imperfecta. It is also demonstrated that the receptor for Eda Edar and an adaptor protein of Edar EDARADD are responsible for HED. Spontaneous mutations of the Eda, Edar or EDARADD gene in mice also develop HED like phenotype. These results indicate a critical role of Eda/Edar/EDARADD signal in the tooth development. Eda/Edar/ EDARADD transduces signals into cytoplasm through TRAF6. Mice deficient in TRAF6 gene showed more severe ectodermal abnormalities than tabby mice which have a mutation in the Eda gene. We therefore hypothesized that an additional pathway plays a role in the tooth development through TRAF6. Because a TNF receptor family member, TROY, which has 33% homology to Edar and directly binds to TRAF6, is expressed in tooth germ, we examined whether TROY plays a critical role in tooth development associated with TRAF6.
We first determined the expression of TROY in tooth germs. RT-PCR analyses demonstrated the expression of TROY in tooth germs of E13 or 14 days mouse embryos. In addition, we also found that Nogo Receptor 66-1 and LINGO-1, both of which function as co-receptors of TROY, were expressed in tooth germs, suggesting an involvement of TROY in the tooth development. To understand the role of TROY in the tooth development, we generated soluble TROY (sTROY), which consists of only an extracellular domain of TROY, and thus is expected to function as a decoy receptor for TROY, and determined its effect on the development of tooth germs of mice in organ culture system. Treatment with sTROY clearly impaired growth of tooth germs. Furthermore, sTROY markedly inhibited expression of phenotypic markers of ameloblast such as ameloblastic amelogenin, and Sp6. These results suggest that TROY is involved in ameloblast differentiation. To determine downstream pathways of TROY involved in the tooth development, we next examined whether p38 and JNK pathways, which are well-known downstream pathways of TRAF6, are involved in the tooth development. We observed that p38 inhibitor (SB203580) and JNK inhibitor (SP600125) dramatically inhibited the development of tooth germs and reduced expression of ameloblastin at transcriptional level. In conclusion, our data suggest that TROY plays an important role in the tooth development, especially in ameloblast differentiation, presumably through p38 and JNK pathways.
Disclosures: N. Yamaguchi, None.
Sclerostin Overexpression Impairs Limb Patterning.N. M. Collette*1, R. M. Harland*2, G G Loots11Biosciences and Biotechnology Division, Lawrence Livermoe National Laboratory, Livermore, CA, USA, 2Department of Molecular and Cellular Biology, University of California, Berkeley, CA, USA.
Scterostin (SOST) is a negative regulator of bone formation that has been described as both a BMP- and WNT- antagonist. Loss of SOST function causes sclerosteosis (MIM 269500), a condition of severe progressive bone overgrowth. Using transgenic mice expressing human SOST from a bacterial artificial chromosome (BAC) we have demonstrated that SOST over-expression causes decreased bone formation and results in osteopenia, similar to the over-expression of other BMP antagonists such as Noggin and Gremlin. These mice also exhibit severe limb patterning defects that are dose-dependent and range from the loss of a single posterior digit to the loss and/or fusion of many distal limb skeletal elements. The apical ectodermal ridge (AER) relays cell-cell signals to the underlying differentiating limb mesenchyme, a place of SOST expression initiated as early as E9.5. To determine how elevated levels of SOST impair proper limb patterning, we have examined AER and cartilage markers by measuring mRNA levels by in situ hybridization. We find FGF8, a major AER signaling molecule and underlying mesenchymal markers BMP2, Glil, Paxl, HoxD12 and gremlin expression to be perturbed in SOST transgenic mice. In addition, skeletal analysis of double mutant animals show that over-expression of SOST fails to complement loss of BMP antagonists (Gremlin and Noggin) essential for limb patterning and chondrogenesis, resulting in more dramatic skeletal defects than single mutants alone, suggesting that SOST action in the limb parallels the BMP-pathway. This work is supported by NIH ROl HD047853 and the work has been performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory Contract No. W-7405-Eng-48. UCRL-ABS-229990
Disclosures: N.M. Collette, None.
This study received funding from: NIH R)l HD047853.
Lysophosphatidic Acid Induces Osteocyte Dendrite Outgrowth.S. A. Karagiosis*, N. J. Karin. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.
Osteocytes form an extensive mechanosensory network in bone matrix and communicate intercellularly via gap junctions established at dendrite termini. These membrane extensions are thought to be essential for osteocyte function, but the mechanisms that govern dendrite formation are not known. We previously demonstrated that osteoblasts, the progenitors of osteocytes, are highly responsive to the lipid growth factor lysophosphatidic acid (LPA). LPA-treated MC3T3-E1 osteoblastic cells developed membrane structures that resemble osteocyte dendrites [Masiello et al. (2006) Bone 39:72-82], and we predicted that LPA would promote osteocyte membrane outgrowth. We developed a method to measure osteocyte dendritogenesis in vitro using a modified transwell assay and determined that LPA is a potent stimulator of dendrite outgrowth in MLO-Y4 osteocytes. The stimulatory effects were dose-dependent with maximal outgrowth observed within a physiological range of LPA. The LPA-induced increase in dendrite formation was blocked by the specific LPA-receptor antagonist Ki 16425 and by pertussis toxin. LPA-treated osteocytes exhibited distinct rearrangements of the actin cytoskeleton and a more stellate morphology than control cells. These data are the first evidence for the regulation of dendrite formation by a soluble factor. Osteocytes in vivo are encased in bone matrix, rendering them non-motile. However, LPA promoted MLO-Y4 cell chemotaxis, suggesting that commonalities exist between the intracellular signaling pathways that regulate dendrite outgrowth and cell motility. Bone cells in vivo encounter platelet-derived LPA in regions of bone damage, and we postulate that this lipid factor is important for re-establishing osteocyte connectivity during fracture repair. We also found that MC3T3-E1 cells, primary mouse calvarial osteoblasts and MLO-Y4 osteocytes express autotaxin, a lysophospholipase that catalyzes the generation of LPA. This indicates that bone cells may be exposed to LPA under conditions other than the response to bone damage, and that this lipid growth factor may have broader roles in the control of skeletal homeostasis.
Disclosures: S.A. Karagiosis. None.
This study received funding from: U.S. Department of Energy.
TNF-alpha Upregulates Aortic BMP2-Msx2-Wnt Signaling in Diabetic LDLR-/- Mice.J. S. Shao, C. F. Lai, Z. Al-Aly*, J. Cai*, E. Huang*, S. L. Cheng, D. A. Towler. Dept. of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.
Aortofemoral calcification is prevalent in type II diabetes (T2DM), tracking metabolic syndrome parameters and increasing the risk for lower extremity amputation. LDLR-/-mice fed high fat diets (HFD) develop obesity, T2DM, and accumulate aortic calcium - the latter mediated via osteogenic mechanisms that resemble craniofacial mineralization. HFD upregulate aortic BMP2 and Msx2-Wnt signaling cascades that promote mineralization in neural crest-derived skeletal tissues. Administration of recombinant purified BMP2 augments aortic Msx2-Wnt signaling in TOPGAL reporter mice, and promotes aortic calcium deposition in LDLR-/- mice. Low-grade inflammation, including elevated circulating TNF-alpha, is characteristic of obesiry with T2DM, therefore, we examined the relationships between TNF-alpha and aortic BMP2-Msx2-Wnt signaling in the LDLR-/-model. HFD feeding promotes obesity, hyperglycemia, and hyperlipidemia - and upregulated serum TNF-alpha and haptoglobin in male LDLR-/- mice. Oxidative stress, reflected in serum 8-F-alpha-isoprostane (8-IsoP) levels, was increased, with concomitant upregulation of aortic BMP2 (2.5-fold), Msx2 (2.5-fold), Wnt3a (10-fold), and Wnt7a (14-fold) gene expression (all p < 0.01, 2-tailed t-test). Treatment of diabetic LDLR-/- mice with the TNF-alpha antagonist infliximab (10 ug / gm twice weekly, 5 animals per group) did not reduce obesity, hyperleptinemia, or hyperglycemia; however, serum 8-IsoP and haptoglobin levels were significantly decreased. Moreover, aortic BMP2, Msx2, Wnt3a, and Wnt7a expression and aortic calcium accumulation were concomitantly and significantly reduced by infliximab treatment (all p < 0.05). Administration of sodium salicylate, an alternative anti-inflammatory strategy, also reduced serum haptoglobin and aortic Msx2 gene expression. Finally, C57BL/6 mice with arterial smooth muscle TNF-alpha expression augmented by a SM22- TNF-alpha transgene accumulate significantly higher levels of aortic BMP2, Msx2, Wnt3a, and Wnt7a mRNAs vs. their non-transgenic sibling cohorts Furthermore, 80% of SM22-TNFalpha; TOPGAL mice exhibit aortic beta-galactosidase reporter staining vs. 0% of their non-transgenic TOPGAL siblings (p=0.05 with Yates' chi-square correction), indicating net enhanced mural Wnt signaling by the SM22-TNFalpha transgene. Thus, inflammatory TNF-alpha signals promote pro-calcific BMP2-Msx2-Wnt programs in the aortic tissues of diabetic LDLR-/- mice. Strategies that inhibit inflammation-induced arterial BMP2-Msx2-Wnt signals may improve aortofemoral physiology and reduce lower extremity amputation risk in T2DM.
Disclosures: D.A. Towler, National Institutes of Health 2: Barnes-Jewish Hospital
Foundation 2: Wyeth 5: GlaxoSmithKline 5.
This study received funding from: National Institutes of Health.
1GF-I Engineered Bone Marrow Mesenchymal Stem Cells Improve the Fracture Healing Process.F, Granero-Molto1, J, A, Weis*1, L. D, O'Rear*1, M. I. Miga*2, A. Spagnoli11Pediatrics, Vanderbilt University, Nashville, TN, USA, 2Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
Healing failure occurs in 20% of the fractures The reasons why fractures do not heal are mostly unknown Limitations in adult stem cells (MSC) and growth factor imbalance play critical roles The purpose of this study is to determine the regenerative ability of bone marrow (BM) MSC engineered to express 1GF-I in a rodent fracture healing model MSC, isolated from BM of syngenic FVB male mice, were retrovirally infected and selected either to stably express human des-IGF-I (MSC-IGF-I) or empty vector (MSC) Syngenic female mice (8-10 weeks old) were subjected to a three point bending stabilized tibia fracture and then transplanted either with 106 MSC-IGF-I or MSC by IV tail injection Fractured females were also used as controls (no cells) Fractured tibias were analyzed longitudinally by μCT scan (Scanco 40 urn) Fracture mechanoproperties were determined by distraction biomechamcal testing (BMT) (ELF 3100, Bose) Callus cross-sections were subjected to histological and in-situ hybridization studies for cartilage and bone markers As shown in Table I, fractured mice transplanted with MSC-IGF-I showed a significant improvement of the biomechamcal properties of the callus with an increase m peak force, modulus of elasticity and energy to peak compared to controls.
As determined by μCT analyses, fractured mice transplanted with MSC-IGF-I showed a significant increase in total, new high and low mineralized bone volumes (Table II).
In-situ hybridization showed that in mice transplanted with MSC-IGF-I, the increase in biomechamcal properties and bone callus volumes was associated with an increase of collagen-1 and 10 expression We conclude that MSC-IGF-I transplant improves fracture repair increasing the fracture mechanoproperties, bone volumes and bone/cartilage gene expression Our studies provide critical data to implement a novel therapeutic approach in patients with fracture healing failure transplanting MSC engineered to express IGF-I.
Disclosures: F. Granero-Molto, None.
This study received funding from: NIH-NIDDK
E-Selectin Ligand 1 Negatively Regulates TGFβ in the Golgi During Skeletogenesis.T. Yang*1, R. Mendoza-Londono*1, H. Lu*1, K. Li*1, B. Keller*1, M. Jiang*1, Y. Chen*1, T. Benin*1, B. Dabovic*2, D. B. Rifkin*3, J. Hick*4, A. L. Beaudet*1, B. Lee11Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 2Department of Cell, New York University Medical Center, New York, NY, USA, 3New York University Medical Center, New York, NY, USA, 4Dept. Pathology, Baylor College of Medicine, Houston, TX, USA.
Transforming growth factor β (TGFβ) signaling is critical for the regulation of growth and differentiation during development and disease Its context dependent action is specified by numerous control mechanisms at the extracellular level and downstream of ligand-receptor interactions, but little is known about the regulation of its post-translational trafficking E-Selectin Ligand-1 (ESL-1), the cysteine rich protein originally isolated as a ligand for E-Selectin, was found to interact with FGFs and to be co-punfied with TGFbl in a large protein complex To elucidate the in vivo function of ESL-1, we mutated Esl-1 gene in mice by conventional knock-out strategy The newborn and adult Esl-1−/− mice are notably smaller with narrow chests and generalized shortening and thinning of the long bones, nbs and spine By histological analysis, PI Esl-1−/− mice showed shortening of the growth plates in both the proliferating zone and hypertrophic zone Further molecular biology assays show that ESL-1 acts as a negative regulator of TGFβ production by binding TGFβ precursors in the Golgi in a cell autonomous fashion In vivo, loss of ESL-1 function causes increased TGFβ signaling resulting in decreased cell proliferation and delayed terminal differentiation in the cartilaginous growth plate, independent of effects on BMP and FGF signaling Moreover, in vivo genetic models of gain vs loss of TGFβ signaling in the growth plate confirm this effect These data identify a novel cellular mechanism for regulating TGFβ during skeletogenesis and cartilage homeostasis.
Disclosures: T. Yang, None.
This study received funding from: Arthritis Foundation.
Klotho Ablation Completely Reverses the Biochemical and Skeletal Alterations in FGF23(R176Q) Transgenic Mice.X. Bai, H. Fu*, D. Oiu*, D. Goltzman, A. C. Karaplis. Department of Medicine, McGill University, Montreal, PQ, Canada.
Recently, we described the generation of a murine model of FGF23(R176Q) overexpression (FGF23Tg) and the associated biochemical and bone histological changes consistent with abnormal vitamin D metabolism and rickets. In this study, we have sought to use the mouse genetic approach to clarify the role of Klotho in the metabolic derangements associated with FGF23(RI76Q) overexpression. To this end, we have crossed mice heterozygous for the hypomorphic Klotho allele (Kl−/−+/−) and Fgf23Tg mice to obtain Fgf23Tg transgenic mice homozygous for the Kl−/−-hypomorphic allele (Fgf23Tg/Kl−/−−/−). Mice were sacrificed on day 50 post partum and serum, urine, and tissues were procured for analysis and comparison to Fgf23Tg WT, and Kl−/−−/− controls.
From 4 weeks onward, Fgf23Tg/Kl−/− mice were clearly distinguishable from Fgf23Tg mice and exhibited a striking phenotypic resemblance to the Kl−/− controls. Their average body weight was 12.5 g ± 0.7 (N=12), similar to that of Kl−/− mice (12.7 g ± 0.6; N=12) and significantly different from Fgf23Tg (18.6 g ± 0.3; N=12) and WT controls (23.5 g ± 0.5; N= 12). Similarly, their average life span was dramatically diminished compared to Fgf23Tg mice (59.6 days vs. ∼2 years) but comparable to that of Kl−/− mice (62.4 days).
Serum analysis for calcium (12.04 ± 0.30 vs. 11.19 ± 0.12 vs. 10.27 ± 0.11 mg/dl), phosphorus (13.74 ± 0.66 vs. 15.87 ± 0.44 vs. 6.43 ± 0.27 mg/dl), PTH (46.67 ± 0.58 vs. 48.24 ± 1.19 vs. 84.23 ± 11.37 pg/ml), 1,25(OH)2 vitamin D3 (164.30 ± 13.71 vs. 159.3 ± 14.11 vs. 73.33 ± 8.49 pg/ml), and alkaline phosphatase activity (163.30 ± 15.59 vs. 125.40 ± 26.20 vs. 611.3 ± 66.04 IU/L) further confirmed the serum biochemical resemblance between the Fgf23Tg/Kl−/− and Kl−/− mice and their distinct difference from that of Fgf23Tg controls, respectively. Serum intact FGF23 levels also paralleled this pattern (371,500 ± 16,500 vs. 336,000 ± 32,300 vs. 2,544 ± 463 pg/ml) among animals of the three genotypes. The characteristic bony changes associated with FGF23(R176Q) overexpression were also dramatically reversed by the absence of Klotho. Hence, frontal view of 3D reconstructed proximal end of Fgf23Tg/Kl−/− tibia obtained using microCT showed complete reversal of the wide, unmineralized growth plate observed in the Fgf23Tg mice. Laproved skeletal mineralization was also confirmed on histological sections of the Fgf23Tg/Kl−/− epiphyseal region of femurs with complete absence of the profound osteomalacic changes apparent in trabecular and cortical bone in animals expressing FGF23(RI76Q).
In summary, our findings confirm the pivotal role of Klotho in the mechanism of action of FGF23 as its concomitant ablation fully reverses the complete spectrum of biochemical and skeletal disturbances attributed to FGF23.
Disclosures: X. Bai. None.
l,25(OH)2D3 Inhibits Bone Nodule Mineralization Through the FGF23-Mediating ERK Pathway in Rat Calvaria Osteoblast Cultures.T. Minamizaki*1, Y. Yoshiko1, H. Wang*1, S. Suzuki*1, K. Kozai*2, J. E. Aubin3, N. Maeda*11Oral Growth & Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan, 2Pediatric Dentistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan, 3Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada.
Vitamin D, or its active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), plays a major role in the regulation of mineral homeostasis and affects bone metabolism, but separating direct effects on osteoblasts and bone from indirect effects via other cell types and organs has been difficult in humans and animal models. However, osteoblasts possess a specific receptor for l,25(OH)2D3 and l,25(OH)2D3 affects many osteoblast functions including proliferation, extracellular matrix synthesis, etc. In the well-established rat calvaria (RC) osteoblast model, we found that l,25(OH)2D3 inhibits bone nodule mineralization, concomitant with upregulation of production of fibroblast growth factor (FGF)23, a phosphate-regulating factor responsible for several phosphaturic disorders; we also found that overexpression of FGF23 in RC cells inhibits bone nodule mineralization. We therefore hypothesized that FGF23 may be involved in 1,25(OH)2D3-dependent mineralization defects in the RC osteoblast model. To test this hypothesis, we first confirmed that RC osteoblasts expressed Klotho, a senescence-related protein and FGF receptor (FGFR)s, which together are necessary for FGF23-specific signaling. l,25(OH)2D3 (0.1∼10 nM) upregulated FGF23 production in nodule-forming osteoblastic cells but not proliferating progenitor cells, and this activity was enhanced by 10 mM β-glycerophosphate, a stimulator of mineralization. We next prepared conditioned media from 10 nM l,25(OH)2D3-treated (CM-D; FGF23, ≥10,000 pg/ml) and -untreated (CM-C; FGF23, approx. 20 pg/ml) RC osteoblast cultures. As predicted, CM-D but neither CM-C nor 1,25(OH)2D3 at reduced concentrations in CM-D (≤0.4 nM) inhibited bone nodule mineralization in a dilution dependent manner (1:50–1:200). Neutralizing anti-FGFR antibody, SU5402 (an inhibitor of tyrosine kinase activity of FGFR1) and U0126 (an inhibitor of extracellular signaling-related kinase (ERK)) all rescued the mineralization defects seen in both CM-D and l,25(OH)2D3 (10 nM)-treated RC osteoblast cultures. Theses effects were, at least in part, mimicked by either small interfering (si)RNA knockdown of FGF23 or siRNA knockdown of Klotho. Thus, our observations suggest that 1,25(OH)2D3 inhibits bone nodule mineralization through an FGF23-induced ERK pathway in RC osteoblast cultures.
Disclosures: T. Minamizaki, None.
FGF-7 Is a Potent In Vivo Phosphaturic Agent in Rats.A. Shaikh*, T. Berndt*, R. Kumar. Nephrology Research, Mayo Clinic Rochester, Rochester, MN, USA.
FGF-7 has been identified as a tumor-derived phosphatonin on the basis of its ability to inhibit Na+ dependent phosphate uptake in cultured renal epithelia. Its bioactivity in vivo is unknown. The purpose of this study was to compare the phosphaturic effect of fibroblast growth factor-7 (FGF-7) to that of FGF-23 in rats with intact parathyroid glands. Rats were anesthetized and prepared for clearance studies to determine the glomerular filtration rate and fractional excretion of phosphate following infusion of protein or vehicle. After a 90-minute stabilization period, a control 30-minute clearance sample was collected and then either vehicle (saline, n=8), recombinant FGF-7 (0.1 nmol/kg/hr, n=4) or recombinant FGF-23 (1 nmol/kg/hr, n=8) were infused intravenously. Glomerular filtration rate remained stable in all groups. The fractional excretion of phosphate (FEP) was unchanged in rats receiving vehicle alone (control vs. experimental period, 21±4 to 26±4%). Infusion of full length FGF-23 at a dose of 1 nmol/kg/hr increased FEP from 14±3% to 32±5%. Infusion of 0.1 nmol/kg/hr of FGF-7 increased FEP from 25±7% to 42±5%.
We conclude that FGF-7 increases phosphate excretion in vivo at a 10-fold lower dose than FGF-23. FGF-7 is likely to be responsible for the abnormal phosphate metabolism seen in some patients with tumor-induced osteomalacia in which tumors elaborate FGF-7 but not FGF-23.
Disclosures: A. Shaikh, None.
The Role of Npt2c in Renal Pi Reabsorptive Process of Npt2a KO Mice.Y. Tomoe*, H. Segawa*, S. Sugino*, M. Ito*, S. Tatsumi, M. Kuwahata*, K. Miyamoto. Molecular Nutrition, Health Biosciences. The University of Tokushima Graduate School, Tokushima, Japan.
The proximal tubule is the major site of renal Pi reabsorption. The rate-limiting step in Pi reabsorption is a sodium-dependent step that occurs in the luminal brush border membrane (BBM). Two transporters (Npt2a and Npt2c) are expressed exclusively in the proximal convoluted tubule and are regulated by Pi intake, PTH, and FGF23 levels. The type IIa Na/Pi cotransporter (Npt2a) play a major role in reabsorption (70–80%), while type IIc Na/Pi transporter (Npt2c) is more important in weaning animals and less so in adult animals. Recently, mutation of the human Npt2c gene has shown to cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), implicating that the NPT2c plays an important role in renal Pi reabsoiption in human and may be a key determinant of plasma Pi concentration. In this study, we examined administration of FGF23, which is a strong Pi diuresis factor, to Npt2a KO mice to determine the role of Npt2c in renal Pi reabsorptive process using naked DNA methods. As described previously, the levels of plasma FGF23 concentration were kept extremely low in the Npt2a KO mice when compared with wild-type (WT) mice. The levels of plasma FGF23 protein was markedly increased in Npt2a KO mice injected FGF23R179Q. FGF23R179Q induced severer hypophosphatemia, and markedly deceased renal Na/Pi transport activity, and Npt2c protein levels in Npt2a KO mice when compared with WT mice (FGF23R179Q group) 4 days after administration. FGF23R179Q suppressed the levels of intestinal Na/Pi transport activity in WT mice. However, intestinal Na/Pi transport activity were similar when compared Npt2a KO (FGF23R179Q group), and Npt2a KO (Mock group). These results suggested that Npt2a KO mice retain the capacity to reabsorb Pi at a rate that can be explained by the presence of Npt2c. Renal Npt2c is a major Na/Pi transporter in Npt2a KO mice.
Disclosures: Y. Tomoe, None.
Protein-PTH mRNA Interactions that Determine PTH mRNA Stability in Secondary Hyper- and Hypoparathyroidism.M. Nechama*, I. Z. Ben-Dov*, J. Silver, T. Naveh-Many. Minerva Center for Calcium and Bone Metabolism, Hadassah Hebrew University Medical Center, Jerusalem, Israel.
Hypocalcemia, uremia and hypophosphatemia regulate PTH gene expression, PTH secretion and parathyroid cell proliferation. Gene expression can be regulated at the transcriptional or post-transcriptional level. Serum Ca2+ and P regulate PTH gene expression post-transcriptionally by changes in mRNA stability. A low serum Ca2+ and uremia result in increased protein-PTH mRNA binding of a stabilizing complex and increased PTH mRNA levels in vivo. A low P has the opposite effects. We have previously identified two trans acting proteins, AU rich binding factor 1 (AUF1) and Upstream of N-ras (Unr) that bind a specific conserved cis element in the PTH mRNA 3′- untranslated region (UTR) and stabilize PTH mRNA. Here we use both in vitro degradation assays with rat parathyroid extracts and transfected heterologous cells to characterize the mechanisms of PTH mRNA decay and to identify the ribonucleases that determine PTH mRNA stability. mRNA decay can be exonucleolytic from either the 5′ or 3′ end of the mRNA or endonucleolytic by cleavage in the mRNA body. We show that PTH mRNA is endnucleolytically cleaved by parathyroid extracts and is a substrate for the endonuclease PMR1. PMR1 co-immunoprecipitates with the 3′ to 5′ exonucleolytic complex, the exosome. Knock-down of an exosome component by siRNA prevents PMR1-mediated degradation of PTH mRNA. Similarly, exosome immunodepletion in parathyroid extracts prevents PTH mRNA endonucleolytic degradation. PMR1 also interacts with the mRNA decay-promoting protein KSRP that binds PTH mRNA and decreases PTH mRNA levels in transfected cells. Significantly, the association of KSRP with PTH mRNA is increased in parathyroid glands from rats fed a phosphorus restricted diet where PTH mRNA is unstable, compared to parathyroid glands from rats fed a calcium-restricted diet or uremic rats, where PTH mRNA is stable. We suggest that KSRP recruits a degradation complex to degrade PTH mRNA and determine PTH mRNA stability and levels in response to changes in Ca2+ and P. Phosphorous restriction increases PTH mRNA association with KSRP generating a degradation complex that accelerates PTH mRNA decay and decreases PTH mRNA levels. Calcium depletion and uremia lead to formation of a stabilizing complex on PTH mRNA, consisting of AUF1 and Unr. This complex competes for the association of PTH mRNA with the degrading complex and leads to increased PTH mRNA stability and levels. This is the first identification of the molecular mechanisms whereby protein-RNA interactions determine PTH mRNA stability and levels in response to changes in serum Ca2+ and P and experimental uremia.
Disclosures: T. Naveh-Many, None.
The Calcium-Sensing Receptor Regulates PMCA2 Activity in Mammary Epithelial Cells: A Mechanism for Calcium-Stimulated Calcium Transport into Milk.J. N. VanHouten, J. J. Wvsolmerski. Yale University, New Haven, CT, USA.
Milk contains a great deal of calcium. Mammary epithelial cells secrete calcium into milk via a high capacity transepithelial transport system that is only partly understood. Prior experiments have demonstrated that calcium can stimulate its own transport from the maternal circulation into milk. Activation of the calcium-sensing receptor (CaR) at the basolateral surface of mammary epithelial cells promotes transepithelial calcium transport and also stimulates bulk milk production in mice. In this study, we investigated the mechanism by which CaR signaling affects calcium transport across mammary epithelial cells. The plasma membrane calcium ATPase, isoform 2 (PMCA2), has been reported to be the major calcium pump responsible for the transport of calcium into milk. We confirmed that expression of the PMCA2bw splice variant is upregulated in the mammary gland during lactation. Immunofluorescence and immuno-electron microscopy demonstrated that PMCA2 is found only at the apical surface of mammary epithelial cells. Milk calcium was reduced by 70% in homozygous Dfw-2J mice, which carry a loss-of-function mutation in the PMCA2 gene (Atp2b2). These data confirm that PMCA2 is critical for calcium transport into milk. Therefore, we sought to determine whether PMCA2 is a downstream target of CaR signaling in mammary epithelial cells. In cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation by extracellular calcium or the calcimimetic, gadolinium, had no discernable effect on PMCA2 expression, mRNA splicing, or localization. However, activation of the CaR increased calcium-dependent ATPase activity in mammary epithelial cell plasma membrane preparations. Knockdown of PMCA2 expression with siRNA showed that PMCA2 accounts for the preponderance of calcium-ATPase activity in mammary epithelial cells. Furthermore, siRNA-mediated ablation of CaR expression in mammary epithelial cells eliminated the ability of extracellular calcium or gadolinium to elicit an increase in plasma membrane calcium-dependent ATPase activity. These results demonstrate that activation of the CaR on die basolateral aspect of mammary cells increases PMCA2 activity in the apical membrane and provide a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland.
Disclosures: J.N. VanHouten, None.
Conditional Ablation of the Osteoblast Calcium-Sensing Receptor Causes Abnormalities in Skeletal Development and Mineralization.M. M. Dvorak1, C. Tu*1, H. Elalieh*1, T. Chen*1, B. Liu*1, B. E. Kream*2, D. D. BiKle1, W. Chang1, D. M. Shoback11Department of Medicine, University of California, San Francisco, CA, USA, 2Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA.
The calcium-sensing receptor (CaR) is a G protein-coupled receptor essential for maintenance of calcium homeostasis. Defining the role for the osteoblast CaR in vivo, using global full-length CaR knockout models, is complicated by metabolic disturbances and the potential for compensation by a CaR splice variant identified in these mice. To circumvent these issues, we generated Flox-CaR+/+ mice, in which loxP sites flank exon 7 that encodes the transmembrane and signaling domains of the receptor. Osteoblast-specific inactivation of the CaR was achieved by mating Flox-CaR+/+ mice to transgenic mice expressing Cre-recombinase, under control of the 3.6 kb fragment of the rat al(I) collagen promoter (Coll-Cre). Coll-Cre+/-/Flox-CaR+/+ mice exhibited growth delay from birth and died within four weeks. At 21 days, the skeletal phenotype was hallmarked by hypomineralization, evident by von Kossa staining and micro-computed tomography (μCT) analysis, which revealed significant reductions in bone volume/tissue volume (BV/TV; ↓ ∼75%), bone mineral density (BMD; ↓>90%), segmented BMD (↓ ∼20%), trabecular number (↓ ∼ 40%), trabecular thickness (↓ ∼ 30%) and connectivity density (↓ ∼80%) in the secondary spongiosa of the distal femur (n=6, p<0.05, ANOVA), compared to controls. The changes were comparable, although less extensive in the L4 vertebra. The cortical compartment of the femur was also affected, with significant decreases in BV (↓ ∼60%), cortical thickness (↓ ∼70%), BMD (↓ ∼45%), and segmented BMD (↓ ∼15%) and markedly increased cortical porosity (∼70%), compared to controls (n=6, p<0.05, ANOVA). Histology of femora from Coll-Cre+/-/Flox-CaR+/+ mice revealed severe hyperosteoidosis (Goldner staining) as well as trabeculation of the cortex (Von Kossa staining). Reduced mineral content could be secondary to increased turnover, with inadequate mineralization of newly made osteoid. This is consistent with quantitative realtime PCR (qPCR) analysis of humeri and calvaria that indicate significant increases in markers of proliferation (ccndl), osteoblast differentiation (collagen I, alkaline phosphatase, osteopontin), mineralization (ankylosis protein, ectonucleotide pyrophosphatase/phosphodiesterase 1) and regulators of osteoclastogenesis (RANK-L, osteoprotegerin); (n=3–5, p<0.05, ANOVA). Our findings indicate a critical role for the skeletal CaR in the control of bone mineralization in early postnatal skeletal development.
Disclosures: M.M. Dvorak, None.
Calcium Sensing Receptor Plays Significant Role in Protection Against Hypercalcemia.L. Kantham1, S. Ouinn*1, O. Egbuna1, J. Pang*1, R. Butters*1, M. Pollak*2, E. M. Brown11Medicine / Endocrine, Brigham & Women's Hospital, Boston, MA, USA, 2Medicine / Renal division, Brigham & Women's Hospital, Boston, MA, USA.
The calcium sensing receptor (CaR) plays a pivotal role in controlling the secretion of parathyroid hormone (PTH), which, in turn, via its direct and indirect actions on kidney, bone and intestine restores and maintains normal extracellular ionized calcium levels. To date, there is only limited understanding of the functions of CaR expressed in tissues other than the parathyroid. Mice homozygous for knockout of the CaR were unsuitable for such studies as they die from lethal hyperparathyroidism. As an alternative, we employed single and double knock out (KO) mouse models, specifically mice lacking PTH alone (CaR+/+PTH−/−), both CaR and PTH (CaR−/− PTH−/−), and wild type (CaR+/+PTH+/+) of the same genetic background for studies to gain insight into CaR-specific functions in the absence of confounding actions of CaR-mediated changes in PTH.
In the first set of experiments, 3–4 month old adult male mice were fed ad lib with regular chow containing 0.8% calcium and 0, 1 or 2% CaCl2 containing water for one week. Measurement of serum calcium (SCa) levels revealed marked differences between the three genotypes. As expected, CaR+/+PTH+/+ mice maintained their SCa levels close to 9.5 mg/dL under all conditions. The CaR+/+PTH−/− and CaR−/− PTH−/− mice were hypocalcemia showing SCa of 6.8 and 6.2 mg/dL, respectively, in the absence of added calcium in the water. However, the SCa level rose to 8.3 in the CaR+/+PTH−/− mice and to over 10 mg/dL in the CaR−/−PTH−/− mice when they received water containing 1% CaCl2. On 2% CaCl2 water, the CaR+/+PTH−/− mice showed a SCa (9.5 mg/dL) in the normal range. In contrast the SCa increased markedly to 14 mg/dL in the CaR−/−PTH−/− mice. The results indicate that despite the lack of PTH, CaR is able to defend the mice against hypercalcemia in the face of an increased oral load of calcium. It is known that severe phosphate deficiency induces hypercalcemia and we tested the response of these mice to a phosphate deficient diet (containing 1% calcium) and plain water for one week. Once again we noted significant differences between mice expressing the CaR and those that did not. The wild type and CaR+/+PTH−/− mice maintained near normal SCa levels of 9–10 mg/dL, whereas those in the CaR−/−PTH−/− mice reached 15 mg/dL. Further investigation revealed that the calcitonin response to hypercalcemia induced by increased oral calcium load was normal in the control mice but was severely blunted in the CaR−/−PTH−/− mice. Thus the CaR defends effectively against hypercalcemia induced by a calcium load even in the absence of CaR-mediated changes in PTH secretion. Ongoing studies are directed at further characterization of the mechanism(s) by which it does so.
Disclosures: L. Kantham, None.
The Small GTPase RhoA and its Effector Kinase ROCK Mediate Actin Cytoskeleton Reorganization Leading to Osteocyte Anoikis by Glucocorticoids.L. I. Plotkin, K. VYas*, S. C. Manolagas, T. Bellido. Center for Osteoporosis and Metabolic Bone Diseases, Univ Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA.
Glucocorticoids (GC) induce osteocyte apoptosis by activating the focal adhesion-related kinase Pyk2 and its downstream target, the kinase JNK, which in turn counteract pro-survival signaling mediated by the focal adhesion kinase FAK. This results in osteocyte detachment-induced apoptosis or anoikis. We report herein that the retraction of osteocyte cytoplasmic processes and cell rounding that precede GC-induced apoptosis of osteocytic MLO-Y4 cells are associated with rapid disruption of stress fibers and formation of peripheral actin rings, as visualized by confocal microscopy of cells stained with Alexa Fluor-conjugated phalloidin. The small GTPase family of proteins has been shown to transduce extracellular signals into actin cytoskeleton reorganization, ultimately controlling cell morphology and diverse cellular functions, including apoptosis. Consistent with a role of small GTPases in GC actions on osteocytes, the GC-induced apoptosis, changes in cell shape as well as actin reorganization were abolished by pharmacological inhibition of the small GTPase RhoA or its effector kinase ROCK with p75NTR or Y-27632, respectively. On the other hand, inhibition of another small GTPase Racl with NSC23766 did not prevent GC effects. Furthermore, whereas GC did not induce apoptosis in cells expressing a kinase deficient dominant negative Pyk2 (K- Pyk2), expression of a constitutively active (ca) RhoA mutant, as well as two different ca JNK mutants, rescued GC-induced apoptosis in cells expressing K- Pyk2. These results indicate that Pyk2 activation by GC results in activation of both RhoA and JNK. Moreover, the ca RhoA rescued GC-induced apoptosis in cells expressing a dn JNK mutant, whereas the ca JNK mutants were unable to rescue GC-induced apoptosis in cells treated with the ROCK inhibitor. These results indicate that activation of JNK precedes RhoA/ROCK activation. We conclude that the small GTPase RhoA and its downstream kinase ROCK act downstream of Pyk2 and JNK activation by GC in a pathway that leads to rapid reorganization of the actin cytoskeleton. The resulting changes in cell morphology lead to osteocyte detachment-induced apoptosis or anoikis.
Disclosures: L.I. Plotkin, None.
This study received funding from: N1H.
Col3.6-HSD2 Transgenic Mice: A Glucocorticoid Loss-of-Function Model Spanning Early and Late Osteoblast Differentiation.M. Yang*1, J. R. Harrison2, P. J. Adams3, B. E. Kream11Medicine, University of Connecticut Health Center, Farmington, CT, USA, 2Craniofacial Sciences, University of Connecticut Health Center, Farmington, CT, USA, 3Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT, USA.
Glucocorticoids (GC) exhibit complex regulatory effects on bone remodeling. To elucidate the role of endogenous GC signaling in mature osteoblasts, we previously developed Col2.3-HSD2 transgenic (TG) mice in which a 2.3-kb Collal promoter fragment drives expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which metabolizes biologically active GC. Col2.3-HSD2 TG mice showed decreased vertebral trabecular and femoral cortical bone mass, due in part to impairment of osteoblast differentiation. The goal of the present study was to characterize the bone phenotype of Col3.6-HSD2 TG mice in which a longer 3.6-kb Collal promoter fragment drives 11β-HSD2 expression more widely in the osteoblast lineage. Serum corticosterone was unchanged in 7-week-old TG females. Skeletal parameters were assessed by dual energy X-ray absorptiometry and microcomputed tomography. Compared to WT littermates, TG mice showed reductions of 20% in vertebral trabecular bone volume, 10–17% in femoral and tibial sub-periosteal area, and 20–40% in sub-endosteal area. TG calvarial osteoblast (mCOB) and bone marrow stromal cell (BMC) cultures had decreased alkaline phosphatase and mineral staining, and reduced pOBCol3.6-GFP transgene expression, a marker of early osteoblast differentiation. Northern blot and real time PCR showed that Collal, bone sialoprotein and osteocalcin expression were decreased by 56%, 73% and 72%, respectively, in TG cultures. To study osteoclast formation, BMCs were treated with RANKL and mCSF for 3-7 days. Col3.6-HSD2 TG cells showed lower osteoclast number at day 4, but higher osteoclast number at day 6 and 7, suggesting delayed osteoclast formation and/or suppression of osteoclast apoptosis. To determine gene pathways affected by 11β-HSD2 transgene, RNA of 7-week-old WT and TG female vertebrae was assessed by microarray analysis. Modest (1.5- to 2-fold) but significant effects were seen on genes of the TGFβ, IGF and FGF pathways. TG bone had increased cathepsin K and c-fms expression, markers of the osteoclast lineage. In summary, Col2.3-HSD2 and Col3.6-HSD2 mice showed a similar low bone mass phenotype and impaired ex vivo osteoblast differentiation; in addition, Col3.6-HSD2 mice showed an altered tempo of ex vivo osteoclast formation. Based on the phenotypic similarities between the Col2.3-HSD2 and Col3.6-HSD2 models, we conclude that endogenous GC signaling is required for optimal bone mass acquisition by acting primarily on the later stages of osteoblast differentiation.
Disclosures: M. Yang, None.
This study received funding from: National Institutes of Health.
Intermittent PTH Stimulates Osteoblastic Cells Even in the Absence of Osteoclasts.P. H. Luiz de Freitas1, M. Li2, T. Ninomiva*3, M. Nakamura*3, K. Oda*4, R. Takagi*1, N. Udagawa*3, T. Maeda*5, N. Amizuka21Oral and Maxillofacial Surgery, Niigata University, Niigata, Japan, 2Center for Transdisciplinary Research, Niigata University, Niigata, Japan, 3Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan, 4Biochemistry, Niigata University, Niigata, Japan, 5Oral and Maxillofacial Anatomy II, Niigata University, Niigata, Japan.
PTH anabolic actions affect both components of the bone remodeling cycle. We aimed to determine if the increase on osteoblastic activity seen after intermittent PTH treatment in vivo is solely result of the hormone's actions on osteoblasts, or if it also depends on coupling to, and/or enhancement of, osteoclastic activities. Eight-weeks-old ICR male mice were divided in control and PTH injection groups. Age-matched c-fos knockout female mice, an osteopetrotic strain without osteoclasts, were similarly grouped. Injection groups received PTH (120μg/kg, sc) daily for 14 days, while controls received only saline. Under anesthesia, animals were fixed with an aldehyde solution and had their bones extracted for histological processing, i.e., tartrate-resistant acid phosphatase (TRAP) reaction, alkaline phosphatase (ALP) and PTH/PTHrP receptor (PTH-R) immunohistochemistry, as well as for transmission electron microscopy (TEM). The picture of PTH treatment in ICR mice was one of increased bone content with longer, thicker and interconnected trabeculae. Compared to their controls, injection samples had a thicker ALP-positive preosteoblastic layer lining their bone surfaces. Number of TRAP-positive cells did not differ considerably between control and injection groups, while PTH-R positivity was increased in the latter. TEM imaging revealed at least two categories of cells of the preosteblastic layer according to their morphology: endoplasmic reticulum (ER)-rich cells and ER-poor ones. Collated to their controls, PTH-injected c-fos knockouts exhibited more ALP-positive cells on their bone surfaces. It has been suggested that osteoclastic cells may play an active role on the anabolic actions of PTH, but our data pointed to another direction: PTH-driven osteoblastic anabolism seems to be independent of osteoclast presence and/or activity, yet the outcomes of hormonal administration might be less dramatic when bone resorption is hindered. We have shown that intermittent PTH stimulates cells of the osteoblastic line even if osteoclasts are ablated, substantiating the idea that anabolic effects of PTH are independent of osteoclastic presence.
Disclosures: P.H. Luiz de Freitas, None.
Ablation of IGF-I Signaling Disrupts the Communication Between Osteoblasts and Osteoclasts.Y. Wang1, H. Z. ElAlieh*1, W. Chang1, T. L. Clemens2, D. D. BiKle11Endocrine Unit, University of California, San Francisco/ VA Medical Center, San Francisco, CA, USA, 2Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.
We and others have shown that insulin-like growth factor-I (IGF-I) has been regulates the functions of both osteoblasts (OB) and osteoclasts (OCL). Our previous studies demonstrated that IGF-I was required for normal OBs proliferation, differentiation and stimulated osteoclastogenesis both directly and/or through the interaction between OBs and OCL precursors to support OCL formation. Furthermore, IGF-I signaling is required for FTH stimulation of bone formation, an action that appears to require the OCL as well as IGF-I. However, the mechanisms by which IGF-I signaling is responsible for the cellular communication between OBs and OCLs has not been evaluated. Recent studies identified bidirectional signaling between OBs and OCLs mediated by ephrin B2 in OCLs and EphB4 in OBs. To address whether such signaling was involved in IGF-I action, we used Cre-mediated conditional gene targeting techniques to delete IGF-I receptor (IGF-1R) in vitro and in vivo. Bone marrow stromal cells (BMSC) were isolated from the mice bearing the loxP-flanked IGF-I receptor (IGF-IR) alleles in exon 3 (IGF-IR flox mice). To inactivate IGF-IR, BMSCs were infected with 10 PFU/cell recombinant adenoviruses expressing a functional Cre (Cre-ADV) at day 12 for 2 days. Quantitative real-time PCR (Q-PCR) analysis demonstrated that the mRNA levels of IGF-IR in the BMSCs were reduced by 95% with Cre-ADV infection. mRNA levels of ephrin B2 and Eph B4 were decreased by 78% and 71%, respectively in the cultures infected by Cre-ADV (IGF-IR−/−). PTH treatment (100 ng/ ml, 2 hrs) significantly increased the mRNA levels of both ephrin B2 (2.6 fold) and EphB4 (4 fold) in the in the cultures infected by vehicle (IGF-IR+/+), but these effects were abolished in the IGF-IR−/− cultures. For the in vivo studies, 3 month old mice with a bone specific IGF-IR null mutation (OB IGF-IR−/−) (floxed IGF-IR × osteocalcin promoter driven Cre recombination) and their normal littermates (controls) were treated with vehicle or PTH (80 μg/kg bw/day for 2 weeks). As analyzed by Q-PCR, mRNA levels of ephrin B2 and EphB4 were decreased by 44% and 48% respectively in bone tissue of the OB IGF-IR−/− mice. PTH significantly increased the mRNA levels of ephrinB 2 and EphB 4 in the control mice (1.6 fold and 3.5 fold, respectively), but induced no changes in the mRNA levels of these 2 molecules in the OB IGF-IR−/− mice. Our data indicate that the IGF-IR is critical for the communication between OBs and OCLs mediated by bidirectional ephrin signaling to stimulate OBs differentiation and osteoclastogenesis, suggest that this action is required for PTH stimulated bone turnover.
Disclosures: Y. Wang, None.
Androgenic Effects on Bone and Body Composition Depend on Concerted and Additive Activation of both AR and ERα: Evidence from a New Male Mouse Model with Combined Disruption of both AR and ERα.F. Callewaert*, K. Venken*, J. Ophoff*, K. De Gendt*, S. Boonen, R. Bouillon, G Verhoeven*, D. Vanderschueren. Katholieke Universiteit Leuven, Leuven, Belgium.
During puberty, androgens increase bone and muscle mass but lower fat mass. Both androgen receptor (AR) and estrogen receptor-α (ERα) activation are likely to be involved in these characteristic male phenotypic changes, but the relative contribution of these sex steroid receptors is unclear. In the present study, we characterized bone and body phenotype of male AR-ERα double KO (AR-ERαKO) mice in comparison with ARKO, ERaKO and wildtype (WT) littermates. Tibial bone mineral density and geometry and whole-body composition were assessed weekly by in vivo pQCT and DEXA, respectively, until 16 weeks of age. Results were analyzed by repeated measures ANOVA and expressed as % difference vs. WT. Disruption of the AR decreased body weight gain (-14%, p < 0.01) without additional effect of ERα disruption. Interestingly, body composition was independently affected in ARKO or ERαKO mice; ERα disruption (but not AR disruption) increased fat mass in ERαKO or AR-ERαKO mice from 8 weeks of age (+36% and +30%, resp., p < 0.01), whereas combined AR and ERα disruption caused additive reductions of lean body mass (-24%, p < 0.01). However, ARKO and ERαKO mice showed divergent effects on trabecular BMD; disruption of the AR or ERα decreased (-64%, p < 0.01) and increased (+14%, p < 0.01) cancellous bone density, respectively. The cancellous bone gain in ERαKO mice appeared enterily AR dependent since no increase was observed in AR-ERαKO mice. In contrast, cortical bone mass acquisition was impaired in ARKO or ERαKO mice as reflected by a significantly decreased cross-sectional area (-8% and −6%, resp., p < 0.01) and periosteal perimeter (-4% and −3%, resp., p < 0.01). Moreover, combined disruption of the AR and ERα further reduced cortical bone expansion; an additional decrease in cross-sectional area (-14%, p < 0.01), periosteal perimeter (-5%, p < 0.01) and strength strain index (-29%, p< 0.01) was observed in AR-ERαKO mice. Disruption of the ERα, not AR, also lowered serum IGF-I in ERαKO (-23%, p < 0.01) and AR-ERαKO mice (-10%, p < 0.01), which may explain its additive negative impact on periosteal bone expansion.
Our data indicate that combined disruption of the AR and ERα has an additive effect on cortical bone growth as well as lean body mass compared to ARKO or ERαKO mice. Therefore, we conclude that activation of both AR and ERα are needed for optimal acquisition of cortical bone mass and lean mass. However, AR activation alone is able to maintain cancellous bone mass, whereas ERα activation lowers fat mass and increases serum IGF-I.
Disclosures: F. Callewaert, None.
Factors Produced in Ovary Inhibit Musculoskeletal Growth and Promote Adipogenesis During Puberty: Potential Mediation by Non Estrogenic Actions.K. E. Govoni, R. B. Chadwick*, J. E. Wergedal, S. Mohan. JL Pettis VAMC and Loma Linda University, Loma Linda, CA, USA.
To test if the pubertal surge in estrogen is essential for the increased growth hormone (GH)/insulin-like growth factor (IGF) action and bone accretion during puberty, we evaluated the consequence of ovariectomy in prepubertal mice on skeletal changes and GH/IGF axis during puberty. C57BL/6J mice were ovariectomized (OVX) or SHAM operated at 3 weeks of age (n = 6 to 7) and skeletal changes measured prior to and 3 weeks after surgery. A 57% reduction (P = 0.01) in uterine size confirmed the effectiveness of OVX. Prepubertal OVX caused a 12% increase in the body weight (P = 0.01), 15% increase in lean total body mass (P < 0.01), and 18% reduction in percent total body fat (P < 0.01). Abdominal body fat was reduced by 43% in OVX group (P = 0.01). PIXImus analysis revealed a 12% increase in total body BMC and bone area (P < 0.02). pQCT analysis of femur mid diaphyseal region revealed an 8% increase in cross sectional area (P = 0.07), but no effect on vBMD. Femur length was increased by 3% (P < 0.01). We next determined if the absence of ovarian hormones during puberty influences puberty-induced increase in serum IGF-I level. We found that neither serum level of IGF-I (298 ± 21 vs. 277 ± 19 ng/ml in SHAM vs. OVX, respectively) or local IGF-I expression (1.12 ± 0.14 and −1.23 ±0.13 fold change vs. SHAM in bone and muscle, respectively) were altered in OVX mice at 3 weeks post surgery (P ≥ 0.30). To identify the potential pathways by which ovarian factors negatively regulate their effects on the musculoskeletal system, we performed a whole genome microarray analysis of mRNA from the femurs of OVX and SHAM mice at 3 weeks post surgery. A total of 223 genes were significantly (P ≤ 0.05) regulated in the bones of OVX mice. We observed significant upregulation (P ≤ 0.05) of expression of regulators (osterix, ephrinA1, formin2) and markers (Colla2, thrombospondin2, lumican) of osteoblast differentiation in the bones of OVX mice. In contrast, OVX resulted in significant downregulation (P < 0.05) of genes (lipoprotein lipase, stearoyl-Coenzyme A desaturase 1, caveolinl) which are known to be upregulated in adipocytes. In conclusion: 1) Female mice exhibit male-like features (bone, muscle and adipose) in the absence of ovarian hormones during puberty; 2) The increase in GH/IGF production during puberty is not dependent on estrogen production; 3) Expression of genes involved in osteoblast and adipocyte differentiation was altered in OVX mice during puberty; and 4) Based on these data and the published data that bone size is not increased in estrogen receptor-αβ knockout female mice, we speculate that factors besides estrogen may be responsible for the inhibitory effects of ovarian factors on the musculoskeletal system during puberty.
Disclosures: S. Mohan. None.
This study received funding from: National Institue of Health Grant AR048139.
The Purinergic P2X7 Receptor Is Responsible for Ovariectomy-induced Bone Loss in Mice.S. Petersen*1, S. Syberg*1, Z. Henriksen*1, P. Schwarz2, J. B. Jensen*1, O. H. Sorensen*1, N. R. Jorgensen11Endocrinology and Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark, 2Research Center for Ageing and Osteoporosis, Copenhagen University Hospital Glostrup, Glostrup, Denmark.
The purinergic P2X7 receptor is an ATP-gated cation channel that is primarily expressed in cells of hematopoietic origin such as osteoclasts and macrophages, but also in mature osteoblasts. It has been shown to have important functions both in the regulation of osteoblastic bone formation and in the formation and activity of osteoclasts. The aim of this study was to elucidate possible gender-differences in bone mass between P2X7 genotypes as well as the role of the receptor in the bone loss accompanying estrogen-withdrawal.
Two groups of C57BL mice with the following P2X7 genotype: P2X(Wt), P2X7+/− (He) P2X7−/− (Ho) were examined. The first group (A) was sacrificed at 120 days of age (n=159, 76 males, 83 females). The second group (B) contained all females and were ovariectomiced (OVX) (n=36) at 120 days of age with one month additional follow up for effect of OVX. At sacrifice, both groups had determination of bone mineral density (BMD) and bone mineral content (BMC) using a PIXImus densitometer as whole body BMD/ BMC and right femur BMD/BMC.
In order to determine the role of the P2X7 receptor in gender-differences in bone mass we compared bone mass values for the genotypes in group A. For the Wt animals, males had highly significant BMC/BMD than females (p-values< 0.01), while in the P2X7 null mice only slight differences between genders could be found, and only for BMC of the femur (Mean +/- SEM: 0.037 +/- 0.001 vs. 0.035 +/- 0.0005, p-value: 0.04), despite significantly higher weight in males compared to females in all genotype groups.
For group B, a significant bone loss (5%) was found one month after OVX in the Wt group compared to baseline, but no change in BMD/BMC values could be detected in the He or Ho groups in response to OVX. In conclusion, as expected differences were found in bone mass between male and female mice. Interestingly, these differences were not present in mice with a deletion of the P2X7 receptor. Furthermore, P2X7 null mice were protected against OVX-induced bone loss compared to their Wt littermates. Thus, the P2X7 receptor appears to be involved in the effects of sex hormones on the regulation of bone metabolism.
Disclosures: S. Petersen, None.
Classical ERα Signaling Is Essential for Estrogen Mediated Enhancement of Osteogenesis but not for Inhibition of Adipogenesis.
F. A. Syed, D. G. Fraser*, M. J. Pursier, S. Khosla. Endocrinology, Mayo Clinic College of Medicine, Rochester, MN, USA.
Previous studies have demonstrated that estrogen (E) enhances osteoblastic and inhibits adipocytic differentiation of bone marrow stromal cells (BMSCs). ERα can signal either via the “classical” pathway requiring direct binding to E response elements (EREs) or through indirect, “non-classical” pathways involving protein-protein interactions. We have previously demonstrated (JBMR 20:1992, 2005; Endocrinology 148:1902, 2007) that mice with a deleted ERα allele and a knock-in of a mutant ERα that cannot bind EREs on the other allele (ERα-/NERKI) have osteopenia and decreased bone formation rates, suggesting that ERE signaling may be essential for E effects on bone formation. Interestingly, in contrast to the generally observed inverse relationship between bone mass and marrow fat, ERα-/NERKI mice have osteopenia but no increase and, in fact, a decrease in bone marrow fat: adipocyte volume/total volume (AV/TV) at the proximal tibial metaphysis of 2.3 ± 0.2% vs. 14.6 ± 1.6% for wild type (WT) mice, P <0.05, suggesting dissociation of ERα signaling on bone vs. fat. To test this, we exposed primary BMSC cultures from female WT, ERα-/ERα-, and ERα-/NERKI mice to osteogenic (dex, β-glycerolphosphate, and ascorbate) and adipogenic (rosiglitazone) media in the absence or presence of estradiol (10−8M). Mineralization was assessed using von kossa staining and adipogenesis using oil red O staining. As is evident (Figure, panel A), while E clearly increased the number of osteoblastic colonies in WT cultures, it was totally ineffective in doing so in the ERα-/ERα- or ERα-/NERKI cultures. By contrast, while E inhibited adipogenesis in WT and was ineffective in the ERα-/ERα- cultures, it was able to inhibit adipogenesis in the ERα-/NERKI cultures (panel B). Collectively, our in vivo and in vitro findings demonstrate that ERE signaling is essential for E effects on enhancing bone formation but not for inhibition of adipogenesis, consistent with dissociation of the signaling pathways involved in these effects. Further studies to characterize the different pathways by which E mediates effects on osteogenesis vs. adipogenesis are currently underway.
Disclosures: F.A. Syed, None.
Internalization of Vitamin D Binding Protein (DBP) by Human Monocytes.
R. F. Chun, S. Ren, J. S. Adams, M. Hewison. Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Convention holds that 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3) enter target cells by passive diffusion in their ‘free’ form. However, recent studies have shown that DBP-bound 25D3 can also enter cells by facilitated endocytosis. In proximal tubule and breast epithelial cells this mechanism provides a conduit for delivery of substrate 25D3 to the vitamin D-activating CYP27b1α-hydroxylase. Here we used fluorescent Alexa-tagged DBP, which retains the capacity to bind 25D3 specifically, to show that DBP is also internalized by monocytes, a cell type that exhibits classical extra-renal metabolism of 25D3 to 1,25D, via the 1α-hydroxylase. Human peripheral blood mononuclear cells were cultured in the presence of granulocyte-macrophage colony stimulating factor (10 U/ml) for 1, 3 or 7 days. During this time monocytes showed increased expression and activity of the CYP27blα-hydroxylase with sustained Alexa-DBP uptake at levels 2–4-fold higher than control cell autofluorescence. Uptake of Alexa-DBP was observed when fluorescence-activated cell sorting (FACS) was gated for monocyte sub-populations, but no uptake was observed when cell populations were gated for lymphocyte sub-populations, indicating that uptake was specific for human monocytes. In day-1 and day-3 populations of cells DBP uptake was more pronounced in cells positive for the macrophage marker CD14. However, in day-7 populations, DBP uptake was also observed in CD14-negative dendritic cells. To determine whether uptake of DBP by monocytes was megalin-mediated, as observed in kidney and breast cells, monocytes were preincubated with receptor-associated protein (RAP), an antagonist of megalin-mediated endocytosis. RAP reduced Alexa-DBP internalization in megalin-rich BN16 rat yolk epithelial cells but had no effect on DBP uptake in monocytes, highlighting a megalin-independent means for DBP uptake in these cells. In summary, these data indicate that: 1] human monocyte/macrophages and DCs are capable of internalizing DBP by megalin-independent means; and 2] internalization of DBP occurs in monocytes, macrophages and dendritic cells. We propose that DBP-mediated uptake of 25D3 is a crucial mechanism in defining the immunomodulatory actions of 25D3. Furthermore, the presence of this mechanism in different types of human mononuclear cells suggests that DBP-mediated uptake of 25D3 is important for vitamin D effects on both innate and adaptive immunity.
Disclosures: R.F. Chun, None.
Curcumin: A Novel Nutritionally-Derived Ligand of the Vitamin D Receptor with Implications for Colon Cancer Chemoprevention and Bone Health.
L. Bartik*1, G. K. Whitfield2, M. J. Kaczmarska*1, C. A. Haussler2, M. R. Haussler2, P. W. Jurutka3. 1Biochemistry & Molecular Biophysics, University of Arizona, Tucson, AZ, USA, 2Basic Medical Sciences, University of Arizona College of Medicine, Phoenix, AZ, USA, 3Integrated Natural Sciences, Arizona State University, Phoenix, AZ, USA.
The nuclear vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D3 (1,25D) to alter gene transcription in several target tissues, including intestine and bone. Recently, the secondary bile acid, lithocholate, was recognized as a VDR ligand. Using reporter gene and mammalian two-hybrid systems, we identified curcumin (CM), a turmeric-derived bioactive polyphenol, as a likely additional novel ligand for VDR. CM (10−5 M) activated transcription of a luciferase plasmid containing the distal vitamin D responsive element from the human CYP3A4 gene comparably to 1,25D (10−8 M) in transfected human colon cancer cells (Caco-2). While CM also stimulated transcription via a retinoid × receptor (RXR) responsive element, activation of the glucocorticoid receptor by CM was negligible. Competition binding assays with radiolabeled 1,2SD confirmed that CM binds directly to VDR, obviating the possibility that it acts only via RXR. In mammalian two-hybrid assays employing transfected Caco-2 cells, CM (10−5 M) increased the ability of VDR to recruit RXR and the steroid receptor coactivator (SRC-1) by 10- and 9.6-fold, respectively, an action consistent with the promotion of transcriptional initiation of VDR-regulated genes. Small intestine and colon are important VDR-containing tissues, and frequent consumption of curry-containing foods can result in chronic, long-term exposure to significant levels of CM at these sites. Therefore, it is hypothesized that such diets can result in modest and sustained VDR/CM-mediated transcriptional activation of key target genes in the intestine. If this effect extends to genes involved in calcium transport, such as TRPV6, this may explain the beneficial effects of CM on bone, and our preliminary quantitative real time PCR (RT-PCR) data reveal CM-mediated upregulation of TRPV6 in Caco-2. Moreover, numerous studies have shown intestinal chemoprotection by CM via a variety of mechanisms; 1,25D has known anticancer properties in the intestine potentially due, in part, to VDR activation of CYP-mediated xenobiotic detoxification and/or up-regulation of p21. Using RT-PCR, we demonstrate a significant increase in the levels of p21 mRNA in Caco-2 cells treated with either 1,25D or CM. Thus, our results raise the novel possibility that nutritionally-derived CM facilitates intestinal chemoprevention and enhanced bone health via direct binding to VDR and activation of strategic target genes.
Disclosures: P. W. Jurutka, None.
Molecular Analysis of Two Novel Mutations in the Vitamin D Receptor Corepressor, Hairless, and Expression of Rat Hairless in E. coli.
J. C. Hsieh1, C. A. Sheedy*1, D. R. Mathern*1, S. A. Slater*1, G. K. Whitfield2, C. A. Haussler2, M. R. Haussler2. 1Biochemsitry & Molecular Biophysics, University of Arizona, Tucson, AZ, USA, 2Basic Medical Sciences, University of Arizona, Phoenix, AZ, USA.
Mammalian hairless (hr) is expressed primarily in skin and brain tissues. Its gene product (Hr) is postulated to be involved in hair follicle progression and regeneration, as well as in brain development. Hr interacts with the thyroid hormone receptor (TR) and the orphan nuclear receptor, RORα, inhibiting the ability of these receptors to activate transcription. Our recent work demonstrated that Hr also inhibits vitamin D receptor (VDR)-mediated transactivation in human keratinocytes. Two novel autosomal recessive Hr mutations, G985W and ΔAK (a C-terminal deletion), have been reported to cause hair loss in mice. The mutated residues are highly conserved in human, macaque, mouse, and rat species. To test whether the G985W and ΔAK rat Hr (rHr) mutants inhibit VDR and TR action, plasmids expressing the relevant proteins were cotransfected into COS-7 cells. Data revealed that the G985W mutation abolished the ability of Hr to repress TR- and VDR-mediated transactivation. On the contrary, the ΔAK mutant lost the ability to repress TR-mediated transactivation, but retained strong repression of 1,25-VDR-mediated transcription. Pull-down assay demonstrated that both rHr mutants interact well with human VDR. These results provide new insight into the Hr repression mechanism, including the functional, but not VDR-association, involvement of Gly985 in the repression of VDR- and TR-mediated transactivation. Second, the requirement of the C-terminal Ala-Lys residues in rHr for repression of TR- but not VDR-mediated transactivation, implies a clear difference in how Hr modulates the activity of these two nuclear receptors. Third, the hair loss phenotype exhibited by the ΔAK mutant is presumably caused by a defect in Hr function that is downstream of VDR-Hr binding. To further probe Hr-VDR interactions, we next overexpressed rHr. Previous characterization of Hr has been hampered by the limited availability of sufficient quantities of this large, labile protein. Consequently, full-length rHr was overexpressed in E. coli. A 4 kb fragment of the rHr cDNA was subcloned into the pT7–7 expression vector and transformed into E. coli BL21(DE3)plys S cells. The 130 kDa rHr was well expressed in this bacterial system and its authenticity was confirmed by mass spectrometry. The expression yield of rHr in this system was approximately 2–3% of total protein. This E. coli-expressed rHr will provide a resource for future functional studies of the molecular mechanism whereby Hr influences VDR- and TR-mediated target gene transcription.
Disclosures: J.C. Hsieh, None.
This study received funding from: National Institutes of Health NIDDK.
Novel Vitamin D3 Analogs (DLAMs) Antagonize Bone Resorption Via Suppressing RANKL Expression in Osteoblasts.
M. Inada, K. Tsukamoto*, M. Takita, M. Hirata, A. Hoshino*, T. Tominari*, K. Nagasawa*, C. Miyaura. Department of Biotechnology and Life Scence, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
Active vitamin D3, 1α, 25(OH)2D3 (D3), has been proposed to regulate bone remodeling through bone formation and resorption that is maintaining calcium levels in blood serum. Recently new aspects of D3 activities have been reported in bone resorption, inhibition of osteoclast differentiation via interference of c-fos and NFATc1 expression. The other aspect was found in D3 analogs, synthetic modification at C2 position of D3 that is enhancing bone formation and bone mass. Here we show newly synthesized analogs, 1α,25(OH)2D3–26,23-lactam, (23S,25S)-DLAM-1P and DLAM-2P, that has a lactam moiety in the side chain, perfectly antagonized D3-induced osteoclast (Oc) differentiation. In computer docking simulation estimated DLAM-1P bind to VDR, lactam moiety in DLAM-1P may interfere VDR helix-12 folding at the site of Phe-422 (ligand binding domain). Oc formation was assessed by mouse co-culture system. We first examined antagonistic effects of DLAMs on Oc formation induced by D3. When simultaneous treatment of DLAM-1P (10–7M to 10–5M) and D3 (10–8M), DLAM-IP clearly suppressed the number of TRAP+ Ocs in a dose-dependent manner. To understand the mechanism of action of DLAM-1P, we analyzed mRNA expression of RANKL, a sole molecule for Oc differentiation. DLAM-1P clearly suppressed the D3-induced expression of RANKL mRNA in osteoblasts. In organ culture using mouse calvana. bone resorbing activity (calcium release) induced by D3 was clearly suppressed by adding DRAM-1P that is associated with less induction of RANKL mRNA. The other analog DRAM-2P has shown similar activities to DRAM-1P. Therefore, DLAM analogs act on osteoblasts as an antagonist of D3 to suppress RANKL dependent Oc differentiation in the cell and the organ, suggesting the DLAMs are novel candidate for the treatment of pathological bone loss such like osteoprosis. Further modification will require for the therapeutic compound, enhancing bone formation and inhibition of RANKL expression beside minimum modification in DLAMs structure, e.g. installing substituents on its C2 position.
Disclosures: M. Inada. None.
Targeted Inactivation of the Vitamin D Receptor by Caspase-3.
P. J. Malloy*, D. Feldman. Department of Medicine, Stanford University, Stanford, CA, USA.
Calcitriol inhibits the growth of many cells including breast, colon, ovarian, pancreatic, and prostate cancer cells by causing cell cycle arrest and in some cells by inducing apoptosis. In breast cancer cells, calcitriol induces apoptosis by a caspase independent mechanism. On the other hand, in prostate cancer cells, calcitriol induction of apoptosis involves activation of caspase activity. Calcitriol actions are mediated by the vitamin D receptor (VDR) a member of the steroid-thyroid-retinoid receptor superfamily of nuclear transcription factors. In LNCaP prostate cancer cells induction of apoptosis by staurosporine abolished [3H]1,25(OH)2D3-binding and VDR protein suggesting to us that the VDR might be targeted for inactivation by caspases, a family of cysteine proteases that are activated during apoptosis. We identified a potential caspase-3 cleavage site (DxxDS) in helix H2n (D195MMDS199) in the VDR ligand-binding domain. Mutations (D195A, D198A and S199A) were generated in the putative caspase-3 cleavage site and their affects on caspase cleavage of VDR examined. In reporter gene assays, all of the VDR mutants exhibited transactivation activity similar to the WT VDR. COS-7 cells transfected with WT and mutant VDR cDNA expression vectors were then treated with staurosporine and VDR examined by western blot. A 22 kDa cleavage fragment was detected on western blot from cleavage of the WT VDR and the S199A mutant VDR but not the D195A or D198A mutant VDRs. Addition of the caspase-3 specific inhibitor z-DEVD-FMK prevented cleavage of the WT VDR in vivo. Treatment of transfected COS-7 cells with calcitriol also resulted in cleavage of the WT VDR but not the D198A mutant VDR. Calcitriol-induced cleavage of the WT VDR was blocked with z-DEVD-FMK demonstrating that calcitriol activates caspase-3 in these cells. In vitro treatment with caspase-3 resulted in the cleavage of WT VDR and S199A but not D195A or D198A mutant VDRs. WT VDR was also cleaved by caspase-6, and −7 but not caspase-8 in vitro. In conclusion, our results demonstrate that the VDR is cleaved by caspase-3 in vivo. We further demonstrate that calcitriol induces caspase-3 activity that results in the cleavage of the VDR. Our results suggest that activation of caspase-3 by calcitriol, while contributing to apoptosis may also may limit the apoptotic effects of calcitriol in cells that express caspase-3. Since caspases also regulate the activity of many proteins under non-apoptotic conditions, the inactivation of VDR by caspases may be of general importance as a mechanism to limit calcitriol activity.
Disclosures: P.J. Malloy, None.
Phe377del Mutation in the Ank Gene Causes Craniometaphyseal Dysplasia (CMD)-like Phenotype in Knock-in Mice.
I. Chen*, J. C. Wang*, E. J. Reichenberger. Reconstructive Sciences, UCHC, Farmington, CT, USA.
Craniometaphyseal dysplasia (CMD) is a rare craniotubular disorder characterized by progressive thickening of craniofacial bones concurrent with widened and radiolucent metaphyses in long bones. Mutations for autosomal dominant CMD have been identified in the human ANK gene (ANKH). ANK serves as a pyrophosphate (PPi) transporter regulating intra and extracellular PPi levels. To date, little is known about the pathogenesis of CMD and medical therapies of CMD show limited effect. We have generated a knock-in (KI) mouse model expressing a deletion mutation (Phe377del) in the Ank gene, the most common mutation found in our CMD patients. The purpose of this study is to characterize the progression of bone phenotype in these mice.
Ank+/k1 and AnkK1/K1 mice appear normal at birth. AnkK1/K1 mice begin to weigh less than their respective wild type and Ank+/K1 littermates after weaning. Within four to five weeks they develop a stiff, flat-footed gait, similar to phenotypes in Ankank/ank and Ank null mice. Decreased mobility of joints becomes more severe with age and they die around 6 months of age. Radiographically, bones from 1, 3 and 6 month-old AnkK1/K1 mice show increased radiopacity of cranial vault, base, facial bones, and mandibles; widening of metaphyses with increased radiolucency in femurs; fusion of joints in paws; narrowing of inter-disc space of vertebrae. Most Ank+/K1 mice are indistinguishable from wild type mice but bones from 3 or 6 month-old heterozygous mice develop an intermediate phenotype. Skulls and jaws from 3 month-old AnkK1/K1 mice show significantly increased bone mineral density and bone mineral content. MicroCT results from skulls, femurs and mandibles of 3 month-old AnkK1/K1 mice show hyperostosis of calvariae and cranial base, narrowing of cranial neural foramina; significant decrease in trabecular number and bone volume fraction (BVF) of club-shaped femurs; increased BVF but decreased cortical density of mandibles compareding to wild type and Ank+/K1 mice. Dynamic histomorphometry analysis of femurs from 10 week-old AnkK1/K1 mice shows a marked decrease in mineral apposition rate. These observations suggest that the Ank mutation causing the CMD-like phenotype may be in a dose and time-dependent manner. These Ank knock-in mice can be used as a model for CMD.
Based on the similar joint phenotype to Ank null mice, we hypothesize that the CMD-causing mutant Ank is a loss of function mutation. The unique CMD-like phenotype in these mice suggests that a second molecular mechanism, rather than solely an abnormality in the extracellular pyrophosphate level, is involved in CMD pathogenesis.
Disclosures: I. Chen, None.
This study received funding from: AR495S9.
BMP Signaling in Osteoblasts Negatively Regulates Canonical Wnt Signaling to Reduce Bone Mass During Embryonic Bone Development
N. Kamiya1, L. Ye2, T. Kobayashi3, Y. Mochida4, M. Yamauchi4, H. Kronenberg3, J. Feng2, Y. Mishina*1. 1LRDT, NIEHS/NIH, Research Triangle Park, NC, USA, 2Oral Biology, University of Missouri-Kansas City, Kansas City, MO, USA, 3Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA, 4Dental Research Center, University of North Carolina, Chapel Hill, NC, USA.
Bone morphogenetic proteins (BMPs) have been believed to be osteogenic inducers over 40 years, because of their ability to induce ectopic endochondral bone formation after subcutaneous implantation. However, physiological function of BMP signaling in osteoblasts remains largely unknown, because mice with germline mutations in genes encoding BMPs or BMP receptors that are predominantly expressed in bone (i.e., BMP2, BMP4, BMPRIA, and ACVRI) die in early embryonic stages before bone develomment. In the present study, we focused on function of BMP signaling during embryonic bone development using loss-of-function and gain-of-function strategies for BMP receptor IA (BMPRIA) in mice. We unexpectedly found that BMP signaling in osteoblasts restrained bone mass through regulation of canonical Wnt signaling.
BMP receptor type IA (BMPRIA or ALK3) is one of three type I receptors for BMPs and is predominantly expressed in bone. Since homozygous mice for a null mutation in Bmprla die at embryonic day 7.5, we rescued the embryonic lethality by generating a bone-specific knockout of Bmprla using tamoxifen inducible Cre-loxP system. Interestingly, under the physiological toss of BMPRIA signaling in osteoblasts, bone mass was increased and osteoclastogenesis was reduced concomitantly. Moreover, canonical Wnt signaling was upregulated due to a dramatic reduction in some of the Wnt inhibitors. Treatment with one of the Wnt inhibitors ex vivo rescued the phenotype of Bmpr1a-deficient bones with concomitant reduction of canonical Wnt signaling. In addition, upregulation of BMPRIA signaling in osteoblasts increased the expression of the Wnt inhibitor and increased osteoclastogenesis through OPG/RANKL pathway. Lastly, the bone phenotype of Bmpr1a-deficient mice was rescued by upregulation of BMPRIA signaling in mice with concomitant reduction of canonical Wnt signaling and an increase in osteoclastogenesis. These data indicate that BMP signaling in osteoblasts negatively regulates canonical Wnt signaling and positively regulates osteoclastogenesis through OPG/RANKL pathway to reduce bone mass during embryonic bone development. This study provides the first evidence of the molecular mechanism how these two major signaling pathways, BMP and Wnt, interact specifically in bones. We believe this study will provide innovative understanding of the physiological function of BMP signaling in bone biology.
Disclosures: N. Kamiya, None.
Characterization of the Bone Phenotype in CIC-7 Deficient Mice.
A. V. Neutzskv-Wulff*1, K. Henriksen1, A. Snel*1, T. J. Jentsch*2, J. Fuhrmann*2, P. Lange*2, C. Christiansen3, M. A. Karsdal1. 1Nordic Bioscience, Herlev, Denmark, 2MDC/FMP, Berlin, Germany, 3CCBR, Ballerup, Denmark.
Loss of the chloride channel CIC-7 leads to severe osteopetrosis. CIC-7 is believed to play a role in the ability of the osteoclasts to acidify the resorption lacuna, and thereby their ability to resorb bone. We therefore examined the bone phenotype of CIC-7 knockout (KO) mice in vitro and in vivo in detail, and compared it to the phenotype of oc/oc mice.
CIC-7 KO, oc/oc mice, and their corresponding wildtype littermates (WT) were sacrificed at 4–5 weeks of age. Bones and spleens were dissected and used for isolation of osteoclasts. The isolated cells were differentiated into mature osteoclasts on bone using M-CSF and RANKL. Cell culture supernatants were collected for measurements of CTX-I, TRACP and gelatinase activity by zymography. Cells were fixed and TRACP stained and the resorption pits were counted. Biochemical markers of resorption (CTX-I), osteoclast number (TRACP 5b), and osteoblast activity (ALP) were measured in serum of CIC-7 KO, oc/oc mice and the corresponding WT. Osteoblastogenesis in vitro was investigated using calvarial osteoblasts. In addition, bones were used for histological examination of TRACP positive osteoclasts with respect to number and morphology. Furthermore, osteoblast numbers and morphology were examined.
The osteoclasts from the CIC-7 KO mice were unable to resorb bone, as measured by CTX-I and by counting of resorption pits. Measurements of TRACP activity, as well as TRACP staining, showed the presence of equal numbers of osteoclasts in WT and KO cultures. Gelatinase activity was similar in both genotypes. Furthermore, the morphology of the KO cells was normal. Histological investigation of TRACP stained bone sections of both CIC-7 KO and oc/oc mice, indicated an elevated number of large osteoclasts present compared to WTs. The serum TRACP levels were increased by 250% in CIC-7 KO and oc/oc mice, whereas the resorption per osteoclast was reduced to 50% of the WT level. Finally, the serum ALP level in KO and oc/oc mice was increased by 30%, whereas no differences in osteoblast function were observed in vitro.
In summary, the osteoclasts from CIC-7 KO mice differentiate normally and form actin rings, but fail to resorb bone in vitro. In vivo, the osteoclasts are larger and more numerous, however, show no signs of resorption. Interestingly, an elevation of TRACP, a marked reduction in resorption per osteoclast, and elevated ALP level were observed in serum from both CIC-7 KO and oc/oc mice, showing increased signs of bone formation despite the low resorption. These findings indicate that the osteoclasts, and not their activity, control osteoblastic activity.
Disclosures: A. V. Neutzsky-Wulff, None.
Identification of Single Nucleotide Polymorphisms (SNPs) Contributing to Bone Mineral Density and Fractures: A DNA Microarray Based Study.
L. Gennari1, D. Tejedor*2, D. Merlotti1, V. De Paola1, A. Cadaval*2, G. Martini1, F. Vallesgi1, B. Franci*1, S. Campagna*1, B. Lucani*1, L. Simon*2, A. Martinez*2, R. Nuti1. 1Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry, University of Siena, Siena, Italy, 2Progenika Biopharma SA, Derio, Spain.
Osteoporosis is a complex polygenic disorder determined by the interaction between multiple genes and environmental risk factors, each with a small to modest effect. Traditional single candidate gene approach was only able to figure out a very small fraction of the whole genetic background of bone mass and fractures. We performed a DNA microarray-based analysis allowing the simultaneous study of 113 single nucleotide polymorphisms (SNPs) of 54 genes, which have been selected by their potential impact on bone metabolism. BMD and other phenotypes relevant to the pathogenesis of osteoporosis, such as ultrasound properties of bone, bone volume, bone turnover markers and sex hormone levels were evaluated in a population-based cohort of 904 postmenopausal women and elderly men. Moreover, 125 consecutive patients with non traumatic fractures were also investigated. Relationship between the selected clinical outcomes and the patient's pattern of SNPs was analysed by logistic regression. For each clinical outcome the optimal predictive model, based on a weighted combination of SNPs and disease characteristics and aimed resulting in a very high (95%) specificity, was selected. We identified 19 major SNP associated with BMD of whom 7 were non -sex-specific and 12 sex-specific. In particular SNPs at LRP-5, CYP19, ESR2, PPARalpha, and CYP1B1 genes were preferentially involved in determining BMD in males and SNPs at ESR1, SRD5A2, AR, IGF1, and IL-6 appeared to be female specific. Moreover, in females 2 SNPs (ESR1 and IL-6) were specific for spine BMD and 2 for hip BMD (AR and SRD5A2). In males, LRP-5 and CYP19 SNPs affected both femoral and lumbar BMD, while PPARalpha SNP mainly regulated BMD at the spine. Interestingly, 7 SNPs were associated with nontraumatic fractures independently of BMD. The most consistent associations with non-vertebral fractures were observed in IntegrinBeta3 and COL1A2 SNPs. In contrast, ESR1, TNFalpha, ADRB2, COMT, IL-6, and CYP19 were associated with both vertebral and non vertebral fractures. Importantly, different associations of SNPs were able to discriminate between the presence or the absence of non-traumatic fractures in the analysed population with a specificity of 95%, sensitivity 57% or 55%, and a likelihood ratio of 11,4 or 11,0 in males and females, respectively. The validation of these associations in larger samples could result useful for the identification of patients at higher fracture risk.
Disclosures: L. Gennari, None.
Genome-Wide Measure of Pleiotropy Among Osteoporosis-Related Traits in the Framingham Study.
D. Karasik1, Y. K. Cho2, L. A. Cupples*2, D. P. Kiel1, S. Demissie*2. 1IFAR, Hebrew Senior Life, Boston, MA, USA, 2Biostatistics, BU Sch Public Health, Boston, MA, USA.
There have been no genome-wide association studies (GWAS) for osteoporosis-related traits using high-density genotyping platforms published to date. Such a GWAS may be used to examine pleiotropic associations of single nucleotide polymorphisms (SNPs) with BMDand bone geometric (BG) indices.
We used the Affymetrix 100K SNP GeneChip marker set in the Framingham Heart Study (FHS) to examine genetic associations with BMD and BG indices of the hip. We evaluated 70,987 autosomal SNPs with genotypic call rates ≥80%, HWE p≥0.001, and MAF ≥10%. In 1141 phenotyped members of FHS families (495 men and 646 women, mean age 62.5 yrs), using multivariable-adjusted residual trait values, we used linear regression analysis with generalized estimating equations (GEE), as well as family-based association tests (FBAT), to test associations between SNPs and BMD and BG traits. Variance components analysis (SOLAR) was performed to estimate genetic correlations (RhoG) among these traits.
We searched for SNPs associated with pairs of bone traits and presented the proportion of SNPs associated with the first trait that are also associated with the second trait.
We found that at a pre-specified significance threshold α, BMD traits (measured at femoral neck, FN, and trochanter, TR) share 23% at α = 0.001 (36% at 0.01 level) associated SNPs in GEE analysis, as is expected from the high RhoG=0.84 between them (Table). There are almost no such SNPs that share associations with both hip BMD and BG traits (femoral neck and shaft width, N and S WID, and neck length, FNL), despite substantial RhoG among these traits (ranging −0.25 to −0.44). Also, the BG traits shared a surprisingly small percentage of associated SNPs.
In conclusion, GWAS offers an unbiased strategy to identify new candidate genes for osteoporosis, but also may be used to better understand how related phenotypes potentially share the same genetic determinants. These results need to be replicated in other GWAS samples.
Disclosures: D. Karasik, None.
This study received funding from: NHLBI/NIAMS/NIA.
LRP5 G171V Mutation and Age-Related Bone Fragility.
M. P. Akhter, D. M. Cullen, R. R. Recker. Medicine, Creighton University, Omaha, NE, USA.
It is well documented that aging has significant impact on the mechanical properties of bone in the human skeleton. In an animal model, we characterized bone structural and strength properties to determine if the LRP5 G171V mutation will protect against bone loss associated with Aging. Seventy two female mice representing two genotypes [(all bred to C57BL/6 mice), WT (Lrp5+/+, wild type), HBM (High bone mass with LRP5 G171V mutation)] and three age groups (4, 18, 22 month) were used (Table). At the time of necropsy fourth lumbar (L4) vertebral bodies were collected and then scanned using micro-CT (μCT-40, Scanco) to quantify trabecular bone structural properties. The vertebral bodies were then mechanically tested in compression (along cranial-caudal direction) at 3mm/min displacement rate using an Instron-5543 testing device. We analyzed the trabecular bone structure and strength data using the General Linear Model (SPSS, IL) for univariate analysis to test for differences (P < 0.05) due to age and genotype. There was age-related loss in vertebral body structure and strength (Table). By 18 months of age, vertebral body trabecular bone structure (BV/TV) declined 28% in HBM mice and 54% in WT mice. In addition, HBM (33–60% decrease) and WT (47–57% decrease) mice lost significant structural strength (ULT, YLD, STIFF) by 18 months (Table). However, the decline between the 18 and 22 month age groups was not significant for either bone structure or strength within each genotype. HBM's trabecular bone structure (BV/TV) and strength remained significantly elevated at each age group (Table). These data suggest that the HBM mice (Lrp5 G171V mutation) experienced similar rates of bone loss with age but maintained both structural and strength advantage over the WT mice.
Disclosures: M.P. Akhter, None.
This study received funding from: The State of Nebraska LB595 Funds.
LRP5 Mutations Are Associated with Osteoporosis, Impaired Glucose Tolerance and Hypercholesterolemia.
A. Saarinen*1, T. Saukkonen*2, M. Somer*3, C. M. Laine*2, S. Toiviainen-Salo*4, A. Lehesjoki*1, O. Mäkitie2. 1Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland, 2Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland, 3The Family Federation of Finland, Helsinki, Finland, 4Helsinki Medical Imaging Center, Helsinki University Hospital, Helsinki, Finland.
LRP5 and LRP6 are coreceptors in the Wnt signaling pathway. Homozygous mutations in LRP5 cause osteoporosis-pseudoglioma syndrome (OPPG), characterized by severe osteoporosis and blindness. Heterozygous LRP5 mutations may also result in reduced bone mass. Recently mutations in LRP6 have been associated with coronary artery disease and metabolic syndrome. We have assessed bone health and glucose and lipid metabolism in a large Finnish OPPG pedigree with several heterozygous carriers of LRP5 mutations.
DNA samples were collected from 30 family members, two of whom had OPPG. The exons and exon-intron boundaries of LRP5 were sequenced. Skeletal phenotype was assessed in 29 subjects by fracture history, bone mineral density (BMD) and spinal radiographs. Fasting blood samples were collected and oral and intravenous glucose tolerance tests were performed to assess glucose and lipid metabolism in individuals with LRP5 mutations.
Two different missense mutations were identified. The subjects with OPPG (aged 52 yrs and 58 yrs) were homozygous for R570W. One subject (age 85 yrs) was a compound heterozygote for R570W and R1036Q; she had normal vision but severe osteoporosis. Six subjects were heterozygous for R570W and three, for R1036Q; their median age was 59 yrs (15 — 72 yrs) and median BMI 26 kg/m2. No mutations were found in 18 family members; their median age was 48 yrs (20 — 67 yrs) and median BMI 26 kg/m2. Patients with two LRP5 mutations had multiple spinal and peripheral fractures and low BMD. The nine heterozygous LRP5 mutation carriers had significantly lower median BMD Z-scores at the lumbar spine (-1.0 vs +0.4, P=0.002) and femoral neck (-0.8 vs +0.75, P=0.009), more often vertebral fractures (44% vs 17%) and more peripheral fractures than those 18 without mutations. Of the 11 subjects with LRP5 mutation(s) six subjects (55%) had abnormal glucose tolerance: five had diabetes and one, impaired glucose tolerance. Intravenous glucose tolerance tests suggested impaired beta cell function; no insulin resistance was observed. Six subjects (55%) had hypercholesterolemia; three of them were on medication.
In addition to osteoporosis we found an unexpectedly high prevalence of diabetes and hypercholesterolemia in subjects with LPR5 mutations. These results link mutations in LRP5 to risk factors of coronary artery disease and implicate LPR5 as a novel gene that plays a role in glucose dependent insulin secretion from pancreatic beta cells.
Disclosures: A. Saarinen, None.
Association Study Between Polymorphism Across the Osteoprotegerin Gene and Osteoporotic Phenotypes.
S. Jurado*1, N. Garcia-giralt*1, L. Agueda*2, M. Bustamante*2, A. Supervia*1, M. A. Checa*1, L. Perez-edo*1, G. Salo*1, M. J. Peña*1, I. Aymar*1, J. M. Garces*1, S. Balcells*2, D. Grinberg*2, L. Melliboysky*1, A. Diez-perez1, X. Nogues1. 1URFOA IMIM Universitat Autonoma de Barcelona, Hospital del Mar, Barcelona, Spain, 2Genetics Department, Universitat de Barcelona, Barcelona, Spain.
Osteoprotegerin (OPG) is a key regulator of bone resorption in the OPG/RANK/RANKL system. Therefore, the study of polymorphisms in the OPG gene is relevant in relation to osteoporosis. The aim of the present study is to evaluate common variation across the OPG genomic region using a haplotype-based approach.
A total of 964 postmenopausal women from Hospital del Mar of Barcelona (BARCOS cohort) were recruited and anthropometric features, together with bone mineral density (BMD) and osteoporotic fracture data, were recorded. Cases were consecutive unselected women attending to the Menopause Unit outpatient clinics in our institution. Secondary causes of metabolic bone disease were ruled out. BMD was measured with HOLOGIC QDR 4500 SL at lumbar spine (LS) and femoral neck (FN) sites. Twenty four single nucleotide polymorphisms (SNPs) were selected from haplotypic blocks obtained from the second phase of HapMap (CEU). Genotyping was performed by SNPlex at CeGen facilities. Biomedical Research Park of Barcelona. Haplotype frequencies were obtained using the PHASE software. Statistical analyses included ANCOVA for BMD and logistic regression for fractures (SPSS for windows statistical package v.12.0). Additive, recessive and dominant models were considered in all cases.
Twelve out of the 24 SNPs yielded significant statistical results (p value < 0.05) for LS BMD, FN BMD and/or osteoporotic fracture. These SNPs are located between the promoter region and intron 1, and belong to five different haplotypic blocks. Haplotypes within these blocks were also found to be associated with the different bone phenotypes. While none of the SNPs was associated with the three phenotypes, three of them were associated with both FN BMD and fracture, and two with FN and LS BMD.
In conclusion, genetic variants at the promoter and intron 1 of the OPG gene were found related to BMD and fracture risk in Spanish postmenopausal women, suggesting a role for regulatory SNPs in this gene for bone mass and / or fragility determination.
Disclosures: X. Nogues, None.
Inhibition of Osteolytic Bone Metastases in Breast Cancer with a Cathepsin K Inhibitor.
G. Wesolowski1, M. Pickarski1, G. Neusch*1, P. Leung*1, R. Oballa*2, M. Percival*2, W. Black*2, L. T. Duong1. 1Bone Biology & Osteoporosis, Merck Res. Labs., West Point, PA, USA, 2Merck Res. Labs., Pointe-Claire-Dorval, PQ, Canada.
Breast and prostate cancer commonly metastasize to bone, resulting in bone loss, bone pain and fracture. Orally active cathepsin K inhibitors are currently being developed as potent bone resorption inhibitors for the treatment of osteoporosis. The standard of care, I.V. bisphosphonates, provides proof-of-concept for the use of potent antiresorptives for the treatment of metastatic bone disease. High expression of Cat K has also been detected in metastatic breast carcinoma, which suggests a role of Cat K in tumor invasion. L-006235 is a potent inhibitor of Cat K, with IC50 of 0.2, 0.5, 12 and 22 nM against human, rabbit, rat and mouse Cat K, respectively, and of rabbit osteoclastic bone resorption, with IC50 of 5 nM. Previously, L-235 was shown to be lysosomotropic, resulting to potential inhibition of other cathepsins, including Cat B, at high concentration in vivo. In this study, oral L-006235 was evaluated up to 100 mg/kg/day and compared to zoledronic acid (ZOL, 7.5 μg/kg, sc, wkly) for the prevention of osteolytic lesions and tumor growth in the intra-tibial engraftment model of MDA-MB-231 breast carcinoma cells in nude rats. Progression of osteolysis and tumor burden were monitored by radiography and evaluated by μCT and histological analyses over 6 weeks. Compared to vehicle, L-006235 at 10, 30 and 100 mg/kg dose-dependently inhibited tumor-induced bone volume loss by 18%, 22%, and 43% (p<0.001), respectively, with full protection at the highest dose which was comparable to the efficacy of ZOL (45%). Compared to vehicle, L-006235 also reduced tumor growth by 29%, 40% and 63% (p<0.001), versus a 56% reduction with ZOL. In this study, L-006235 at 30 and 100 mpk also reduced cortical disruption, tumor necrosis, and tumor cell migration from the original injection site. Taken together, these data suggest that cathepsin K inhibitors provide potent anti-resorptive efficacy and, compared to the current standard of care, may potentially have added benefits in reducing tumor invasion, and preventing bone metastases associated with breast cancer.
Disclosures: L. T. Duong, Merck & Co. 3.
Parathyroid Hormone-Related Protein Induces Bone Pain Through Stimulation of Proton-Secretion in Osteoclasts.
L. Wang*, H. Wakabayashi*, T. Hiraga*, T. Yoneda.. Department of Biochemistry, Osaka University Graduate School of Dentistry, Osaka, Japan.
Bone pain is the most common complication associated with bone metastases. Of note, clinical studies reported that inhibitors of osteoclasts such as bisphosphonates (BPs) efficiently reduced bone pain, suggesting a causative role of osteoclasts. Parathyroid hormone-related protein (PTH-rP), a potent stimulator of osteoclasts, has been implicated in bone metastasis. Activated osteoclasts are known to release protons via the a3 type vacuolar-H+-ATPase (a3 V-H+-ATPase) to dissolve bone minerals, thereby inducing acidosis in the neighboring environment. Acidosis is a well-known cause of pain. These results collectively suggest that PTH-rP is associated with bone pain through stimulating osteoclastic bone resorption. Repeated subcutaneous injections of PTH-rP caused increased osteoclastic bone resorption in the metatarsal bones and hyperalgesia in the hind-paw. Immunohistochemical examination revealed that the hyperalgesia was associated with increased protein expression of the transient receptor potential channel-vanilloid subfamily member 1 (TRPV1) and phosphorylated-ERK (p-ERK) in the ipsi-lateral dorsal root ganglions (DRGs) and c-Fos in the spinal dorsal horn. The hyperalgesia and elevated protein expression were significantly reduced by a most potent BP zoledronic acid, recombinant human OPG a specific inhibitor of the V-H+-ATPase FR167356 and an antagonist of TRPV1 activation 1-RTX, respectively. Finally, TRPV1 knockout mice exhibited reduced PTH-rP-induced hyperalgesia. Relevant to bone pain in bone metastases of breast cancer, we developed an animal model in which the MDA-MB-231 human breast cancer cells that produces large amounts of PTH-rP were directly inoculated into the tibial marrow cavity in nude mice. These mice showed hyperalgesia in the ipsi-lateral hind-paw and a monoclonal antibody to PTH-rP reduced the hyperalgesia. These results suggest that PTH-rP produced by metastatic cancer cells plays a part in causing bone pain through stimulation of proton-secretion in osteoclasts, thereby activating the acid-sensing receptors such as TRPV1 that are expressed in the nociceptive neurons innervating bone.
Disclosures: L. Wang, None.
Runx2 Regulates the Indian Hedgehog-PTHrP Pathway in Breast Cancer Cells.
J. Pratap, J. Dobson*, A. J. van Wijnen, J. L. Stein*, G. S. Stein, J. B. Lian. Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA, USA.
Runx2 is expressed in hypertrophic chondrocytes and osteoblasts and is essential for bone formation. However, Runx2 is also highly expressed in bone metastatic cancer cells compared to non-metastatic cells. Importantly, Runx2 upregulates several metastatic markers (MMPs and VEGF) in cancer cells and a loss of function mutation of Runx2 prevents the osteolytic lesions formed by breast cancer cells in vivo. To further understand how Runx2 participates in the osteolytic process mediated by cancer cells, we investigated the effect of Runx2 on key components of the “vicious cycle”. The vicious cycle of metastatic bone disease involves overproduction of PTHrP (Parathyroid hormone related protein) by tumor cells in the bone microenvironment, resulting in osteoclastic bone resorption. The resorbed bone releases TGF-β thereby stimulating tumor-cell proliferation and consequently continuing the vicious cycle. In the growth plate chondrocytes, Indian hedgehog (IHH) regulates PTHrP expression. Here, we show, following transduction of Runx2 expressing adenovirus that Runx2 activates endogenous levels of IHH more than 10 fold compared to 3–4 fold activation of other Runx2 target genes (VEGF, MMP9, MMP13 and osteocalcin) in MDA-MB-231 cells detected by real time PCR analysis. Runx2 also increased PTHrP levels 2–3 fold. The C-terminal deletion mutant of Runx2 (lacking the activation domain) failed to induce IHH and PTHrP levels in MDA-MB-231 breast cancer cells, as well as Runx2 target genes. In a time course analysis, we find Runx2 was able to activate IHH levels within 6hr of Runx2 expression in MDA-MB-231 cells. Consistent with these findings, depletion of Runx2 by siRNA decreases endogenous IHH and PTHrP levels produced by breast cancer cells. Blocking the Hedgehog signaling pathway using cyclopamine also inhibits Runx2 mediated activation of PTHrP which suggests that PTHrP upregulation by Runx2 is via IHH signaling events. We confirmed Runx2 recruitment on the IHH promoter in breast cancer cells by chromatin immunoprecipitation assays. Runx2 binds to a proximal (-0.5Kb) and distal site (-1.4Kb) of human IHH promoter. We propose that metastatic cancer cells by having higher levels of Runx2 are able to activate components of the vicious cycle as well as target genes that promote tumor progression in bone and increases bone loss. Thus this master regulator of bone formation is also a significant mediator of the metastatic activities of cancer cells and deserves consideration for targeted inhibition in tumor cells as a therapeutic approach to prevent the osteolytic bone disease.
Disclosures: J. Pratap, None.
Osteonecrosis of Jaw Under Bisphosphonate Therapy: Patient Profile and Risk Assessment.
T. I. Jung*1, J. von der Gablentz*1, B. Hoffmeister*2, S. Mundlos*3, P. Fratzl*4, M. Amling*5, M. J. Seibel*6, D. Felsenberg1. 1Zentrum für Muskel- und Knochenforschung (ZMK), Charite — Universitaetsmedizin, Berlin, Germany, 2Clinic for Oral, Maxillofacial and Plastical Surgery, Charite — Universitaetsmedizin, Berlin, Germany, 3Institute for Medical genetics, Charite — Universitaetsmedizin, Berlin, Germany, 4Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam-Golm, Germany, 5Centre for Biomechanics and Skeletal Biology, University Hospital, Hamburg-Eppendorf, Germany, 6ANZAC Research Institute, University of Sydney, Sydney, Australia.
The objective of the ongoing German Register for Osteonecrosis of the Jaw (ONJ) was to explore the development of ONJ as a serious adverse event of bisphosphonate (BP) treatment. A standardized questionnaire was used to analyze all cases that were officially reported within Germany up until 31 Dec 2006. Focus was placed on 1) demographics, 2) primary disease incl. its therapy 3) BP intake 4) dental status 5) diagnosis of ONJ and outcome of ONJ therapy. We included cases with osteomyelitis or ONJ while cases due to radiotherapy were excluded.
In 383 reported cases, patients were 63.4 ± 8.3 years old and women were affected twice as often. They suffered from malignant disease (78.4%), osteoporosis (8.2%), or a combination of both (13.4%). Common malignant diseases were breast cancer (46.7%), multiple myeloma (30.1%) and prostate cancer (13.5%). Patients had previously received chemotherapy (68.9%), radiotherapy (62.3%) and glucocorticosteroids (47.5%). The majority (67.2%) was treated with a single BP: Zoledronate (79.7%) or Pamidronate (12.4%). Two BPs were administered consecutively to 30.7% of the patients: Pamidronate/ Zoledronate (53.5%). ONJ occurred after 23.8 ± 17.6 (Zoledronate) or 36.4 ± 20.0 (Pamidronate) months of BP treatment. Patients presented clinically with infection (33.3%), mucosal changes (21.4%), wound healing disturbance (21.4%), exposed bone (11.9%) and pathological dental status (46.2%). Histological signs were infection (51.9%) and osteonecrosis (34.2%) while radiologists diagnosed osteolysis (19.5%) and osteomyelitis (14.3%). Dental management included surgical (45.3%) or conventional (13.2%) methods, or a combination of both (41.6%). Success rate was highest (17.0%) for the combination therapy.
The etiopathogenesis of ONJ remains unclear but is likely to be complex. A discrete increase in osteoporotic patients was noted but high- risk patients received BPs due to their malignant disease. The occurrence of ONJ might be reduced by adjusting the guidelines of bisphosphonate treatment and by defining algorithms for the dental management of high-risk patients.
Disclosures: T.I. Jung, Elsbeth Bonhoff Foundation 2.
This study received funding from: Elsbeth Bonhoff Foundation.
The SABRE (Study of Anastrozole with the Bisphosphonate Risedronat E) Study: 12-Month Analysis.
R. Eastell1, C. Van Poznak*2, R. A. Hannon1, G. Clack3, M. Campone*4, J. R. Mackey*5, J. Apffelstaedt on behalf of the SABRE Investigators*6. 1Academic Unit of Bone Metabolism, University of Sheffield, Sheffield, United Kingdom, 2Dept of Internal Medicine, University of Michigan, Michigan, MI, USA, 3AstraZeneca, Macclesfield, United Kingdom, 4Site Hospitalier Nord de Nantes, Centre Renè Gauducheau, Nantes, France, 5Dept of Oncology, University of Alberta, Edmonton, AB, Canada, 6Dept of Surgery, University of Stellenbosch, Tygerberg, South Africa.
The SABRE study (NCT00082277) evaluated the effects of the bisphosphonate risedronate on bone mineral density (BMD) and bone turnover in postmenopausal women with hormone receptor-positive early breast cancer treated with anastrozole. Patients were stratified according to baseline risk of fracture by lumbar spine and hip T-scores. High-risk (H) patients (T-score <−2.0 for spine or hip, or those deemed high-risk by the investigator) received anastrozole (A) 1 mg/day plus risedronate (R) 35 mg/week orally. Moderate-risk (M) patients (T-score <−1.0 for spine or hip but ≥-2.0 for both) were randomized in a double-blind manner to receive A plus either R or placebo (P). Low-risk (L) patients (T-scores ≥-1.0 at both sites) received A alone. All patients received calcium and vitamin D. Bone turnover was assessed using serum procollagen type I amino terminal peptide (PINP), bone alkaline phosphatase (bALP), and serum C-terminal crosslinking telopeptide of type I collagen (sCTX). Percentage change in BMD is shown in the table. BMD increased significantly in H and decreased significantly in L. In M, a significant difference was seen between treatment groups for lumbar spine (p < 0.0001) and total hip (p=0.002, analysis of covariance).
In M, there was a significant difference in the percentage changes in bone markers between treatment groups (A + R sCTX −43.73, PINP −47.56, bALP −23.91 vs A + P 5.18, −7.65, 1.61 respectively; p<0.0001 for all). Incidences of adverse events (AEs) were comparable between M treatment groups (A + R 88%, A + P 86%; serious AEs A + R 5%, A + P 5%). In postmenopausal women with breast cancer who are already at moderate to high risk of fragility fracture, and are scheduled for treatment with anastrozole, bone health can be managed according to established guidelines. In this study, for patients already at risk, risedronate at the licensed dose was sufficient to prevent a decrease in BMD and an increase in bone turnover.
Disclosures: R. Eastell, Astra Zeneca 2, 5, 8; Alliance for Better Bone Health 2, 5, 8.
This study received funding from: SABRE was funded by AstraZeneca and Aventis.
In Vivo Inhibition of Osteosarcoma Growth by Notch Pathway Inhibition.
T. Setoguchi*, M. Tanaka*, S. Komiya*. Department of Orthopaedic Surgery, Kagoshima University, Kagoshima, Japan.
The Notch signaling pathway functions as an organizer in embryonic development. Genetic analysis has demonstrated a critical role for the Notch pathway in morphogenesis. Recent studies have shown constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear whether this pathway is activated in human bone tumor. Here, we examined the expression of the Notch pathway components by RT-PCR and immunohistochemical staining. RT-PCR and immunohistochemistry for Notch1–4, Jagged, Dll, Hes. and Hey was performed using human osteosarcoma cell lines and human osteosarcoma samples. And also, we investigated the effects of Notch pathway inhibition on osteosarcoma growth using pharmacologic inhibitors of γ-secretase (GSI) in vitro and in vivo. Cell viability was examined by MTT method. In order to determine the effects of Notch blockade in vivo, we examined the formation of tumor xenografts using osteosarcoma cell line and nude mice. RT-PCR revealed high expression of Notch-1,2, Jagged1 Hes, and Hey in all of osteosarcomas cell lines. And also, 7 osteosarcoma human samples also showed high expression of notch signaling molecules mRNAs. In addition, immunohistochemistry showed high expression of Notch-IC in nucleus of osteosarcoma cell lines and human osteosarcoma samples. And also, expression of Jagged and Hes was observed in osteosarcoma cell lines and human osteosarcoma samples. MTT assay showed that GSI suppresses the growth of the osteosarcoma cell lines in vitro (Fig.1). In addition, intraperitonealy GSI administration dramatically inhibited the osteosarcoma xenografts growth in vivo (Fig. 2). Our data indicate that the Notch pathway is a new candidate for therapeutic target of osteosarcoma.
Disclosures: T. Setoguchi, None.
This study received funding from: Japan Society for the Promotion of Science.
Transforming Growth Factor β Receptor I Kinase Inhibitor Reduces the Development and Progression of Melanoma Bone Metastases.
K. S. Mohammad1, D. Javelaud*2, E. G. Stebbins*3, M. Niewolna*1, C. R. McKenna*1, X. Peng*1, L. A. Kingsley1, P. G. J. Fournier1, L. S. Higgins*3, D. H. Wong*3, A. Mauviel*2, T. A. Guise1. 1Internal Medicine, University of Virginia, Charlottesville, VA, USA, 2INSERM U697, Paris, France, 3Scios Inc., San Francisco, CA, USA.
Melanoma often metastasizes to bone, where it is exposed to high concentrations of TGF-β. Constitutive Smad signaling occurs in human melanoma. Because TGF-β promotes metastases to bone by several solid tumors, we hypothesized that TGF-β could promote melanoma metastases to bone. Consistent with this, overexpression of the inhibitory Smad7 blocked the capacity of 1205Lu melanoma cells to form bone metastases by preventing the TGF-β induction in vitro of genes known to mediate osteolytic breast cancer bone metastases such as PTHrP, IL-11, CXCR4 and osteopontin and by blocking the induction of TGF-β target genes PTHrP, IL-11 and CTGF in bone metastases in vivo. We next tested the effect of a small molecule inhibitor of TGF-β receptor I kinase (TGF-βRI), SD-208 on development and progression of bone metastases due to 1205Lu melanoma. This inhibitor effectively reduced bone metastases due to MDA-MB-231 breast cancer. In vitro, SD-208 blocked TGF-β induction of Smad3 phosphorylation relative activity of the TGF-β (Smad)-responsive promoter CAGA in several melanoma lines: 1205Lu, WM852, 501mel and 888mel as well as invasion through matrigel by 1205Lu. We therefore hypothesized that pharmacologic blockade of TGF-β signaling using SD-208, would be effective treatment for melanoma metastasis to bone. Nude mice were inoculated with 1205Lu melanoma cells into the left cardiac ventricle and treated with SD-208 (20 and 60 mg/kg po qd administered via oral gavage) in either prevention or treatment protocol. In the former, SD-208 (60mg/kg/d), started 2 days before tumor inoculation, prevented the development of osteolytic bone metastases compared with vehicle. In the latter, mice given SD-208 (60/mg/kg/d) after confirming the presence of osteolytic lesions by radiography, had significantly smaller osteolytic lesions vs. vehicle treated mice (p=0.0009). These data indicate that therapeutic targeting of TGF-β may decrease the osteolytic bone metastases caused by malignant melanoma by mechanisms similar to those implicated in breast cancer osteolysis.
Disclosures: K.S. Mohammad, None.
RANKL Inhibition Blocks Lung Cancer-induced Osteolytic Lesions and Reduces Skeletal Tumor Burden in Both RANK-Expressing and RANK-Negative Lung Cancers.
R. Miller1, J. Jones*1, M. Tometsko*1, M. Chaisson-Blake*1, M. Roudier*2, W. Dougall1. 1Department of Hematology/Oncology Research, Amgen Inc., Seattle, WA, USA, 2Department of Pathology, Amgen Inc., Seattle, WA, USA.
Bone metastasis is a serious complication of lung cancer and occurs in 30% of patients. Tumor cells interact with the bone microenvironment to induce osteoclastogenesis, leading to bone destruction and release of growth factors that have the potential to feed back to the tumor growth. RANK ligand (RANKL) is essential for osteoclast formation, function, and survival. Tumor cell-mediated osteolysis occurs ultimately via induction of RANKL within the bone stroma, and inhibition of RANKL in animal models of breast or prostate cancer bone metastasis blocks tumor-induced osteolysis and prevents the increase in bony tumor burden. Another role for RANK and RANKL in promoting metastasis to bone has recently been proposed, in which RANK-expressing tumor cells migrate into the bone environment via RANKL acting as a chemotactic factor (Holstead-Jones et al. 2006). We examined a panel of lung cancer cell lines to develop a model of lung cancer in bone.
Lung cancer cell lines that expressed RANK included H146, H2107, H2108, H1975, H2126, while H526 and H1299 were negative for RANK, as determined by flow cytometry. Using bioluminescent imaging (BLI) and intracardiac challenge, we observed that several lung cancer lines became established and progressed in the bone, resulting in osteolytic lesions. The growth in bone and development of osteolytic lesions occurred with both RANK-expressing (H2126, H1975) and RANK-negative (H1299) cell lines. We treated mice bearing either H1299 (RANK-negative) or H1975 (RANK-positive) with the RANKL inhibitor OPG-Fc at early or late timepoints. OPG-Fc completely blocked the radiographically-evident osteolytic lesions induced by either cell line at all time points tested. In addition, RANKL inhibition blocked the skeletal tumor progression of the H1299 lung cancer line as determined by longitudinal BLI analysis and histomorphometry.
In conclusion, these results clearly show that both RANK-expressing and RANK-negative lung tumor cells lead to bone metastasis. The ability of RANK expression on tumor cells to further promote bone metastasis will require additional studies. These results demonstrate that, irrespective of RANK expression on tumor cells, RANKL is a required factor for the tumor-induced osteolytic lesions caused by each lung cancer tested in vivo. RANKL inhibition reduced skeletal tumor burden of the H1299 lung cancer line, presumably via the indirect mechanism of blocking tumor-induced osteoclastogenesis and resultant production of growth factors from the bone microenvironment.
Disclosures: R. Miller, Amgen Inc. 1. 3.
This study received funding from: Amgen, Inc.
Myeloma Cells Decrease EphB4 Expression in Osteoblasts: A Novel Mechanism for Regulation of Bone Formation in Multiple Myeloma.
A. L. Bates*, G. R. Mundy, C. M. Edwards. Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, TN, USA.
Multiple myeloma is associated with a destructive osteolytic bone disease, characterized by an increase in osteoclastic bone resorption and a reduction in osteoblastic bone formation. The increase in bone resorption in myeloma is well characterized; however the precise cellular and molecular mechanisms which mediate the reduction in bone formation and the uncoupling of bone resorption from bone formation are poorly understood. Recently, a novel mechanism has been suggested for coupling between osteoblasts and osteoclasts during normal bone homeostasis due to bidirectional signaling between the ligand ephrin B2, expressed by osteoclasts, and its receptor EphB4, expressed by osteoblasts. The interaction between EphB4 and ephrin B2 resulted in inhibition of osteoclast activity by reverse signaling through ephrin B2 on osteoclasts and stimulation of osteoblast differentiation and bone formation by forward signaling through EphB4 on osteoblasts (Zhao et al. 2006). Since the normal coupling of bone resorption to bone formation is dysregulated in multiple myeloma, we hypothesized that this may be mediated by modifications in the EphB4/ephrin B2 receptor/ligand interaction. To study bone formation in multiple myeloma in vivo, we used the 5TGM1 murine model of myeloma. 5TGM1 myeloma cells were inoculated by i.v. injection into C57BlKaLwRij mice, resulting in homing of myeloma cells to the bone marrow, and development of an osteolytic bone disease. In addition to the well characterized increase in osteoclastic bone resorption, the bone disease was associated with a significant reduction in osteoblast number and rates of bone formation (p<0.01). Real time PCR demonstrated expression of EphB4 mRNA in 5TGM1 myeloma cells, C2C12 and 2T3 osteoblasts, with a 2-fold increase in expression in 5TGM1 myeloma cells when compared with C2C12 or 2T3 cells. Western blotting demonstrated expression of EphB4 protein in C2C12 and 2T3 cells. Treatment of C2C12 or 2T3 cells with conditioned media from 5TGM1 myeloma cells resulted in a significant reduction in expression of EphB4 in both C2C12 and 2T3 cells, suggesting that myeloma cells release a soluble factor which down-regulates EphB4 in osteoblasts. In conclusion, we have shown that the development of myeloma bone disease in the 5TGM1 murine model of myeloma is associated with a reduction in bone formation, and that 5TGM1 myeloma cells can down-regulate EphB4 expression in osteoblasts. This raises the possibility that the reduction in bone formation associated with myeloma bone disease is mediated by a reduction in EphB4 expression and thus disruption of the normal coupling of bone resorption and bone formation.
Disclosures: C.M. Edwards. None.
Prevention and Treatment of Hypogonadism-associated Bone Loss and Bone Metastases by OPG-Fc in a Model of Mixed Osteolytic/Osteoblastic Metastases.
S. S. Padalecki1, B. Grubbs*1, B. Goins*1, A. Soundarajan*1, G. R. Mundy2, W. Dougall1. 1UTHSCSA, San Antonio, TX, USA, 2VUMC, Nashville, TN, USA, 3Amgen, Inc., Cambridge, MA, USA.
Bone resorption plays an important role in the development of osteoblastic metastases. Osteoclastic bone resorption results in the release of potent growth factors from bone. Androgen ablation, the standard treatment for men with advanced prostate cancer, increases systemic bone resorption and releases these growth factors from bone. As a result it also likely makes the skeleton a more fertile environment for prostate cancer metastasis. We have previously shown that increased osteoclastic bone resorption due to hypogonadism causes a more fertile environment for mixed osteolytic/osteoblastic bone metastases utilizing a mouse model which mimics the clinical situation of men rendered hypogonadal as a result of treatment for prostate cancer. To investigate the potential of OPG-Fc in preventing and treating bone loss due to hypogonadism and mixed bone metastases, male nude mice underwent orchiectomy or sham surgery 4 weeks prior to intracardiac inoculation with TSU-PR1 bladder cancer cells that cause mixed bone metastases. The potential of OPG-Fc in prevention of bone loss and mixed bone metastases was assessed by treating mice 3X weekly with OPG-Fc or vehicle from the time of surgery. To determine the efficacy of treating existing bone metastases with OPG-Fc, treatments were not started until 75% of the mice had evidence of bone metastases by radiograph. Radiographs, bone mineral density, 99mTc-MDP MicroSPECT imaging and bone histomorphometry were used to assess bone turnover and tumor osteolysis. As expected, hypogonadism resulted in lower bone mineral density and increased bone turnover compared with sham-controls. This was prevented by OPG-Fc. TSU-PR1 bone metastases were increased and developed faster in hypogonadal mice treated with vehicle compared to sham-controls. OPG-Fc decreased bone metastases in hypogonadal TSU-PR1-inoculated mice when used in a preventative fashion but had no effect on soft tissue metastases or survival. When OPG-Fc was used to treat existing bone metastases, it resulted in a decrease in radiographic lesion number and area.
This work provides in vivo evidence that OPG-Fc not only prevents bone loss due to androgen deprivation and reduced bone metastases in a prevention scenario, but suggests it also may lessen the severity of existing bone metastases. These data support the hypothesis that increased bone resorption due to androgen deprivation may result in a more fertile environment for the development of bone metastases. Bone resorption inhibitors, such as OPG-Fc, may benefit hypogonadal men with advanced prostate cancer to prevent and treat bone loss as well as skeletal metastases.
Disclosures: S.S. Padalecki, Amgen, Inc. 2.
This study received funding from: Amgen, Inc.
Serum Pentosidine Level Is Positively Associated with the Presence of Vertebral Fractures in Postmenopausal Women with Type 2 Diabetes.
M. Yamamoto, T. Yamaguchi, M. Yamauchi, S. Yano, T. Sugimoto. Department of Internal Medicine 1, Shimane University Faculty of Medicine, Izumo, Japan.
Despite higher BMD than healthy subjects, patients with type 2 diabetes mellitus (2DM) have an increased risk of fractures, suggesting that BMD was not sensitive enough to assess the risk of fractures. However, there are no useful methods for assessing the bone fragility in 2DM. Recently, bone content of pentosidine, which is one of well-known advanced glycation end-products, is known to be negatively associated with bone strength. In this study, an association between serum pentosidine levels and the presence of vertebral fractures were examined in diabetic patients whose bone fractures lack their predictive markers.
Ninety-four male and 76 postmenopausal female patients with 2DM within normal creatinine levels were examined by bone mineral density (BMD) at the lumbar spine, femoral neck and one-third of radius as well as spine radiographs, and by measurement of biochemical parameters including serum pentosidine, serum bone-specific alkaline phosphatase (BAP) and urinary levels of N-telopeptide (uNTX). Thirty-three male and 20 female had vertebral fractures. Serum pentosidine level significantly increased with age in the diabetic men and women (r = 0.232, p = 0.026 and r = 0.319, p = 0.006, respectively). Comparison between diabetic subjects with and without vertebral fractures revealed no significant differences in BMD values at any sites or biochemical parameters such as BAP or uNTX in both genders. In contrast, serum pentosidine levels in women with vertebral fractures were significantly higher than in those without fractures (0.0440 ± 0.0136 μg/ml vs 0.0321 ± 0.0118 μg/ml, p < 0.001). Multivariate logistic regression analysis adjusted for age, body weight, body height, HbA1c, serum creatinine and diabetic duration showed that serum pentosidine level was identified as an independent factor associated with the presence of vertebral fractures in postmenopausal women with diabetes as shown in Table.
Disclosures: M. Yamamoto, None.
Factors Predicting Osteoporosis Treatment Initiation in a Regionally-Based Clinical Cohort.
W. D. Leslie1, J. F. Tsang*2, L. M. Lix*3. 1Dept of Medicine, University of Manitoba, Winnipeg, MB, Canada, 2University of Manitoba, Winnipeg, MB, Canada, 3Manitoba Centre for Health Policy, University of Manitoba, Winnipeg, MB, Canada.
BMD testing is used by many clinical practice guidelines to identify those at high fracture risk and to guide osteoporosis therapy (OTx) initiation. We hypothesized that OTx initiation would show large step increases at discrete cutoffs corresponding to proposed T-score intervention thresholds (e.g., −1.5, −2.0 or −2.5).
Women age 50 y or older who had not been dispensed any OTx medication in the year prior to baseline DXA (1998–2002) were identified in a regionally-based database which contains all clinical DXA test results for the Province of Manitoba, Canada. Women with independent indications for OTx (previous glucocorticosteroid use or non-traumatic fractures of the hip, spine, wrist or humerus) were excluded. The rate of OTx initiation in the year after BMD (1 or more pharmacy dispensations) was studied using descriptive and logistic regression methods.
3826 of the 8654 women (44.2%) were dispensed OTx in the year after BMD. OTx initiation increased progressively as BMD decreased (8.2% normal, 41.0% osteopenic, 78.5% osteoporotic, p-for-trend<.00000l). Contrary to expectation, there was a gradient response to OTx initiation rather than step increases at conventional T-score intervention thresholds. A small step increase occurred at a T-score of −1.0 but this is not generally considered as an OTx initiation threshold in the absence of other indications. In a logistic regression model BMD was strongly associated with OTx (p<.00001) while age (p>.2), weight or BMI (p>.2), and fracture (excluding hip, spine, wrist or humerus) in the last year (p>.2) were not. Similar results were seen when spine and hip T-scores were considered separately, or when OTx initiation was defined as 2 or more pharmacy dispensations. We conclude that physicians rely heavily on the BMD T-score to decide on OTx initiation. Although many clinical practice guidelines suggest that age in addition to other risk factors should be considered in OTx decision-making, we did not see evidence of this. More explicit methods of reporting fracture risk may be required to assist physicians in selecting patients likely to derive the largest benefit from OTx.
Disclosures: W.D. Leslie, Merck Frosst Canada Ltd; Sanoβ-Aventis; Proctor &. Gamble Pharmaceuticals Canada, Inc. 2, 5, 8.
Utility of Lumbar Spine DXA Scanning in Women Undergoing Asymptomatic Osteoporosis Screening.
V. Pao*, D. E. Sellmeyer. Medicine/Endocrinology, University of California San Francisco, San Francisco, CA, USA.
The International Osteoporosis Foundation recommends osteoporosis screening with bone mineral density (BMD) be conducted at the hip. The planned WHO fracture risk assessment model includes femoral neck BMD. Previous studies of research populations have suggested that lumbar BMD may be affected by degenerative changes and contribute little additional information to fracture risk assessment. To determine the utility of spine BMD in asymptomatic postmenopausal women undergoing routine clinical osteoporosis screening, we conducted a retrospective chart review of women over age 50 years who were referred for screening via dual x-ray absorptiometry (DXA) scanning at the UCSF Osteoporosis Center during 12 consecutive months. Women were excluded if they had a known secondary cause of bone loss or were on medications known to affect bone. We categorized each DXA as normal, osteopenia, or osteoporosis at the lumbar spine, femoral neck, and total hip according to the WHO T-score definitions. We also recorded patient demographics and specialty of referring physician. Patient characteristics were analyzed across categories of bone density using ANOVA.
The average age of the 744 participants was 65.7 ± 9.4 (mean ± SD) years; 65% were Caucasian and 21% were Asian. DXA results showed 18.5% of the women had normal bone mineral density (BMD) at all three sites while 57.2% had osteopenia (T-score between −1 and −2.5) at one or more sites and 24.2% had osteoporosis (T-score < −2.5) at one or more sites. Compared to women with normal BMD, women with osteoporosis were more likely to be of Asian or Caucasian ethnicity (p<0.001), have a smaller BMI (p<0.001), and have a past history of back pain (p=0.009). Osteoporotic patients were less likely to be taking calcium and vitamin D (p=0.02) and more likely to have a history of fracture (p=0.003). The majority of the patient population was referred by their primary care provider (64.1%), followed by gynecologists, endocrinologists, and rheumatologists. Orthopedic surgeons referred only 0.1% of patients. Of women who had normal BMD readings at both the total hip and femoral neck, 9.3% had osteopenia or osteoporosis at the lumbar spine. The average lumbar spine T-score among these women was −1.6 ± 0.5. Many health care providers limit screening for osteoporosis to hip and femoral neck measurements. However, in our study a significant proportion of women had osteopenia or osteoporosis at the lumbar spine despite having normal BMD at the hip and femoral neck. These women would be expected to have an increased risk for vertebral fracture and would be missed if screening were limited to the hip site. Consideration should be given to regularly including the lumbar site when conducting routine osteoporosis screening in asymptomatic women.
Disclosures: D.E. Sellmeyer, None.
In Vivo Assessment of 3-Dimensional Bone Micro Architecture with HR-pQCT in Patients with and without Fractures.
H. Radspieler1, I. Frieling2, M. Neff3, R. Fischer*4, M. Zulliger*3, M. A. Dambacher5. 1Center of Osteoporosis Munich, Muenchen, Germany, 2Center of Osteoporosis Hamburg, Hamburg, Germany, 3Center of Osteoporosis Zurich, Zurich, Switzerland, 4Klinik Eppendorf, Hamburg, Germany, 5ZORQ Zurich, Switzerland.
Measurement of areal bone mineral density (BMD/DXA) with derived T-scores may not be sufficient to evaluate the individual fracture risk. HR-pQCT reveals volumetric cortical and trabecular bone density and structural parameters as BV/TV, trabecular thickness and separation and cortical thickness.
We examined more than 200 women in the centres of osteoporosis in Hamburg and Munich with normal (T-Score > — 1SD), osteogenic (- 2,5 SD > T-score < — 1,0 SD) or osteoporotic (T-score < — 2,5 SD) BMD measured with DXA. In all patients volumetric BMD and trabecular architecture of the radius and tibia were measured by HR-pQCT (XtremeCT®, SCANCO Medical AG).
Women with and without fractures showed no significant differences in height and weight but in age (68 vs. 63 y). The median T-scores from DXA measurements in women with and without fractures showed no significant differences (lumbar spine: −2.2 and −2.1, total hip: −2.2 and −2.2, neck: −2.0 and −1.8, respectively).
However, selective and region specific trabecular densities as well as structure parameters measured by HRpQCT differed significantly in osteopenic and osteoporotic women with and without fractures. The mean total density was lower in women with fracture than in women without fractures (D100, 247 vs. 291 mg/ml, p < 0.001), as well as the mean trabecular density (Dtrab, 94 vs. 123 mg/ml, p < 0.001) and the mean inner trabecular density (Dinn, 49 vs. 75 mg/ml, p < 0,001). Furthermore structure parameters in patients with fractures showed significantly increased trabecular separation (TbSp, 678 vs. 548 μm, p < 0.001) and significantly decreased trabecular number (TbN, 1,3 vs. 1,6/mm, p < 0,001) as well as BV/TV (7,8 vs. 10,3%, p < 0,001) and significantly decreased cortical thickness (CtTh, 623 vs. 735 μm, p < 0,05) with visible inhomogeneity of trabecular structure. In conclusion, high-resolution peripheral computed tomography might help to predict fracture risk in osteopenic and osteoporotic patients. Especially in patients with osteopenia (according WHO-definition), the T-score seems to underestimate the fracture risk.
Disclosures: H. Radspieler, None.
Use of DXA-Based Structural Engineering Models of the Proximal Femur to Predict Hip Fracture.
L. Yang1, N. Peel2, J. Clowes1, E. V. McCloskey1, R. Eastell1. 1Academic Unit of Bone Metabolism, University of Sheffield, Sheffield, United Kingdom, 2Metabolic Bone Centre, Northern General Hospital, Sheffield, United Kingdom.
Several DXA-based structural engineering models of the proximal femur have been developed to estimate stress due to sideway falls. Their usefulness in predicting hip fracture has not yet been established and we therefore evaluated these models as diagnostic tools for hip fracture.
We recruited 51 consecutive female patients presenting to the orthopaedic ward with low-trauma hip fracture (ages 58–81); 30 of the fractures had occurred at the femoral neck (FN), 17 were classified as trochanteric (TR) and 4 fracture sites were not specified. For each case we selected 3 age-height and weight-matched controls from a population-based cohort. The hip DXA scans were analysed using a special version of Hologic's software that produces a pixel-by-pixel BMC map. Curved-beam, curved composite beam and finite element (FE) models were generated and the stress calculated at each pixel. The elastic modulus and yield stress of the models were determined according to Keyak (Keyak et al. Clin Orthop Relat Res 437: 219–28). Index of fracture risk (IFR) was defined at each pixel as the stress divided by the yield stress. The maximum and mean IFR in the FN, narrowest FN, middle trochanter (MT), trochanter and total hip were calculated. The usual hip structure analysis (HSA) parameters were also calculated. Logistic regression was performed followed by ROC analysis, expressed as area under the curve (AUC), to identify the best discriminators between cases and controls. Analysis was performed regarding hip fracture as a homogeneous condition and repeated separately for FN and TR fractures.
TH BMD, FN axial length (AL), MT cortical thickness (CTH) and mean IFR (avgIFR) from the FE models were identified as best predictors. Specificity for hip fracture was consistently high (93–99%). The sensitivity and AUC are shown in the Table where * indicates AUCs that are significantly (p<0.05) different from that with TH BMD alone as predictor.
In conclusion, this study suggests that HSA and FE models can enhance hip fracture discrimination over TH BMD alone. Preliminary analyses suggest their contribution may differ between FN and TR fractures but further evaluation is required.
Disclosures: L. Yang, None.
Longitudinal Rate of Change in Bone Mineral Density by Age for Men and Women.
L. Langsetmo*1, C. Berger*1, D. A. Hanley2, J. Prior3, W. P. Olszynski*4, J. Brown*5, L. Joseph*6, A. Tenenhouse*6, D. Goltzman6. 1CaMos, Montreal, PQ, Canada, 2University of Calgary, Calgary, AB, Canada, 3University of British Columbia, Vancouver, BC, Canada, 4University of Saskatchewan, Saskatoon, SK, Canada, 5Laval University, Ste-Foy, PQ, Canada, 6McGill University, Montreal, PQ, Canada.
The objective of this study was to estimate the average rate of change in bone mineral density (BMD) of the lumbar spine and hip as a function of age for men and women 25–85 years old. The source population was a longitudinal cohort of 9423 community-dwelling Canadian adults recruited from randomly selected households within a 50 km radius of nine cities across Canada. BMD of the lumbar spine and hip (total hip, femoral neck, trochanter, and Ward's triangle) was measured at baseline and 5-year follow-up for all age groups and at 3-year follow-up for those 40–60 years old. The study excluded anyone without repeat BMD measurement (n=2665), over 85 years (n=179), or with more than 3 months corticosteroid use (n=211). A direct calculation or linear regression was used to estimate individual rate of change. A separate analysis was done for men and women and further stratified by use of anti-resorptives. The average rate of BMD change was calculated based on 5-year age intervals and used to construct age trajectories for the rate of bone loss.
The main age-dependent changes for women are a period of accelerated bone loss in those 40–54 years old, increased loss at hip sites in those over 70 years old, and a sustained increase in BMD of the lumbar spine in those over 65 years old. The main age-dependent changes for men are increased loss at all sites in those 25–39 years old, increased loss at hip sites in those over 65 year old, and a sustained increase in lumbar spine BMD in those over 40 years old. Different hip sites had similar but not parallel trajectories. The maximal femoral neck BMD loss for women not using anti-resorptives was an average 0.0077 (95% CI: 0.0062–0.0091) g/cm2 per year in the 50–54 year age group and for men not using anti-resorptives was an average 0.0060 (95% CI: 0.0032–0.0088) g/cm2 per year in the 25–29 year age group. In contrast, the femoral neck BMD loss for women using anti-resorptives was an average 0.0026 (95% CI: 0.0017–0.0035) g/cm2 per year in the 50–54 year age group. Mean rates of bone loss for anti-resorptive users were lower than non-users for both men and women 50–79 years old. The differences between users and non-users were statistically significant for women but not men. The overall pattern of bone loss with age is substantially different in men and women with different periods of increased bone loss. Understanding the timing of maximal bone loss may enable better strategies in prevention and management of osteoporosis.
Disclosures: L. Langsetmo, None.
Impact of Herbal Remedies for Menopause on Bone Mineral Density: The Herbal Alternatives for Menopause (HALT) Trial.
K. M. Newton1, A. Z. LaCroix2, S. D. Reed*3, L. C. Grothaus*1, J. W. Lampe*2, K. Ehrlich*1. 1Group Health Center for Health Studies, Seattle, WA, USA, 2Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 3Obstetrics and Gynecology, University of Washington, Seattle, WA, USA.
While estrogen therapy is associated with decreased fracture risk, evidence regarding the effects of soy isoflavones on bone mineral density (BMD) is mixed, and the influence on BMD of herbal approaches used for menopause symptoms is unknown. The purpose of this study was to evaluate the effects of herbal approaches for menopause symptoms on hip and spine BMD.
We enrolled 351 peri and postmenopausal women, aged 45–55, in Washington state, into the HALT study, a 5-arm randomized controlled trial comparing 3 herbal therapies and hormone therapy to placebo. Entry criteria included ≥ 2 hot flashes and/or night sweats per day. Over 95% completed the month 6 BMD, 93% the month 12. Women were randomly assigned to: 1) black cohosh (160 mg daily); 2) multibotanical with black cohosh (200 mg daily) and 9 other ingredients; 3) multibotanical plus dietary soy counseling; 4) conjugated equine estrogen 0.625 mg ± medroxyprogesterone acetate 2.5 mg daily (HT); or 5) placebo. Outcome measures were BMD of the lumbar spine and total hip measured by DXA at baseline, 6, and 12 months. We used maximum likelihood (mixed models) to compare mean percent change from baseline for herbs and hormone therapy vs. placebo, adjusted for age, body mass index (kg/m2), dietary calcium and baseline BMD. Compared to placebo, the multibotanical plus dietary soy was associated with a significant decrease in hip and spine BMD at 6, but not 12 months. Compared to placebo, HT had no significant effect on hip BMD but was associated with a significant increase in spine BMD at 6 and 12 months.
Black cohosh and the multibotanical with black cohosh had no impact on hip or spine BMD, Table 1.
We conclude that black cohosh alone or as part of a multibotanical preparation has no impact on BMD of the total hip or lumbar spine, and that black cohosh taken in combination with other herbs and soy diet may negatively impact spine BMD.
Disclosures: K.M. Newton, None.
Effects of Functional Single Nucleotide Polymorphism in the Tissue-Nonspecific Alkaline Phosphatase Gene (787T>C) Associated with BMD.
M. Goseki-Sone1, N. Sogabe*1, K. Oda*2, H. Nakamura*3, H. Orimo*4, T. Hosoi5. 1Department of Food and Nutrition, Japan Women's University, Tokyo, Japan, 2Division of Biochemistry, Niigata Univerisity, Graduate School of Medical and Dental Sciences, Niigata, Japan, 3Department of Chemistry, Gakushuin University, Tokyo, Japan, 4Division of Molecular Genetics and Nutrition, Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan, 5Department of Advanced Medicine, National Center for Geriatrics and Gerontology, Aichi, Japan.
Human alkaline phosphatases (ALPs) are classified into four types: tissue-nonspecific, intestinal, placental, and germ cell types. Previously, we identified polymorphisms of the TNSALP gene 787T>C (Y246H) associated with BMD among 501 postmenopausal women. There was a significant difference in the BMD and the score of BMD adjusted for age and body weight (Z-score) among haplotypes, which was lowest among 787T homozygotes, highest among 787T>C homozygotes, and intermediate among heterozygotes. In subgroups divided by age, haplotypes were significantly associated with the BMD in older postmenopausal women. These results indicated that the effect of haplotypes on the BMD depended on age. In the present study, we have investigated the effects of amino acid substitution on the biosynthesis and the catalytic properties of the protein translated from the 787T>C gene, and three-dimensional structure-related effects on calcium. Furthermore, we explored the possibility that the TNSALP gene may contribute to phosphate metabolism in humans by measuring serum concentrations of bone-specific ALP (BAP), osteocalcin, calcium, phosphorus, and FGF-23 (phosphatonin). As the results, TNSALP (787T) and TNSALP (787T>C) were synthesized similarly as a high-mannose-type 66-kDa form, and became an 80-kDa form in an in vitro translation system. Expression of the 787T>C TNSALP gene using mouse marrow stromal cells (ST2) demonstrated that the protein translated from 787T>C exhibited an ALP-specific activity similarly to that of 787T. Interestingly, the Km value for TNSALP in cells transfected with the 787T>C TNSALP gene was decreased significantly compared to that of cells bearing the 787T gene (p<0.01). Moreover, we demonstrated the associations among the levels of serum BAP and phosphorus in 60 healthy young males. The level of serum BAP was significantly correlated with serum phosphorus in 787T (p<0.01), but not in 787T>C homozygotes. These results suggest that TNSALP variation may be an important determinant of age-related bone loss in humans, and that the phosphate metabolism pathway may provide a novel target for the prevention and treatment of osteoporosis.
Disclosures: M. Goseki-Sone, None.
The Effect of Nutritional Calcium on the Risk of Postmenopausal Fractures Varies with Age. A 15-year Follow-up of the OSTPRE Cohort.
R. J. Honkanen1, K. Salovaara2, M. Kärkkkäinen1, M. T. Tuppurainen3, H. P. Kröger1, E. M. Alhava4. 1BCRU, Clinical Research Center, University of Kuopio, Kuopio, Finland, 2Surgery, Kuopio University Hospital, Kuopio, Finland, 3Gynaecology, University Central Hospital of Kuopio, Kuopio, Finland, 4Surgery, University Central Hospital of Kuopio, Kuopio, Finland.
We have previously shown that nutritional calcium (Ca) prevents early postmenopausal distal forearm fracture (DFF). The purpose was to examine the stability of this Ca effect with age.
The study population consisted of those 9403 OSTPRE cohort women (bom in 1932-.41) who responded to all four enquiries: in 1989 (N= 13100), in 1994 (N=11798), in 1999 (N=10977) and in 2004 (N=9403). Self-reported follow-up fractures were validated by perusal of patient records: 777 women recorded fractures in 1989–94, including 279 women with DFF and 6 women with hip fracture; the number of women with fractures in 1999–2004 was 835, including 322 women with DFF and 28 women with hip fracture. Self-reported dairy Ca intake (forming about 75 % of overall nutritional Ca intake) was used as a measure of nutritional Ca intake. Logistic regression was used as the statistical method.
Mean Ca intake was 827(SD 391) mg/day in 1989 and 848 (SD370) mg/day in 1999. Ca intake did not predict fractures in general in 1989–94 or in 1999–2004. It protected from DFF in 1989–94 (p=0.001 for continuous Ca variable) (with OR of 0.68 (95% CI 0.51.0.92) at Ca intake of 500–999 mg and OR of 0.57 (0.40–0.81)) at Ca intake >999 mg compared to women with Ca intake <500 mg) but not in 1999–2004 (p=0.353 for continuous Ca variable). However, a joint effect of high Ca intake and HRT on the incidence (%) of DFF was seen even in late postmenopausal women in 1999–2004:
In addition, Ca intake strongly protected from hip fracture in 1999–2004 (p=0.001 for continuous Ca variable) with OR of 0.27 (0.13–0.59) at Ca intake of <500 mg and OR of 0.08 (0.02–0.34) at Ca intake >999 mg compared to Ca intake of <500 mg. Adjusting for weight, height, fracture history, HRT, time of menopause and number of health disorders did not affect these fracture risks. Ca intake did not protect from fractures at other sites.
The protective effect of nutritional Ca on distal forearm fracture risk is more easily seen in early than late postmenopausal women, whereas the effect on the risk of hip fracture becomes apparent later with increasing incidence of hip fractures.
Disclosures: R.J. Honkanen, None.
This study received funding from: Academy of Finland.
Leg Lean Mass and Risk of Hip Fracture: The Framingham Study.
M. T. Hannan, K. E. Broe*, R. R. McLean*, D. P. Kiel. HSL Institute for Aging Research, Boston, MA, USA.
While bone density is a potent risk factor for fracture, few data exist on lean muscle mass and risk of hip fracture. Low lean mass, or ‘sarcopenia’, has been associated with reduced muscle strength and lower functioning in elders. We examined whether leg lean mass predicted risk of hip fractures in women of the Framingham Original Cohort with the hypothesis that lower lean mass of the legs would be associated with increased hip fracture risk even after adjustment for major confounders. Men were not included as too few hip fractures occurred in men and their lean mass measures were different from women.
432 women of the Original Cohort had leg lean mass (kg) measured in 1992–93 using whole body DXA (Lunar DPX-L) and were followed for incident hip fracture. Fracture information through 12/31/2003 was provided by the Framingham Fracture Registry (incident hip fractures ascertained by interview, record reviews and confirmed by x-ray). Number of person-years was determined from DXA scan date to hip fracture, last contact, death, or end of follow-up. Cox proportional hazards regression was used to calculate hazard ratios (HR) and 95% confidence intervals (CI) estimating the risk of hip fracture for each kg increase in leg lean mass. Covariates (baseline) included age, weight, height, physical activity (PASE score), calcium and vitamin D intake and femoral neck bone mineral density (BMD).
Mean age was 78 yrs (range: 72–92 y); 49 hip fractures occurred over follow-up. Mean leg lean mass for these women was 11.38 kg (range 7.2–17.0). Leg lean mass was significantly inversely correlated with age, and significantly positively correlated with weight, height, physical activity and BMD. In Cox regression model adjusting for age, weight and height, leg lean mass predicted subsequent hip fracture, HR=1.46 (95% CI: 1.15, 1.85). Further adjustment for physical activity, calcium and vitamin D intake did not change these results. Adding femoral neck BMD to the model attenuated the risk somewhat: for each kg increase in leg lean mass in women, the risk of hip fracture increased by 30% (HR=1.30, 95% CI: 1.01, 1.67).
In sum, leg lean mass predicted hip fracture in elderly women even after adjusting for BMD. While not supporting our hypothesis, these unexpected results may reflect that those elderly women with greater lean mass were more physically active and were more apt to fall and fracture (ie, adjusting for physical activity may not have fully adjusted for increased opportunity to fall & fracture). Also, perhaps a lower fracture risk exists for middle-age adults but not the geriatric women we studied (ie, hip fracture risks change with age). Further studies should examine lean mass and fractures in men as well as change in physical activity and falls for risk of hip fractures in elders.
Disclosures: M. T. Hannan, None.
This study received funding from: NIH.
Burden of Hip and Wrist Fractures: Incidence Estimates of Non-traumatic Fractures in Older Women and Men in North America, Western Europe, Australia, and Japan.
C. Strader*1, C. W. Shinoff2, M. S. Anthony2, L. A. Fitzpatrick2. 1The Mattson Jack Group, St. Louis, MO, USA, 2Amgen Inc., Thousand Oaks, CA, USA.
Fractures in older men and women lead to increased risk of subsequent fractures and mortality. Current international estimates of non-traumatic fracture incidence are not readily available, but are needed to estimate the burden of osteoporotic disease. We estimated the annual incidence of non-traumatic hip and wrist fractures in women >50 and men >60 years of age. A literature review and web-search was conducted to find studies that quantified fracture incidence in the United States, Canada, United Kingdom, Germany, France, Italy, Spain, Australia, and Japan. Data from the National Health and Nutrition Examination Survey (NHANES) 1999–2002 were used to estimate the ratio of site-specific non-traumatic fractures to total hip and wrist fractures. Non-traumatic fractures were defined as a fall from standing height or less. If the country-specific fracture studies did not exclude traumatic fractures, the site-specific NHANES ratio of non-traumatic to total fractures was used to estimate site and country-specific non-traumatic fracture incidence rates. Census data were used to standardize the age-, gender-, and country-specific incidence data to the 2005 country-specific population. Non-traumatic hip fracture incidence was 1–6 fold higher and wrist fracture incidence 3–8 fold higher in women compared to men (Table 1). Hip fracture incidence was highest for women in Germany and wrist fracture rates were highest in US women. Hip and wrist fracture rates increased with age for both men and women.
Fractures in older women and men represent a significant problem globally. Projected to the 2005 census, there are about 500,000 non-traumatic hip fractures and 537,000 non-traumatic wrist fractures annually in women older than 50 and men older than 60 years of age in these 9 countries. Differences in rates between countries could be due to inherent biologic differences, differences in fracture ascertainment, or differences in environmental factors, such as strategies for preventing fractures. As the population ages, these rates will continue to increase.
Disclosures: C. Strader, Amgen Inc. 5.
This study received funding from: Amgen Inc.
Chronic Proton Pump Inhibitor Use Is Not Associated with an Increased Risk of Osteoporotic Fracture.
L. E. Targownik*1, W. D. Leslie1, L. M. Lix*2, C. J. Metge*3, H. J. Prior*2, S. Leung*1. 1Internal Medicine, University of Manitoba, Winnipeg, MB, Canada, 2Community Health Sciences, University of Manitoba, Winnipeg, MB, Canada, 3Pharmacy, University of Manitoba, Winnipeg, MB, Canada.
Proton pump inhibitors (PPIs) are commonly prescribed medications used for the treatment and prevention of a variety of gastrointestinal diseases. They are believed to decrease gastrointestinal absorption of dietary calcium, and are also postulated to stimulate osteoclast activity. Therefore, PPIs are a putative risk factor for the development of osteoporosis and osteoporotic fracture. Results of studies linking PPI use to osteoporotic fracture are conflicting.
We used the Manitoba Health Research Data Repository to perform a nested case-control study. Cases, defined as patients admitted for a hip/proximal femoral fracture between 1996–2004, were matched to controls with no history of osteoporotic fracture (hip, spine, wrist) on age, sex, medical comorbidity and ethnicity (Aboriginal vs. non-Aboriginal). PPI exposure was obtained from the provincial pharmacy database, and tracked for either 1, 2, or 4 years prior to the index date. Subjects were considered to be continuously exposed to PPIs if there was documented PPI use over >70% of days in the exposure period of interest (1, 2, and 4 years prior to event), and unexposed if PPIs were used <10% of the time during the exposure period of interest. Conditional logistic regression was performed to obtain odds ratios and confidence intervals, adjusted for pertinent covariates.
Numbers of cases and controls for each exposure period and both the unadjusted and adjusted odds ratios are displayed in the table below. We did not detect an association between PPI use and hip fracture over 1, 2 or 4 years of prior exposure.
Disclosures: L.E. Targownik, None.
This study received funding from: Canadian Institutes of Health Research.
Vertebral Fracture Lines Are Not Smooth, but Mixtures of Multiple Schmorl's Nodes and Endplate Perforations.
T. Okamoto*1, S. Okamoto1, H. Noguchi*2, H. Suzuki*1, N. Otsu*1, A. Itabashi4. 1SORT Okamoto Clinic, Oita, Japan, 2Noguchi Thyroid Clinic and Hospital Foundation, Beppu, Japan, 3Suzuki Orthodontic Office, Nagasaki, Japan, 4Saitama Center of Bone Research, Saitama, Japan.
Although vertebral fracture cannot occur without mechanical compression, discussion on what exactly is the primary causative factor of vertebral depression has been conspicuously absent. The 3-D models revealed that the vertebral endplates of fractured vertebra are rarely smooth and are compromised with multiple Schmorl's nodes and endplate perforations. The TV X-ray fluoroscopy was performed on 2,600 female patients with osteoporosis in one outpatient clinic in Oita, Japan. Care was taken to adjust the posture of each patient to align the vertebrae in right angle to the film. Also we created computerized 3-D renderings of 1280 cases comprised of post-menopausal women aged 45–85, primarily of patients of osteoporosis and osteoarthritis, and healthy female controls aged 20–40. The digital 3-D models were constructed from helical CT data and examined for morphologic characteristics. The 3-D models revealed that the vertebral endplates of post-menopausal women are frequently compromised with perforated indentations. We calculated the ratio of the perforations to the area of the endplates and found that the there was a significant correlation between the perforation/endplate area ratio and the number of years after menopause. The space enclosing the vertebral disc actually expands while the rims of the vertebrae converge. What has conventionally been known as fish-shaped vertebral fractures may in fact be secondary to the herniation of the disk nucleus into the weakened vertebral body of the osteoporotic spine. The so-called wedge type fractures appear to be the result of rim destruction due to direct contact between the vertebral rims after narrowing of intervertebral space. The multiple perforations which appears just after menopause, and gradually enlarge, are eroding vascular pathways which nourish the vertebral disc through the vertebral medulla. To avoid inaccurate diagnosis of vertebral fractures, a better grasp of the vertebral structure as a complex three-dimensional entity is definitely necessary.
Disclosures: T. Okamoto, None.
Computer Based Measure of Kyphosis Predicts Fractures in the Thoracic Spine of Postmenopausal Women.
P. C. Pettersen1, M. de Bruijne*2, J. Chen1, Q. He*1, L. B. Tankó*3, C. Christiansen4. 1Radiology, Center for Clinical and Basic Research, Ballerup, Denmark, 2Department of Computer Science, University of Copenhagen, Copenhagen, Denmark, 3Clinical Research, Center for Clinical and Basic Research, Ballerup, Denmark, 4Nordic Bioscience, Herlev, Denmark.
The aim of this study is to investigate whether recently introduced computer-based measures of the degree of kyphosis and irregularities in vertebral alignment of the thoracic spine are independent contributors to fragility fractures.
This was a case control analysis including 126 healthy elderly women, 63 of whom maintained skeletal integrity, whereas 63 sustained a thoracic fracture over the mean observation period of 4.8 years. None of the women received ongoing osteoporosis treatment and the cases and controls were matched for age, follow-up, spine BMD L1-L4 (DEXA), and body weight. Lateral X-rays acquired at baseline and follow-up were digitised and the corner points on each vertebra from Th4 to L1 were marked. Spine curvature at each vertebra level was characterized by the curvature (1/radius) of the circle connecting its center with the centers of neighbors. The degree of kyphosis is defined as the mean curvature of the thoracic spine (Th5 to Th12). The sum of differences between the individual curvatures and the mean curvature defined irregularity of vertebral alignment.
Kyphosis presented a statistically significant difference between the two groups at baseline (fig1 p=0.002), and the measure of kyphosis was increased from baseline in the fracture group (p=0.02). The analyses of irregularities along the thoracic spine did not show differences between the two groups at baseline (fig1). However, the measure of irregularity of vertebral alignment increased in those sustaining a vertebral fracture (p=0.0001). The mean values and SD are shown in Figure 1.
The measure of kyphosis, but not the measure of irregularity, can predict fragility fractures in the thoracic spine of postmenopausal women, independent of age, body weight and spine BMD. The observation of increasing irregularity of vertebrae alignment is considered as a consequence of irregular anterior wedge accentuation due to either remodeling or fragility fractures. Secondly, this observation indicates that the herein presented computer based diagnostic tool could be a useful supplement to existing approaches to fracture prediction.
Disclosures: P.C. Pettersen, None.
Endogenous Sex Hormones and Incident Fracture Risk in Older Men: The Dubbo Osteoporosis Epidemiology Study.
C. Meier1, T. V. Nguyen2, D. J. Handelsman*3, C. Schindler*4, M. M. Kushnir*5, A. L. Rockwood*5, W. A. Meikle*6, J. R. Center2, J. A. Eisman2, M. J. Seibel7. 1Division of Endocrinology, University Hospital Basel, Switzerland, 2Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney, Australia, 3Department of Andrology, ANZAC Research Institute, Sydney, Australia, 4Institute of Social and Preventive Medicine, University of Basel, Switzerland, 5ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA, 6Department of Medicine and Pathology, University of Utah, Salt Lake City, UT, USA, 7Bone Research Program, ANZAC Research Institute, Sydney, Australia.
One third of osteoporotic fractures occur in men. Data on the influence of gonadal hormones on incident fracture risk in elderly men are limited. The present study examined the prospective relationship between serum levels of testosterone (T) and estradiol (E2) and future fracture risk in elderly men as part of the long-term observational Dubbo Osteoporosis Epidemiology Study of community-dwelling men over 60 years followed prospectively since 1989. This analysis included men who had serum samples for baseline measurements (n=609, 70.6%) with follow-up of 5.8 years (up to 13 yrs) closing in 2005. Clinical risk factors, including bone mineral density (BMD) and lifestyle factors were assessed at baseline. Serum T and E2 were measured by tandem mass spectrometry (LC-MS/MS). The incidence of low-trauma symptomatic fracture was ascertained by X-ray record.
During follow-up, 113 men suffered at least one low-trauma fracture. Fracture risk was increased in men with reduced T levels (hazard ratio [HR] 1.33; 95%CI: 1.09, 1.62). After adjustment for SHBG, both low serum T (HR 1.48; 95%CI: 1.22, 1.78) and serum E2 (HR 1.21: 95%CI: 1.00, 1.47) were associated with increased overall fracture risk. After further adjustment for major fracture risk factors (age, weight or BMD, fracture history, smoking status, calcium intake and SHBG), lower T was still associated with increased risk of hip (HR 1.88; 95%CI: 1.24, 2.82) and non-vertebral (HR 1.32; 95%CI: 1.03, 1.68) fracture. By contrast, lower E2 was only associated with increased fracture risk in the presence of body weight (HR 1.25; 95% CI: 1.02, 1.54), but not at any site after adjustment for BMD (HR 1.19; 95% CI: 0.69, 1.03).
In community-dwelling men over 60 years of age, serum T, but not E2, is an independent predictor of osteoporotic fracture and its measurement may provide additional clinical information for the assessment of fracture risk in elderly men.
Disclosures: C. Meier, None.
Respiratory Function Is Associated with Bone Ultrasound Measures and Hip Fracture: European Prospective Investigation Into Cancer-Norfolk Population Cohort Study.
A. Moayyeri*1, R. N. Luben*1, S. Bingham*2, A. Welch*1, N. J. Wareham*3, S. Kaptoge1, J. Reeve1, K.T. Khaw*1. 1Department of Public Health and Primary Care, Institute of Public Health, School of Clinical Medicine, The University of Cambridge, Cambridge, United Kingdom, 2MRC Dunn Human Nutrition Unit, The University of Cambridge, Cambridge, United Kingdom, 3MRC Epidemiology Unit, The University of Cambridge, Cambridge, United Kingdom.
Forced expiratory volume in 1 second (FEV1), an easily obtainable measure of respiratory function in clinics, has been shown to be associated with physical activity. We hypothesized that FEV1 is linked with bone health. In the context of the European Prospective Investigation into Cancer-Norfolk study, 14,800 participants aged 42–81 in 1997–2000 were evaluated by spirometry and heel ultrasound and were followed for fracture outcomes up to July 2007. After excluding participants with history of pulmonary diseases, among 5,555 men and 6,935 women (mean age 62.1+/- 9.0), FEV1 significantly correlated with heel broadband ultrasound attenuation (BUA; Pearson r=0.403; p<0.001) and velocity of sound (VOS; r= 0.269; p<0.001). The association remained significant in sex-stratified linear regression models after adjustment for age, history of fracture, height, body mass index, smoking status and alcohol consumption (standardized Beta coefficient=0.057 and p<0.001 in men; Beta=0.075 and p<0.001 in women). Mean adjusted FEVI among 109 hip fracture patients (2.00+/-0.60 liter) was significantly lower than that of other participants (2.49+/-0.71 liter; t-test p<0.001). In a Cox proportional hazards regression model, FEV1 was a significant predictor of hip fracture after adjustment for age, sex, history of fracture, height, body mass index, smoking and alcohol consumption (hazard ratio for 1 standard deviation [700 ml in 1 second] decrease in FEV1 = 1.39, 95%CI 1.03–1.88, p=0.029). Among 6197 current and former smoker participants, hazard ratio for I standard deviation decrease in FEV1 was l.67(95%CI 1.11–2.57, p=0.014). Middle aged and older men and women with lower respiratory function appear to be at increased risk of osteoporosis and hip fracture. The observed association might be related to the level of physical activity or deformities in thoracic spine related to osteoporosis. Given the feasibility and affordability of spirometry in general practices, it can be used to improve the identification of high risk groups at the first point of care.
Disclosures: A. Moayyeri, None.
This study received funding from: Medical Research Council and Cancer Research UK.
Competing Risk of Subsequent Fracture and Death After Hip Fracture in Nursing Home (NH) Residents.
S. Berry, K. Broe, D. Kiel. IFAR, Hebrew SeniorLife, Boston, MA, USA.
Prior studies have demonstrated an increased risk of subsequent fracture among community dwellers with hip fracture. It is unclear whether this is also true in NH residents given the high burden of co-morbidities and associated competing risk of mortality. Therefore, we described the incidence of second fractures and the competing risk of death following a hip fracture in a large NH.
Participants were 218 consecutive residents (49 men, 169 women) of Hebrew Rehabilitation Center, a 725 bed long term care facility in Boston, MA, with a hip fracture between 1/1/1999 and 12/31/2006. Fractures were identified from a review of mandated injury files (excluding fractures associated with malignancy, Paget's disease, or prosthesis; n=3). Second fractures included any fracture occurring after the initial fracture, excluding toe and hand fractures. Baseline characteristics were obtained from administrative databases in the three months preceding the initial hip fracture. Subjects were followed from initial hip fracture until second fracture, death, discharge, or 12/31/2006. A Wilcoxon rank sum compared baseline age among subjects with second fracture and subjects who died without second fracture. Cumulative incidence estimates, taking into account the competing risk of death, described the incidence of second fractures.
At baseline, 82% of subjects had 0–1 major co-morbidities, and 35% were functionally intact or had mild impairment. Over a median follow-up of 11 months (range, 2 d- 8 yrs), 17% of subjects (7 men, 31 women) experienced a second fracture (17 hip, 5 leg, 2 ankle, 5 arm, 5 pelvic, 2 clinical vertebral, 1 skull, 1 clavicle), and 55% of subjects (30 men, 90 women) died. Subjects with second fracture were younger than subjects who died without second fracture (median 88 yrs vs. 91 yrs, p=0.02). Five percent of subjects experienced a second fracture within six months, and 10% experienced a second fracture within 1 yr of the initial hip fracture. In contrast, 27% of subjects died within 6 months, and 37% died within 1 yr of the initial hip fracture (Kaplan-Meier estimates). In conclusion, the striking mortality among NH residents with minimal co-morbidities and hip fracture highlights the importance of hip fractures as a sentinel event in the NH. Among NH residents who survive a hip fracture, second fractures are common. Future studies should focus on identifying likely survivors with sufficient quality of life to target secondary fracture prevention.
Disclosures: S. Berry. None.
Factors Influencing the Diagnosis and the Treatment of Osteoporosis Following a Fragility Fracture.
L. Bessette1, J. P. Brown1, S. Jean*2, K. S. Davison1, M. Beaulieu*3, M. Baranci*4, J. Bessant*5, L. Ste-Marie*6. 1Laval University, Quebec, PQ, Canada, 2INSPQ, Quebec, PQ, Canada, 3Merck Frosst, Montreal, PQ, Canada, 4sanofi aventis, Montreal, PQ, Canada, 5P&G Pharma, Montreal, PQ, Canada, 6U. of Montreal, Montreal, PQ, Canada.
ROCQ (Recognizing Osteoporosis and Its Consequences in Québec) revealed that 73% of women 50y and over are not provided anti-fracture therapy following fragility fracture. This study's objectives were to determine predictors of osteoporosis (OP) diagnosis (DX) and treatment (TX) 6 to 8 months after fragility fracture.
At phase 1, women were recruited at cast or outpatient clinics in 17 hospitals 0 to 16 weeks following fracture. Consenting patients were contacted by phone to answer a short questionnaire to classify them as having either experienced a fragility or traumatic fracture. During the first phone contact, there was no reference to the possible association between the fracture and OP and no investigation or intervention was proposed. At phase 2, 6–8 months following fracture, women were contacted again by phone to complete a questionnaire on demographic features, clinical characteristics and risk factors for OP. The DX (informed of OP and/or BMD measurement with diagnosis of OP) and TX (bisphosphonates, raloxifene, nasal calcitonin or teriparatide) rates of OP were determined via this questionnaire. This analysis included only women with a fragility fracture who were not receiving OP TX at phase 1.
Of the 1273 women who completed phase 1, 1001 (79%) sustained a fragility fracture; 818 were not on treatment at phase 1 and completed the phase 2 questionnaire. Overall, 79% of these participants had not received a DX of osteoporosis or were without OP TX at phase 2. The highest rate of DX and TX of OP was observed between 0–5 months following fracture and decreased considerably thereafter. In multivariate analyses, the results of BMD tests before or after the fracture event (p<0.0001) and mobility problems (p=0.03) were the only variables that influenced the DX of OP. The BMD test result was the strongest predictor (p<0.0001) of TX followed by the fracture site (hip, femur and pelvis; p=0.015) and vitamin D supplements at the time of fracture (p=0.035). All other risk factors for OP such as age, fracture history after age 40, family history of OP, and comorbidities did not influence the DX or TX rate. No demographic and clinical features or OP risk factors were significantly associated with the decision to send women for a BMD testing following fracture.
Despite the evidence clearly showing that the occurrence of a fragility fracture represents a greater risk of future fragility fracture than a low BMD measurement, physicians based their decision to treat on BMD results and not on the clinical event (fragility fracture).
Disclosures: L. Bessette, Merck Frosst Canada 2, 5, 8; The Alliance for Better Bone Health (P&G and sanofi-aventis) 2, 5, 8: Eli Lilly 2, 5. 8: Novartis Pharma 2. 5. 8.
This study received funding from: Merck Frosst, P&G Pharmaceuticals, sanofi-aventis, Eli Lilly, Novartis.
A Comparison of the Effects of Thiazide Diuretics and Angiotensin Converting Enzyme Inhibitors on Fracture Risk — A Randomised Controlled Trial.
T. Winzenberg1, K. Willson*2, P. Ryan*2, M. Nelson*1, G. Jones1. 1Menzies Research Institute, Hobart, Australia, 2Adelaide University, Adelaide, Australia.
Observational evidence suggests that antihypertensives, particularly thiazide diuretics may affect bone and fracture risk. However head-to-head comparisons of the effects of different antihypertensives on fracture risk to inform choice of antihypertensive are lacking. We aimed to prospectively compare the effects of thiazide diuretics and angiotensin converting enzyme (ACE) inhibitors on fracture risk.
The second Australian National Blood Pressure trial is a prospective, randomized, open-label study which was conducted in 6083 hypertensive Australian family practice patients aged 65–84 years. Subjects were followed for a median of 4.1 years. Family practitioners (FPs) were responsible for the stepwise management of antihypertensive treatment, guided by study protocols for drug choices and the study's blood pressure treatment goals. Management was to conform to the randomized treatment assignment. Patients were randomised to initially receive either diuretic (hydrochlorothiazide recommended first-line) or ACE inhibitor (enalapril recommended first-line), but the choice of the specific agent and dose was made by the FP. Fractures were a secondary outcome, and were ascertained from FPs' records. Relative risks (RR) were calculated using log-binomial Generalized Estimating Equations, using both intention-to-treat and per-protocol analyses. In the diuretic group, 251 subjects had a fracture (8.3%), compared to 236 (7.8%) in the ACE group. There was no evidence of a difference in fracture rates between those patients randomised to diuretic and those to ACE with the age- and sex-adjusted RR for fracture in the diuretic compared to ACE groups being 1.06 (95% CI 0.90–1.25) for all fractures and 1.16 (95% CI 0.72–1.86), 1.02 (95% CI 0.66–1.57) and 1.15 (95% CI 0.82–1.62) for hip, vertebral and upper limb fractures respectively, in intention-to-treat analyses. Per protocol analysis examining treatments received and comparing use of thiazides, indapamide or either of these diuretics with use of neither of these agents demonstrated similar results, again with no differences in fracture rates.
There is no evidence to suggest that either thiazide diuretics or ACE inhibitors are superior to each other for reducing fracture risk in elderly hypertensive patients, or that choice of hypertensive therapy should be influenced by concerns about effects on fracture risk. Treatment of hypertension should still be guided by current best practice recommendations taking into account other significant co-morbidities.
Disclosures: T. Winzenberg, None.
Vasomotor Symptoms Are Related to Lower Bone Mineral Density (BMD): A Longitudinal Analysis.
C. J. Crandall1, S. Crawford*2, R. Thurston*3, E. Gold*4, J. Johnston*3, G. Greendale1. 1University of California, Los Angeles, CA, USA, 2University of Massachusetts, Worcester, MA, USA, 3University of Pittsburgh, Pittsburgh, PA, USA, 4University of California, Davis, CA, USA.
Vasomotor symptoms (hot flashes or night sweats, VMS) are common in perimenopausal and postmenopausal women. We determined within each menopause transition stage whether women with vasomotor symptoms had lower BMD than women without VMS.
We analyzed longitudinal data from 2136 participants of the Study of Women's Health Across the Nation, a cohort study of Caucasian, African American, Japanese, and Chinese women. At baseline, women were aged 42 to 52 years, had intact uterus and >1 ovary, and were pre- or early peri-menopausal. Menopausal stage and VMS were assessed by annual questionnaire. We defined frequent VMS as reporting VMS for >5 days in the past 2 weeks. Menopause stages were premenopausal (no change in menstrual regularity), early perimenopausal (decreased regularity in past 3 months), late perimenopausal (no menses in past 3–11 months), or postmenopausal (no menses for >12 months). Using data from baseline to annual follow-up visit 5 in repeated measures mixed models, we determined the association between VMS (any vs. none, frequent vs. infrequent) and BMD (by DXA) within each menopause status category.
After controlling for age, menopausal stage, cumulative time in each menopausal stage, ethnicity, study site, and baseline menopause stage, postmenopausal women with any VMS had lower lumbar (0.007g/cm2 lower, P=0.002) and lower total hip (0.004 g/cm2 lower, P=0.04) BMD than postmenopausal women without VMS. Compared to early perimenopausal women without VMS, early perimenopausal women with any VMS had lower femoral neck BMD (0.003g/cm2 lower, P<0.0001). Premenopausal women with any VMS had lower femoral neck BMD (0.003g/cm2 lower, P=0.02), compared to premenopausal women without VMS. Results from analyses of women with frequent vs. infrequent VMS were similar. Lumbar and total hip BMD were not different among premenopausal women with any VMS compared to premenopausal without VMS, or among early perimenopausal women with any VMS compared to early perimenopausal women without VMS. Results from analyses additionally controlled for baseline weight, height, and weight change were similar for any vs. no VMS, and frequent vs. infrequent VMS.
Within each menopause transition stage, women with VMS had lower BMD than women without VMS. Effects varied by anatomical site, being most evident in postmenopausal women at the lumbar spine and total hip, and among premenopausal and early perimenopausal women at the femoral neck. Even at the earliest menopause transition stages, women with VMS are at risk for greater bone loss than women without VMS.
Disclosures: C.J. Crandall, None.
This study received funding, from: SWAN has grant support from the NIH, DHHS, through the NIA, the NINR and the NIH ORWH. (Grants NR004061; AG012505, AG012535, AG012531, AG012539, AG012546, AG012553, AG012554, AG012495). Crandall was supported by NIH grant #s 5K12AG01004 from the NIA.
Comparison of Sex Steroid Measurements in Men by Mass Spectroscopy vs. Immunoassay: Relationships with Cortical and Trabecular Volumetric BMD.
S. Khosla, R. J. Singh*, L. J. Melton, S. Achenbach*, J. Peterson*, B. L. Riggs. Mayo Clinic, Rochester, MN, USA.
While immunoassays (IA) have been used extensively for measurement of serum testosterone (T) and estradiol (E2) levels, there is concern about their specificity, particularly at low E2 levels as present in men. Thus, we developed and validated mass spectroscopic (MS) measurements of T and E2 and used this method to measure T and E2 levels in a population-based sample of men (n = 313), age 22 to 91 yrs. We compared the MS levels with values previously obtained using IAs and, to test the biological relevance of E2 measured by the two techniques, related bioavailable (bio, or non-SHBG bound) E2 to cortical and trabecular volumetric BMD (vBMD) at various skeletal sites using QCT. Panels A and B show the relationships between the bio T and bio E2 levels determined using either the MS or IA for the T and E2 measurements. Bio T measurements using the two methods were highly correlated, but the correlation was weaker for bio E2.
Since, based on IAs, we previously postulated the existence of a “threshold” bio E2 level for E effects on cortical, but not trabecular, bone we reanalyzed these relationships for values below vs. above the median levels for bio E2 in men > 60 yrs (8.6 pg/ml and 8.1 pg/ml for the MS and IA, respectively); the Table shows the respective correlation coefficients. As is evident, for both assays, vertebral trabecular vBMD showed similar correlations with bio E2 in subjects with low or high bio E2 levels, whereas femur neck cortical vBMD was correlated with bio E2 at low, but not high bio E2 levels. In summary, although E2 measurements with IAs correlate less well with the MS measurements than do the T measurements in men, these data indicate that the fundamental relationships with BMD for E2 observed with the IAs are also present with the MS measurements. Thus, while MS is considered the “gold standard”, the lower cost and greater accessibility of IAs justifies their ongoing use to assess sex steroid levels, at least in men.
Disclosures: S. Khosla, None.
Morphometric Vertebral Fracture Status and WHO Predictors of Fracture Risk.
P. Chen*1, J. H. Krege*1, D. Goltzman*2, J. C. Prior*3, A. Tenenhouse*2, J. P. Brown*4, M. Papadimitropoulos*1, N. Kreiger*5, W. P. Olszynski*6, R. G. Josse*5, J. P. Adachi*7. 1Eli Lilly and Company, Indianapolis, IN, USA, 2McGill Univ, Montreal, PQ, Canada, 3Univ of British Columbia, Vancouver, BC, Canada, 4Laval University, Ste-Foy, PQ, Canada, 5Univ of Toronto, Toronto, ON, Canada, 6Univ of Saskatchewan, Saskatoon, SK, Canada, 7McMaster Univ, Hamilton, ON, Canada.
The WHO tool considers BMD, age, prior clinical fragility fracture, current smoking, alcohol use, family history of osteoporosis, and glucocorticoid use to assign a 10-year absolute fracture risk [Kanis et al. Osteopor Int 2002; 13:527–36]. We assessed the information gained from consideration of baseline morphometric vertebral fracture (MVF) status and some of the likely WHO risk factors. Data were from 4744 subjects aged >50yr (3452 women, 1292 men) with baseline and 5-yr radiographs from the Canadian Multicentre Osteoporosis Study (CaMos), a prospective, randomly-selected, population-based cohort. Using the CaMos method [Jackson et al. Osteopor Int 2000; 11:680–87], MVF status was defined as: a) present or absent; b) number; c) maximum severity (normal=0, mild=1, moderate or severe=2); and d) spinal deformity index (SDI, sum of severity scores of T4 to L4). Incident vertebral fractures were identified via baseline and 5-year lateral spine radiographs and non-vertebral fractures by an annual questionnaire with medical confirmation. Logistic regression analyses were performed to assess the prediction of 5-yr risk of any fragility fractures (vertebral or non-vertebral) using individual WHO risk factors and MVF status. In univariate analyses, age, BMD, family history, glucocorticoid use, prior clinical fragility fracture and MVF status were statistically significant predictors. As the results were similar, multivariate analyses were performed in men and women together. This analysis of the statistically significant factors showed MVF status, especially greater number or severity of vertebral deformities or higher SDI, provided the greatest discrimination of risk (Table). MVF was the most important predictor of 5-yr fracture risk in this population-based cohort.
Odds ratios (95% CI) for baseline significant risk factors. Analyses were repeated with MVF status defined as yes/no, maximum severity, number, and SDI.
Disclosures: P. Chen, Eli Lilly and Company 1, 3.
This study received funding from: Eli Lilly and Company.
Changes in Hip Geometric Structures with Aging—Longitudinal Data Analysis from the Women's Health Initiative Observational Study.
T. Bassford*1, T. J. Beck2, G. Wu*1, J. A. Cauley3, A. Z. LaCroix*4, C. E. Lewis5, Z. Chen1. 1University of Arizona, Tucson, AZ, USA, 2Johns Hopkins University, Baltimore, MD, USA, 3University of Pittsburgh, Pittsburgh, PA, USA, 4Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 5University of Alabama-Birmingham, Birmingham, AL, USA.
Fracture risk increases with age. In addition to bone loss, change in the distribution of bone mass is another significant factor associated with bone fragility. The objective of this study was to investigate the relationship between aging and changes in hip geometry derived from hip structure analysis (HSA). A prospective study in an ethnically diverse subcohort of 5,856 postmenopausal women enrolled in the Observational Study of the Women's Health Initiative was conducted. Eligible subjects were between 50 and 79 years of age and had completed bone scans using Dual-energy X-ray Absorptiometry (DXA). The Beck HSA software was used to estimate bone mineral density (BMD), cross-sectional area (CSA), outer diameter (OD), section modulus index (SM), average cortical thickness (CT) and buckling ratio (BR) for the femoral narrow neck (FNN), intertrochanter (IT) and proximal femoral shaft regions (FS). Analyses showed that BMD, CSA, SM and CT were all significantly lower and the OD and BR measures were significantly higher in the older age group in comparison to the younger age group at baseline (p<0.01). Results from paired t-test indicated significant changes in all HSA measures from baseline to year 6 (Table). Interestingly, both the bone bending strength (SM) and the likelihood of cortical failure under compression (BR) increased significantly over this same time frame, suggesting a complex process with changes in the hip geometry associated with aging. The rates of changes in HSA measures varied by age group and by femoral site. For example, OD expansion in all regions was significantly larger in the 60–69 or 70–79 years age group in comparison to the 50–59 years age group. While significant age differences in the change of other HSA measures were only observed in the FNN region. In conclusion, hip geometric structures change significantly with aging and the rate of changes varies by age and femoral site. The impacts of these changes on hip bone strength are complicated and remained to be investigated.
Disclosures: T. Bassford, None.
This study received funding from: NIAMS R01-AR049411.
Relationships Between Lower-Limb Physical Function and Bone Mineral Density in Community-Dwelling Older People.
T. Komatsu*1, H. Park1, D. Nozaki*1, S. Kashiwaguchi*2, T. Kamitani*2, C. Okada*1, Y. Mutoh*1. 1Department of Physical and Health Education Graduate School of Education, The University of Tokyo, Tokyo, Japan, 2Department of Orthopedic Surgery, Tokyo Koseinenkin Hospital, Tokyo, Japan, 3Department of Rehabilitation, Tokyo Koseinenkin Hospital, Tokyo, Japan.
The Fall Prevention Program was established in 1997 to decrease falls and fall-related fractures in community-dwelling older people. We investigated the relationship between lower-limb physical function, such as gait function and single-leg stance, and osteoporosis (Komatsu, 2006). Four hundred and eighty-four older adults (average age 71.0±3, 52.5±8.9kg, BMI 22.3±3.3) participated in the Fall Prevention Program between December, 1997 and January, 2007. The bone mineral density (BMD) of the femoral neck was measured by dual-energy X-ray absorptiometry using the Hologic co. QDR2000. The subjects were classified into four groups according to BMD by the WHO criteria: normal, osteopenia, osteoporosis and severe osteoporosis. For the linear relationship, that calculated the partial correlation coefficients. Analyses of covariance (ANCOVA) assessed independent associations between the BMD and the lower-limb physical function after controlling the effect of the age. Moreover the logistic regression analysis was used to determine odds ratios and their 95% confidence intervals adjusted by age for the BMI in order to assess association between the osteoporosis and the lower-limb physical function. The probability less than 5% significance was assumed to be significant by using the Statistical Package for Social Science (Ver.15.0). A significant correlation was observed between the BMD and the lower-limb physical function (r=0.567, p=0.013). Subjects who are in the lowest quartile of the maximum gait speed were 1.96 folds more at risk of osteoporosis than the highest quartile (Table 1). Therefore, we concluded that gait speed is an important factor in determining fall and fracture risk (Kanis.2005). In this study of community-dwelling older people who participated in the Fall Prevention Program, the relationship between gait speed and osteoporosis was explained.
Disclosures: T. Komatsu, None.
Associations Between Body Composition and Hip Geometry in Postmenopausal Women in the Women's Health Initiative.
S. Going*1, T. Beck*2, L. Arendell*1, J. Cauley*3, B. Lewis*4, T. Bassford*1, Z. Wang*5, A. LaCroix*6, T. Lohman1, Z. Chen*1. 1Univ of AZ, Tucson, AZ, USA, 2Johns Hopkins Univ, Baltimore, MD, USA, 3Univ of Pittsburgh, Pittsburgh, PA, USA, 4Univ of Alabama, Birmingham, AL, USA, 5Columbia Univ, New York, NY, USA, 6Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Body weight (wt) and composition are associated with bone mass and bone mineral density (BMD) and likely also affect bone strength through associations with other structural features. While ethnic differences in mass and BMD have been described, differences in structural parameters and their association with soft tissue composition are not well studied. The aim of this study was to assess the associations among measures of body composition and hip geometry (HG) in non-Hispanic White, African American, Hispanic and Native American postmenopausal women (n=5,761) aged 50 to 79 y, in the observational arm of the WHI. Participants' mean age, wt and height (ht) at baseline was 63.7 ± 7.5 y, 72.7 ± 17.1 kg, and 161.4 ± 6.5 cm respectively. Whole-body fat (FM) and lean soft tissue (LSTM) masses and BMD were measured by dual energy x-ray absorptiometry. HG measures, estimated with Hip Structural Analysis (HSA), included femur narrow neck, inter-trochanter, and shaft cross-sectional area (CSA), outer diameter (OD), section modulus (SM), cortical thickness (CT) and buckling ratio (BR). The means and standard deviations of HG measures in the femoral narrow neck region by the quartile of baseline LSTM (table) suggest increased bone strength with higher LSTM.
Linear regression was used to assess the association of baseline LSTM and FM with baseline, year 3 and year 6 HG, with baseline wt, ht, physical activity (PA), age and ethnicity as covariates. Body wt and composition, age and ethnicity were significantly correlated with HG LSTM and FM were related positively to BMD, CSA, OD, SM and CT, and negatively to BR at all sites at all years. LSTM explained more variance in HG (adj R2=.04 to .41) than FM (A adj R2=.01-.03). The associations were not confounded by age, ethnicity, PA or baseline ht and wt. In conclusion, HSA measurements are significantly related to soft tissue composition, especially LSTM in postmenopausal women.
Disclosures: S. Going, None.
This study received funding from: NIH/NIAMS.
Recognition of Postmenopausal Women at Risk of Fracture Using BMD and Clinical Risk Factors: Results from a Northern Italian Multicenter Study.
A. Giusti1, A. Barone*1, G. Pioli2, G. Girasole3, G. Bianchi3, E. Palummeri*1, M. Pedrazzoni4. 1Gerontology, Galliera Hospital, Geneva, Italy, 2Gerontology, ASMN, Reggio Emilia, Italy, 3Reumatology, La Colletta Hospital, Arenzano, Italy, 4Internal Medicine, University of Parma, Parma, Italy.
Objectives: To estimate the prevalence of 4 internationally validated independent clinical risk factors (CRFs) (low body mass index (BMI), prior history of fragility fracture, family history of fracture, current smoking) for fragility fractures in a cohort of Italian postmenopausal women and evaluate the impact of the combined assessment of CRFs with bone mineral density (BMD) measurement on risk stratification.
Subjects: Postmenopausal women examined in 3 osteoporosis centers from Northern Italy (Arenzano, Genoa, Parma) over a period of 2 years.
Methods: All women answered a standardized questionnaire to assess smoking habits, history of prior fragility fractures at any site and fractures of the spine, ribs, distal forearm, humerus and hip in first-degree relatives. Each subject underwent anthropometric assessment to calculate BM1. BMD measurements of lumbar spine (L1-L4) and total hip were obtained by dual-energy x-ray absorptiometry. The relative risk (RR) of fracture versus the general population was modeled as a function of BMD only and BMD plus CRFs, using published data from Kanis et al. (Bone, 2004; Osteoporos Int, 2005).
Results: 10068 post-menopausal women were included in the study (mean age±standard deviation, 63.3±8.7 years; range 45–97). The overall population was divided in subgroups according to the number of prevalent CRFs: the proportion of women with 0, 1, 2 and ≥3 prevalent CRFs was 53.1%, 36.1%, 9.8% and 1%, respectively. Overall 4702 women (46.9%) presented at least one risk factor. The most frequent risk factor was a prior history of fragility fracture (21.8%), followed by a parental history of fracture (16.7%), current smoking (14.7%) and BMI <20 kg/m2 (5.6%). As expected, the relative prevalence of some risk factor varied with age. In particular, the proportion of women with a prior fragility fracture increased progressively with age; whereas the percentage of current smokers decreased.
The addition of CRFs to BMD improved risk stratification as compared to BMD alone. The effect was modest in women with only one CRF (RR based on BMD: 1.2; RR based on BMD plus CRFs: 1.7), but more marked in women with 2 CRFs (a 2-fold increase in RR from 1.4 to 2.9) and in women with ≥3 prevalent CRFs (a 3-fold increase from 1.7 to 5.4)
Conclusion: The prevalence of risk factors in this cohort of postmenopausal Italian women is similar to that observed in international population-based studies. The addition of CRFs to BMD resulted in a clinically significant increase in the gradient of risk in about 10% of subjects.
Disclosures: A. Giusti, None.
Hip Geometric Structure Is Weaker in Anemic Women—Results from the Women's Health Initiative Observational Study.
G. Wu*1, T. J. Beck2, T. Bassford*1, J. A. Cauley3, A. Z. LaCroix*4, C. E. Lewis5, Z. Chen1. 1University of Arizona, Tucson, AZ, USA, 2Johns Hopkins University, Baltimore, MD, USA, 3University of Pittsburgh, Pittsburgh, PA, USA, 4Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 5University of Alabama-Birmingham, Birmingham, AL, USA.
Anemia is a common health problem in the U.S. older population and it is associated with increased hip fracture risk. The influence of anemia to bone geometric structures is unknown. This study used baseline data from participants of the Women's Health Initiative Observational Study to examine the association between hip geometric structure and anemia. Participants were age 50–79 years and postmenopausal women who came from different ethic backgrounds. Only participants who had hemoglobin measurements (Hb) and hip bone density scans on dual-energy x-ray absorptiometry at one of the three WHI bone mineral density (BMD) centers were included in this study (n = 5,617). The Beck's hip structure analysis (HSA) method was used to measure BMD, cross-sectional area (CSA), outer diameter (OD), section modulus (SM), cortical thickness (CT) and buckling ratio (BR) in the femoral narrow neck (FNN), intertrochanter (IT) and femoral shaft (FS) region. Anemia was defined as Hb concentrations less than 12 gm/dl based on the definition by the World Health Organization (WHO). Overall, 6.8% of the participants were anemic and the rate of anemia varied by age and ethnicity. The results from multiple regression analyses showed that, after adjustment for age, height, weight, ethnicity, hormone use, physical activity and total body percent lean mass, women with anemia tend to have lower BMD, CSA, SM, CT, and higher BR in all the three femoral regions compared to women with a normal Hb level. However, statistically significant differences by anemic status were only observed for the SM and BR measure in the IT region (Table 1). Compared to the FNN and FS region, the differences in HSA measures were more obvious at the FN. In conclusion, hip geometric structure differs by anemic status and the differences seem bigger in the IT region than in the FNN and FS region.
Disclosures: G. Wu, None.
This study received funding from: NIAMS R01-AR049411.
How Should Hypercalciuria Be Defined?
N. M. Maalouf, M. A. Cameron*, K. Sakhaee. Charles and Jane Pak Center for Mineral Metabolism, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Background: Although hypercalciuria is a well-established risk factor for nephrolithiasis and is frequently considered in the differential of secondary osteoporosis, its definition has been debated and the “normal range” for urinary calcium excretion is not clearly defined. The most commonly used definitions for hypercalciuria are based on absolute calcium excretion and weight-based calcium excretion. We examined whether the prevalence of hypercalciuria differs according to the definition used in a cohort of healthy individuals.
Methods: 24-hour urinary calcium excretion was measured in 149 healthy individuals with no history of kidney stones, while on an unrestricted diet. Absolute hypercalciuria (AHC) was defined as urinary calcium excretion exceeding 250 mg or 300 mg of calcium per day for women and men respectively. Weight-based hypercalciuria (WBHC) was defined as calcium excretion greater than 4 mg/kg of body weight per day. The prevalence of hypercalciuria was evaluated using both definitions, and was examined within the following body mass index (BMI) categories: normal, BMI < 24.9 Kg/m2, overweight, BMI 25–29.9 Kg/m2, and obese, BMI > 30 Kg/m2.
Results: The cohort was composed of 46 individuals with normal BMI, while 52 were overweight and 51 were obese. For the entire cohort, mean BMI was 28.3±6.6 Kg/m2, mean 24-hr urine calcium was 163.0±94 mg/day, or 2.09±1.10 mg/Kg/day. The prevalence of AHC was 15.4% while the prevalence of WBHC was 6.7% (p=0.016). Weight based urine calcium excretion did not vary significantly between the 3 groups of BMI (2.10±1.12 mg/Kg/day for normal BMI, 2.06±1.10 mg/Kg/day for overweight, and 2.10±1.11 mg/Kg/day for obese). On the other hand, absolute urine calcium excretion increased significantly with increasing BMI (125.5±66.0 mg/day for normal BMI, 157.7±73.3 mg/day for overweight, and 202.0±117.9 mg/day for obese). Accordingly, the prevalence of hypercalciuria varied significantly between BMI categories using the AHC but not the WBHC definition.
Conclusions: The prevalence of hypercalciuria depends on the definition used, particularly in obese patients. These results should be considered in the evaluation of calcium excretion in patients with nephrolithiasis and osteoporosis. Further studies are needed to establish the optimal definition of hypercalciuria.
Disclosures: N.M. Maalouf, None.
Oral Treatment with the Calcium Receptor Antagonist SB-423557 Causes PTH Release in Multiple Species and Positive Bone Forming Effects in the Rat.
S. Kumar1, X. Liang1, J. A. Vasko*1, G. B. Stroup2, S. J. Hoffman1, V. R. Vaden*2, H. Haley*2, J. Fox3, E. F. Nemeth3, A. M. Lago*2, J. F. Callahan*2, P. Bhatnagar*1, W. F. Huffman*1, M. Gowen2. 1GlaxoSmithKline, Collegeville, PA, USA, 2GlaxoSmithKline, King of Prussia, PA, USA, 3NPS, Salt Lake City, UT, USA.
Antagonists of the parathyroid calcium receptor (calcilytics) stimulate the secretion of PTH. Previously, we demonstrated the ability of an orally active calcilytic compound to cause sustained increases in circulating levels of endogenous PTH and to stimulate bone formation and resorption (without a net increase in bone formation) in the ovariectomized (OVX) rat. In the present study, a prodrug approach has been used to preserve oral bioavailability and yield a calcilytic with a shorter half-life in vivo. SB-423557 is the ethyl ester prodrug of SB-423562 that, when administered orally to rats, dogs or monkeys, caused a dose-dependent, transient increase in circulating levels of endogenous PTH. In order to examine the bone forming effect of SB-423557, six-month-old female rats received OVX or sham surgery and were untreated for 6 weeks to allow osteopenia to develop and then treated orally daily with either vehicle, SB-423557 (50 mg/kg), or with rat PTH(1–34) (5 μg/kg SC) for 12 weeks. Plasma levels of PTH peaked at 10–60 min following oral administration of 50 mg/kg SB-423557 (3-fold, Cmax of 40–60 pM) and returned to baseline by 2–3 hours. SC administration of rat PTH(1–34) resulted in a systemic Cmax of 127–240 pM at 10 minutes post-injection.
SB-423557 significantly and completely prevented additional OVX-induced loss of BMD in the lumbar spine and partially prevented trabecular BMD loss in the proximal tibia by 39% (ns) compared to OVX controls. Histomorphometric analysis indicated greater trabecular bone area (36%, ns) in the spine and increased cortical area (72%) and endocortical bone formation rate (220%) with no effect on the eroded perimeter of the distal tibia in the SB-423557 treated rats compared to vehicle-treated OVX animals. Serum osteocalcin increased (ns) with SB-423557 treatment with no effect on urinary deoxypyridinoline levels. In addition, treatment with SB-423557 resulted in greater ultimate strength (ns), toughness, and elastic modulus of a lumbar vertebral body and at the femoral diaphysis compared to OVX controls. Treatment with PTH(1–34) also completely prevented the OVX-induced loss in bone mass, BMD, and strength.
These data provide a proof of principle for stimulation of bone formation following daily brief antagonism of the calcium receptor in the OVX rat and support the potential use of these agents to treat disorders of bone metabolism such as osteoporosis.
Disclosures: S. Kumar, GlaxoSmithKline 3.
Prior Alendronate Treatment Does Not Inhibit the Early Stimulation of Osteoblast Activity in Response to Teriparatide.
R. Lindsay1, F. Cosman1, H. Zhou2, J. W. Nieves1, M. Bostrom3, N. Barbuto*1, D. W. Dempster2. 1Clinical Research Center, Helen Hayes Hospital, West Haverstraw, NY, USA, 2Regional Bone Center, Helen Hayes Hospital, West Haverstraw, NY, USA, 3Department of Orthopaedics, Hospital for Special Surgery, New York, NY, USA.
Several studies suggest that the bone density response to PTH treatment is blunted in subjects previously treated with alendronate. The present study evaluates the effects of teriparatide (TPTD) on bone formation variables from iliac crest biopsies in subjects who were either treatment naive or pretreated with alendronate (ALN). We recruited 25 postmenopausal women with osteoporosis (12 treatment naive; 13 pretreated with alendronate for at least 1 yr (mean age 63). The two groups were randomized to control or TPTD (20mcg/day). All underwent iliac crest bone biopsy 7.5 weeks after randomization. We used quadruple tetracycline labeling as previously described (JBMR 2006) to provide longitudinal data on bone formation in each subject. Biopsies were read with the observer blinded to treatment group. Groups were well matched for demographics and indices of cancellous and cortical bone structure. In ALN- treated patients, mineral apposition rate (MAR), mineralized perimeter (Md.Pm) and bone formation rate (BFR) were lower at baseline than in naïve patients.
After TPTD, the absolute increment in MAR was dramatic in the ALN group, increasing approximately 80–120% above baseline in cancellous bone and on the endocortical surface (Table). MAR also increased significantly with TPTD in the naïve group (21–25%), but the increments were not as marked as in ALN subjects. The absolute increases in BFR and Md.Pm were similar in ALN and naïve groups in cancellous bone but were larger in the naïve group on the endocortical surface due to the higher baseline Md.Pm. However, the percentage increments in all bone formation variables were greater on both envelopes in the ALN compared to the naïve group. We conclude that the increments in the work rate of osteoblast teams (MAR) in response to early treatment with TPTD are greater following ALN pretreatment than in naïve subjects. However, the extent of bone forming surface is overall larger in naïve subjects, perhaps providing an explanation for the possible blunting of the BMD effect in ALN treated women given TPTD.
Disclosures: R. Lindsay, Eli Lilly & Company 2, 8.
This study received finding from: Primary finding, NIH: Product, Eli Lilly.
β2 Adrenergic Receptor Deficiency Enhances Bone Mass in by Antagonizing Against Aging-induced Bone Loss and Blunts Anabolic Effects of PTH on Osteoblasts.
R. Hanyu*1, Y. Saita*1, J. Nagata*1, Y. Izu*1, T. Hayata*1, H. Hemmi*2, S. Takeda*3, Y. Ezura*1, K. Nakashima*1, H. Kurosawa*4, M. Noda*1. 1Department of Molecular Pharmacology, 21st Century COE Program, MRI, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, 2MTT Program, MRI, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, 3Department of Orthopedics, 21st Century COE Program, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, 4Department of Orthopedics, Juntendo University School of Medicine, Bunkyo-ku Tokyo, Japan.
Soaring of the aged population in modern world increases the fraction of aged population with diseases and thus in bone field, leads to increase in osteoporosis incidence. Loss of bone mass is accelerated with age regardless of gender. Osteoporotic patients with their bone mass levels at their nadir require treatment to increase bone mass through the activation of bone formation. PTH has been proven to be efficacious in increasing bone mass. However, its efficacy in the highly aged population such as those over 80 years of age in human have not yet been fully elucidated. In mice, aged models of over one year old, indicate lower bone mass than younger adult animals regardless of the gender. Recently, sympathetic nervous system was reported to regulate bone mass in mice. However, these reports always indicate the effects of adrenergic system on young adult mice. Therefore, the purpose of this paper is examine the effects of β2 adrenergic system and PTH on the bone mass in aged mice. Aged (54 weeks old) female mice null for β2 adrenergic receptor (Adrβ2-/-) and wild type (WT) were used. Mice were treated with vehicle or PTH (80μg/kg/day sc) for 4 weeks (5 days/week). The base line bone mass levels in these aged mice were significantly reduced compared to young adult mice. Aged Adrβ2-/- mice exhibited higher total body BMD, trabecular bone volume fraction(BV/TV), cortical bone volume compared with aged WT mice. In WT, PTH treatment significantly increased the levels of total body BMD, 3D- BV/TV, mineral apposition rate (MAR) and bone formation rate (BFR). In contrast, in aged Adrβ2-/- mice, PTH failed to increase the levels of BMD and BV/TV. Analyses on aged Adrβ2-/- mice also revealed that PTH failed to enhance BFR and MAR. Importantly, Adrβ2 deficiency increased cortical bone volume compared to WT while this parameter was no longer enhanced by PTH in the mutant mice. Regarding bone resorption side, Oc.S/BS, N.Oc/BS were similar regardless of the genotype or PTH treatment. In conclusion, we found that in one year old aged female mice, β2 adrenergic receptor signal operates to reduce bone mass, while β2 adrenergic signaling is required for the anabolic actions of PTH on bone formation.
Disclosures: R. Hanyu, None.
PTH Improves Bone Strength in an Osteoporosis Model Through Increasing Mineralized Matrix Yet the Elastic Modulus of the Trabecular Surface Is More Heterogeneous than Observed with Anti-Resorptive Agents.
Z. Cheng1, W. Yao1, K. J. Koester*2, M. Balooch*2, R. O. Ritchie*2, N. E. Lane1. 1Center for Healthy Aging, UCDavis Medical Center, Sacramento, CA, USA, 2Materials Science and Engineering, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Current approved medical treatments for osteoporosis reduce fracture risk to a greater degree than predicted from changes in bone mineral density (BMD). Based on these observations, we hypothesize that these bone active agents improve bone strength by altering material properties of the bone.
Methods. 18-month-old female Fischer rats were ovariectomized (OVX) or sham-operated and were untreated for 60 days to induce osteopenia before they were treated with single doses of risedronate (iv., 500μg/kg), zoledronic acid (iv., 100μg/kg), continuous raloxifene (2mg/kg, po., 3x/wk), hPTH (1–34) (25μg/kg, sc., 3x/wk) or vehicle. Groups of animals were sacrificed after d60 and d180 of treatment. Measurements of bone quality from the proximal tibial metaphyses (PTM) included μCT, the degree of bone mineralization (DBM), mineral heterogeneity index (MHI); localized elastic modulus (EM) mapping of select trabecular surface by scanning probe microscopy, and bone compression testing from a lumbar verterbral body (LVB).
Results. The bone volume (BV/TV), DBM, EM and compressive bone strength were significantly lowered at d60 post-OVX (pretreatment, day 0 study) than at baseline. At d60 all treatments restored bone material measurements (BV/TV, DBM, EM and compressive bone strength) and no differences between OVX + anti-resorptives and OVX + PTH were observed. However, at d180 OVX + PTH further increased BV/TV (+30% from d 60), DBM, MHI and bone strength to baseline levels. EM mapping of PTM from the OVX + vehicle and OVX + PTH at d180 had significant heterogeneity with higher percentage of high and low EM areas than sham. Moreover, at d180 there was a higher percentage of high EM in OVX + PTH compared to other OVX + anti-resorptives and whole bone strength was higher. Therefore, these data suggest that localized changes in the material properties of bone after PTH, differs from anti-resorptive agents, and while the EM of the matrix is more heterogeneous, the overall result in greater bone strength. Additional studies are needed to correlate these preclinical findings with the clinical efficacy of these agents in osteoporosis.
Disclosures: Z. Cheng, None.
This study received funding from: R01 AR043052–078, 1K12HD05195801, DE AC-02 05CH11231.
The Use of RAP-011, a Novel Bone Anabolic Agent, in Combination with Parathyroid Hormone or Zoledronic Acid Using a Mouse Model of Established Bone Loss.
M. Cornwall-Brady*, M. Mangini*, D. Barbosa*, L. Mazzola*, T. Marvell*, J. Milling*, B. Haigis*, R. Kumar*, K. Underwood*, R. S. Pearsall. Acceleron Pharma, Cambridge, MA, USA.
RAP-011, a novel soluble receptor fusion protein based on the activin receptor type IIA was previously reported to increase bone density in vivo (Pearsall et al, J Bone Min. Res. 21(S1) 2006). Here, we investigate the efficacy of RAP-011 compared with parathyroid hormone (PTH) or zoledronic acid (ZOL).
Eight week old C57BL/6 mice were ovariectomized (OVX) or sham operated (SHAM). 8 weeks after surgery all mice were analyzed by dual-energy x-ray absorptiometry (DEXA) prior to treatment. Mice were administered either RAP-011 (IP, 10 mg/kg, biw), PTH (SC, 80 μg/kg/day) or a single dose of ZOL (IP, 20 μg/kg).
Study 1 included 48 OVX (16 each treated with PBS, RAP-011, PTH) and 8 SHAM mice. After six weeks of treatment, half of each group received a single IP dose of the ZOL. The bone mineral density (BMD) of each animal was measured 0, 6, 10, 14 and 18 weeks after treatment initiation. This study demonstrated an increase in total body BMD of 3.6%, 4.2%, 7.6%, and 10.0% in SHAM VEH, OVX VEH, OVX-RAP-011 and OVX PTH respectively after 6 weeks of treatment. Four weeks after ZOL dosing DEXA results showed an additional increase (relative to week 6 results) of 4.7%, 4.6%, and 1.3% in OVX groups treated with PBS, RAP, and PTH respectively. In groups that did not receive ZOL treatment the BMD changes were 2.4%, 1.9%, 2.0% and −3.6% for SHAM VEH, OVX VEH, OVX RAP-011 and OVX PTH mice respectively.
Study 2 included 32 OVX (8 each treated with PBS, RAP, PTH, and a combination of both RAP-011 and PTH), 24 Sham (8 each PBS, RAP-011 and PTH). DEXA was performed at weeks 0 and 6. In this study we show an increase in total body BMD of 2.8%, 4.6%, 9.8% and 13.3% for OVX groups treated with PBS, RAP-011, PTH and the combination of both RAP-011 and PTH, respectively. SHAM groups had total body BMD changes of −0.1%, 8.0% and 6.9% for PBS, RAP-011 and PTH respectively.
Consistent with our previously reported results, these data show that total body BMD increased significantly after 6 weeks of treatment with either RAP-011 or PTH. Subsequent treatment with ZOL resulted in equivalent increases in BMD in groups that were previously treated with RAP-011 or PBS. These results indicate that RAP-011 does not alter the bone microenvironement in such a way as to preclude the use of anitresorptive therapy. DEXA results also indicate that the total body BMD increase as a result of a combination treatment with both RAP-011 and PTH was larger than either the RAP-011 or PTH treatment alone in both SHAM and OVX mice. The results of these two studies provide evidence that RAP-011 can be used in combination with other currently approved therapies.
Disclosures: M. Cornwall-Brady, Acceleron Pharma 1, 3.
Endogenously Produced n-3 Fatty Acids (Fat-1 transgene) Protects Bone after Ovariectomy in Mice.
J. Banu1, A. Bhattacharya*2, G. Halade*1, M. Rahman1, G. Fernandes*1. 1Med/Clinical Immunology & Rheumatology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 2Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Aging is associated with bone loss leading to escalating the risk of fractures. Recently, there is growing interest in identifying nutritional supplements that can be more effective in preventing age-related as well as postmenopausal bone loss with minimum side-effects. There is increasing evidence on the beneficial effects of n-3 fatty acids in the prevention of postmenopausal bone loss. In 2004, a novel transgenic mouse model (fat-1) that could convert n-6 fatty acids to n-3 fatty acids was produced. We studied bone loss after ovariectomy in this mouse model and their wild type counterparts (C57BL/6).
Fat-1 transgenic and wild type C57BL/6 mice were used in this study. Female mice were 8 weeks old when they were either ovariectomized or sham operated. They were maintained in the laboratory for 6 months and then sacrificed. Serum alkaline phosphatase and osteocalcin were measured. The femur, tibia and lumbar were analyzed using peripheral quantitative computed tomography (pQCT) densitometry.
Serum alkaline phosphatase (ALP) was decreased significantly in the wild type mice after ovariectomy. The fat-1 mice had lower ALP when compared to the wild type mice, but after ovariectomy there was no further decrease in the enzyme levels. Osteocalcin levels were not significantly altered after ovariectomy in the wild type mice and fat-1 mice.
In wild type mice, there was significant loss of bone after ovariectomy in the neck of the femur and proximal tibial metaphysis. However, in the fat-1 transgenic mice there was significantly less bone lost after ovariectomy in the neck of the femur and proximal tibial metpahysis. In the femoral diaphysis, fat-1 transgenic mice had significantly higher bone mass when compared to that of the wild type mice. The tibia fibular junction did not show any differences between the wild type and transgenic mice. Similarly, ovariectomy did not affect the pure cortical bone in both wild type and transgenic mice.
We conclude that endogenously produced n-3 fatty acids can attenuate postmenopausal bone loss significantly in the femur followed by the tibia and this bone loss is mainly due to decreased endocortical resorption.
Disclosures: J. Banu, None.
This study received funding from: NIH.
Vitamin K Supplementation in Postmenopausal Women with Osteopenia: The ECKO Trial.
A. M. Cheung1, L. E. Tile*1, Y. Lee*2, G. Tomlinson*3, G. Hawker4, J. Scher*1, H. Hu*1, R. Vieth*5, L. Thompson*6, S. A. Jamal*2, R. Josse2. 1Osteoporosis, University Health Network, Toronto, ON, Canada, 2Osteoporosis, St. Michael's Hospital, Toronto, ON, Canada, 3Medicine, University Health Network, Toronto, ON, Canada, 4Osteoporosis, Women's College Hospital, Toronto, ON, Canada, 5Pathobiology and Laboratory Medicine, Mt. Sinai Hospital, Toronto, ON, Canada, 6Nutrition, University of Toronto, Toronto, ON, Canada.
BACKGROUND: Vitamin K has been widely promoted as a supplement for decreasing bone loss in postmenopausal women, but the long-term benefits and potential harm are unknown.
METHODS: We performed a 2-year single-centre placebo-controlled double-blind randomized trial involving 440 postmenopausal women with osteopenia: 217 received 5mg of vitamin K1 daily and 223 received placebo daily. Outcome measures included bone mineral density (BMD), bone turnover markers, fragilitiy and non-fragility fractures, long-term adverse effects and health-related quality of life. The study was extended for earlier participants for a maximum duration of up to 4 years because of interest in long-term safety and fractures.
RESULTS: Bone mineral densities decreased by −1.28% and −1.22% (p=0.84) at the lumbar spine and −0.69% and −0.88% (p=0.51) at the total hip in the vitamin K and placebo groups over two years, respectively. There were no significant differences in changes in BMDs at any site between the vitamin K and the placebo groups over the 2 to 4-year duration of the trial. Vitamin K supplementation did increase serum vitamin K levels (22.6% versus 2.0%, p<0.0001) and carboxylation of osteocalcin (-21.4% versus −2.0%, p<0.0001) and lowered total osteocalcin levels (bone formation marker) (-13.2% versus — 0.1%, p<0.0001); however, it did not change C-telopeptide (6.2% versus −1.2%, p=0.10). There were fewer cancers (3 versus 12, p=0.02) and fewer clinical fractures in the vitamin K group (9 versus 20, p=0.04). Health-related quality of life was not significantly different between the two groups.
CONCLUSIONS: Daily vitamin K supplementation for 2 to 4 years did not protect against age-related decline in BMD in postmenopausal women with osteopenia. There were lower incidences of fractures and cancer with vitamin K supplementation. Further studies are required to confirm these findings. (ClinicalTrials.gov number, NCT00150969)
Disclosures: A.M. Cheung, None.
This study received funding from: Canadian Instute for Health Research.
Bisphosphonate Treatment During Long-Term Disuse Blunts the Recovery Response of Bone to Restored Weight-Bearing.
J. C. Fritton*1, C. Y. Li*1, X. Zhang*1, D. Laudier*1, R. Mann*2, M. B. Schaffler1. 1Leni & Peter W. May Department of Orthopaedics, Mount Sinai School of Medicine, New York, NY, USA, 2Bronx VA Medical Center, New York, NY, USA.
Bone retention with bisphosphonates (BIS) may set the stage for improved recovery from disuse osteoporosis. We previously found that risedronate (RIS) treatment attenuates approximately half of cortical bone loss associated with long-term immobilization. The purpose of this study was to test whether bone that remains after RIS-treatment during long-term immobilization better recovers its architecture after restoration of mechanical usage. Right forelimbs of 5–7 year-old retired breeder female Beagles (N=27) were immobilized (IM) with splints as in our previous studies. Dogs were treated daily with either RIS (0.5 mg/kg, p.o., provided by P&G Pharmaceuticals) or sterile-water vehicle for 6 months, after which tissues were obtained. In remaining animals, RIS treatment was stopped and splints removed. Remobilized (RM) dogs were allowed 12 months of unrestricted weight-bearing. Age-matched dogs served as controls. Cortical bone structure was assessed by μCT scanning of third metacarpals at 20-μm resolution. Calibrations determined bone mineral density (BMD) values; structural analyses used a global BMD threshold of 600 mg/cc. Cross-sectional properties obtained at the mid-shaft included cortical bone, marrow cavity and total areas, and polar moments of inertia. Effects were tested by ANOVA with post-hoc PLSD comparisons (p<0.05, data shown as Means ± SD). RIS treatment prevented approximately 60% of the bone loss due to IM. RM of vehicle-treated dogs restored significant amounts of cortical bone lost during disuse. Surprisingly, despite discontinuation of RIS, a similar remobilization response was absent in the animals drug-treated during IM.
BIS have long half-lives (on the order of years) and thus have effects after active dosing has stopped. This may underlie the impaired response of RIS-treated bone to restored loading. The decision to use BIS in prevention of bone loss during long-term immobilization (e.g., bed rest or space flight) must include consideration of side effects. Our results indicate that these side effects include an impairment of the ability of cortical bone to respond appropriately to the reintroduction of mechanical loading.
Disclosures: J.C. Fritton, None.
This study received funding from: NSBRI & NIH.
Combination Treatment with a Selective Androgen Receptor Modulator (SARM) and a Bisphosphonate has Additive Effects in Osteopenic Female Rats.
E. G. Vajda, A. Hogue*, K. N. Griffiths*, W. Y. Chang*, K. Burnett*, K. Marschke*, D. E. Mais*, B. Pedram*, Y. Shen*, A. van Oeveren*, F. J. López*. Ligand Pharmaceuticals, San Diego, CA, USA.
Recent clinical trials with bisphosphonates and PTH have not supported the hypothesis that combination treatments with antiresorptive and anabolic agents would lead to synergistic activity. We hypothesized that combination treatment with a SARM, LGD3303, and a bisphosphonate would be beneficial. In vitro, LGD3303 is a potent androgen that shows little or no cross-reactivity with related nuclear receptors. In male rats, LGD3303 has tissue selective properties, increasing the levator ani muscle weights above eugonadal levels at high doses, but having greatly reduced activity on the prostate, never increasing the ventral prostate weight above eugonadal levels. To assess bone effects, three month-old female SD rats were ovariectomized (OVX) and allowed to develop osteopenia for 8 weeks. Rats were orally dosed with vehicle, LGD3303, alendronate sodium, or the combination from week 8 to 20 post-OVX (all dosed at 3 mg/kg/day). LGD3303 and combination treatment significantly increased gastrocnemius muscle weight, unlike alendronate. All three treatments increased whole femur BMD and BMC measured by DEXA. BMC was significantly higher with LGD3303 and combination treatment (19.0% and 19.9% increase, respectively) compared to alendronate (8.3% increase). At the lumbar spine, LGD3303 and alendronate increased BMD (10.8% and 7.6% increase, respectively), but combination treatment restored it to near sham levels (18.0% increase). Similarly, elevations in bone strength at the femur and lumbar spine were also observed with all treatments; however the combination had the largest increase, particularly at the lumbar spine. Histomorphometry confirmed that LGD3303 and combination treatment significantly increased mid-femur periosteal bone formation (75.9% and 74.2%, respectively), whereas alendronate (6.3%) was nearly inactive. In contrast, all three treatments suppressed cancellous bone remodeling at the lumbar spine and increased trabecular bone volume. Collectively, these results indicate that LGD3303 and alendronate have positive effects on cancellous bone, but the combination treatment is the most effective at suppressing bone turnover and improving spinal strength and bone density. Muscle and cortical bone were unaffected by alendronate, whereas LGD3303 has anabolic activity which is unaltered by combination treatment with alendronate. At every measured site, combination treatment was as effective as either single agent and in some cases showed significant added benefit.
Disclosures: E.G. Vajda, Ligand Pharmaceuticals 3.
Decreased Bone Turnover and Porosity Are Associated with Improved Bone Strength in Ovariectomized (OVX) Cynomolgus Monkeys Treated with Denosumab, a Fully Human RANKL Antibody.
M. S. Ominsky1, J. Schroeder*1, J. Jolette*2, S. Y. Smith2, D. J. Farrell*2, J. E. Atkinson1, P. J. Kostenuik1. 1Amgen Inc., Thousand Oaks, CA, USA, 2Charles River Laboratories Preclinical Services Montreal, Inc., Montreal, PQ, Canada.
Denosumab (DMab), a fully human monoclonal antibody against RANKL, was previously shown to decrease biochemical markers of bone turnover and increase bone mineral density in adult OVX cynomolgus monkeys. We now report from that study the effects of DMab on bone turnover at the histologic level, and their relationships with bone strength.
One month after surgery, OVX cynos (9–16 years old) were treated with either vehicle (OVX-Veh) or DMab (25 or 50 mg/kg, SC, once/month) for 16 months (n=14–20/group). Sham controls were treated with vehicle (n=17). Double fluorochrome labels were injected prior to iliac and rib biopsies (at month 6 and 12), and prior to sacrifice. Histomorphometry was performed on these biopsies, the tibial diaphysis, and cancellous bone in L2 vertebra and the proximal femur.
Cancellous bone turnover was increased in OVX-Veh animals, based on significantly greater mineralizing surface (MS/BS), bone formation rate (BFR), and activation frequency at the lumbar spine, iliac crest, and femur neck (P<0.05 vs Sham). Both doses of DMab fully prevented these OVX-related changes while reducing values significantly below those of sham controls (P<0.05). Osteoclast and osteoblast surfaces were 78–100% lower in the lumbar spine, iliac crest, and femur neck of DMab groups, while eroded surface was decreased by 45–93% (P<0.05 vs OVX-Veh).
Cortical bone turnover was also increased in OVX-Veh cynos, as shown by significantly greater cortical porosity, labeled perimeter, and BFR at the endocortical and haversian surfaces of the tibial diaphysis and rib (P<0.05 vs Sham). Both doses of DMab fully prevented these OVX-related changes, while reducing labeled perimeter and BFR significantly below sham control levels (P<0.05).
Prolonged DMab-related turnover suppression was associated with significant increases in strength parameters at the lumbar vertebrae, femur neck, and femur diaphysis (P<0.05 vs OVX-Veh). Regression analysis of all groups combined demonstrated significant inverse relationships between the strength of L5-L6 cores (yield load) and mineralizing surface (r2=0.44) or eroded surface in L2 (r2=0.31; P<0.001). Cortical porosity in the tibia was negatively correlated with peak load at the femur diaphysis (r2=0.21; P<0.001).
In summary, denosumab treatment of OVX cynos was associated with significant decreases in histomorphometric indices of bone turnover. These changes were accompanied by significant improvements in bone strength at cortical and cancellous sites.
Disclosures: P.J. Kostenuik, Amgen, Inc. 1, 3.
This study received funding from: Amgen Inc.
Bone Effects of Cathepsin K Inhibitor in Ovariectomized Rhesus Monkeys.
P. Masarachia, S. Pun*, D. Kimmel. Molecular Endocrinology and Bone Biology, Merck Research Laboratories, West Point, PA, USA.
Cathepsin K (CatK), a cysteine protease highly expressed in osteoclasts, degrades type I collagen. CatK inhibitors, by slowing bone matrix degradation that follows the demineralization phase of bone resorption, may be useful as anti-resorptive agents for treating osteoporosis. The objective of this study was to evaluate bone mineral density (BMD) and bone turnover in newly-ovariectomized (OVX) non-human primates treated with CatK inhibitor MK-0822. Rhesus monkeys (age 12–19 yrs) were assigned to four groups (N=10–11): (1) vehicle-treated intact; and OVX treated with (2) vehicle, (3) 6mg/kg/d or (4) 30 mg/kg/d MK-0822. Daily oral dosing started on ∼day 11 following OVX. BMD of spine and hip were measured (DXA, Hologic DiscoveryA) at baseline and 11 months. Calcein (12 mg/kg, SC) was given on the 18th and third days before obtaining right transilial (TIBx) and ninth rib (RBx) biopsies. Specimens were embedded undecalcified and sectioned at 6μm or 100μm. Both trabecular (TB) and endocortical surfaces (EC) were used for standard histomorphometric endpoints. BMD declined significantly at the spine and femoral neck after OVX. This BMD decline was prevented by 30mg/kg/d MK-0822. In both TIBx and RBx, double-labeled surface (DLS/BS) was elevated by OVX and reduced vs. OVX by ∼80–90% (P<.01) by both 6 and 30 mg/kg/d MK-0822. In RBx, Haversian turnover tended to be elevated by OVX and reduced by 30 mg/kg/d MK-0822. In TIBx, mineralizing surface (MS/BS, %) and mineral apposition rate (MAR) decreased vs. OVX for both doses in both TB and EC compartments (P<.01). All treatment effects were significant except MAR in the TB compartment. Bone formation rate (BFR) tended to increase with OVX, and was decreased relative to OVX by MK-0822; differences were significant for both doses in the TB compartment but only for 6 mg/kg in EC compartment. Osteoid thickness (Os.Th, μm) and surface (OS/BS, %) were increased 7ge;50% (P<.05) by OVX and decreased vs. OVX for both MK-0822 doses (P<.05) in both compartments. The distribution of osteoid thicknesses was shifted to thicker seams by OVX, but was similar to intact animals with MK-0822. In RBx, both osteoclast covered (OcS/BS) and non-osteoclast covered eroded surface (ES/BS) were significantly lower with MK-0822 treatment. In animals with higher amounts of osteoid surface, osteoclasts were seen adjacent to osteoid surfaces with the same low frequency in all groups. MK-0822 reverses the OVX-induced rise in bone turnover in the TB and EC bone of the rib and ilium at 6 and 30mg/kg/d, but reduces rib cortical bone turnover only at 30mg/kg/d, the only dose that preserved spine and hip BMD. We conclude that both doses of MK-0822 reduced TB turnover in biopsy sites and the higher dose tested prevented OVX- induced loss of BMD at osteoporosis fracture sites.
Disclosures: P. Masarachia, None.
A Comparison of Three High Dose Oral Vitamin D, Supplementation Regimens.
C. J. Bacon*, G. D. Gamble*, A. M. Horne*, I. R. Reid. Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
Vitamin D insufficiency is recognized as a major clinical problem in the elderly. Most previous supplementation studies have provided daily calciferol doses of 400–800 IU/day. It may take considerable time on these doses for many elderly people to attain recommended levels of 25-hydroxyvitamin D [25(OH)D]. The purpose of this study was to compare the magnitude and time-course of 25(OH)D response to three high dose vitamin D3 regimens. Sixty-three elderly people, aged 81.6±6.7 years (mean±sd), were recruited on discharge from general medical wards. They were randomly assigned to supplementation with either a single dose of 500 000 IU (Stat), 500 000 IU stat plus 50 000 IU monthly (Stat+Monthly), or 50 000 IU monthly alone (Monthly), in a double-blind trial. The figure shows 25(OH)D levels throughout the study. Baseline 25(OH)D was 58±32 nmol/L and was not different between groups. Mixed-model analysis of variance showed significant Month, Group, and Month × Group interaction effects (p<0.001). At one month, 25(OH)D in the Stat and Stat+Monthly groups rose to 120±33 nmol/L compared to 67±24 nmol/L in the Monthly group. The maximum 25(OH)D level reached at one month was 220 nmol/L, from a baseline level of 136 nmol/L. Three other individuals attained levels >150 nmol/L. The Monthly group reached a plateau of approximately 80±20 nmol/L after five months. At the end of the study only the Stat+Monthly and Monthly groups maintained mean levels >80 nmol/L. In order to rapidly increase 25(OH)D to recommended levels and sustain these levels in frail elderly, a loading dose followed by regular supplementation is preferable to regular supplementation alone.
Disclosures: C.J. Bacon, None.
This study received funding from: New Zealand Health Research Council.
Teriparatide Administration Following Antiresorptive Therapy (BBB-Study): Changes in 3D Microarchitecture, Mineralization and Remodeling at the Iliac Crest After 6 Months.
B. Jobke*1, B. Muche2, A. J. Burghardt1, J. Semler2, G. Delting*3, T. M. Link1, S. Majumdar1. 1MQIR — Dept. of Radiology, University of California, San Francisco, CA, USA, 2Dept. of Osteology, Immanuel-Hospital, Berlin, Germany, 3Inst. for Pathology, Hannover, Germany.
The purpose of this investigation is to obtain more information regarding the onset and duration of new bone formation that result in architectural changes under recombinant human parathyroid hormone 1–34 (rhPTH = teriparatide) following inadequate response to long-term bisphosphonate (BP) therapy for osteoporosis.
Materials and Methods: 25 female osteoporotic patients (age 69 ±9 years) self-administered teriparatide (20μg/d), for an ongoing treatment period of 18 months, after long-term BP use (mean duration 40±22months). In this single center clinical trial, to date, 13 paired iliac crest biopsies (Jamshidi technique) were obtained at baseline (MO) and 6 months (M6) at alternating sides. Tetracyline double-labeled and PMMA embedded biopsies were analyzed semi-quantitatively using light microscopy. Bone volume fraction (BV/TV), structural parameters (Conn.D, SMI, Tb.N, Tb.Th, Tb.S, TBPF) and tissue mineralization (mgHA/ccm) were assessed using μCT (MicroCT 40).
Results: Teriparatide application led to a significant increase in bone remodeling units at M6 compared to M0: in 10/13 (77%) patients an increase was found, in 2/13 there was no change and in 1/13 a decrease in bone turnover was documented. 4 patients showed minor forms of local endosteal fibrosis and 3 had a moderate surface osteoidosis at M6. Pairwise changes in the degree of tissue mineralization did not differ from M0 (p=0.945; mean change: −1.5% to 1087mgHA/ccm ±30), independent of bone turnover status. Pairwise changes in BV/TV and ‘structure model index’ (SMI) improved significantly (p=0.014 and p=0.046, respectively). The average increase in bone volume fraction was +1.8%. Increases in bone volume fraction >2% compared to M0 were observed in 62% (8/13).
Discussion: Despite previous long-term suppression of bone turnover with BP, rhPTH considerably stimulated or balanced bone turnover after 6 months of treatment. In contrast to previous reports there was no detectable decrease in tissue mineralization under osteoanabolic rhPTH treatment. This observation may be due to a relatively small gain in new mineralized bone at M6. It appears that an early gain in bone volume at M6 was best represented by BV/TV whereas most changes in structural parameters were not significant yet. Our results from the iliac crest are consistent with findings from previous clinical trails which found high levels of bone formation markers at M6 with a subsequent improvement in bone stability.
Disclosures: B. Jobke, Travel grant from: Eli Lilly 9.
This study received funding from: Eli Lilly.
Pulmonary Delivery of the Parathyroid Hormone Analogue Ostabolin-C™ Stimulates Markers of Bone Formation in Postmenopausal Women.
P. Morley*1, J. Bishop*1, R. Anderson*1, C. L. Barclay*1, D. Krause*1, G. Morelli*2, M. Newhouse*3, N. Sadrzadeh*4, M. Eldon*4. 1Zelos Therapeutics Inc., Ottawa, ON, Canada, 2MDS Pharma Services, Montreal, PQ, Canada, 3McMaster University, Hamilton, ON, Canada, 4Nektar Therapeutics, San Carlos, CA, USA.
Ostabolin-C is a cyclic analogue of human parathyroid hormone (PTH) (1–31) that has demonstrated marked increases in bone mineral density of the lumbar spine and hip in a 1-year subcutaneous (SC) injection Phase II clinical trial. Pulmonary delivery will improve compliance with PTH therapy, especially in patients averse to self-injection. In the present study, the pharmacokinetic (PK), safety and efficacy profile of Ostabolin-C inhalation powder (OCIP) was evaluated in healthy postmenopausal female volunteers using a randomized, double-blind, placebo-controlled single and 28-day repeat escalating dose study design. Seventy two subjects (49–105 kg; 47–74 years of age) in 9 cohorts of 6 OCIP and 2 placebo treated subjects received a single inhalation of OCIP (dose range 0.1–1.6 mg/day) or placebo powder followed by 28-days of daily inhalation of OCIP or placebo for 7 of the 9 cohorts. Pre- and post-dose urinary cAMP, P1NP, osteocalcin, CTx, NTx, ECG, vital signs, spirometry, adverse events (AEs) and plasma Ostabolin-C levels were recorded. Following administration of OCIP using the Nektar T326 inhaler, Cmax and AUC increased proportionally with dose while Tmax and t1/2 were dose independent. There was no accumulation with repeat dosing and PK profiles of OCIP and SC Ostabolin-C were similar. Biological activity, measured by urinary cAMP, showed significant dose-dependent increases from pre- to post-dose on Day 1 that were maintained with repeat dosing. Significant dose-dependent increases in P1NP and osteocalcin were observed on Day 28. There was no change in the markers of bone resorption CTx or NTx. The safety profile was consistent with PTH class effects, i.e., headache and nausea were the most commonly reported AEs and a dose-dependent increase in heart rate was recorded. There were no treatment-emergent pulmonary or cardiovascular AEs, no effect on pulmonary function and no clinically relevant laboratory abnormalities. Tolerability was poor in subjects receiving 1.2 mg or greater due to PTH class AEs. Serial total serum calcium (0.25–24 hours post-dose) in subjects dosed with 0.8 or 1.2 mg demonstrated a pharmacological effect on serum calcium only at the 1.2 mg dose. This first-in-human trial of inhaled Ostabolin-C demonstrates that it is well tolerated and stimulates cAMP and markers of bone formation to the same extent as SC Ostabolin-C. Inhaled Ostabolin-C represents a convenient alternative to daily injections for patients who require PTH therapy.
Disclosures: P. Morley, Zelos Therapeutics Inc. 1, 3, 4.
This study received funding from: Zelos Therapeutics Inc.
Efficacy of Adding Teriparatide Versus Switching to Teriparatide in Postmenopausal Women with Osteoporosis Previously Treated with Raloxifene or Alendronate.
F. Cosman1, R. A. Wermers2, C. Recknor3, K. F. Mauck*2, L. Xie*4, E. V. Glass*4, J. H. Krege4. 1Helen Hayes Hospital, West Haverstraw, NY, USA, 2Mayo Clinic, Rochester, MN, USA, 3United Osteoporosis Centers, Gainesville, GA, USA, 4Eli Lilly and Company, Indianapolis, IN, USA.
In patients previously treated long term with antiresorptive drugs, information regarding the relative efficacy of adding teriparatide (TPTD, 20 mcg/d) versus switching to TPTD is not available. Postmenopausal women with osteoporosis previously treated for at least 18 months with alendronate (ALN, 70 total mg/week, median treatment duration 37.3 months) or raloxifene (RLX, 60 mg/d, median treatment duration 36.9 months) were randomized to either add TPTD or switch to TPTD for 6 months. A preplanned 12-month extension of this study is currently ongoing. Efficacy results included markers of bone turnover and DXA BMD. Baseline BMD and other characteristics were well matched with the exception that previous ALN patients had lower baseline bone turnover than previous RLX patients (Table). Adding TPTD conferred smaller increases in bone turnover versus switching to TPTD; these differences were more marked between the groups previously treated with ALN. However, adding TPTD conferred greater increases in BMD versus switching to TPTD; these differences were again more marked between the groups previously treated with ALN. All regimens were well tolerated. In conclusion, an anabolic response to TPTD was observed in patients previously treated with ALN or RLX regardless of whether the previous antiresorptive drug was continued or discontinued. In general, greater bone turnover was achieved by switching from antiresorptive to TPTD, while greater BMD increase was achieved by continuing antiresorptive during TPTD treatment.
For bone markers, values are median baseline, median absolute change from baseline (% change). For BMD, values are mean baseline, mean absolute change from baseline (% change). BL, baseline; PINP, amino-terminal propeptide of type I collagen; BSAP, bone specific alkaline phosphatase; CTx, C-terminal telopeptide of type I collagen; LS, lumbar spine; FN, femoral neck; TH, total hip. P-values are based on percent changes. *P<0.001 and **P<0.05 within group from baseline; †P<0.001 and ‡P<0.05 between groups within a stratum.
Disclosures: F. Cosman, Eli Lilly and Company 2, 5, 8; Merck 5, 8.
This study received funding from: Eli Lilly and Company.
Correlations between Bone Turnover Markers and BMD in Patients treated with Teriparatide or Alendronate for Glucocorticoid-induced Osteoporosis.
A. L. Burshell1, R. Möricke2, R. Correa-Rotter3, P. Chen*4, M. R. Warner*4, J. H. Krege4. 1Endocrinology, Ochsner Clinic Foundation, New Orleans, LA, USA, 2Clinical Research Laboratory, Magdeburg, Germany, 3Nephrology and Mineral Metabolism, National Medical Science and Nutrition Institute, Mexico City, Mexico, 4Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA.
Correlations between bone turnover markers and BMD were assessed in a double-blind, active-comparator study of anabolic versus antiresorptive treatment for glucocorticoid-induced osteoporosis (GIOP). Men and women taking 7ge;5 mg/d prednisone equivalent for ≥3 months prior to screening were randomized to teriparatide (TPTD) 20 mcg/d (N=214) or alendronate (ALN) 10 mg/day (N=214) for 18 months. Markers of bone formation included N-terminal and C-terminal propeptide of type I procollagen (PINP and PICP) and bone-specific alkaline phosphatase (BSAP), and a resorption marker, carboxy-terminal telopeptide of type 1 collagen (CTX). Morning fasting blood specimens were measured at baseline and at 1 and 6 months. Lumbar spine (LS) and femoral neck (FN) BMD were measured by DXA at baseline and 18 months. Spearman correlation coefficients were calculated for baseline and changes from baseline in biomarkers at 1 and 6 months with changes from baseline in BMD at 18 months (Table). Baseline median glucocorticoid dose was 7.8 and 7.5 mg/d in the ALN and TPTD group, respectively; mean (±SD) LS BMD T-score was −2.5±1.0 and −2.4±1.0 in the ALN and TPTD group, respectively. In the ALN group, baseline markers were correlated with change in FN BMD, 1 and 6 month change in PINP and CTX were negatively correlated with increases in FN BMD, and 1 month change in CTX was negatively correlated with increased LS BMD. In the TPTD group, increases in PINP were correlated with gains in LS and FN BMD. In conclusion, bone turnover at baseline was correlated with FN BMD response to ALN but not to TPTD. In the ALN group, decreases in markers were correlated with gains in FN BMD. In the TPTD group, increased PINP was correlated with increased LS and FN BMD. These results demonstrate the opposite mechanism of action of TPTD and ALN in patients treated for GIOP.
Disclosures: A.L. Burshell, Eli Lilly and Company 2.
This study received funding from: Eli Lilly and Company.
Antagonism of Calcium Sensing Receptor Stimulates Dose-related Release of Endogenous Parathyroid Hormone in Normal Volunteers: A Proof of Concept Study.
D. Ethgen1, T. Danoff*1, M. Schultz*1, D. Tenero*1, A. Anderson*1, G. B. Stroup2, S. Kumar1, A. M. Lago*2, J. F. Callahan*2, P. Bhatnagar*1, W. F. Huffman*1, M. Gowen2, L. A. Fitzpatrick1. 1GlaxoSmithKline, Collegeville, PA, USA, 2GlaxoSmithKline, King of Prussia, PA, USA.
Calcium sensing receptor antagonists (calcilytics) are a new class of drugs that stimulate bone formation through a PTH-mediated mechanism. Preclinical investigation has shown that briefly antagonizing the calcium receptor (CaR) on the parathyroid gland results in a transient secretion of endogenous PTH, a profile that stimulates bone formation.
SB- 423562 is a small molecule with potent short- lived CaR antagonist activity. This was the first clinical study administering this compound to healthy male volunteers. Safety and tolerability, as well as preliminary pharmacokinetics and pharmacodynamics of a single IV administered rising doses were investigated.
A single 10-minutes IV infusion of SB-423562 (dose range 0.02 to 5.0 mg) in 28 healthy adult male humans resulted in a strong dose-related transient PTH release at varying dose levels.
Pharmacokinetic measurements showed that SB-423562 was quantifiable for 0.25 h post-dose (155 μg dose) up to 3 h post-dose (5.0 mg dose). SB-423562 area under the concentration-time curve (AUC) and maximal observed plasma concentration (Cmax) increased in an approximately dose-proportional manner. The elimination half-life, where estimated, was <1 h. Total clearance was moderate (approximately 500 mL/min). Volume of distribution was approximately 20 L.
SB-423562 produced a strong endogenous PTH release profile, particularly at 1.25 mg, 2.5 mg and 5.0 mg. Mean Tmax was 15 minutes or less. Duration of effect was typically less than 1 h. A 3- to 5.5-fold increase in PTH levels relative to baseline was observed with the 1.25, 2.5 and 5.0 mg doses at 10–15 minutes post infusion. At the higher doses (0.625 to 5 mg), a PTH profile was produced that is consistent with bone forming effects observed with previous experience with SC administered PTH (1–34). A dose related effect was observed on total and ionized serum calcium levels.
SB-423562 was generally safe and well tolerated in healthy male volunteers, no deaths, serious adverse events or withdrawals due to adverse events were reported during the study. There were no apparent differences observed across regimen for safety laboratories values, blood pressure and heart rate.
These data provide a proof of principle that a small molecule with CaR antagonizing properties delivered to healthy male subjects can generate endogenous PTH release in a profile compatible with a long term bone forming effect. Further work is ongoing to develop a formulation allowing the oral delivery of this class of drugs to osteoporotic patients.
Disclosures: D. Ethgen. GlaxoSmithKline 3.
Low Energy Femoral Diaphyseal Fractures Associated with Alendronate Use.
B. Lenart*, A. Neviaser*, M. G. Peterson*, F. Edobor-Osula*, B. Schreck*, C. Chang*, D. G. Lorich*, J. M. Lane. Orthopedic Surgery, Hospital for Special Surgery, New York, NY, USA.
The purpose of this study was to elucidate a correlation between prolonged alendronate use and low energy subtrochanteric and shaft fractures in postmenopausal women.
We performed a retrospective case-control study to investigate our hypothesis. Cases were defined as postmenopausal women with low energy subtrochanteric/shaft fractures verified by xray who presented from 2000–2007 at a university hospital. Controls were postmenopausal women matched by age, race and body mass index with low energy intertrochanteric or femoral neck fractures. Forty one cases were identified and matched to 82 controls, one intertrochanteric and femoral neck fracture each. Patients with diseases or drugs known to affect bone metabolism were excluded.
Alendronate use occurred at a rate of 36.6% in the cases, significantly higher than the 11% observed in controls (p<.001) with an odds ratio of 4.68 for subtrochanteric/shaft fractures with alendronate use (95% CI, 1.83–11.98). Furthermore, alendronate was the bisphosphonate prescribed in each of the cases, which was significantly different than that expected based on 2003 estimates of proportion of bisphosphonates prescribed (p=.042). Logistic regression analysis showed that calcium and alendronate were most significantly associated with cases (p=.09 and p=.003 respectively). Baseline characteristics of cases and controls were similar except serum calcium, which was significantly lower in the cases (p=.02). Patients with subtrochanteric/shaft fractures taking alendronate also shared a common xray fracture pattern, defined as a simple or oblique fracture with cortical thickening and beaking of the cortex on one side. This xray pattern was highly associated with alendronate use (p<.001) with an odds ratio of 15.33 (95% CI, 3.06–76.90). Patients with the xray pattern had an average duration of alendronate use of 7.3 years, while that for those on alendronate without the xray pattern was 2.8 years (p<.001). Wilcoxon survival analysis yielded significantly different cumulative survival without fracture between these two groups (p=.003). By Spearman's rank correlation, duration of alendronate use was significantly correlated to the ratio of the cortical thickness to diameter (p<.001). Alendronate treatment historically reduces the rate of hip fracture by 50%. Our study demonstrates that alendronate may actually increase the risk of low energy subtrochanteric and shaft fractures with prolonged use. While this data does not prove a causal relationship, it provides strong evidence that a correlation exists between prolonged alendronate use and femoral diaphyseal fractures.
Disclosures: B. Lenart, None.
The Effect of Risedronate on Risk of Clinical Fracture Among Patients with Prior Hip Fracture.
M. R. McClung*1, A. Grauer2, X. Zhou*2, P. D. Miller*3, S. Boonen*4. 1Oregon Osteoporosis Center, Portland, OR, USA, 2Procter & Gamble Pharmaceuticals, Mason, OH, USA, 3Denver Osteoporosis Center, Denver, CO, USA, 4University Hospital Leuven, Leuven, Belgium.
Previous hip fracture is a risk factor for subsequent fractures. In this analysis, the anti-fracture efficacy of risedronate was examined among patients with previous hip fracture at baseline.
A total of 339 postmenopausal women between ages 70–79 years with low BMD in HIP trials (McClung, MR et al. 2001 NEJM) had a history of at least one hip fracture prior to study entry. These subjects were treated with either placebo (PLC) or risedronate (RIS) 2.5mg or 5 mg daily. The incidence of osteoporosis-related clinical fracture, defined by the occurrence of the radiographically confirmed clinical vertebral fracture or radiographically confirmed non-traumatic non-vertebral fracture was calculated using Kaplan-Meier survival estimates over a 3 year period and was compared between placebo- and risedronate-treated subjects. Treatments were compared using log rank test, and the risk ratio and its 95% confidence interval were obtained using a Cox regression model stratified for study.
Subjects' mean age was 75 years, and their mean femoral neck (FN) and lumbar spine (LS) T-scores were −3.1 and −3.2 SD, respectively. Baseline characteristics were well balanced between the 3 treatment groups. Over the 0–3 year period, the clinical fracture incidences were 28.4%, 14.9% and 13% for the PLC, RIS 2.5mg and RIS 5mg groups, respectively. Relative to the placebo group, RIS 5mg and 2.5 mg statistically significantly reduced the risk for clinical fracture over 0–3 year period (p<0.05). The risk ratio of clinical fracture was 0.5 for both the risedronate 5mg and 2.5 daily groups relative to the placebo group.
Risedronate treatment was well tolerated in HIP trial.
Disclosures: M.R. McClung, Procter & Gamble Pharmaceuticals 5.
The Effects of Zoledronic Acid 5 mg Once-Yearly on Bone Remodeling and Structure in Osteoporotic Women Are Consistent Across Age.
R. R. Recker1, P. Delmas2, I. Reid3, S. Boonen4, J. Halse5, P. Garcia*6, J. Supronik*7, M. Lewiecki8, L. Ochoa*9, P. Miller10, F. Hartl11, J. A. Gasser11, P. Mesenbrink*12, H. Hu*12, E. Eriksen11. 1Creighton Univ. Osteoporosis Res. Center, Omaha, NE, USA, 2Univ. of Lyon, INSERM Res. Unit 831, Lyon, France, 3Univ. of Auckland, Auckland, New Zealand, 4Univ. Ziekenhuizen K.U., Leuven, Belgium, 5Osteoporoseklinikken, Oslo, Norway, 6Hosp. Univ., Monterrey, Mexico, 7Sniadecki Hosp., Bialystok, Poland, 8New Mexico Clin. Res. & Osteoporosis Center, Albuquerque, MN, USA, 9OSTEOSOL COMOP, Col. Hipodromo, Mexico, 10Colorado Center for Bone Res., Lakewood, CO, USA, 11Novartis Pharma AG Basel, Switzerland, 12Novartis Pharmaceuticals Corp, East Hanover, NJ, USA.
In the pivotal fracture trial establishing the antifracture efficacy of once-yearly infusions of zoledronic acid (ZOL) 5 mg in women with postmenopausal osteoporosis aged 64–89, we tested whether tissue level responses to ZOL differed with age by analyzing changes in bone remodeling and bone structure between treatment and placebo (PLB) in 3 age groups: < 70, 71–74 and ≥ 75 yrs. Bone remodeling indices were assessed in tetracycline labeled biopsies obtained from 43 patients on ZOL and 49 on PLB (Table 1). Prior to histomorphometric analysis, trabecular bone structure and cortical thickness were assessed on intact biopsy cores by μCT in 50 patients on ZOL and 49 on PLB.
In the PLB group bone turnover, reflected in activation frequency (Ac.f) and mineralizing surface (MS/BS) decreased with increasing age. Despite this, ZOL induced consistent reductions by 71%, 69%, and 76% across the 3 age groups. Differences between other histomorphometric indices: osteoid surface (OS/BS), mineral appositional rate (MAR), and volume referent bone formation rate (BFR/BV), were stable across age groups. Structural parameters (bone volume (BV/TV), trabecular number (Tb.N), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th) and cortical thickness (Ct.Th)) indicated preservation of bone structure in patients on ZOL in all age groups, albeit the numerical differences were lower in the oldest age group.
In conclusion, changes in bone remodeling and preservation of bone structure after 3 yrs of treatment with once-yearly doses of ZOL 5 mg iv are similar across all age groups in postmenopausal women aged 64–89 yrs.
Disclosures: R.R. Recker, Novartis Pharma AG 5.
This study received funding from: Novartis Pharma AG.
Mechanistic Bases of Bone Mineral Density Increase During Alendronate Therapy.
D. Vashishth1, P. Chavassieux2, G. Boivin2, P. D. Delmas2. 1INSERM Unite 831 Universite de Lyon France & Rensselaer Polytechnic Institute, Troy, NY, USA, 2INSERM Unite 831 Universite de Lyon, Lyon, France.
An increase in the mean degree of tissue mineralization (DMB) occurring through secondary mineralization has been proposed to increase bone mineral density (BMD) during bisphosphonate (BP) therapy . In this study we conducted additional analyses on human iliac crest biopsies obtained as part of alendronate (ALN) clinical trials  to identify the mechanistic bases of BMD increase.
Out of a group of 16 patients on a three-year ALN-therapy , we identified two groups of 5 patients each showing lower (8.5 %) and higher (13.3%) bounds of BMD increase but no difference in baseline BMD. For all 10 patients, previously prepared microradiographs were reanalyzed to measure the mean degree of tissue mineralization (DMB) at the osteonal and interstitial compartments in both cortical and cancellous bone tissues. Based on a moving average analysis, six fields each of cortical and cancellous bone tissues were randomly selected for measurement from each biopsy. The average values for patients in each group within osteonal and interstitial compartments were compared between and across cortical and cancellous bone tissues. All DMB measurements were also tested for correlation with standard histomorphometric measures of bone structure (BV/TV, Tb.Th, Tb. Separation, Tb.N), osteoclast activity (EV/BV, E-Depth, Oc#/BS), osteoid (OS/BS, OV/BV, OTh), and bone formation (MAR, BFR/BS, FP) reported previously . The low-BMD-gain group demonstrated no difference between the osteonal and interstitial bone compartments within cortical or cancellous bone tissues but demonstrated a higher DMB in cancellous than in conical tissue (p<0.05). In contrast, the high-BMD-gain group showed higher DMB in interstitial than in osteonal compartment for both cortical and cancellous tissues as well as a higher DMB in cancellous than in cortical tissue (p<0.05). Out of all the DMB measures in cortical and cancellous tissues, only cortical bone interstitial level DMB correlated to bone formation rate (BFR/ BS) (r = −0.86; p = 0.006) and formation period (FP) (r = −0.75; p = 0.04).
In conclusion, this study demonstrates that the effects of ALN-therapy are more evident in cancellous than in cortical bone. Moreover since the interstitial level DMB is at least partially dependent on the duration of secondary mineralization, and negatively correlated to formation parameters, slow bone formation rate and longer bone formation period produce conditions conducive to complete secondary mineralization and consequently larger BMD gain.
References:  Boivin et al. Bone. 2000 5: 687–94.  Chavassieux et al. JCI 1997 100:1475–80.
Disclosures: D. Vashishth, Merck 5.
This study received funding from: INSERM. France & NIH Grants AR49635, AG20618.
Mineralization Status of Bone Matrix in Postmenopausal Women During a 10-year Alendronate Treatment Period.
P. Roschger1, G. Mair*2, N. Fratzl-Zelman1, P. Fratzl*2, D. Kimmel3, K. Klaushofer1, A. LaMotta*3, A. Lombardi3. 1Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Med. Dept., Hanusch Hospital, Vienna, Austria, 2Max Planck Institute of Colloids and Interfaces, Dept Biomaterials, Potsdam, Germany, 3Merck Research Laboratories, Rahway, NJ, USA.
Knowledge of the effects of long-term antiresorptive treatment on bone material quality is an important issue for the safety and optimal duration of treatment. Mineralization density distribution (BMDD) as well as mineral particle thickness, both important composite parameters of bone material, were studied by quantitative backscattered electron imaging (qBEI) and small angle x-ray scattering (SAXS). Previously published data of biopsies from Phase III alendronate studies of 2 or 3 years treatment duration (placebo, n=12, alendronate (ALN), n=12) were compared to data obtained from the FLEX study (fracture intervention trial long-term extension) (5 yrs ALN + 5 yrs placebo, n=14 and 10 yrs ALN, n=16). The mean (CaMean) and the most frequent calcium (CaPeak) concentration, the heterogeneity in mineralization (CaWidth) and the amount of low mineralized bone area (primary mineralization) (CaLow) were determined. ALN treatment for 5 yrs, followed by 5 yrs of placebo treatment resulted in an increase of CaMean (+3.5%) and CaPeak (+2.4%) compared to the group treated with placebo for 2 or 3 yrs. Continuous treatment with ALN for 10 years did not further increase CaMean and CaPeak. Remarkably, the decrease in CaWidth (-14.5 %) observed with 2 or 3 yrs ALN treatment was reversed after 5 yrs and 10 yrs ALN treatment, respectively. This transient narrowing of the BMDD around 3 yrs is consistent with computer modeling of BMDD and indicates the effect on BMDD by the sudden decrease in bone turnover during ALN treatment. In none of the investigated cases CaMean and CaPeak values did exceed those of normal adult trabecular bone, nor did the mean mineral particle thickness parameter get out of the normal range (3.5 to 4.2 nm). Our data support the hypothesis that antiresorptive treatment with ALN should be maintained for 3 to 5 years and that there are no negative effects on bone matrix mineralization even with longer treatment.
Disclosures: P. Roschger, None.
This study received funding from: Merck.
Bisphosphonates Adherence and Fracture Risk: Time-Dependent Relationships from 103,038 Bisphosphonate Users in the U.S.
J. R. Curtis1, A. Westfall*1, H. Cheng1, E. Delzell*1, K. Lyles2, K. G. Saag1. 1University of Alabama at Birmingham, Birmingham, AL, USA, 2Duke University, Durham, NC, USA.
Background: There is high interest in determining if there is a ‘threshold’ level of real-world adherence to bisphosphonate (BP) therapy, after which additional fracture (fx) risk reduction is minimal.
Methods: Using the claims and pharmacy databases of a large consolidated health plans database, we identified persons initiating alendronate or risedronate (no use in prior 6 months) and calculated adherence as a Medication Possession Ratio (MPR: number of days of BPs dispensed divided by the calendar time since BP initiation (the index date)). Claims data identified non-vertebral fxs occurring after the index date. Survival analyses compared fx rates of persons with MPR ≥ 80% (adherent) vs. those with MPR ≤ 50% (nonadherent) at 90 days, stratifying by history of fx in the 6 months prior to the index date.
We then examined fx rate by 10% increments of MPR and characterized MPR in a time-dependent fashion in sequential 3 month intervals. This analysis was repeated excluding those who did not have BPs dispensed for at least 90 days with the expectation that these ‘very non-adherent’ individuals might confound the adherence-fx relationship.
Results: We identified 103,038 new BP users, mean age 62.2 ± 8.2 (SD) years. Overall, there was a significant relationship between adherence and fx risk (p < 0.0001 between adherence groups); survival curves stratified by prior fx (yes/no) are shown (Figure). At 3 years, the absolute fx rate of the adherent vs. the non-adherent was 4.6% vs. 5.6% (number needed to treat = 103). Among those with prior fx, the rate was 35.8 vs. 38.9% (number needed to treat = 32).
The relation between adherence (in 10% increments) and fx was linear with no inflection point observed. After excluding patients who did not have BPs dispensed for at least 90 days, the relation between adherence and fx was curvilinear with an inflection point at MPR = 40–50%.
Conclusions: Adherence with BPs is an important determinant of fx rates, although the number of adherent patients needed to treat to prevent one fx is strongly determined by additional risk factors such as prior fx. The overall relation between adherence and fx is monotonic; although, likely subject to unmeasured confounding. However, even after partially accounting for this confounding, the incremental benefit in fx risk reduction with increasing adherence remained.
Disclosures: J.R. Curtis, Novartis 2: Amgen 2: Merck 2, 5, 8; Procter & Gamble 5, 8.
This study received funding from: Novartis.
Modeling the Contribution of Long-term Persistence with Weekly or Yearly Bisphosphonate Treatment to Fracture Outcomes.
S. Rietbrock*1, M. Olson2, T. van Staa*1. 1General Practice Research Database, London, United Kingdom, 2Novartis Pharmaceuticals, Basel, Switzerland.
Purpose: The objective of this study was to evaluate the impact of improving long-term persistence on fracture outcomes.
Methods: The study population included patients prescribed alendronate or risedronate in the UK General Practice Research Database (GPRD). Individualised probabilities of fracture and death during bisphosphonate therapy and of treatment persistence were estimated by age, gender, dosage regimen, calendar year and clinical risk factors using Cox-regression. Persistence was calculated by measuring repeat prescribing. A unique patient-based pharmacoeconomic model was then developed using these probabilities. By varying the persistence probabilities in a simulation, the fracture outcomes with different scenarios of persistence were then evaluated. The model included seven different fracture types, with weighting of different types by post-fracture mortality. The outcomes were simulated over a 4-year period (maximum of 3 years of bisphosphonate use followed by 1 year of offset). It was assumed that the bisphosphonate users had experienced similar fracture reductions as observed in clinical trials and that weekly and yearly bisphosphonates had similar relative rates of fracture risk reduction.
Results: The study population included 43,525 patients. The 1-year persistence for weekly bisphosphonate in the model was 56.7% and 3-year persistence 35.3%. Modeling showed that improvement of the 3-year persistence by 20% (over current persistence in GPRD) would prevent an additional 41.3 hip fractures per 10,000 patients with weekly treatment. If weekly treatment (with refill at 4 week periods) was substituted with yearly treatment (refill once a year), an additional 28.7 hip fractures (per 10,000 patients) would be prevented. If 3-year persistence improved by 20% with yearly treatment, an additional 75.7 hip fractures would be prevented compared to monthly bisphosphonates. The effects of this substitution were largest in elderly patients and in women. Yearly bisphosphonates would prevent an additional 64.8 hip fractures in women aged 80+ compared to weekly bisphosphonates (with equal 3-year persistence), while this number was 3.5 in men aged 40 to 49. Conclusion: This study found that the switch of weekly to yearly bisphosphonates may prevent additional fractures due to the longer treatment.
Disclosures: T. van Staa, Novartis Pharmaceuticals 2.
This study received funding from: Novartis Pharmaceuticals.
Bone Material Characteristics in Osteoporotic Postmenopausal Women after 3-year Treatment with Strontium Ranelate.
C. Li*1, P. Roschger2, R. Zoehrer*2, I. Manjubala*1, E. P. Paschalis2, N. Fratzl-Zelman2, P. Fratzl*1, K. Klaushofer2. 1Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, 14424 Potsdam, Germany, 2Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Medical Department, Hanusch Hospital, Vienna, Austria.
Three-year treatment of postmenopausal osteoporotic women with strontium ranelate is reported to reduce the risk of vertebral and hip fractures by 41% and 36%, respectively. The underlying mechanism of strontium may involve an effect on bone material properties. Biopsies (kindly provided by Servier) from osteoporotic women treated for 3 years with strontium ranelate (n = 6) or placebo (n = 6) were investigated using complementary nondestructive scanning methods like quantitative backscattered electron imaging (qBEI), x-ray microanalysis (EDX), scanning small- and wide-angle x-ray scattering (sSAXS/sWAXS), and Fourier transform microspectroscopy (FTIRM). qBEI gray level histograms of strontium ranelate-treated subjects showed a distinct shift to bone material with higher average atomic number compared with placebo-treated subjects, which is consistent with the incorporation of Sr (Z = 38), which has a higher atomic number than calcium (Z = 20). EDX analysis of small selected areas (10 × 10 μm) within individual bone packet areas revealed that Sr was present in a molar fraction up to 6 %, exclusively in bone packets newly formed during strontium ranelate treatment, while the old bone packets (most likely generated before strontium ranelate treatment was started) had only marginal amounts of Sr (close to the detection limit of the method). As previously shown by others, global mean bone Sr content obtained by destructive chemical analysis was 1.56% ± 0.30% (Sr/Sr+Ca %mol/mol). WAXS line scans across the bone packets detected an increase in the hydroxyapatite crystal lattice spacing (c-axis) consistent with the number of Ca atoms replaced by Sr atoms (up to 0.5 of the 10 calcium ions composing each unit cell replaced by 0.5 Sr ions), indicating that Sr is incorporated into the bone mineral. Area scans by sSAXS failed to reveal any Sr-dependent changes in mean mineral particle thickness (3.1 to 3.6 nm). FTIRM analysis of trabecular bone did not detect any differences in collagen cross-link ratio (pyr/deH-DHLNL) between placebo and strontium ranelate treatment. In conclusion, after 3 years of treatment with strontium ranelate, strontium was only found in newly formed bone, with preservation of examined bone material characteristics.
Disclosures: K. Klaushofer, None.
This study received funding from: Servier.
Vitamin-D 800 IU/Day + Calcium 1000 mg Supplementation Decreases the Risk of Falling Among Postmenopausal Ambulatory Women — A Population-based, Randomized, 3-Year Study (OSTPRE-FPS).
M. Kärkkäinen*1, M. Tuppurainen1, T. Rikkonen*1, K. Salovaara2, J. Sirola*2, R. Honkanen1, E. Alhava*3, H. Kröger2. 1Bone and Cartilage Research Unit, University of Kuopio, Kuopio, Finland, 2Department of Orthopaedics and Traumatology, Kuopio University Hospital, Kuopio, Finland, 3Department of Surgery, Kuopio University Hospital, Kuopio, Finland.
Vitamin D insufficiency and subsequent secondary hyperparathyroidism are common especially in Northern latitude. Vitamin D insufficiency has been related to low bone mass, falls and fractures. In this 3-year population-based randomized trial (OSTPRE-FPS) we hypothesized that Vitamin-D and Calcium supplementation improves muscle strength, functional capacity and decreases falls in ambulatory postmenopausal women.
The present study population consisted of 606 ambulatory women age varied from 66 to 71 years (mean (SD) 68.5 (1.7)) who belong to the OSTPRE-FPS cohort (n = 3432). The intervention group (n = 292) obtained Cholecalciferol 800 IU/d + Calcium 1000 mg/d supplementation (Calcichew-D3 Forte, Leiras-Nycomed Ltd) for 3 years and the control group (n = 314) received no supplementation.
Falls (ICD10 W00-W19) were recorded based on phone inquiry done at 4 months intervals. The functional capacity tests included: standing-on-one-foot, squat down, chair rising, standing eyes closed 10s, fast regular walk 10m, tandem walk 6m, grip and leg extension strength measurements.
A total of 873 falls in 364 subjects were recorded during the 3-year follow up. There were 381 falls in 165 women in Vitamin D-supplementation group and 492 falls in 199 women in the control-group. Thus, the vitamin-D and calcium supplementation decreased the incidence of falls by 23 % (p = 0.039). Results in chair rising test and leg extension strength improved in the supplementation group (p = 0.002 and p < 0.001, respectively) but the difference between the groups did not reach statistical significance.
In conclusion, Cholecalciferol 800 IU + Calcium 1000 mg supplementation is effective in fall prevention among ambulatory postmenopausal women. The positive effect may be mediated through improvements in lower extremity muscle strength and balance.
Disclosures: M. Kärkkäinen, None.
This study received funding from: Leiras-Nycomed Ltd, Academy of Finland, EVO grant of the Kuopio University Hospital.
Antiinflammatory Role of Osteoprotegerin (OPG) in the Pathophysiology of Osteoporosis and Arteriosclerosis.
C. Kasperk1, U. Liegibel*1, U. Sommer*1, U. Wolf*1, I. Grafe*1, L. Basler*2, P. Nawroth*1, P. J. Meeder*2. 1Internal Medicine I, Endocrinology, University of Heidelberg, Heidelberg, Germany, 2Orthopedic Surgery, University of Heidelberg, Heidelberg, Germany.
Osteoporosis and arteriosclerosis are associated diseases which have numerous genetic, cellular and epidemiological characteristics in common, e.g. OPG-/- mice develop osteoporosis and arteriosclerosis (Bucay et al. Genes Develop 12:1260, 1998). Previously, we demonstrated that mechanical stress by cellular elongation exerts a stimulatory effect on OPG production of osteoblastic cells Liegibel et al. J Exp Med 196:1387, 2002). We hypothecized that there is a biphasic response of the OPG gene to mechanical stress in osteoblastic (OC) and endothelial cells (EC) and that OPG exerts an antiinflammatory action on bone and endothelial cell metabolism implicating a direct effect of OPG on OC and EC. We exposed primary human OC and EC (HUVEC) to elongation stress (ES) using a Flexer cell device. ES up to 2,5 % of initial cell length stimulated OPG production in OC (200 % of control) and had no effect on OPG in EC. ES > 10 % inhibited OPG production in OC down to 50 % and in EC to 15 % of control. OPG increased ERK-1 and −2 phosphorylation and alphavß3 integrin production in OC and EC, effects which could be inhibited by antiRANKL antibodies and the MAP kinase inhibitor PD98059, respectively. The proinflammatory cytokine TNFalpha stimulated IL-6 production in OC and EC which could be inhibited by an OPG co-treatment in both cell types. Interestingly, TNFalpha and IL-1ß decreased alkaline phosphatase activity (AP) in OC but increased AP in EC. However, a combined treatment of OC and EC with the proinflammatory cytokines TNFalpha or IL-1ß together with OPG converted the cytokines' effects on AP and thus exerted a stimulation of AP in OC whereas in EC the AP activity was significantly decreased by the cytokine-OPG co-treatment. In order to clarify antiinflammatory OPG effects we examined a possible action of OPG on IkB and observed a stimulatory action of OPG on IkB production in OC and EC. In summary, in both OC and EC (1) ES exerts a biphasic effect on OPG production, (2) OPG directly activates MAP kinases and stimulates alphavß3 integrin and IkB production, (3) TNFalpha and IL-1ß stimulate IL-6 production which can be inhibited by OPG co-treatment, and (4) TNFalpha and IL-1ß decreased AP in OC but inceased AP activity in EC; OPG-cotreatment converted the inhibitpry action of TNFalpha and IL-1ß and thus resulted in a stimulation of AP in OC and decreased AP activity in EC. In conclusion, OPG is an antiinflammtory cytokine which exerts direct effects on OC and EC metabolism. Locally decreased OPG production may contribute to localized osteoporosis in bone and to mineralization processes closed to branching blood vessels.
Disclosures: C. Kasperk, None.
Alpha/Beta-T Cells Do Not Modulate Androgen Withdrawal-induced Bone Loss in Aged Rats.
R. G. Erben, K. Mildner*, C. Schüler*, M. Skalicky*. Dept. of Natural Sciences, University of Veterinary Medicine, Vienna, Austria.
T lymphocytes are thought to play an important role in the regulation of bone turnover. To explore further the role of T lymphocytes in sex steroid deficiency-induced bone loss, we developed a T cell depletion model in aged Fischer rats. Twelve-month-old male Fischer 344 rats were either thymectomized (TX) or sham-operated (SHAM-TX). Beginning 2 weeks postsurgery, TX rats received 3 intraperitoneal injections (every 3 days) of 20 mg/kg of a monoclonal mouse anti-rat antibody directed against the rat α/β-T cell receptor. SHAM-TX animals received 20 mg/kg of a non-immune monoclonal mouse isotype control antibody. T cell depletion was monitored by FACS analysis of peripheral blood. Three weeks after T cell depletion, TX and SHAM-TX rats were orchiectomized (ORX) or sham-operated (SHAM). All animals were killed 2 months post-ORX after fluorochrome double labeling. The data were analyzed by two-way analysis of variance (ANOVA), determining the influence of the factors TX and ORX and their mutual interaction.
FACS analysis revealed that intraperitoneal injection of TX rats with high dose anti-rat α/β-T cell receptor antibody resulted in a lasting depletion of circulating α/β-T cells. As expected, 2-way ANOVA showed that ORX induced pronounced loss of total and trabecular bone mineral density (BMD) of the proximal tibial metaphysis and of the fourth lumbar vertebral body measured by peripheral quantitative computed tomography (pQCT), 2 months post-ORX. TX by itself had no influence on tibial or vertebral BMD, and significant two-way interactions between the factors TX and ORX were lacking. The ORX-induced bone loss and the increase in bone resorption as measured by urinary collagen crosslink excretion were identical in T cell-depleted TX and in T cell replete SHAM-TX rats. Thus, our study provides evidence that circulating α/β-T cells are not involved in the up-regulation of bone turnover and the subsequent bone loss induced by androgen withdrawal in aged male rats.
Disclosures: R.G. Erben, Schering 2, 5; Procter & Gamble 2; Amgen 2; AiCuris 5.
This study received funding from: German Research Foundation.
High Cardiovascular Risk in Men with Increased Bone Resorption or Low Bone Mass.
P. Szulc, P. D. Delmas. INSERM 831, Hopital E. Herriot, Lyon, France.
Recent studies suggest the existence of an association between bone mineral density (BMD) and cardiovascular disease. We studied the predictive value of BMD and biochemical bone turnover markers (BTM) for major cardiovascular events (myocardial infarction, stroke) in 628 men aged 50 years and over in whom 61 such events occurred during an 8-year follow-up. At baseline, BMD was measured at the lumbar spine, hip, whole body and distal forearm. After adjustment for age and co-morbidities, lower BMD at all sites of measurement was associated with an increased risk of cardiovascular event (O.R. = 1.25 to 1.55 per 1 standard deviation decrease, p < 0.04–0.002). Low BMD, defined by T-score < −1, was associated with a twofold increase in the cardiovascular risk, e.g. whole body BMD — O.R. = 2.63, 95% C.I.: 1.49–4.65, p < 0.001). Elevated levels of the urinary markers of bone resorption were defined by 1 standard deviation above the mean in men aged 50 to 60 years. After adjustment for age, co-morbidities, serum creatinine concentration and season, elevated levels of urinary markers of bone resorption were associated with a higher risk of the cardiovascular event (total deoxypyridinoline — O.R. = 2.90, 95% C.I.: 1.41–5.98, p < 0.005; free deoxypyridinoline — O.R. = 2.24, 95% C.I.: 1.13–4.44, p < 0.03; β-CTX-1 — O.R. = 2.37, 95% C.I.: 95% C.I.: 1.09–5.16, p < 0.03). Bone formation markers did not predict the cardiovascular event.
To the best of our knowledge, this is the first study showing that the increased bone resorption is associated with an increased risk of myocardial infarction and stroke in elderly men. We also confirm that low BMD is associated with an increased cardiovascular risk in elderly men.
Disclosures: P. Szulc, None.
Homocysteine Levels and Risk of Hip Fracture in Postmenopausal Women with Poor Renal Function: The Women's Health Initiative Observational Study.
M. S. LeBoff1, R. Narweker*1, A. LaCroix*2, L. Wu*2, R. Jackson*3, J. Lee*4, D. C. Bauer*5, C. Kooperberg*2, J. Cauley*6, S. Cummings*7. 1Endocrine-Hypertension, Brigham and Women's Hospital, Boston, MA, USA, 2Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 3Ohio State University, College of Medicine and Public Health, Columbus, OH, USA, 4University of California, Davis, CA, USA, 5San Francisco Coordinating Center, CPMC Research Institute, San Francisco, CA, USA, 6University of Pittsburgh, Pittsburgh, PA, USA, 7University of California, San Francisco, CA, USA.
Hip fractures are the most serious osteoporotic fractures leading to disability and increased mortality. Recent studies suggest that high homocysteine levels are associated with an increased risk of fractures, although the mechanisms are not fully understood. Women with decreased renal function, as measured by cystatin-C, have elevated homocysteine levels and an increased risk of hip fractures. To assess the relationship between homocysteine levels and renal function on hip fracture risk, we conducted a nested case-control study in the Women's Health Initiative Observational Study (WHI-OS). The WHI-OS, is a multiethnic study among American women (n = 93,676) aged 50–79 years at study entry. We selected 400 incident cases of hip fracture and 400 controls matched on age, ethnicity, and blood draw date among women not on treatment for osteoporosis including hormone use at baseline. These analyses were adjusted for body mass index, parental hip fracture history, treated diabetes, alcohol use, smoking, history of stroke, and calcium intake. Serum homocysteine levels were measured using a high-performance liquid chromatography assay. Serum cystatin-C was measured using the Dade Behring BN-II nephelometer with a particle-enhanced immunonepholometric assay (interassay co-efficient of variation = 5.7%, sensitivity = 0.02 mg/L). The risk of hip fracture increased 1.36-fold (1.13, 1.63) for each SD increase in serum homocysteine level. In multivariate models adjusted for potential confounders, women in the highest quartiles of both homocysteine and cystatin-C (n = 111) had a 2.77-fold higher risk of hip fracture (1.60, 4.81) as compared to women in the lowest three quartiles of both homocysteine and cystatin-C (p = 0.025 for the interaction). However, women in the highest quartile of homocysteine but lower three quartiles of cystatin-C had an OR = 0.85 (0.53, 1.37) of hip fracture. In summary, high homocysteine levels confer a substantial increased risk of subsequent hip fracture mainly among women with poor renal function.
Disclosures: M.S. LeBoff, None.
This study received funding from: NIH.
Mutations in p62 Linked to Paget's Disease Make the Bone Microenvironment Highly Osteoclastogenic.
Y. Hiruma1, N. Kurihara1, M. A. Subler*2, H. Zhou3, D. W. Dempster3, S. Ishizuka4, G. D. Roodman5, J. J. Windle*2. 1Medicine/Hem-Onc, University of Pittsburgh, Pittsburgh, PA, USA, 2Human Genetics, Virginia Commonwealth University, Richmond, VA, USA, 3Regional Bone Center, Helen Hayes Hospital, New York, NY, USA, 4Teijin Biomedical Research, Tokyo, Japan, 5Medicine/Hem-Onc, VA Pittsburgh Healthcare System and University of Pittsburgh, Pittsburgh, PA, USA.
Bone lesions in Paget's Disease (PD) are characterized by abnormal osteoclasts (OCL), markedly increased bone resorption and formation and increased RANKL expression in the bone microenvironment. Genetic factors contribute to the pathogenesis of PD, with mutations in the p62 gene being the most frequently linked to PD. p62 plays a critical role in NF-κB and p38 MAPK activation induced by TNF-α and RANKL, but the contributions that mutations in p62 make to the abnormalities in OCL and the marrow microenvironment in PD are unclear. To address this question, we generated p62P394L/P394L knock-in (KI) mice that express the most frequent p62 mutation in PD and compared these animals to wild-type (WT) mice. KI mice did not develop PD-like lesions in vivo. However, OCL formation in unfractionated marrow cultures from KI mice was increased and OCL formed at much lower concentrations of RANKL, TNF-α and 1,25-(OH)2D3, similar to marrow cultures from PD patients. Since the p62P394L mutation in KI mice was expressed in both OCL precursors and marrow stromal cells, we then cultured highly purified OCL precursors (CFU-GM) and marrow stromal cells from KI mice separately to determine their respective contribution to the enhanced OCL formation. Purified KI OCL precursors were hyper-responsive to RANKL and TNF-α and formed increased numbers of OCL compared to WT. However, unlike PD patients, KI OCL precursors were not hyper-responsive to 1α,25-(OH)2D3. We then treated stromal cells from WT and KI mice with 1α,25-(OH)2D3 and determined the levels of RANKL expression. KI marrow stromal cells expressed high levels of RANKL and RANKL mRNA compared to WT. To determine the mechanism responsible for increased RANKL expression, we then examined the effects of the p62P394L on activation of downstream signaling pathways induced by 1α,25-(OH)2D3 in stromal cells. 1α,25-(OH)2D3 increased p38 MAPK activity in KI stromal cells, and p38 inhibitors as well as a VDR genomic antagonist decreased RANKL expression. 1β,25-(OH)2D3, which blocks nongenomic actions of 1α,25-(OH)2D3, also decreased RANKL expression. These results show that expression of p62P394L in marrow stromal cells markedly increases RANKL production via p38 MAPK and genomic and nongenomic actions of VDR. They also suggest that p62P394L enhances RANKL production by the marrow microenvironment in PD and predisposes but is not sufficient to induce PD.
Disclosures: Y. Hiruma, None.
This study received funding from: NIH.
Mast Cell Migration to Bone Surfaces Precedes and Appears Essential for PTH-induced Peritrabecular Fibrosis.
K. Marley1, M. B. Lowry*2, S. Lotinun*1, U. T. Iwaniec1, R. T. Turner1. 1Oregon State University, Corvallis, OR, USA, 2Microbiology, Oregon State University, Corvallis, OR, USA.
Increased mast cell number is associated with osteoporosis, osteopetrosis and marrow fibrosis. However, no cause and effect relationship has been established between the mast cell and any of these metabolic bone diseases. The presence of mast cells at sites of bone turnover and degranulating mast cells in the immediate vicinity of osteoclasts in rats led to investigation of the mast cell's role in the skeletal response to parathyroid hormone (PTH). Intermittent increases in PTH (iPTH) are anabolic to bone whereas chronic increases in PTH (cPTH) result in multiple skeletal abnormalities (parathyroid bone disease), including focal bone resorption, impaired mineralization and peritrabecular marrow fibrosis. We now report compelling evidence that the mast cell mediates many of the actions of PTH on bone: 1) cPTH induced severe bone disease in rats but not in 3 mouse strains (C57BL6, DBA2, WBB6F1) lacking mature bone marrow mast cells. In addition, the effects of iPTH and cPTH on osteoblasts (OB) and osteoclasts (OC) were greatly attenuated in mice compared to rats. 2) Both iPTH and cPTH resulted in rapid migration of mast cells to bone surfaces in rats with the mast cells adhering to OB in response to iPTH, and to fibroblasts in response to cPTH. In a time-course study, cPTH induced rapid mast cell migration to bone surfaces. By day 3, mast cells at the bone surface were increased 3-fold but peritrabecular fibrosis was not detected until day 5. This suggests that mast cell signaling stimulates fibroblast proliferation and facilitates migration of fibroblasts to bone surfaces. In support of this hypothesis, mast cells produce PDGF, a profibrotic cytokine. Administration of Trapidil, a PDGF receptor antagonist, did not impede mast cell migration to bone surfaces but suppressed cPTH-induced fibrosis, suggesting a critical chemotactic role for this mast cell-derived factor on fibroblasts. 3) Kit signaling mediates mast cell migration. OB lineage cells produce kit ligand (stem cell factor) which is chemotactic to mast cells. The Kit antagonist Gleevec prevented mast cell migration to bone surfaces and inhibited fibrosis. 4) Kit signals through phosphatidlylinositol 3-kinase (PI3K) to initiate mast cell degranulation. The PI3K inhibitor wortmannin dramatically reduced fibrosis in rats treated with cPTH, suggesting a critical role for this pathway in cPTH-induced fibrosis. Taken together, these findings indicate that mast cell migration to bone surfaces precedes and is obligatory for cPTH-induced peritrabecular fibrosis and suggest that mast cells potentiate the bone anabolic effects of iPTH as well as the catabolic effects of cPTH.
Disclosures: K. Marley, None.
Marked Improvement of Cortical Bone Geometry After Parathyroidectomy in Postmenopausal Women with Primary Hyperparathyroidism.
M. Yamauchi1, R. Nomura*2, H. Kaji2, S. Yano1, T. Yamaguchi1, T. Sugimoto1. 1Internal Medicine 1, Shimane University Faculty of Medicine, Izumo, Japan, 2Division of Endocrinology/Metabolism, Neurology and Hematology/Oncology, Kobe University Graduate School of Medicine, Kobe, Japan.
Cortical bone geometry is one of the most important factors of bone quality. It has been reported that recombinant human PTH(1–34) treatment induced beneficial changes in cortical geometry and our cross-sectional study from women with primary hyperparathyroidism (pHPT) revealed that endogenous PTH excess markedly affected cortical bone geometry (JCEM 2003). On the other hand, our six-year longitudinal study in postmenopausal women with pHPT revealed that parathyroidectomy (PTX) led to marked and sustained increase in bone mineral density (BMD) (Clin Endocrinol 2004). However, no data have been available about geometric changes of cortical bone after PTX in pHPT. In the present study, we assessed the longitudinal effects of removal of endogenous PTH excess on cortical bone geometry in pHPT postmenopausal patients with PTX and compared them with age-, body height and weight-, postmenopausal period-matched normal women. Twenty pHPT postmenopausal women (mean: age 63.6yr, Ca 12.0mg/dl, iPTH 267pg/ml) and thirty control women (age 64.6yr) participated in this study. We assessed volumetric BMD, architectural parameters of cortical bone and SSIp (polar strength strain index) by pQCT at the site of radius. Measurements of pQCT were performed at the time of enrollments and were repeated 1 year after PTX in pHPT group, and 1 year thereafter in control group. During 1-year period, total BMD and cortical BMD significantly decreased in control group (-2.1%, −1.3%), but increased in pHTP group (+2.9%, +1.6%), respectively. Significant decreases in cortical thickness and area were also observed in control group (-3.0%, −2.5%). In contrast, pHPT group showed significant increases in cortical thickness and area (+8.5%, +7.6%) as well as SSIp (+6.2%) during the follow-up period. In conclusion, the present longitudinal study showed that there were significant beneficial changes in vBMD, cortical bone geometry and bone strength in pHPT postmenopausal women, whereas age-related thinning of cortical bone as well as decrease of vBMD were observed in control group. The present findings suggest that the removal of sustained PTH excess markedly affects cortical bone geometry and that PTX in pHPT improve the bone strength by amelioration of cortical bone geometry as well as increase in BMD.
Disclosures: M. Yamauchi, None.
Long-Term Propranalol Improves Bone Mineral Content and Lean Body Mass in Pediatric Burn Patients.
L. K. Branski*1, D. N. Hemdon*1, W. B. Norbury*1, M. G. Jeschke*1, R. P. Mlcak*1, G. L. Klein2. 1Surgery, University of Texas Medical Branch and Shriners Burns Hospital, Galveston, TX, USA, 2Pediatrics, University of Texas Medical Branch and Shriners Burns Hospital, Galveston, TX, USA.
Bum injury over 40% total body surface area (TBSA) causes marked hypermetabolism and episodic increases in catecholamine production as part of the stress response. The aim of our study was to evaluate the effects of propranalol (Prop) given over the first 12 months post-bum on total body bone mineral content (TBMC) and lean body mass (LBM). Forty-nine (49) children, ages 2–18 yr with burns ≥ 40% TBSA were enrolled and randomized to receive Prop (n = 24) or placebo (Cont, n = 25). Prop was titrated to achieve a reduction in heart rate by 15%. TBMC and LBM were measured by dual energy x-ray absorptiometry (DXA) using a 4500A absorptiometer (Hologics, Waltham MA) at hospital discharge, 6,9, and 12 months post-burn. Results were calculated as % discharge (baseline) values. Statistical analysis used two-way ANOVA with post-hoc Tukey's test for inter-group comparisons and one-way repeated measures ANOVA with post-hoc Bonferroni's t-test for intra-group comparisons to baseline values. Prop increased LBM and TBMC and body weight (p < 0.02) vs Cont at 12 months post-burn. Within the Prop group, LBM and body weight increased vs baseline at 9 months, while TBMC increased at 12 months compared to baseline (p < 0.02). Within the Cont group, patients showed an increase in LBM and body weight at 12 months (p < 0.05) compared to baseline. We conclude that long-term use of propranalol has a beneficial effect on bone acquisition due to its effects on increased lean body mass and secondary skeletal loading and/or an inhibition of the episodic increase in endogenous catecholamine production that may promote bone resorption and inhibit bone formation.
Disclosures: G.L. Klein. None.
This study received funding from: NIH.
Bone Morbidity at Diagnosis among Children with Acute Lymphoblastic Leukemia.
L. M. Ward1, N. Shenouda*1, N. Alos2, S. Atkinson3, C. Clarson*4, R. Couch*5, E. Cummings*6, R. Grant*7, C. Rodd8, D. Stephure*9, S. Taback*10, M. Matzinger*1, F. Rauch8, and the STOPP Consortium*11. 1University of Ottawa, Ottawa, ON, Canada, 2Université de Montréal, Montréal, PQ, Canada, 3McMaster University, Hamilton, ON, Canada, 4University of Western Ontario, London, ON, Canada, 5University of Alberta, Edmonton, AB, Canada, 6Dalhousie University, Halifax, NS, Canada, 7University of Toronto, Toronto, ON, Canada, 8McGill University, Montréal, PQ, Canada, 9University of Calgary, Calgary, AB, Canada, 10University of Manitoba, Winnipeg, MB, Canada, 11Canadian Pediatric Bone Health Working Group, Ottawa, ON, Canada.
Children with acute lymphoblastic leukemia (ALL) can manifest vertebral compression at diagnosis; however, the frequency and pattern of vertebral fractures, as well as their relationship to bone mineral density (BMD), has not been systematically studied. We therefore evaluated bone morbidity in 116 newly diagnosed children (age (mean ± SD) 6.6 ± 4.1 years; 62 boys) with ALL (pre-B cell ALL: N = 102; T-cell ALL: N = 14), who were enrolled in the STeroid-induced Osteoporosis in the Pediatric Population (STOPP) study, a prospective research program comprising 11 Canadian pediatric tertiary care centers. All patients underwent lateral thoracolumbar spine x-rays and BMD studies within 30 days of diagnosis. Forty-one patients (35%) had a total of 113 vertebral compression fractures (90 thoracic, 23 lumbar), as independently assessed by two radiologists at the coordinating site. Twenty-eight patients (68%) had one or two vertebral fractures, 10 patients (25%) had three to seven fractures and 3 patients (7%) had between nine and twelve fractures. Vertebrae T6 and T7 were most commonly affected and accounted for 41 of the 113 fracture events (36%). Ninety-two of the fractures (81%) were graded as mild, 17 (15%) as moderate and 4 (4%) as severe. Eighty-six of the compressed vertebrae (76%) had an anterior wedge configuration. Children with vertebral compression fractures had lower mean lumbar spine areal BMD z scores than those without fractures (–1.5 ± 1.4 versus −0.67 ± 1.3; P < 0.05). Mean height z scores did not differ between the two groups (0.3 ± 1.19 for those with fractures; 0.31 ± 1.25 for those without; P = 0.9). Only 14 of the 41 patients with vertebral compressions (34%) reported back pain. Thus, significant overt skeletal morbidity is common in newly diagnosed children with ALL, but is asymptomatic in the majority of cases.
Disclosures: L.M. Ward, None.
This study received funding from; Canadian Institutes for Health Research.
Structural Implications of Low-Magnitude Mechanical Stimulation in a Pilot Study of Patients with Renal Osteodystrophy Evaluated with the MRI-based Virtual Bone Biopsy.
C. E. Jones*1, F. W. Wehrli1, J. F. Magland*1, C. T. Rubin2, S. A. Nihtianova*3, M. B. Leonard3. 1University of Pennsylvania, Philadelphia, PA, USA, 2State University of New York, Stony Brook, NY, USA, 3Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Renal osteodystrophy (ROD) is a pervasive disorder of bone metabolism that results in impaired trabecular architecture, loss of cortical bone, and increased fracture rates. In this double blind, placebo-controlled pilot study we examined the hypothesis that low magnitude mechanical stimulation (LMMS) increases bone volume fraction and improves the structural integrity of the trabecular bone in ROD. A total of 30 dialysis patients were randomized to daily (20 min/day) LMMS treatment with an active device (0.3g, 30 Hz,) or a placebo device. Micro-MRI scans were performed in the distal tibial metaphysis at a voxel size of 137 × 137 × 410 μm3 using a custom designed 3-D spin echo pulse sequence at baseline and after 6 months of treatment. The images subsequently underwent a cascade of processing steps, comprising the virtual bone biopsy, resulting in a final voxel size of 45 × 45 × 135 μm3 and BV/TV was computed. After skeletonization of the images was performed (so that trabecular plates were converted to surfaces and rods to curves), the data were subjected to digital topological analysis. This algorithm establishes the topological identity of each voxel as belonging to a curve, surface or junction between the two fundamental types. To date, repeat MRI scans have been obtained on 10 LMMS and 11 placebo-treated subjects. Fig. 1 illustrates the wide range of structural abnormalities in some of the study subjects at baseline. In patients treated with the active LMMS device, TB/TV was increased 2.8% (p = 0.02) while the surface-to-curve ratio (S/C, ratio of all surface-type divided by all curve-type voxels) was increased 8.0 percent (p = 0.03), indicating a relative increase in plates over rods. Similarly, the erosion index (a measure of loss of connectivity) was decreased 6.9% (p = 0.03) suggesting improved trabecular connectivity. No significant changes were noted in the placebo group. The observed changes are commensurate with the treatment's anabolic effect resulting in improved bone quality and hence potentially reduced risk of fracture. These data are the first showing structural implications of LMMS treatment in patients with renal disease assessed with in vivo structural imaging.
Disclosures: C.E. Jones. None.
Thioredoxin-1 Overexpression Attenuates Streptozotocin-induced Diabetic Osteopenia in Mice: A Novel Role of Oxidative Stress and Therapeutic Implications.
Y. Hamada*1, H. Fujii*1, R. Kitazawa2, S. Kitazawa2, M. Fukagawa1. 1Division of Nephrology and Kidney Center, Kobe University School of Medicine, Kobe, Japan, 2Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University School of Medicine, Kobe, Japan.
Diabetes mellitus is associated with increased risk of osteopenia and bone fracture. However, the mechanisms accounting for diabetic bone disorder still remain to be clarified. Moreover, there are few effective treatments for this disease. We have previously reported that streptozotocin-induced diabetic mice develop low turnover osteopenia associated with increased oxidative stress in diabetic condition (BONE 2007). Therefore, in order to determine the role of oxidative stress in the development of diabetic osteopenia, we investigated the effect of thioredoxin-1 (TRX) overexpression, a major intracellular antioxidant, on the development of diabetic osteopenia using TRX transgenic mice (TRX-Tg). TRX-Tg was C57BL/6 mice that carry the human TRX transgene under the control of β-actin promoter.
Eight-week-old male TRX-Tg and wild type littermates (WT) were intraperitoneally injected with either streptozotocin or vehicle alone. Mice were classified into four groups: 1) non-diabetic WT, 2) Non-diabetic TRX-Tg, 3) diabetic WT, and 4) diabetic TRX-Tg. After 12 weeks of streptozotocin treatment, the physical properties of femora, and the parameters of bone histomorphometry of tibiae were assessed. Oxidative stress in the whole body as well as in the bone was evaluated.
TRX overexpression did not affect either body weights or hemoglobin A1c levels both in the diabetic mice and in the non-diabetic mice. There were no significant differences in renal function, and serum levels of calcium, phosphate, and intact parathyroid hormone among four groups. Urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was significantly elevated in diabetic WT, which was attenuated in diabetic TRX-Tg. Immunohistochemical staining for 8-OHdG was clearly intensified in the bone tissue of diabetic WT compared with non-diabetic WT. In contrast, staining was attenuated in diabetic TRX-Tg. TRX overexpression partially restored reduced bone mineral density and prevented the suppression of bone formation (OV/BV, OS/BS, Ob.S/BS, O.Th, MAR, and BFR/BS) observed in diabetic WT. These results suggest that increased oxidative stress in diabetic condition contributes to the development of diabetic osteopenia. Furthermore, our findings indicate that suppression of increased oxidative stress by TRX induction can be a therapeutic approach for treatment of diabetic osteopenia.
Disclosures: Y. Hamada, None.
Time Sequence of Secondary Mineralization and Microhardness of Bone in an Ewe Model.
Y. Bala*, D. Farlay*, C. Simi*, P. J. Meunier, P. D. Delmas, G. Boivin. INSERM Unité 831, Universite de Lyon, Lyon, France.
The degree of mineralization is a major determinant of the mechanical resistance of bone (Follet et al. 2004, Bone 34:783–9). Mineralization begins by a rapid primary mineralization followed, after the full completion of the Basic Structural Units (BSUs), by a secondary mineralization phase, i.e., a slow and gradual maturation of the mineral component leading to complete mineralization of newly formed BSUs (Meunier & Boivin 1997, Bone 21:373–7). The duration of primary mineralization has been evaluated by several autors but the time sequence of secondary mineralization is still poorly investigated [Fuchs et al. 2005, J Bone Miner Res 20(Suppl. 1):325]. Our aim was to determine the time course of secondary bone mineralization in ewe, an animal model having an Haversian bone tissue with a remodeling activity close to the Human one (Chavassieux et al. 1997, Bone 20:451–5). 18 ewes (4.5 ± 0.4 years, INRA, Theix, France) received every six months for 30 months fluorescent labelings following the schedule: TO double tetracycline, T6 single fluorescein, T12 triple tetracycline, T18 double fluorescein, T24 single tetracycline, T30 double alizarin labeling in order to date the age of the BSUs. Transiliac bone samples have been taken on each ewe after T18 and T30 labelings, then embedded in methyl methacrylate. Microradiography (Boivin & Meunier 2002, Calcif Tissue Int 70:503–11) performed on 100 ± 1 μm-thick sections allowed the measurement of the focal Degree of Mineralization of Bone (DMB g/cm3) on 505 different BSUs in which the duration of the mineralization was precisely determined by labeling. Microhardness using a Vickers indenter under a load of 25g for 10s (Hv kg/mm2) was measured on 367 among the 505 BSUs [Bala et al. 2006, J Bone Miner Res 21(suppl.1):S332]. DMB measured at primary mineralization (0.76 ± 0.10 g/cm3) significantly increased during the first 6 months (+26%, p < 0.0001). Then, DMB increased more slowly until 30 months to reach a mean of 1.20 ± 0.12 g/cm3. DMB measured at the end of the primary mineralization corresponded to 64% of the final mineralization. DMB after 6 and 12 months were 85% and 91% of the final DMB, respectively. Secondary mineralization is thus the fastest during the first year. Hv followed a similar trend, with a rapid increase during the first six months (+32%, p < 0.0008), then a slow increase until 30 months. Hv and DMB are strongly and positively correlated (r2 = 0.52, p < 0.0001). The time course of secondary mineralization can be divided into two parts having separated trends, a rapid increase during the first year then a slowdown of mineralization until 30 months. Mineralization explains a great part of the hardness of bone at BSU level.
Disclosures: G. Boivin, None.
Risedronate Reduces Osteonal Cortical Porosity in Osteoporotic Women.
B. Borah*1, T. Dufresne*1, J. Nurre*1, P. Chmielewski*1, R. Phipps1, M. Lundy1, X. Zhou*1, M. Bouxsein2, E. Seeman3. 1Procter & Gamble Pharmaceuticals, Mason, OH, USA, 2Ortho Biomechanics Lab, Harvard Medical School, Boston, MA, USA, 3Austin Health, University of Melbourne, Melbourne, Australia.
Measurements of cortical porosity include 1) Haversian canals (diam. <50 μm), 2) the remodeling spaces of the osteons (diam. ∼100 to 300 μm) in the intra-cortical envelope, and 3) larger cavities (diam. >380 μm) primarily in the endocortical envelope. In this study, we evaluated the effect of risedronate on cortical porosity in transiliac biopsies from osteoporotic women. Paired biopsies taken at baseline and after 5 years risedronate treatment (5 mg/day, n = 28) or placebo (n = 21) were imaged by micro-CT at 8 μm isotropic resolution. The cavities were then stratified by size, as determined by the minor axis length of the holes in the 2D slices. Porosity was averaged over 10 to 14 slices ∼300 μm apart and was calculated as the area of cavities expressed as a percentage of the total cortical bone area. The porosity at baseline was not statistically different between placebo and risedronate for any pore size. Compared to baseline, 5 years treatment with risedronate caused a significant 15% reduction in porosity for cavities of <320 μm diameter (p < 0.05, Figure), an effect likely to be the result of reduced osteonal remodeling. In the placebo group, porosity from cavities in this same size range increased by ∼20% compared to baseline (not significant). The between group difference was significant or approached significance for cavities <320 μm diameter (p = 0.01–0.06). The reduction of porosity by risedronate was not significant when cavities >400 μm were included. These larger cavities produce trabecularization of the inner cortex and cortical thinning. It appears that due to the variability of the age-related changes in the endocortical trabecularization, treatment-related reduction of porosity cannot be reliably assessed from measurements made throughout the cortex. These findings indicate that risedronate reduces cortical porosity by reducing the birth rate of new osteons or by filling in the remodeling spaces in osteons that existed prior to treatment
Disclosures: B. Borah, Procter & Gamble Pharmaceuticals 3.
LRP5 G171V Mutation Protects Against Disuse-Related Trabecular Bone Fragility.
M. P. Akhter, D. M. Cullen, R. R. Recker. Medicine, Creighton University, Omaha, NE, USA.
Fragility fracture risk is greater in people with low bone mass or osteoporosis. In addition, a sedentary (disuse or lack of exercise) lifestyle choice may further deteriorate low bone mass associated with osteoporosis. We have shown (#F171 ASBMR-06) that disuse causes far greater loss in bone volume fraction (BV/TV) in the KO mice (67%) than in the HBM mice (14%). In this study, we characterized trabecular bone strength properties to determine if the LRP5 G171V mutation will protect against bone loss associated with disuse. Forty eight adult (4 mo.) male mice representing three genotypes (all bred to C57BL/6 mice): WT (Lrp5+/+, wild type), KO (Lrp5+/- knockout), HBM (High bone mass with LRP5 G171V mutation) were randomly divided between control and Disuse groups. The mice in the disuse group were hind limb suspended in individual cages for 4 weeks. All mice were inspected daily to assure compliance, adequate food intake, and general health. Femurs were collected and frozen in saline for biomechanical strength testing. A 1.26 mm diameter indenter was used to measure trabecular bone strength at the distal femur site (testing rate of 3mm/tnin, Instron 5543). We analyzed the data using the General Linear Model for univariate analysis to test for differences (P < 0.05) due to disuse within each genotype. Trabecular bone strength declined in all the three genotypes (Table). Bone strength variables (ultimate load [ULT], yield load [YLD], stiffness [STIF]) were lower in the disuse group as compared to their controls within each genotype (HBM, WT, and KO). Disuse-related loss in strength (ULT) was 48%, 51% and 74% in HBM, WT and KO mice respectively. In addition, trabecular bone strength (ULT, YLD, STIF) in HBM mice remained elevated as compared to both WT and KO within each treatment group (Control & Disuse). Similar to trabecular bone structure data (reported previously), the already fragile KO has a large decrease in trabecular bone strength (74%), and the HBM losses were moderate with final bone strength remaining well above normal for the wild type mice. The mice with the LRP5 G171V mutation (HBM) did show a significant response to disuse demonstrating mechanosensitivity. However, the relative advantage in trabecular bone strength (ULT, YLD) between HBM and KO went from 10 fold in the control mice to 20 fold after disuse (Table). These data suggest that the HBM genotype (LRP5 G171V mutation) is relatively protected against disuse-related bone fragility.
Disclosures: M.P. Akhter, None.
This study received funding from; The State of Nebraska LB595 Funds.
Beta-Adrenergic Receptor Agonist Administration During Hindlimb Unloading Attenuates Losses in Bone Geometry, Structure, and Mechanical Properties.
J. M. Swift*1, J. T. Ali*2, J. L. Stallone*1, H. A. Hogan2, S. A. Bloomfield1. 1Health and Kinesiology, Texas A&M University, College Station, TX, USA, 2Mechanical Engineering, Texas A&M University, College Station, TX, USA.
A major challenge to long-duration spaceflight is the significant bone loss that occurs in weightbearing limbs. The rodent hindlimb unloading (HU) model effectively simulates the deleterious effects of microgravity on the skeleton. In addition, HU results in significant declines in blood flow and increases in vascular resistance in unweighted hindlimb bone. Previously, we demonstrated that dobutamine (DOB), a beta-adrenergic receptor agonist (ADRB), given during simulated microgravity significantly blunts decreases in femoral midshaft cortical bone area and polar moment of inertia (CSMI). The purpose of this study was to assess the effectiveness of DOB in attenuating HU-associated losses in tibiae and femora bone content, density, area, and strength during 28 days of unloading. Male Sprague-Dawley rats, aged 6-mo, were assigned to either a normal cage activity (CC) or HU group (n = 24/group). Animals were given one daily bolus dose (4 mg/kg BW/d) of DOB (n = 12) or an equal volume of saline (VEH; n = 12). In vivo peripheral quantitative computed tomography (pQCT) scans were taken at the proximal tibia metaphysis (PTM) and tibial mid-diaphysis on anaesthetized animals on days 0 and 28 to reveal geometric and structural changes. Post-sacrifice, hindlimb bones were excised for determination of mechanical properties using an Instron 1125 device; ex vivo pQCT scans were performed at the femoral neck (FN) and mid-diaphysis. At the PTM, HU resulted in similar reductions in total bone mineral content (BMC) in animals treated with VEH (-17%) and DOB (-15%); however, reductions in total volumetric bone mineral density (vBMD) were only evident in HU+VEH (-9%). Total BMC, total area, and CSMI were significantly higher at FN in HU+DOB vs. HU+VEH (+18%, +23%, and +52%, respectively). Mechanical testing of FN revealed that HU+DOB exhibited a significantly higher ultimate load (+25%) as compared to HU+VEH. No significant changes in mid-diaphyseal tibia bone structure or area during HU were evidenced by in vivo pQCT scans, but 3-point bending to failure revealed a significantly higher ultimate load (+46%) in HU+DOB vs. HU+VEH. Ex vivo pQCT scans of mid-diaphyseal femora revealed significantly higher total BMC (+7%), total area (+5%), and CSMI (+10%) in DOB-treated rats subjected to 28-day HU vs. HU alone. These results suggest that DOB administration during HU prevents significant declines in total vBMD at PTM and produces a higher midshaft tibia ultimate load. We speculate that these positive effects may be attributed to ADRB-mediated vasocirculation, imporoving blood flow to bone during unloading.
Disclosures: J.M. Swift, None.
This study received funding from; The National Space Biomedical Research Institute through NASA NCC 9–58.
Attenuated Response to Skeletal Load in Connexin43 Deficient Mice.
S. K. Grimston, M. D. Brodt*, M. J. Silva, R. Civitelli. Bone and Mineral Diseases, Washington University in St. Louis, St. Louis, MO, USA.
In vitro data suggest that connexin43 (Cx43) plays an important role in mechanotransduction. We reported that the anabolic response to parathyroid hormone is attenuated in mice with conditional ablation of the Cx43 gene (Gja1) in osteoblasts, obtained using a Cx43flox/LacZ gene replacement model and a 2.1 kb fragment of the α1(I) collagen promoter to drive Cre (ColCre;Cx43flox). We tested the hypothesis that the anabolic response of the skeleton to mechanical loading is also dependent on Cx43. Four-month-old female mice were anesthetized and subjected to an in vivo 3-point bending protocol on the tibia, 5 days a week for 2 weeks. ColCre;Gja1−/flox mice had significantly greater marrow area, total tissue area, and thinner cortices than wild type equivalent Gja1+/flox mice. However, area moment of inertia was similar (0.130 ± 0.059 versus 0.128 ± 0.054 mm4, respectively), suggestive of adaptive responses similar to aging bone in Gja1 deficient mice. In Gja1+/flox mice, the loading regimen produced abundant new bone formation at the endocortical surface, as evidenced by double calcein labeling (injected ip on days 5 and 12), proportional to the estimated strain applied (1600 μϵ), with maximal effects at the apex (tension) and on the lateral aspect (compression) (arrows and arrowhead in Fig. 1, respectively). Conversely, there were predominantly single labels in ColCre;Gja1−/flox mice (Fig. 2). Accordingly, mineral apposition rate (MAR) and bone formation rate were significantly lower (p < 0.05) in ColCre;Gja1−/flox (1.02 ± 0.97 μm/day; 83.5 ± 84.12 μmVμm2/yr) relative to Gjal+/flox mice (1.86 ± 0.65 μm/day; 192.23 ± 89.5 μm3/μm2/yr), with heterozygous Gja1+/flox mice showing intermediate values (1.86 ± 0.94μm/day; 150.54 ± 120.89 μm3/μm2/yr). Relative to the unloaded contralateral leg, MAR increased in the loaded leg by 67 ± 16% (p < 0.01) in Gja1+/flox mice, and 19 ± 22% (n.s.) in ColCre;Gja1−/flox mice. Loading also stimulated the formation of periosteal woven bone on the surface subject to pressure, and this was also less evident in the conditionally ablated ColCre;Gja1−/flox (22.7 ± 4.4%) relative to Gja1+/flox mice (31.3 ± 13.1%; p < 0.05). Thus, deletion of Gja1 prevents full anabolic response to in vivo skeletal physical loading. Cx43 is critical for bone's rapid adaptive response to mechanical stimuli.
Disclosures: S.K. Grimston, None.
This study received funding from: NIH AR41255.
The Lrp5 Gain of Function Mutation Is Associated with an Increased Osteogenic Response to Loading in Trabecular as well as Cortical Bone in Female but not Male Mice.
L. K. Saxon, T. Sugiyama, J. Price, L. E. Lanyon. Veterinary Basic Sciences, Royal Veterinary College, London, United Kingdom.
The Lrp5 gain of function G171V mutation (Lrp5+) is associated with increased sensitivity to mechanical loading in cortical bone , however it is not known if the same occurs in trabecular bone. We therefore used our mouse tibial loading model  to investigate the effect of loading on trabecular and cortical bone in male and female Lrp5+ mice.
The right tibiae of 17-week-old male (n = 6) and female (n = 7) Lrp5+ and WT mice were subjected to a short period of dynamic axial loading for 40 cycles/day, 10 Hz, 3 days a week for 2 weeks. Strain gauges bonded to the medial mid-shaft were used to determine the magnitude of the load required to apply similar strains within each group. Differences in trabecular bone volume (BV/TV), number (Tb.N), thickness (Tb.Th), total bone area (TA), cortical area (CA), periosteal perimeter (Pp), endocortical perimeter (Ep), moment of inertia (MMI), and cortical thickness (C.Th) in the loaded (right) versus non-loaded (left) tibia were measured using micro CT.
In male Lrp5+ mice all measured parameters in control bones were higher than in WT except C.Th (p < 0.001). To engender 850μϵ a 19.8N load was applied to Lrp5+ and 10.5N to WT. In WT males loading increased Pp, CA, MMI, Tb.Th (p < 0.05) whilst in Lrp5+ mice loading only increased C.Th (p < 0.05); all other measures showed no loading-related difference. The magnitude of loading-related change between Lrp5+ and WT mice was only different for periosteal perimeter (3.9 vs −1.3%, p < 0.001).
In female Lrp5+ mice all measured parameters were also higher than in WT (p < 0.001). A load of 19.8N applied to the tibia of Lrp5+ mice and 9N to WT mice induced 1100μϵ. All loaded WT tibiae showed gains in Pp, CA, MMI (p < 0.05), and all parameters increased except Tb.N in Lrp5+ mice (p < 0.05). In cortical bone the magnitude of the loading response was higher in the Lrp5+ mice compared with WT (TA 10.1 vs 1.2%, CA 17.7 vs 1.2%; Pp 5.5 vs −3.1%; Ep 10.4 vs −2.4%; MMI 21.7 vs −3.0%; C.Th 9.1 vs 4.1%, p < 0.05). The gains in BV/TV were also greater in Lrp5+ mice. This was due to increased trabecular thickness not number (BV/TV 16.8 vs 5.6%; Tb.Th 17.5 vs 2.7%, p < 0.05; Tb.N −0.5 vs 3.1%).
In conclusion, the G171V mutation results in a more robust bone phenotype in male mice. However, at the strain levels we used there was no difference from WT in their osteogenic response to loading in trabecular bone, and only a small difference in cortical bone. By contrast, in females the G171V mutation results in both a more robust bone phenotype and increased sensitivity to loading in trabecular and cortical bone.
- 1.Akhter et al Bone 2004
- 2.DeSouza et al Bone 2005
Disclosures: L.K. Saxon, None.
Bone Adaptation to Impact Loading — Significance of Loading Intensity.
A. Vainionpää*1, R. Korpelainen*2, J. Leppäluoto*3, T. Jämsä*1. 1Department of Medical Technology, University of Oulu, Oulu, Finland, 2Department of Sports Medicine, Oulu Deaconess Institute, Oulu, Finland, 3Department of Physiology, University of Oulu, Oulu, Finland.
It is important to find efficient strategies for prevention of osteoporosis and fragility fractures as the prevention with medication is impossible at the population level. Exercise is one of the major prevention strategies even though the optimal loading patterns of bone have not yet been clearly defined in terms of bone loading forces in clinical settings. Our aim was to study the effects of impact loading on bone and assess the intensity and amount of efficient impact loading.
We conducted a 12 month population-based exercise intervention in 120 premenopausal women aged 35 to 40 years. The women were randomized equally to exercise (EG) and control groups (CG). The exercise regimen consisted of supervised, progressive high-impact exercises three times per week and an additional home program. Bone mineral density (BMD) in the proximal femur was measured by dual x-ray absorptiometry and bone geometry in the mid-femur was quantified by computed tomography (QCT). Also bone turnover was examined by using biochemical markers (TRACP-5b, PINP and Uncoupling Index). Daily impact loading was continuously recorded with a novel accelerometer-based measurement device and analyzed as the daily number of impacts within five acceleration ranges between 0.3 g to 9.2 g (g = acceleration of gravity, 9.81 m/s2).
Regular impact exercise increased the femoral neck BMD by 1.5 % (p = 0.003) and led to geometric adaptations by increasing the mid-femur periosteal circumference by 0.2 % (p = 0.03) when compared to controls. It also altered bone turnover balance in favour of bone formation. The women with the highest number of impacts at the intensity level above 4 g had the greatest increment in proximal femur BMD. The number of impacts needed exceeding this level was 60 impacts per day. Impacts exceeding 2.5 g were associated with increased periosteal circumference and enhanced bone turnover favouring bone formation.
According to our results, impact loading has significant dose- and intensity dependent effect on weight-bearing bones. The threshold for improving BMD in the hip is 60 impacts per day exceeding 4 g, while geometric adaptation and bone turnover activation can be achieved with lower intensity levels. Impacts required for osteogenic effects can be obtained during habitual exercise training. These thresholds for osteogenic exercise give tools to promote efficient exercise patterns to be included in everyday routines and personal activities, thus increasing the potential to affect in community level.
Disclosures: A. Vainionpää, None.
This study received funding from; Tekes, Finland; Newtest Ltd, Finland.
Gene Polymorphisms, Bone Mineral Density and Bone Mineral Content in Children: The Iowa Bone Development Study.
M. C. Willing1, T. L. Burns*2, J. C. Tomer*2, E. Letuchy*2, S. Santiago-Parton*1, K. F. Janz*3, T. Marshall*4, J. M. E. Gilmore*4, J. J. Warren*4, S. M. Levy4. 1Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA, USA, 2Epidemiology, College of Public Health, Iowa City, IA, USA, 3Health and Sports Studies, College of Liberal Arts, Iowa City, IA, USA, 4Preventive and Community Dentistry, College of Dentistry, Iowa City, IA, USA.
We examined associations of candidate gene polymorphisms with bone mineral density (BMD) and content (BMC) in a cohort of 412 healthy non-Hispanic white children participating in the Iowa Bone Development Study. Whole body (WB), hip and spine BMD and BMC were determined using a Hologic QDR 4500a densitometer for 213 girls and 199 boys age ∼11 years. Candidate genes included the type I collagen genes (COL1A1 and COL1A2), osteocalcin, osteonectin (SPARC), osteopontin, vitamin D receptor (VDR), estrogen receptor (ER), androgen receptor (AR), IGF-1, IGF-2, PTR, PTHR and LRP5. Gender-combined and gender-specific prediction models for bone measures that included age, weight, height, dichotomous Tanner stage (and gender) were developed using multiple linear regression analysis and residuals were calculated. COL1A2 genotype (identified by one or more of the following: Rsa1, Intron 12 VNTR, 5′(CA)1 — CGCACA(CG)m(CA)n GTx 3′) was found to have the strongest and most consistent association with adjusted BMD/BMC measures. When stratified by gender, COL1A2 genotype was associated with WB BMC, spine BMC, and hip BMD in both boys and girls, as well as with hip BMC in girls. IGF-2 (6815 A/T), osteocalcin (C/T promoter), PTHT3 (AAAG) and VDR (Bsm-I) genotypes were also associated with some bone measures in the combined analyses, but the associations differed by gender. In girls, osteocalcin and IGF-2 genotypes were associated with hip BMC and BMD and with WB BMC and hip BMD, respectively. PTHR genotype was associated with all bone measures except spine BMC in the combined analyses, but results only reached significance in girls. In contrast, VDR (Bsm-I) genotype was associated with hip BMD only in boys, and COL1A1 (Rsa1) genotype was associated with spine BMD, hip BMC and BMD only in boys. Gender-specific quartiles of the BMD/BMC residuals were determined and an association analysis using the recursive partitioning technique represented by the nonparametric classification tree approach was used to identify candidate genotypes associated with high vs. low quartile status. Significant gene x gene interaction effects involving PTHR and each of the following: IGF-1, IGF-2, COL1A2, and osteonectin were identified for spine BMD, particularly in girls. Our data suggest that genetic variation at multiple genetic loci is important in bone accrual in children. Combinations of individual genotypes may also impact BMD and BMC.
Disclosures: J.M.E. Gilmore, None.
Jump Starting Skeletal Health: Bone Increases from Jumping Exercise Persist Seven Years Post Intervention.
K. B. Gunter*1, A. Baxter-Jones*2, R. Mirwald*2, H. C. Almstedt*3, S. Durski*1, A. A. Fuller-Haves*1, C. M. Snow1. 1Department of Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR, USA, 2College of Kinesiology, University of Saskatchewan, Saskatoon, SK, Canada, 3Department of Natural Science, Loyola Marymount University, Los Angeles, CA, USA.
Evidence suggests that bone mineral increases attributable to exercise training prior to puberty may confer a significant advantage into adulthood. However, there is a dearth of supportive prospective longitudinal data. We assessed the change in bone mineral content (BMC) at the left proximal femur over eight years in children who participated in a seven-month jumping intervention and who were pre-pubertal at study onset. We hypothesized that jumpers would accrue and retain more BMC compared to controls and therefore be at a considerable advantage in optimizing peak bone mass accrual. Subjects were drawn from the BUGSY study (Building Growing Skeletons in Youth), an ongoing mixed longitudinal study of bone mineral accretion in growing children initiated in the fall of 1997. Participants were recruited from local elementary schools in Corvallis, Oregon and were randomly assigned within classrooms to either an intervention (jumping) or control (stretching) group. Subjects were assessed at baseline, at the end of the intervention (7 months) and at approximately 19 months. Participants were reassessed at 31, 43, 55, 67, 79 and 91 months from study entry for a total of nine measurement occasions over approximately 8 years. Bone mineral content was assessed by dual-energy X-ray absorptiometry. Multi-level random effects models were constructed and used to predict change from study entry in BMC at each measurement occasion. We were able to utilize data from a total of 421 DXA scans on 57 individuals who were measured on 3 or more occasions. At 7 months, the intervention group had 3.6% more total hip bone accrual than the non-intervention group (p < 0.05), once the effects of change in age, height and weight were accounted for. The effects of the intervention, in terms of percentage contribution to Atotal hip BMC, decreased at each measurement occasion thereafter. After an average of 7.6 years, the intervention group had 1.4% more total hip bone accrual than the nonintervention group, once the effects of change in age, height and weight were accounted for (p < 0.05). This provides the first evidence that changes in BMC attributable to short-term exercise undertaken prior to puberty persist more than 7 years following exercise cessation in growing children. If the benefits are sustained into adulthood, effectively increasing peak bone mass, this could have substantial effects on fracture risk.
Disclosures: K.B. Gunter, None.
This study received funding from; National Institutes of Health RO1 AR45655–08.
Regular Physical Activity Has Only Temporary Effect on Bone Gain in Pubertal Girls: A 6.5 Year Follow-up Study.
E. Völgyi1, F. A. Tylavsky2, H. Suominen1, S. M. Cheng1, A. Lyytikäinen1, M. Alén1, U. M. Kujala*1, H. Kröger3, S. Cheng1. 1Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland, 2Health Science Center; Preventive Medicine, University of Tennessee, Memphis, TN, USA, 3Department of Surgery, Kuopio University Hospital, Kuopio, Finland.
The effects of exercise on bone gain in early puberty have been proven in many different short intervention studies. Whether effects found in early puberty can be sustained until early adulthood in children who are not in athletic training is still debatable. The purpose of this study is to evaluate the relationship between physical activity and bone gain in girls and the relationship with their mothers' physical activity level and bone characteristics. The study subjects consisted of 258 10–13-year old girls with maturation status of I-III at the baseline. They were followed on average of 6.5 years and their results reflect the entire follow-up period. In addition, a group of these girls' mothers (n = 108, mean age 44 yrs) had measurement twice within a 3.5 year interval. Bone area (BA), mineral contact (BMC) and density (BMD) of the total femur (TF), femoral neck (FN), and lumbar spine (L2-L4) were assessed by DXA (Prodigy, GE Lunar). The level of leisure time physical activity (PA) was assessed by questionnaire. We only compared those with consistently low (LPA, < 2 times & hours /wk, n = 92 0mo, n = 65 12 mo, n = 92 24mo, n = 25 36mo, n = 26 84mo), to those consistently high (HPA, >4 times & hours/wk, n = 101 0mo, n = 71 12mo, n = 71 24mo, n = 22 36mo, n = 26 84mo) PA groups at each measurement point. No differences were found in age, relative weight and height, and in Tanner stage between the HPA and LPA groups in girls through-out the follow-up. Those girls in the HPA group had higher BMC (p = 0.017, t-test) of the FN at 24mo, and BMD of the FN at 0mo (p = 0.033), 12mo (p = 0.01) and 24mo (p = 0.029) than that of the LPA group. There were no differences in BA from any of the bone sites between PA groups at any follow-up time point. By age 17.9 years the differences in BMC and BMD of FN, TF or L2–4 between the HPA and LPA had vanished. When we compared the girl's PA with the mother's PA (paired), we found PA levels were associated only at baseline but not in other time points. The agreement between mothers and daughters for LPA was 71% and for HPA was 56% (p = 0.043, χ2 analysis). They reached the mean value of the mothers (n = 108 at 36mo) only in FN BMD (p > 0.05). There were no differences in FN BMC, TF BMD, L2–4 BMD, FN BA and L2–4 BA (p > 0.05). Daughters had lower TF BMC (p = 0.001), TF BA (p < 0.001) and L2–4 BMC (p < 0.001) compared to mothers. Our results indicate that the higher BMC and BMD value due to HPA during puberty may be temporary or participating physical activity more than 4 times and 4 hours a week was not sufficient to impact on bone gain.
Disclosures: E. Völgyi, None.
This study received funding from: ASBMR Bridge Funding, Academy of Finland and Finnish Ministry of Education, Juho Vainion Säätiö, Finnish and Hungarian Government Scholarship Pool (CIMO).
Bone Density at Age of Peak Bone Mass in Women Is Not Associated with History of Childhood Fractures.
S. M. Ott1, A. Z. LaCroix2, L. E. Ichikawa*3, L. Spangler*1, D. Scholes4. 1U of WA, Seattle, WA, USA, 2U of WA and Fred Hutchinson, Seattle, WA, USA, 3Group Health, Seattle, WA, USA, 4U of WA and Group Health, Seattle, WA, USA.
A recent large study of ten-year-old children found that low bone mineral density (BMD) predicted fracture incidence. Peak bone mass is attained during late teenage years at the hip and in the mid-twenties at the spine. Whether childhood fractures are related to the bone density during adolescence or young adulthood has not been as well studied. We therefore examined this in a cross-sectional study of 606 adolescents and young women aged 14–30 years. Members of Group Health, a Washington State HMO, were selected from the health plan computerized databases based on prescriptions for oral contraceptive use and age. We mailed letters of invitation to participate in a study of bone health. BMD (DEXA) was measured at the hip, spine, and whole body. Data on previous fractures and other factors related to bone health were collected via interview and a written questionnaire. Potential subjects with severe health problems, known metabolic bone disease, recent pregnancy, or use of prednisone or progesterone-only birth control methods were excluded. Overall, 51% of the cohort was 14–18 years of age. 206 of 606 subjects (34%) reported at least one fracture; there were a total of 336 fractures. Most common sites were ankle, wrist and toe. Fractures were most common at age 11 when 4.2% of the subjects experiences their first fracture. Ninety percent of the fractures occurred before age 17. The fracture history differed by racial background: 9% of Black subjects, 11% of Asian and 39% of Whites and other races reported history of fracture. Previous fractures occurred in 32% of subjects who were currently underweight and 40% of those who were currently obese; 41% who had weight loss greater than ten pounds in the past; in 46% of subjects with asthma, in 33% of those with menarche older than age 13, and in 43% of those who had a relative with a history of fractures.
The BMD was not lower at the hip, spine, or whole body in the adolescents or young women who had a history of fracture: total hip BMD was .993 g/cm2 in those with a fracture history vs .986 in those without; at the spine the results were 1.023 vs 1.019 and at the whole body 1.103 vs 1.096, respectively. Similar findings were seen in teens as in young adults, with slightly higher bone density in the fracture cases at all three measurement sites. Adjusting for age, race, BM1, calcium intake, physical activity, and current smoking status did not alter the findings. Our data show that history of fracture is common, reported in about a third of otherwise healthy young women. At the age of peak bone mass women in this study with childhood fractures did not have lower bone density than those without fractures.
Disclosures: S.M. Ott, None.
This study received funding from: NIH.
PTHrP Induces Mitogen-Activated Protein Kinase Phosphatase-1 in Differentiated Bone Marrow Stromal Cells, Mouse Osteoblast and Cementoblast Cell Lines.
N. S. Datta, R. Kolailat*, G. Pettway*, J. Berry*, L. K. McCauley. Periodontics and Oral Medicine, University of Michigan, Ann Aror, MI, USA.
This study investigated the signaling mechanisms of Parathyroid hormone (PTH) and PTH related protein (PTHrP) during osteogenic cell proliferation and differentiation. Mitogen-activated protein kinases (MAPKs) play a critical role in this process. In order to understand the up-stream signaling pathways, the role of MAP kinase phosphatase-1 (MKP-1) in regulating MAPKs, cyclin D1 and osteoblast cell growth arrest were studied. Analysis of MAPK activity revealed down-regulation of cyclin D1 (5–10 fold), p38 and ERK1/2 phosphorylation after PTHrP stimulation in differentiated mouse MC3T3-E1 osteoblasts. Interestingly, MKP-1 was induced concurrently with the attenuation of p38 and ERK1/2 phosphorylation. Real-time PCR and western blot analysis revealed 4–8 fold increase in MKP-1 expression following PTHrP stimulation (15 min to 4h) compared to vehicle controls in differentiated osteoblasts. Using different pharmacological inhibitors and activators of cAMP/PKA and PKC pathways both cAMP and PKC were implicated in the PTHrP regulation of MKP-1. MKP-1 expression was also induced in differentiated primary bone marrow stromal cells (BMSCs) and OCCM cementoblast cells (tooth root lining cells phenotypically similar to osteoblasts). Microarray analysis of ectopic ossicles (implanted BMSCs) generated in mice, demonstrated down-regulation of MKP-1 in less differentiated ossicles following acute injection of PTH. In contrast, there was an increase in MKP-1 expression by real-time PCR following three weeks of anabolic PTH treatment in more differentiated ossicles. These findings highlight MKP-1 as a potential regulator of the PTH and PTHrP response in osteoblasts and may be an important target gene in the anabolic action of PTH in bone.
Disclosures: N.S. Datta, None.
This study received funding from: NIH grants DE016865 and DK53904.
See Sunday Plenary Number S002
Odd-Skipped Related 2 Functions in Cell Proliferation.
S. Kawai, M. Yamauchi*, A. Amano*. Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan.
The drosophila pair-rule gene Odd-skipped encodes a zinc finger transcription factor required for accurate segment formation in the drosophila embryonic development. Mammalian homologues, Odd-skipped related 1 (Osr1) and Osr2, were expressed in limb, branchial arches, kidney, eye, and dermis. Osr2 is known to play a critical role in the secondary palate development. We previously reported the function of Osr2 in the bone using transgenic mice. In this study, we analyzed more detailed functional role of Osr2. Osr2 was found to be highly expressed in multipotent mesenchymal C3H10T1/2 cells. The expression level of Osr2 by C3H10T1/2 cells was examined under various culture condition. Osr2 expression remarkably increased by the serum starvation along the growth arrest, whereas, the expression decreased promptly by the serum re-addition. Next, the DNA methylation of Osr2 promoter was analyzed with the sodium bisulfite sequencing method, and histone acetylation on Osr2 promoter was also analyzed with the chromatin immunoprecipitation. The expression of Osr2 in the serum starvation, the confluent growth arrest, and the serum re-addition was found to be regulated by DNA methylation in the Osr2 promoter as well as by the histone acetylation. Moreover, Osr2 was observed to be localized in the cellular nuclear euchromatin with the confocal laser scanning microscope. Osr2 was participated in the maintenance at the G0 state that corresponded in the quiescent state at the cell cycle by the flow cytometry. These findings indicate that Osr2 play an important role in cell proliferation, especially in G0 regulation which is thought the entrance of the osteoblastic cell differentiation.
Disclosures: S. Kawai, None.
See Sunday Plenary Number S004
Grx5 Regulates Osteoblast Cell Functions by Protecting Against Oxidative Stress.
G. R. Linares, W. Xing, K. E. Govoni, T. T. Nguyen*, S. Chen*, S. Mohan. JLP VAMC and LLU, Loma Linda, CA, USA.
There is now increasing evidence which suggest an important role for reactive oxygen species (ROS) in a variety of pathological situations including osteoporosis. However, little is known on the molecular components of the oxidative stress pathway or their functions in bone. Based on our recent findings that the newly identified member of glutaredoxin (Grx) family, Grx5, is highly expressed in bone and induced by growth hormone and the published findings that Grx5 protects yeast against oxidative stress, we proposed the hypothesis that Grx5 protects osteoblasts (Obs) from oxidative stress. We found that treatment of MC3T3-E1 Obs with CdCl2 an inhibitor of Grx activity, increased apoptosis in a dose dependent manner with a 2.5 fold increase (P < 0.001) at 1 μM dose. To verify that the CdCl2 effects on Ob apoptosis is caused by inhibition of Grx5 activity, we treated MC3T3-E1 Obs with Grx5 siRNA or control siRNA and evaluated effects on apoptosis. Grx5 expression decreased (P < 0.01) 80 and 40% at 24 & 72 hrs, respectively, after treatment with Grx5 siRNA. In contrast, neither Grx1 nor Grx2 expression was altered by Grx5 siRNA treatment. Grx5 siRNA treatment decreased osteocalcin expression by 50% (P < 0.01) and increased apoptosis by 40% (P < 0.01) as determined by DNA fragmentation assay. To identify the molecular pathway by which Grx5 regulates Ob functions, we tested based on findings in yeast that Grx5 deficiency results in impaired biogenesis of Fe-S cluster, a critical co-factor for a large number of regulatory proteins. Accordingly, mitochondrial aconitase activity, which is dependent on Fe-S cluster, was decreased by 37% (P < 0.01) in Grx5 siRNA treated cells. Since reduced formation of Fe-S cluster would lead to increased level of free iron, a competitive inhibitor of Mn-SOD (a key mitochondrial enzyme that scavenges ROS) we measured MnSOD activity in Grx5 deficient Obs. Mn-SOD activity was significantly reduced in Grx5 siRNA treated cells (0.89 ± 0.11 vs. 2.04 ± 0.05 U/mg protein; P < 0.001). Because estradiol treatment has been shown to protect Obs against oxidative stress and increase Grx5 expression (1.5 fold, P < 0.05), we evaluated the consequence of Grx5 overexpression on Obs. We found that Grx5 overexpression using retroviral vectors protected MC3T3-E1 cells against H2O2-induced apoptosis and formation of ROS (50 & 40% reduction; P < 0.01). Furthermore, Grx5 overexpression increased mineralized nodule formation in MC3T3-E1 cells (23% increase; P < 0.01). Our findings are consistent with the hypothesis that Grx5 is an important determinant of Ob cell functions and acts via a molecular pathway that involves regulation of Fe-S cluster formation, Mn-SOD activity, and ROS generation.
Disclosures: G.R. Linares. None.
ROS Expression in Osteoblasts of NOS1, 2 and 3 Knock Out Mice.
H. Watanabe, Y. Sugawara, T. Yanagisawa*. Ultrastructural Science, Tokyo Dental College, Chibacity, CHlBA, Japan.
Oxygen is converted to reactive oxygen species (ROS) and nitric oxide (NO) with high reactivity, and reacted with proteins and genes, affecting cell metabolism and intracellular transport. Bone tissues are readily affected by the gas environment, but reports on the effects of the gas environment on osteoblasts have been rare. In this study, we examined the protein levels of various NO and ROS synthetases (NOX) in normal mice and NOS1 (n-NOS), NOS2 (i-NOS), and NOS3 (e-NOS) knock-out (KO) mice, and evaluated the interrelationships among various NO and ROS synthetases. After 5-week-old normal mice and NOS1, 2, and 3 KO mice were anesthetized, immunohistochemical evaluations were performed using NOXO, NOXA, NOX4, and NQO antibodies. In osteoblasts of the control mice, the NOXO, NOXA, and NQO expression was negative, but NOX4 expression was weakly positive. Both NOXO and NOXA were expressed in NOS2 and NOS3 KO mice, also suggesting the expression of NOX1. Furthermore, since the expression of NOX4 and NQO were strongly positive in NOS2 and NOS3 KO mice, the presence or absence of i-NOS and e-NOS is considered to be involved in the generation of ROS in osteoblasts.
Disclosures: H. Watanabe, None.
Rapamycin Inhibits Osteoblast Proliferation and Differentiation in MC3T3-E1 Cells and Primary Mouse Bone Marrow Stromal Cells.
U. K. Singha*1, Y. Jiang*2, S. Yu1, M. Luo*1, Y. Lu1, J. Zhang1, G. D. Roodman1, G. Xiao1. 1Medicine, University of Pittsburgh, Pittsburgh, PA, USA, 2Pharmacology, University of Pittsburgh, Pittsburgh, PA, USA.
While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on these parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.
Disclosures: G. Xiao, None.
Effects of Traumatic Brain Injury on Bone Formation/Remodeling During Fracture Healing in a Rat Model.
I. Arango-Hisijara1, E. R. Kulick*2, S. Rehman*3, M. B. Elliott*4, M. F. Barbe4, R. F. Tuma*2, F. F. Safadi1, S. N. Popoff1, W. G. DeLong3. 1Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA, USA, 2Physiology, Temple University School of Medicine, Philadelphia, PA, USA, 3Orthopaedic Surgery and Sports Medicine, Temple University Hospital, Philadelphia, PA, USA, 4Physical Therapy, Temple University, Philadelphia, PA, USA.
Previous studies have established that traumatic brain injury can enhance the process of fracture healing. In this study, we examined the effects of traumatic brain injury on bone formation in the fracture callus and on circulating factors that can influence bone remodeling and/or angiogenesis. Ten week old Male Sprague-Dawley rats were separated into four groups: 1) Control; 2) Standard closed femoral fracture (Fx); 3) Low impact traumatic brain injury combined with Fx (LiHtx/Fx); 4) High impact traumatic brain injury combined with Fx (HiHtx/Fx). Rats were injected with calcein at 5 and 2 days prior to being euthanized at 2 weeks post-treatment. Histological analysis of the brains stained with cresyl violet confirmed diffuse axonal injury in both Htx groups; The HiHtx group displayed more axonal injury and neuronal apoptosis compared to the LiHtx group. Radiographic examination of the fracture callus demonstrated greater bony consolidation in the brain injured LiHtx/Fx and HiHtx/Fx compared to Fx rats. Histological analysis of the fracture callus demonstrated greater amounts of periosteal (intramembranous) and endochondral bone formation occurring in the brain injured LiHtx/Fx and HiHtx/Fx compared to Fx rats. Calcein labeling demonstrated a significant increase in bone formation rates in the brain injured rats, providing direct evidence that brain injury can increase bone formation at the fracture site. Serum osteocalcin was also measured with a significant increase in the LiHtx/Fx and HiHtx/Fx groups compared to the Fx group. Analysis of other circulating factors revealed significant increases in TGFβ1, MCP-1, TNFα, IL-1β, IL-6, and leptin levels in the LiHtx/Fx and HiHtx/Fx groups compared to the Fx group. TNFα, IL-1β and IL-6 are osteoclastogenic factors suggesting that remodeling of the fracture callus occurs earlier in the brain injured LiHtx/Fx and HiHtx/Fx compared to Fx rats. High levels of TGFβ1 and MCP-1 could stimulate proliferation of osteoprogenitors cells, and high levels of leptin have been shown to increase VEGF expression resulting in enhanced angiogenesis that is a requirement for accelerated fracture healing. In conclusion, traumatic brain injury accelerates bone formation and remodeling during fracture healing and changes in circulating levels of factors known to influence bone cell development/function may mediate, in part, the acceleration of fracture repair.
Disclosures: I. Arango-Hisijara, None.
TWEAK Is a Regulator of Human Osteoblast Function.
G. J. Atkins1, C. Vincent*1, K. J. Welldon*1, D. R. Haynes*2, C. Holding*2, T. S. Zheng*3, A. Evdokiou*1, D. M. Findlay1. 1Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia, 2Discipline of Pathology, University of Adelaide, Adelaide, Australia, 3Biogen Idec Inc., Boston, MA, USA.
INF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily is pro-angiogenic, pro-inflammatory and proliferative for endothelial and other cell types. We recently showed that TWEAK is a novel mediator of mouse collagen-induced arthritis and have identified TWEAK expressing cells in human rheumatoid arthritis (RA) tissues. The aim of this work was to investigate a potential role for TWEAK in human bone remodeling.
Human primary osteoblasts (OB) were found, by flow cytometry, to express high levels of the TWEAK receptor, Fn14. OB also expressed abundant TWEAK mRNA. Treatment of OB with exogenous TWEAK was highly proliferative. TWEAK dose-dependently suppressed OCN mRNA expression and increased both the RANKL:GAPDH and the RANKL:OPG mRNA ratios. Time-course studies under mineralising conditions showed that TWEAK suppressed the osteogenesis-related genes OCN and BSP-1 early in cultures, corresponding with the period in which RANKL expression was increased. Whereas RANKL induction was transient (up to day 9), OCN, BSP-1 and type I collagen mRNA expression were inhibited throughout the mineralisation period (up to day 21). Consistent with this, continuous TWEAK exposure inhibited in vitro mineralisation by OB and induced the expression of sclerostin, which has been shown to inhibit bone formation via BMP/Wnt signalling. TWEAK stimulated mitogen-activated protein kinase (MAPK) activity in primary osteoblasts, inducing phosphorylation of ERK-1/2 and JNK, but not P38 MAPK. TWEAK had only a slight effect on NFκB signalling and did not invoke the Akt survival pathway. We also found that TWEAK significantly modifies the osteoblast response to TNFα, suggesting that in an inflammatory situation the outcome may vary, dependent on the relative amounts of these cytokines present. Our results suggest that TWEAK is a novel regulator of bone turnover and are consistent with a role for TWEAK in inflammatory bone loss pathologies, such as RA, where its persistent presence may be anti-anabolic.
Disclosures: G.J. Atkins, None.
See Sunday Plenary Number S010
Lysyl Oxidase Regulates Collagen Quality and Quantity in MC3T3-E1 Cell Culture System.
P. Atsawasuwan*, Y. Mochida, M. Yamauchi. Dental Research Center, University Of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Lysyl oxidase (LOX) is an amine oxidase critical in stabilizing collagen fibrils in tissues including bone. To obtain further insights into the function of this enzyme in osteoblastic cells, we established and characterized the MC3T3-E1 (MC) derived clones stably overexpressing LOX. MC cells were transfected with pcDNA3.1-V5/His containing coding sequence of mouse LOX and several stable clones expressing higher levels of LOX (S clones) were generated. The overexpression of LOX was verified by Western blot analysis with anti-V5 antibody and anti-LOX antibody. Five S clones and two control groups (MC cells and those transfected with an empty vector) were selected and analyzed. Based on MTS assay at day 1, 3 and 5 of culture, all S clones showed the proliferation rate comparable to that of controls. In another set of experiment, the clones and control cells were cultured in α-MEM in the presence of 10% fetal bovine serum, 2mM β-glycerophosphate and 50 μg/ml ascorbic acid for up to 4 weeks. At appropriate time points of culture, cell-matrices were analyzed for collagen content (week 0, 1) and cross-links (wk 2 and 4) by high performance liquid chromatography, fibrillogenesis (wk 3) by transmission electron microscopy and in vitro mineralization (wk 0, 1, 2, 3 and 4) by Alizarin-red S staining. The quantitative cross-link analysis revealed that two major crosslinks (dihydroxylysinonorleucine and pyridinoline), the major aldehyde (hydroxylysine aldehyde) and a total number of aldehydes (free + cross-linked) were significantly increased in S clones in comparison to controls demonstrating higher LOX activities in these clones. However, the collagen content in all S clones was significantly decreased. Consistent with this, the number and diameter of collagen fibrils in S clones were significantly fewer and smaller than those of controls. Furthermore, the onset of mineralization was markedly delayed in S clones. These findings indicate that LOX plays crucial roles not only in collagen stability but also in collagen synthesis. The latter function could be due in part to the ability of LOX to bind and modulate transforming growth factor β1 (TGF-β1) function as we have recently reported. These dual functions of LOX may have significant impact on matrix organization and mineralization in bone.
Disclosures: P. Atsawasuwan, None.
This study received funding from: NIH grants DE10489 and AR052824.
Calcitonin and Teriparatide for Vertebral Fracture Pain.
A. Bazarra-Fernandez*. ObGyn, Juan Canalejo University Hospital, La Corunna, Spain.
Background: Fractures, especially vertebral fractures, are a common complication of osteoporosis, leading to significant pain.
Aim: To compare the pain release induced by osteoporotic vertebral fracture, through teriparatide and teriparatide plus calcitonine.
Methods: We performed a study to compare the analgesic effect of 20 mcg teriparatide versus 20 mcg teriparatide plus metered dose intranasal spray 200IU/activation calcitonin in two groups between postmenopausal women undergoing osteoporosis with vertebral fracture. A 10-point visual analog pain scale (1 = least to 10 = most painful) and a four-point pain grade (grade 1 = least to grade 4 = most painful) were used to measure the pain perception.
Results: The mean pain scores for the teriparatide and teriparatide plus calcitonin were 2.3 ± 1.1 and 8.5 • 1.1, respectively (P < 0.05), while the pain grades for teriparatide and teriparatide plus calcitonin were 1.5 ± 0.3 and 3.5 • 0.4, respectively (P < 0.05). In teriparatide group, analgesics were requested, but in teriparatide plus calcitonin group no analgesics were requested (P < 0.001).
Conclusion: Using teriparatide is more expensive than other osteoporosis treatment. Studies show that taking a bisphosphonate with hormone replacement therapy (HRT), results in increased bone mass when compared to taking either a bisphosphonate or estrogen alone. Besides, calcitonin is better for osteoporotic vertebral fracture pain release than HRT. However, a larger investigation will be needed to achieve more significant case number.
Disclosures: A. Bazarra-Fernandez, None.
Osteoblasts from Serotonin Transporter Knockout Mice Display Reduced Growth and Mineralization Capacity.
M. M. Bliziotes, K. M. Wiren. Medicine, Oregon Health and Science University/pvamc, Portland, OR, USA.
Recent studies in bone have demonstrated the presence of functional pathways for both responding to and regulating the uptake of serotonin (5-HT). This is of high clinical importance given the role of the serotonergic system in affective disorders, and the wide use of pharmacologic agents that target the 5-HT system to manage these disorders. In addition to the presence of 5-HT receptors, osteoblasts, osteoclasts and osteocytes all possess the serotonin transporter (5-HTT). Binding and uptake studies have demonstrated the 5-HTT in osteoblastic and osteocytic cells to be functional and highly specific for 5-HT uptake. Immunohistochemistry of sections of whole bone from rat tibia demonstrated the in situ expression of 5-HT receptors and the 5-HTT in osteoblasts and osteocytes, Additional evidence for a role of 5-HT signaling in the growing skeleton was more recently provided by animal studies. Using two differing animal models, Warden et al. (Endocrinology 2005) found that mice with a life-long null mutation of the gene encoding for the 5-HTT displayed a consistent skeletal phenotype of reduced mass, altered architecture, and inferior mechanical properties. This finding was confirmed in a second model wherein inhibition of the 5-HTT using a SSRI resulted in reduced bone mineral accrual during growth. In both models, the skeletal phenotype resulted from a reduction in bone formation indicating an osteoblastic defect.
We have investigated the phenotype of osteoblastic cells from 5-HTT knockout mice in vitro. Calvaria from wild type and 5-HTT−/− neonatal mice were isolated and osteoblast populations derived with serial collagenase digestion. Cell growth curves, alkaline phosphatase expression and mineralization capacity (as assessed by alizarin red staining of osteoblast cultures) were assessed.
Cells from 5-HTT−/− mice demonstrated reduced growth capacity in the first week of culture, with approximately 30–60% reduction in growth as assessed by cell counts. Alkaline phosphatase activity was also reduced by 20 % in the 5-HTT−/− cells at day 14 of culture. Finally, mineralization capacity was reduced by 20% at day 23 in culture in the 5-HTT−/− cells.
We conclude that the reduced bone formation rates and osteopenia observed in young 5-HTT−/− mice is due, at least in part, to a cell autonomous defect in osteoblasts which results in reduced cell growth, alkaline phosphatase activity and mineralization capacity. The mechanism whereby this defect is expressed in unknown. Given the widespread use of drugs that target the 5-HTT, elucidation of the effects of disruption of this transporter on bone is of major clinical significance.
Disclosures: M.M. Bliziotes, None.
Strontium Ranelate Effects in Human Osteoblasts Support its Uncoupling Effect on Bone Formation and Bone Resorption.
T. C. Brennan*1, M. S. Ryhchyn*1, P. Halbout*2, A. D. Conigrave*3, R. Mason*1. 1School of Medical Sciences (Physiology) and Bosch Institute, University of Sydney, Sydney, Australia, 2Rheumatology, Institut de Recherches Internationales Servier, Courbevoie, France, 3School of Molecular and Microbial Biosciences, University of Sydney, Sydney, Australia.
Strontium ranelate reduces the risk of vertebral and hip fractures in post-menopausal women. Previous studies have shown that strontium ranelate increases bone formation and decreases bone resorption. In the current study, we investigated the uncoupling effect of strontium ranelate in primary human osteoblasts (HOB). For this, we assessed the strontium ranelate effects on indirect markers of bone formation (HOB proliferation, alkaline phosphatase (ALP) activity), on the regulation of osteoclastogenic signals by osteoblasts (OPG mRNA level and RANKL mRNA and protein level) and on HOB lifespan. HOB were cultured in Dulbecco's modified Eagles medium with 10% fetal bovine serum, adapted to serum-free and calcium-free medium for 24h, and then treated with strontium ranelate in physiological Ca2+ (1mM). After a 48h-treatment, strontium ranelate increased HOB proliferation, assessed by thymidine incorporation, in a dose-dependent manner, up to 3.8-fold with 2 mM Sr2+(p < 0.01). ALP activity was increased after 72 h with strontium ranelate by almost 2 fold (1 and 2 mM, p < 0.01). After only 24 h, strontium ranelate dose-dependently increased OPG mRNA expression, by qRT-PCR, up to 1.9-fold with 2 mM Sr2+ (p < 0.001). Under the same conditions, RANKL mRNA expression was dramatically decreased compared with RANKL expression observed in vehicle: remaining expression was below 25% with strontium ranelate concentrations ≥ 0.1 mM (p < 0.001). These results were confirmed at the protein level by RANKL-specific western blotting. Finally, strontium ranelate increased HOB survival under oxidative stress conditions induced by peroxide (0.1 mM, p < 0.05, 1 and 2 mM, p < 0.01), and decreased serum deprivation-induced apoptosis as measured by caspase 3 and caspase 7 activities (0.1 and 1.0 mM, p < 0.05). In conclusion, strontium ranelate, at strontium concentrations close to those observed in patients treated with the therapeutic dose of 2g/day, increases human osteoblast replication, differentiation and the ability to withstand stress, parameters associated with promotion of bone formation. In parallel, human osteoblasts stimulated by strontium ranelate express more OPG and less RANKL, thereby decreasing their capability to stimulate osteoclastogenesis. Overall, these results strongly support the dissociation effect of strontium ranelate on bone formation and bone resorption in human osteoblasts.
Disclosures: T.C. Brennan, None.
Primary Murine Osteoblast Cultures Contain Macrophages that Enhance Osteoblast Mineralisation.
M. Chang*, A. R. Pettit, K, Schroder*, V. M. Ripoll*, D. A. Hume*, L. Raggatt. Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD, Australia.
Delineation of the phenotype and functional capacity of osteoblasts (OBs) has been widely studied using primary OBs harvested via enzymatic digestion of neonatal rodent calvaria (calvarial OBs). Previous studies have suggested heterogeneity within this cell preparation and reported immune functions that are not traditionally performed by mesenchymal cells. These immune functions have been attributed to the OB, however, the heterogeneous nature of the culture has not been considered. We have demonstrated that macrophages are a significant population in standard calvarial OBs using cells isolated from MacGreen mice (macrophages express an eGFP transgene driven by a myeloid restricted promoter). Microarray analysis of differentiating calvarial OB cultures (day 5, 14 and 21) confirmed a large number of macrophage-associated genes at all time points. Clustering analysis using microarray datasets representing nearly all tissues and cell lineages (symatlas.gnf.org) linked the gene expression profiles of calvarial OBs to macrophages and osteoclasts. Immunocytochemistry and flow cytometry confirmed that calvarial OB preparations co-isolated a population of F4/80+ macrophages that persist and expand during OB differentiation in vitro. Multiple passaging did not eliminate macrophages from these cultures. Bone explant cultures were also examined as an alternative approach to generating primary OBs and were similarly shown to contain F4/ 80+ macrophages. Given these observations, and our recent data demonstrating that macrophages are intercalated within bone lining tissues, we hypothesised that macrophages and OBs cooperate in the control of bone metabolism. To delineate the cooperative and distinct functional roles of macrophages and OBs, we used magnetic-assisted cell sorting to generate a population of highly enriched calvarial OBs by removing haematopoietic cells. The majority of cells removed via this method expressed the F4/80 macrophage marker. Strikingly, macrophage removal significantly decreased both osteocalcin mRNA expression levels and in vitro mineralisation (von Kossa staining) in enriched OB differentiation cultures. The presence of a persistent population of macrophages within primary OB cultures raises the possibility that our existing understanding of in vitro OB biology has been influenced by the contribution of this cell population. Our data provide evidence that macrophages regulate OB function and specifically enhance mineralisation in vitro.
Disclosures: M. Chang, None.
See Sunday Plenary Number S016
Atm Regulates Osteoblast Differentiation Through the BMP-Smad1/5/8 Pathway.
J. F. Chau, N. Rasheed*, S. Wang*, B. Li. Institute of Molecular and Cell Biology, Singapore, Singapore.
Ataxia telangiectasia mutated (Atm) is a tumor suppressor that plays a critical role in DNA damage response and is required for genome integrity. In addition, Atm deficient mice have been shown to suffer from osteoporosis (Hishiya et al. Bone 37:497, 2005; Rasheed et al. Hum. Mol. Genet 15:1938, 2006). It was found that Atm deficient osteoblast showed decreased expression of osterix, an essential transcription factor for osteoblast differentiation, leading to the defective osteoblast differentiation in the Atm deficient mice. It is known that osterix is induced by BMPs through the canonical Smad1/5/8 and non-canonical MAPK signaling pathways. As Atm deficiency diminished the BMP induced up-regulation of osterix, we propose that Atm participates in the BMP activated signaling pathways. We found that BMP2 treatment can activate Atm and the activation of Smad1/5/8 was compromised in Atm deficient osteoblast and MEF as detected by Western blotting. This phenomenon was also observed in normal cells treated with wortmannin or caffeine, inhibitors to phosphatidylinositol 3-kinase related kinases (PIKKs), a family of proteins that Atm belongs to. Treatment of wortmannin or caffeine also decreased the protein level of Id1, a known target of BMP-Smad1/5/8 signaling pathway. The results strongly suggest that Atm controls osteoblast differentiation through the BMP signaling pathway.
Disclosures: J.F. Chau. None.
This study received funding from: A-star.
The Effect of Calcium Channel Antagonists on Bone Metabolism in Aged Male and Female Brown Norway Rats.
D. L. DeMoss, B. D. Kidd*, M. J. Harmon*, L. A. Ashley*, G. C. Howard*, A. J. Auxier*, E. D. Nickel*. Biological Sciences, Morehead State University, Morehead, KY, USA.
Bone metabolism is invariably correlated with calcium transport indicating that calcium channels are a potential point of regulation for skeletal remodeling. Calcium channel antagonists are utilized therapeutically and experimentally to decrease the influx of calcium into cells by blocking voltage-regulated L-type calcium channels. Previous experimental evidence has suggested that calcium channel antagonists decrease osteoblastic activity, thus decreasing the activity of the bone forming cells at a time when bone formation is already exceeded by bone resorption, exacerbating the situation. Therefore, the established principle that bone formation decreases following the attainment of peak bone mass, illustrates the need for a more comprehensive understanding of the action calcium channel antagonists have on bone turnover. Experimentation utilized male and female Brown Norway Rats six months of age to compare the effects of estrogen and the antagonists on blood pressure and bone turnover from skeletal compartments. In order to evaluate the positive or negative impact of various calcium channel antagonists on bone loss, bone resorption parameters were compared between normal males and females, and those receiving calcium channel antagonists (diltiazem, nifedipine, verapamil). The models utilized to study bone turnover were the pharmacokinetic loss of the tracer 3H-tetracycline, a compound deposited in the active mineralization front and freely released in urine, the measurement of various bone degradation markers in urine collected throughout the experimental period and organ isotope extraction. The data suggest significant differences between sexes and antagonist impact on the turnover of calcium from multiple calcium pools.
Disclosures: D.L. DeMoss, None.
This study received funding from: NIH-INBRE 5P20RR17481–06.
Protein Kinase D Is an Essential Mediator During Osteoblast Differentiation.
D. N. Y. Fan, W. M. W. Cheung, A. W. C. Kung. Department of Medicine, The University of Hong Kong, Hong Kong, China.
Protein kinase D, also known as PKCμ, is a serine/threonine protein kinase that responds to phorbol esters and diacylglycerol. Using MC3T3-E1 cells, we demonstrated that PKD is activated during DMSO-induced osteoblast differentiation. We further demonstrated that prolonged activation of protein kinase C pathways alone, i.e. in the absence of osteogenic inducers such as BMP-2 and/or DMSO, is sufficient to induce bone formation in preosteoblast cells in vitro. In this study, we examined the functional roles of PKD in osteoblastogenesis. Phorbol 12-myristate 13-acetate (PMA) activates most PKCs including PKD. Treatment of MC cells with PMA enhances osteoblast differentiation as indicated by increased ALP activity and bone nodules formation. Treatment with Gö6976, a specific inhibitor for PKD, at 0.02 and 0.2 μM resulted in a significant dose-dependent decrease in alkaline phosphatase (ALP) activity during osteoblast differentiation of MC cells mediated by DMSO, BMP2 or PMA. Bone nodule formation was attenuated simultaneously. Semi-quantitative RT-PCR analyses demonstrated that Gö6976 treatment resulted in reduced Runx2, Osterix and ALP transcript expression in MC cells. However, a general PKC inhibitor Gö6983 that does not inhibit PKD showed little effect on osteoblast differentiation, suggesting that PKD is crucial in the process. To confirm the functional roles of PKD, we overexpressed wild-type (PKD-WT), constitutively active (PKD-CA) and dominant negative (PKD-DN) PKD in MC cells and examine the effect of PKD modulation on osteoblast differentiation. In the absence of osteogenic inducers, MC cells transfected with PKD-CA resulted in enhanced bone nodules formation as compared to the PKD-DN transfectants. These findings are in good concordance to our previous observations that PKD activation alone is sufficient for inducing osteoblast differentiation. Using both pharmacological and PKD overexpression studies, our findings suggested that PKD plays essential roles in osteoblast differentiation and that osteoblast differentiation can be modulated via the activation of PKD.
Disclosures: D.N. Y. Fan, None.
This study received funding from: AO Research Fund of the AO Foundation, Matching Grant and HKU foundation of the University of Hong Kong.
Tumor Necrosis Factor-α Stimulated Msx2 Expression in C2C12 Cells.
H. Lee*, H. Ryoo, K. Woo, G. Kim*, J. Baek. Department of Cell and Developmental Biology, Seoul National University School of Dentistry, Seoul, Republic of Korea.
Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is involved in local and systemic bone loss through both stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Although previous reports have shown that TNF-α inhibit osteoblast differentiation via enhanced Runx2 degradation, we could observe TNF-α-mediated inhibition of alkaline phosphatase (ALP) activity in Runx2-nulled calvarial cells. Msx2 has been shown to suppress ALP activity via counteracting the ability of Dlx5 to activate ALP promoter. Therefore we examined the effect of TNF-α on Msx2 expression in C2C12 cells. As previously reported, either Msx2 overexpression or TNF-α treatment suppressed BMP2-induced ALP activity in C2C12 cells. TNF-α enhanced the mRNA and protein level of Msx2. TNF-α-mediated Msx2 induction was not suppressed by cycloheximide treatment. BAY-11–7082, an NF-kB inhibitor, suppressed TNF-α-mediated Msx2 induction but SP600125, a JNK inhibitor, did not. BAY-11–7082 partially reversed TNF-α-mediated inhibition of ALP activity. These results suggest that stimulation of Msx2 expression may be involved in TNF-α-mediated inhibition of osteoblast differentiation, at least in part.
Disclosures: J. Baek, None.
See Sunday Plenary Number S021
A Novel R131G RUNX2 Mutation Causing Cleidocranial Dysplasia Disrupts Heterodimerization with Core Binding Factor-β.
H. Kim*1, M. Han*1, K. Lim*1, A. J. van Wiinen2, J. L. Stein2, J. B. Lian2, G. S. Stein2, J. Choi1. 1Biochemistry & Cell Biology, Kyungpook Natl. Univ. School of Medicine, Skeletal Diseases Genome Research Center, Daegu, Republic of Korea, 2Cell Biology, UMASS Medical School, Worcester, MA, USA.
Cleidocranial dysplasia (CCD) is caused by haploinsufficiency of the RUNX2 gene. In previous study (Kim HJ et al, J Cell Physiol 207:114–122, 06), we found a novel RI31G RUNX2 mutation from CCD patient. Here, we showed functional analysis of this RUNX2 RI3IG mutation with respect to molecular mechanism. RUNX2 R131G mutation is located in RUNT DNA binding domain and it also located within putative nuclear localization signal. Therefore, we determined mutant RUNX2 with regard to nuclear localization, DNA binding, heterodimerization with partner protein core binding factor-β (CBF-β) and transactivation function against downstream target gene osteocalcin promoters. In nuclear localization function, RUNX2 R131G mutation had normal nuclear localization function, which was confirmed by transient transfection of HA-tagged mutant RUNX2 into CHO and HeLa cells by in situ immunofluorescent staining. With regard to DNA binding activity, RUNX2 R131G mutant had normal DNA binding activity, which was shown by electrophoresis mobility shift assay. Third, heterodimerization function of mutant RUNX2 with CBF-β was disrupted in cotransfection assays. Finally, RUNX2 R131G mutant showed loss of transactivation function on osteocalcin promoter reporter assay while RUNX2 wild type showed high transactivation function. Collectively, R131G mutation of RUNX2 causes CCD phenotype through the loss of heterodimerization with the partner protein CBF-β without abnormalities of nuclear localization, and DNA binding.
Disclosures: J. Choi, None.
See Sunday Plenary Number S023
Estrogen Suppression of Adipogenesis Is Mediated by TGF-β.
K. B. Clifton, S. Khosla, M. J. Oursler. Endocrine Research Unit, Mayo Clinic, Rochester, MN, USA.
Osteoblasts and bone marrow adipocytes originate from a common mesenchymal precursor. Investigators have hypothesized that a shunting of the common precursor cell away from the osteoblast and towards the adipocyte may be partly responsible for bone loss with aging, and we have hypothesized that estrogen deficiency with age may be responsible for this shift. While estrogen and transforming growth factor-β (TGF-β) have been shown to enhance osteoblast and inhibit adipocyte differentiation, the exact mechanism(s) by which estrogen may regulate these processes is not yet clear. Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor super-family; it is known to be the master regulator of adipocyte differentiation. We tested the hypothesis that estrogen may play a role in regulating adipogenesis by suppressing expression of PPARγ, and that its effects are mediated, at least in part, by TGF-β. The bipotential mouse bone marrow stromal cell line, ST2, was used to address this hypothesis. In time course experiments, ST2 cells were treated with estrogen, TGF-β, or the corresponding vehicle from 1 hour up to 12 days. Expression of PPARγ was significantly reduced (p < 0.05) by TGF-β treated ST2 cells compared to the vehicle treatment at the 24 hour time point and each time point thereafter. Similarly, estrogen suppressed PPARγ expression compared to both the vehicle treated and untreated cells after 48 hours of treatment. ST2 cells were cultured in adipogenic medium for 6 days. Treatment with estrogen or TGF-β reduced PPARγ expression. Moreover, co-treatment with estrogen and a chemical TGF-β signaling inhibitor partially restored expression of PPARγ. To examine possible molecular mechanisms by which estrogen and TGF-β regulate PPARγ expression, cells were infected with dominant negative (dn) Smads 2 and 3, dn MEK, and dn PAK2 to block Smad-dependent and Smad-independent pathways. Both estrogen- and TGF-β-regulated repression of PPARγ were partially disrupted by blocking the Smad 3 and the Smad-independent PAK2 pathways. These results are consistent with the hypothesis that estrogen may play a role in regulating adipogenesis, and that its effects are mediated, at least in part, by TGF-β through both Smad dependent and Smad-independent signaling pathways.
Disclosures: K.B. Clifton, None.
This study received funding from: N1H.
See Sunday Plenary Number S025
Regulation of RUNX2 Translation by 5′- and 3′-UTR Binding Proteins.
N. Elango*, S. Sudhakar*, M. S. Katz. GRECC, VA Medical Center, San Antonio, TX, USA.
RUNX2, the major regulator of osteoblast differentiation, is expressed as two isoforms (RUNX2-I and RUNX2-II) encoded by two mRNAs differing only in their 5′-UTRs and initial coding regions. Recent studies show that RUNX2 gene expression is regulated at the translational level, and RUNX2-I is translated earlier during osteoblast differentiation than RUNX2-II. Since a 3961 nucleotide-long 3′-UTR is common to both mRNAs and the two 5′-UTRs are distinct, we hypothesize that the 3′-UTR contains a common translational regulatory motif and the two 5′-UTRs contain isoform-specific motifs. We have found by deletion analysis that the region between 2000 and 3000 nucleotides of the 3′-UTR contains a negative regulatory motif. Gel retardation assays reveal a 75 kDa protein, expressed in osteoblastic cells (UMR-106, ROS 17/2.8) but not precursors (ST2), binding to a pyrimidine-rich sequence within the 2000–3000 nucleotide region. Using RNA affinity chromatography we have purified six proteins from UMR-106 cells, including a 75 kDa protein, that bind to the pyrimidine-rich sequence. A high level of luciferase expression by RUNX2-I reporter plasmid but not RUNX2-I reporter mRNA in transfected osteoblasts (Elango et al, J Cell Biochem 99:1108, 2006) suggests that nuclear factor(s) bound to the 5′-UTR of RUNX2-I mRNA during transcription (but absent from in vitro transcribed reporter mRNA) are essential for translation. We have purified from UMR-106 cells by RNA affinity chromatography three proteins that bind to the RUNX2-I 5′-UTR. Translation of RUNX2-I but not RUNX2-II in early osteoblastic cells (UMR-106) expressing both RUNX2 mRNAs suggests that RUNX2-II translation is suppressed until later stages of differentiation. Gel retardation assays show that stem-loop structures of RUNX2-II mRNA 5′-UTR bind proteins expressed in early osteoblasts (UMR-106) but not precursors (ST2) or mature osteoblastic cells (ROS 17/2.8); these proteins may function as suppressors of RUNX2-II translation in early osteoblasts. Based on these findings, we postulate: 1) translation of both RUNX2 mRNAs is suppressed in precursors by 3′-UTR binding protein(s); 2) RUNX2-I is translated early in osteoblastogenesis upon expression of a 75 kDa pyrimidine-rich region binding protein and removal of suppression. RUNX2-I translation is also enhanced by RUNX2–1 5′-UTR binding proteins; 3) translation of RUNX2-II is suppressed during early osteoblast differentiation by the transient expression of proteins binding to the RUNX2-II 5′-UTR. Our studies provide a model by which 3′- and 5′-UTR binding proteins control sequential translation of the two RUNX2 isoforms and thereby regulate the course of osteoblast differentiation.
Disclosures: N. Elango, None.
This study received funding from: VA Merit Award.
See Sunday Plenary Number S027
Dlx5 Overexpression Promotes Early Odontoblast Maturation.
V. L. S. Ferrer*1, Y. Kawano2, H. T. Li1, H. Chin*1, L. Wang*3, A. C. Lichtler1. 1Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA, 2Division of Oral Anatomy, Niigata University, Niigata, Japan, 3Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Dlx5 is a member of the Dlx gene family of transcription factors, which have been shown to be important for the differentiation of osteoblasts and chondrocytes and the development of craniofacial structures including teeth. To further understand the role of Dlx5 in vivo, our laboratory generated transgenic mice overexpressing Dlx5 driven by the osteoblast- and odontoblast-directed 3.6 kb fragment of the type I Collagen promoter (3.6Col1a1). We have previously reported that 3.6Col1a1-driven Dlx5 overexpression results in accelerated mandibular enamel and dentin mineralization in 7-week-old mouse incisor. Enamel highlighting of micro-CT 3D reconstruction of the mandible showed initiation of enamel mineralization at the level of the 3rd molar in transgenics in contrast to the wildtype enamel, which begins at the level of the 1st molar. Light micrographs showed that transgenic odontoblasts exhibited advanced maturation, displaying the distinct lack of polarity seen in wild-type early, functional odontoblasts.
To further analyze the phenotype, we examined transgenic mice at earlier time points (1, 3, and 5 weeks) to determine the onset of the phenotype. Micro CT revealed no significant differences in enamel and dentin mineralization at these time points. However, light micrographs showed that the odontoblast phenotype is apparent as early as 1 week.
Further histological analysis of 7-week-old transgenic incisors revealed that as odontoblast differentiation progresses towards the incisal end, the odontoblasts become highly disorganized and eventually are indistinguishable from the pulp. The corresponding dentin lacks the parallel arrangement of the dentinal tubules. Predentin in the incisal area is diminished or absent, leading to jagged and thinner dentin and, ultimately, exposed pulp. Trichrome staining revealed ectopic collagen formation in the pulp adjacent to the dentin. Undecalcified sections of calcein and xylenol orange labeled incisors showed that the formations are ectopic mineralizations. The wavy pattern of labeling, which appears at the incisal tip in the wild-type incisor, begins at the middle third of the transgenic incisor. These observations suggest that 3.6Col1a1-driven Dlx5 overexpression promotes early odontoblast maturation. However, this early maturation results in the eventual loss of recognizable odontoblast morphology and irregularity and depletion of dentin matrix formation, leading to exposed pulp chamber.
Disclosures: V.L.S. Ferrer, None.
This study received funding from: NIH.
See Sunday Plenary Number S029
Positive and Negative Regulation of Osteoblast Differentiation by the SWI/SNF Chromatin Remodeling Complex.
S. Flowers*1, N. G. Nagl*1, H.Guo*1, G. R. Beck2, E. Moran1. 1Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA, 2Division of Endocrinology, Metabolism and Lipids, Emory University School of Medicine, Atlanta, GA, USA.
Osteoblast differentiation requires reprogramming of gene expression to activate tissue specific gene expression and to effect coordinated withdrawal from the cell cycle. Chromatin remodeling is an important part of this process, in which the SWI/SNF complex uses the energy of ATP hydrolysis to disrupt nucleosome architecture, thereby altering the accessibility of gene promoters to transcription factors. The SWI/SNF complex is essential for both positive and negative gene regulation events, which has complicated analysis of its molecular mechanisms of action. A manner by which the complex might distinguish functions is suggested by the occurrence of two subunits as pairs of related alternatives. To test this concept, we used siRNA technology to deplete MC3T3-E1 pre-osteoblast cells of each such subunit. The alternative pairs include the largest subunits of the complex, p270/ARIDIA and its close relative ARID1B, which are members of the ARID family of DNA binding proteins. The other subunit that occurs as a mutually exclusive pair is the ATPase itself, which can be either BRM, or the BRM-related protein BRG1.
This approach successfully distinguished complexes with distinct roles in osteoblast differentiation. The p270/ARID1A subunit was revealed as diagnostic of a complex with a repressor role during cell cycle withdrawal. In contrast, complexes containing the ARID1B subunit play an important positive role in cell cycle activity. The high level of c-myc expression seen in MC3T3-E1 pre-osteoblasts is dependent on the integrity of ARID1B-containing complexes, while differentiation-associated repression of c-myc depends on complexes containing the p270/ARID1A subunit. Chromatin immunoprecipitation (ChIP) assays indicate the c-myc promoter is a direct target of the SWI/SNF complexes, and that depletion of either ARID subunit impairs the association of other important regulators with the c-myc promoter. Likewise, depletion of the ATPase subunits reveals separation of key functions. Both ATPases contribute to cell cycle withdrawal, but they have very distinct functions in tissue specific gene expression and the onset of mineralization. The osteocalcin promoter is a direct target of SWI/SNF complexes, and the ATPases distinguish complexes with different roles in its regulation. These analyses give insight into the molecular mechanisms that underlie lineage determination, and the timing and coordination of the differentiation process.
Disclosures: S. Flowers, None.
See Sunday Plenary Number S031
Regulation of RUNX2 at the Nuclear Lamina.
P. M. Fonseca1, G. Zhou*1, Q. Zheng*1, H. Kingston*2, M. Tassabehji*2, B. Lee1. 1Molecular Human Genetics, Baylor College of Medicine, Houston, TX, USA, 2St. Mary's Hospital, Manchester, United Kingdom.
RUNX2 is a member of the RUNT family of transcription factors. It is essential for osteoblast formation and chondrocyte maturation. Loss of function mutations in RUNX2 result in the human skeletal disorder cleidocranial dysplasia (CCD). RIP (CBFA1/RUNX2-interacting Protein) was isolated in a screening of a human osteosarcoma cDNA library using a yeast two-hybrid approach. Two families with rearrangements involving chromosome 8q — where RIP maps — have been reported to have a CCD-like phenotype. In transient transfection experiments, RIP down-regulated the transactivation by RUNX2 in a dose-dependent manner in both 10T1/2 cells and ROS17/2 cells. RIP is mainly localized in the nucleus and it was identified as a component of the nuclear envelope in proteomic screens. The major components of the nuclear envelope are lamins, which are intermediate filaments with an important role in nuclear organization, cell division, apoptosis and transcriptional regulation. Interestingly, mutations in A-type lamins and lamin-associated proteins result in several laminopathies such as mandibuloacral dysplasia (MAD). MAD presents clinical features such as acroosteolysis, dental crowding, hypoplastic clavicles and delayed closure of the fontanels, partially phenocopying CCD. We hypothesize that the skeletal defects observed in these patients are due to the disruption of lamin interactions with transcriptional regulators relevant in bone formation such as RIP and RUNX2. GST pull-down experiments showed an interaction between LaminA/C and RIP, as well as the RUNT domain of RUNX2. The tail domain, which is mutated in MAD patients, is responsible for the interaction. Moreover, LaminA/C could repress RUNX2 transactivation of reporter genes in COS7 cells in a dosage-sensitive way. Mutant versions of lamin carrying MAD mutations seem to lose their repressive action. These mutants had altered binding properties. These data suggest a novel mechanism for transcription regulation of skeletogenesis involving sequestration of a RUNX2 complex at the nuclear lamina. In order to study the effects of A-type lamins on osteoblast differentiation, we are currently generating a C2C12 stable cell line overexpressing laminA/C and we will also knock-down the gene in pre-osteoblastic cells.
Disclosures: P.M. Fonseca, None.
This study received funding from: Fundacao Ciencia e Tecnologia, Portugal.
See Sunday Plenary Number S033
Metrnl: A New Secreted Protein Inhibit Differentiation of MG-63.
W. Y. Gong*1, Y. Liu*2, R. M. Zheng*1, G. X. Oiu*2, S. O. Lin1. 1Obstetric and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College.Chinese Academy of Medical Scienc, Beijing, China, 2Orthopaedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College.Chinese Academy of Medical Scienc, Beijing, China.
It has been reported that AP-1 transcription factor complex modulates the function of osteoblasts and osteoclasts which participate in bone formation and bone resorption respectively. So in our previous study, we constructed a high-throughput assay method based on the AP-1 signaling pathway dual-luciferase reporter system to screen new function gene expressing in fetal osteoblasts. We found one gene (GMSS555) without reporting function in PUBMED inhibited the activation of AP-1(<50% compare to empty vector). After analysis with basic local alignment tool (BLAST), this gene is noted “Meteorin, glial cell differentiation regulator-like(METRNL)”. Now in this experiment, we cloned this gene and studied whether it could influence the differentiation of MG63. The full-length cDNA of METRNL contains an open reading frame encoding 312 amino acids with a putative signal peptide of 45 aa. Northern blotting revealed that a 1.5kb mRNA was constitutively but lowly expressed in most normal adult tissues. The recombinant METRNL was isolated from BL21 codon plus and used to immunize rabbits to raise polyclonal antibodies. Western blot detected an about 34 kda protein in culture supernatant of CHO cells strablely transfected with ORF of METRNL. Immunohistochemistry analysis demonstrated that METRNL was mainly expressed by the undifferentiated osteoblasts and chondrocytes. To study the function of METRNL in osteoblasts, we established stable MG63 cells that express the METRNL tagged with EGFP at its COOH-terminus. After cultured in osteogenic supplement(containing b-Glycerophosphate/dexamethasone/ascorbic acid-2-phosphate) for 14 days, the METRNL transfected MG63 produces lower matrix than mock-transfected and parent MG63.Real time PCR assays showed METRNL inhibited the ALP and osteocalcin mRNA expression (p < 0.05).
Our study indicated that METRNL expressed mainly in undifferentiated cells and can inhibited transcription activity of AP-1, and reduced the differentiation of human osteoblastic MG63.
Disclosures: W.Y. Gong, None.
TIEG Suppresses Osteoblast Cell Proliferation Through Modulation of the TGFβ/Smad Signaling Pathway and Repression of E2F1 Gene Expression.
J. R. Hawse1, M. Subramaniam1, J. Pirngruber*2, N. M. Rajamannan3, M. J. Oursler1, S. A. Johnsen*2, T. C. Spelsberg1. 1Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, 2Department of Molecular Oncology, Gottingen Center for Molecular Biosciences, Gottingen, Germany, 3Department of Cardiology, Northwestern University Medical School, Chicago, IL, USA.
TGFβ Inducible Early Gene-1 (TIEG) is a member of the Kruppel-like family of transcription factors that was originally identified in osteoblasts. This transcription factor regulates the expression of many genes and plays a critical role in bone formation and maintenance. We have demonstrated that TIEG suppresses the expression of Smad7 following TGFβ treatment of osteoblasts and that this suppression does not occur in the absence of TIEG Further, transient transfection of osteoblasts with a TIEG expression construct results in suppression of Smad7 promoter activity. Through the use of yeast-2-hybrid and co-immunoprecipitation assays, we have shown that TIEG directly interacts with mSin3a, a well characterized co-repressor. In order to determine if TIEG recruits mSin3a to the Smad7 promoter, we performed time course ChIP assays. These studies revealed that TIEG and mSin3a are recruited to the Smad7 promoter within 60 minutes of TGFβ treatment, and that phosphorylated polymerase II is displaced from the Smad7 promoter within 30 minutes of treatment. Interestingly, the HDAC proteins do not appear to be involved in this process since they are not recruited to this promoter region and since TSA, an inhibitor of HDAC activity, does not relieve the repression of Smad7 by TIEG Since we have demonstrated a central role for TIEG in the TGFβ/Smad signaling pathway, and since this pathway is known to be an important regulator of cell proliferation, we sought to determine TIEG's role in the proliferation of osteoblast cells. Overexpression of TIEG in osteoblasts results in a significant decrease in cell proliferation by approximately 50%. Additionally, calvarial osteoblasts isolated from TIEG−/− mice proliferate more rapidly relative to osteoblasts isolated from wild-type littermates. To further characterize this proliferative response, we examined the role of TIEG in regulating the activity of the E2F1 promoter, a well known pro-proliferative gene. Transient transfection assays demonstrate that TIEG drastically decreases the activity of this promoter in osteoblasts. Taken together, these data demonstrate a central role for TIEG in suppressing the expression of Smad7 leading to enhancement of the Smad signaling pathway. TIEG is also shown to play a critical role in regulating osteoblast cell proliferation, and potentially osteoblast differentiation, possibly as a result of TGFβ/Smad signaling and E2F1 expression.
Disclosures: J.R. Hawse, None.
See Sunday Plenary Number S036
TFHA, ATF4, and Runx2 Synergistically Activate Osteoblast-specific Osteocalcin Gene Expression.
S. Yu1, Y. Jiang*2, M. Luo*1, Y. Lu1, J. Zhang1, G. D. Roodman1, G. Xiao1. 1Department of Medicine, University of Pittsburgh Medical Center(UPMC), Pittsburgh, PA, USA, 2Department of Pharmacology, University of Pittsburgh Medical Center(UPMC), Pittsburgh, PA, USA.
Runx2, a member of the runt homology domain family of transcription factors, is a master regulator of osteoblast function and bone formation. Mice lacking Runx2 have no mineralized skeleton due to a complete lack of mature osteoblasts. The expression level of Runx2 protein is regulated by a number of factors including BMPs, FGF-2, IGF-1, TNF-α, TGF-β, and PTH, all of which play important roles in osteoblasts and bone both in vitro and in vivo. In addition, the activity of Runx2 protein is positively or negatively modulated through protein-protein interactions. Activating transcription factor 4 (ATF4) is an osteoblast-enriched factor which regulates the terminal differentiation and function of osteoblasts. ATF4 knock-out mice have reduced bone mass and bone mineral density (severe osteoporosis) throughout their life. To identify proteins interacting with Runx2, we used a yeast two-hybrid system and identified TFIIA, a general transcriptional factor, as a Runx2-interacting factor. While pull-down assays confirmed that TFIIA physically interacted with Runx2 when both factors were coexpressed in COS-7 cells, surprisingly, it did not activate or inhibit Runx2-dependent transcriptional activity. In contrast, TFIIA unexpectedly activated ATF4, which we recently identified as a Runx2-interacting protein, in a dose-dependent manner. Deletion analysis found that this activation required the presence of the C-terminal 15 amino acid residues of ATF4 molecule. Finally, TFIIA, ATF4, and Runx2 synergistically stimulated the 0.657-kb mOG2 (mouse osteocalcin gene 2) promoter activity and endogenous osteocalcin mRNA expression. In summary, this study demonstrates a novel mechanism through which bone-specific transcription factors and general transcription factors cooperate in regulating osteoblast-specific gene expression.
Disclosures: S. Yu, None.
SiRNA Specific Knockdown of Lamin A/C Inhibits Osteogenic Differentiation of Human Mesenchymal Stem Cells.
R. Akter1, D. Rivas1, Y. Dong2, H. Drissi2, G. Duque3. 1Lady Davis Institute for Medical Research, Montreal, PQ, Canada, 2Orthopaedics, University of Rochester, Rochester, NY, USA, 3Medicine/Geriatrics, McGill University, Montreal, PQ, Canada.
Recent studies have indicated the role of lamin A/C, a component of nuclear lamina, in premature ageing with severe bone loss. Mutations in the gene encoding lamin A/C have shown a deficit in the number of both osteoblasts and osteocytes. We have previously shown that lamin A/C expression levels are reduced in the aging osteoblasts in vivo. However, no study has evaluated the direct role of lamin A/C in osteogenic differentiation of mesenchymal stem cells (MSC). In this study, we hypothesize that reduced expression of lamin A/C has a negative impact on osteogenic differentiation of human MSC. We inhibited lamin A/C using different doses of lamin A/C siRNA (200–800 nM) in MSC committed to differentiate into osteoblasts. Cells treated with vehicle but without the siRNA/oligo were used as a control. The level of siRNA was determined by western blot and RT-PCR. We found that up to 400nM did not have any inhibitory effect, but 600 and 800 nM successfully inhibited lamin A/C without affecting cell survival. After 10 days of lamin A/C knockdown, cells were stained with alizarin red (mineralization) and alkaline phosphatase. Additionally, expression of osteocalcin, cbfa1 and lamin A/C were determined by RT-PCR and western blot. Finally, nuclear blebbing, a typical finding of lamin A/C inhibition, was quantified by propidium iodine staining. We found that lamin A/ C knockdown induces higher level of nuclear blebbing and significantly reduces alizarin red staining (p < 0.01) and alkaline phosphatase activity (p < 0.01) in osteogenic differentiating MSC. Additionally, expression of cbfal and osteocalcin was reduced in lamin A/C knockdown cells. Overall, to identify the potential mechanism, gel-shift assay analyses were pursued. We found that lamin A/C knockdown affects the DNA/protein interaction of the binding sites in the cbfal promoter. In conclusion, our results suggest that lamin A/C is a new factor required for the osteogenic differentiation of MSC and that lamin A/C directly interacts with the cbfal transcription pathway.
Disclosures: R. Akter, None.
This study received funding from: Canadian Institutes for Health Research.
See Sunday Plenary Number S039
Osteoformin Enhances Gap Junction Communication of Human Preosteoblastic Cells in Culture.
L. X. Bi, E. Mainous, Y. Zeng*, W. J. Buford*. Dpts. of Surgery & Orthopaedics, UTMB, Galveston, TX, USA.
Gap junctions provide a cell-to-cell low resistance conduction pathway for coordination of tissue function. Composed of nonselective channels they allow passage of ions, nucleotides, and other small molecules. Our previous study has shown that marrow sac cells (preosteoblasts) which separate the bone from marrow display intercellular gap junctions (Bi LX JBMR 17: SA219 Suppl. 1, 2002). To investigate activities of gap junction affected by osteoformin (polyaspartate), we examined the levels of fluorescence recovery after photobleaching (FRAP) using a laser scanning confocal microscope. Human preosteoblast cells were cultured in a-minimum essential medium [a-MEM] and 10% fetal bovine serum with or without osteoformin (5ug/ml) for 3 days. The cells were loaded with 5,6-carboxyfluorescein diacetate, acetoxymethyl ester (5,6-CFDA/AM, 5 μg/ml) for FRAP at 37°C for 30 minutes and then de-esterified by washing with phosphate buffered saline before photobleaching. The FRAP was examined using a laser scanning confocal microscope with a 488-nm wavelength light from an argon laser (LSM microscope) and recorded every 5 seconds in 60 seconds after photobleaching. Osteoformin enhanced levels of intercellular gap junction communication by 68–72% (P < 0.01) compared to that of the control group. To confirm that the fluorescein crosses the gap junction channel, a gap junctional blocker (carbenoxolone, 150 ug ml) was used. Carbenoxolone blocked the effect of osteoformin on human preosteoblasts. We conclude that osteoformin significantly enhances intercellular gap junction communication which is associated with exchange of regulatory ions and osteoblast differentiation in vitro. It might be an important regulator of bone formation by accelerating fracture and bone defect healing, and controlling bone diseases.
Disclosures: L.X. Bi, None.
See Sunday Plenary Number S041
Osteogenic Differentiation of Circulating Endothelial Progenitor Cells.
T. Bick*1, N. Rozen*2, E. Dreyfuss*1, M. Soudry*1, P. Lewinson1. 1Research Institute for Bone Repair, Orthopaedic Surgery A, Rambam-Health Care Campus, Haifa, Israel, 2Research Institute for Bone Repair, Rambam-Health Care Campus, Haifa, Israel.
The dramatic potential of circulating endothelial progenitor cells (EPC) to heal a large bone defect (critical-sized gap in sheep tibiae, see abstract: Healing of Critical-sized Bone Defects by Endothelial Progenitor Cells. By D. Lewinson et al) led us to investigate whether EPC might be induced to transform into osteogenic cells. Peripheral mononuclear fraction of circulating sheep and human mononuclear cells were isolated using Lymphoprep™ (Axis-Shield, Oslo, Norway), seeded on fibronectin-coated plates (Sigma-Aldrich, MS, USA), and cultured in EBM-2 media supplemented with EGM-2MV SingleQuote (Clonetics, Cambrex Bio Science, MD, USA). The endothelial nature of adherent colonies was identified by incorporation of Dil-acetylated LDL (Molecular Probes, Oregon, USA), tube formation on Matrigel (BD Labware, MA, USA) and immunostaining for von Willebrand factor (Dako, Glostrup, Denmark). EPC were expanded and cultured in plastic dishes or seeded into polyethylene glycol/fibrinogen hydrogel scaffolds. Cultures were incubated either in DMEM/F12 (1:1) containing 10% FCS or in an osteogenic differentiation media — DMEM/F 12(1:1) containing 10% FCS, Dexamethasone 10−7 m/L, ascorbic acid 5 × 10−5 m/L and β-glycerophosphate 10−2 m/L. Changes in cell morphology, nodule formation, von Kossa (vK) and alizarin red (AR) staining as well as osteocalcin (OC) immunohistochemistry served to identify osteoblastic differentiation. Nodular aggregates formation was evident in osteogenic media-incubated sheep EPC already by 1 week. These nodular aggregates stained positively by vK, AR and OC. However, human EPC, when cultured in the same conditions showed very intensive staining of equal intensity over all the confluent culture by AR with many more intensively-stained small noduli. vK staining showed many granular deposits dispersed all over the culture plate. In contrast to sheep EPC, not only the nodular aggregates but most of human EPC stained by both vK and AR. Control cultures of both human and sheep EPC showed minimal background staining. Phase contrast microscopy of sheep EPC-seeded scaffolds demonstrated that the cells were distributed homogenously throughout the scaffold and formed tube-like structures by 1 week in both media. However the cell-seeded scaffolds cultured in osteogenic media showed prominent nodular aggregates. vK and AR staining were not informative because the scaffold itself retained the stain. Our results support a possible mechanism that explains the potential of blood derived EPC in tissue engineering for bone regeneration.
Disclosures: T. Bick, None.
See Sunday Plenary Number S043
Differential Effects of Secreted Frizzled-related Proteins (sFRPs) and Wnt Inhibitory Factor (WIF)-1 on Osteoblastic Differentiation of Mouse Pluripotent Mesenchymal Cell Line.
S. W. Cho, S. J. Her*, H. J. Sun*, O.K. Choi*, J. Y. Yang*, H. Y. Cho*, S. W. Kim*, C. S. Shin*. Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
Secreted Frizzled-related proteins (SFRPs) are modulators of Wnt signaling which plays a role in development, apoptosis and tumorigenesis. sFRPs bind directly to the wnts proteins and appear to antagonize the effects of wnts by sequestering them from their receptor. Although overexpression of sFRP-1 in human osteoblasts has been shown to antagonize Wnt-signaling and accelerates apoptosis, a recent report demonstrated that sFRP-3 is a potent stimulator of osteoblast differentiation. This study was undertaken for definitive assessment of contribution of different sFRPs and WIF-1 in osteoblast differentiation and function. Treatment of C3H10TI/2 cells with sFRPl, −2, −3, and −4 in the presence of Wnt-3A conditioned medium did not show significant difference in alkaline phosphatase (ALP) activity compared to vehicle treated cells. However, WIF-1 strongly inhibited the ALP activity in a dose-dependent manner [5 nM, 10 nM, 50 nM, 100 nM; 0.141 ± 0.002, 0.105 ± 0.026, 0.041 ± 0.003, 0.022 ± 0.003 mU/mg protein, p < 0.05]. To further investigate the role of cell-associated sFRPs, we have transduced C3H10T1/2 cells with retroviral vector for sFRPs and WIF-1. Consistent with the previous data, sFRP-1, −2, −3, and −4 did not result in significant difference in ALP activity compared to empty virus transduced cells during osteoblastic differentiation using b-glycerophosphate and ascorbic acid. In contrast, overexpression of WIF-1 significantly inhibited the ALP activity compared to control cells at day 14 of culture [10.078 ± 1.835 vs 18.368 ± 3.848 mU/mg protein, p < 0.05]. BrdU uptake assay and caspase-3 assay showed that treatment of sFRPs and WIF-1 did not affect cell proliferation or apoptosis. In conclusion, we were not able to demonstrate that sFRP1–4 inhibit osteoblastic differentiation of mesenchymal cells. On the other hand, WIF-1 has consistently exhibited negative effects on osteoblastic differentiations of mouse pluripotent mesenchymal cells.
Disclosures: S. W. Cho, None.
See Sunday Plenary Number S045
Mesenchymal Stem Cell Concentration and Bone Repair: Pitfalls from Bench to Bedside.
A. V. Cuomo*1, M. Virk2, F. Petrigliano*1, E. Morgan*3, C. Kang*4, J. R. Lieberman2. 1UCLA, Los Angeles, CA, USA, 2University of Connecticut, Hartford, CT, USA, 3Orthopaedic Surgery, Boston University, Boston, MA, USA, 4Orthopaedic Surgery, UCLA, Los Angeles, CA, USA.
PURPOSE: This is a preclinical trial comparing the osteogenic potential of mesenchymal stem cell enriched bone marrow aspirate (enrBMA) versus unprocessed BMA mixed with demineralized bone matrix (DBM) in a critical-sized athymic rat femoral defect model.
METHODS: The buffy coat was extracted from 8 human BMA donors to obtain the enrBMA. The MSCs concentration was estimated by counting fibroblast colony forming units (f-CFU). Lyophilized DBM allograft (Osteofil-RT™) was mixed with saline, BMA, enrBMA, or recombinant human bone morphogenetic protcin-2 (rhBMP-2) (10 ug) and was used to treat a 6mm critical-sized femoral defect in athymic rats. A total of 59 rats were divided into 6 groups (Table 1). Radiographs at 4, 6, 8, and 12 weeks were graded for bone healing by 3 independent observers. All animals were sacrificed at 12 weeks for micro CT and histomorphometry.
RESULTS: The BMA and enrBMA from all 8 donors averaged 1072 ± 991 f-CFUs/mL and 6503 ± 6953 f-CFU/mL, respectively. All 10 defects healed in the positive control groups (II, VI) with rhBMP-2 (Table 1). One defect healed in each experimental group (III-V). The radiographic scores did not differ between time-points (data not shown) and the bone volume (BV) and mineral density (MD) did not differ between groups IV and V. The wide radiographic variability of defect healing in Groups IV and V did not correlate with the concentration of f-CFU's, the lot number of the DBM, or the age or sex of the BMA donor. Histomorphometry showed more dense trabecular and cortical bone in groups III-V than in group II.
CONCLUSION: Although enrBMA may demonstrate superior bone healing in certain clinical situations, this pre-clinical study cannot support the use of enrBMA when mixed with DBM to reliably heal critical-sized bone defects. The results of this study introduces one or more possibilities: (1) a higher number of MSC may be required for healing critical-sized defects; (2) an enhanced osteoinductive signal is required in the setting of critical-sized defects that cannot be overcome by enrBMA and DBM alone, and (3) the carrier used did not provide the optimal environment to promote cell growth and signaling. Importantly, this study identifies potential pitfalls when adapting the use of MSC to treat humans in a clinical setting.
Disclosures: A. V. Cuomo, Medtronics Sofamor Danek 2.
This study received funding from: Medtronic Sofamor Danek.
See Sunday Plenary Number S047
The Effect of Caloric Restriction on Bone of Aging Rats Is Exerted Through the Stimulation of Sirtuins.
G. Duque1, P. Rivas2, F. Picard*3, S. Miard*3, M. Lafontaine-Lacasse*3, G. Ferland*4, P. Gaudreau*4. 1Medicine/Geriatrics, McGill University, Montreal, PQ, Canada, 2Lady Davis Institute for Medical Research, Montreal, PQ, Canada, 3University of Laval, Quebec, PQ, Canada, 4University of Montreal, Montreal, PQ, Canada.
Caloric restriction (CR) in combination with soy protein is reported to inhibit adipose genes in peripheral fat and to increase bone mass in mice and rats. The mechanisms that explain these effects remain unknown. After the recent finding that Sirt1, a member of the Sirtuins family, acts as a repressor of PPARγ2 in peripheral fat, we hypothesize that CR combined with soy protein diet reduce adipogenesis in bone through the upregulation of Sirt1 and its co-factors. In this study, male Sprague Dawley rats (8 month-old) were fed for 12 months with either casein or soy protein and exposed to a CR of 40% in comparison to the rats fed to satiety. A control group was fed ad libitium. Changes in body mass index (BMI), weight, length and serum leptin were quantified. At 20 months of age, animals (n = 20 per group) were sacrificed and tibiae were obtained for immunohistochemistry, immunofluorescence and in-situ hybridization for Sirt1 and its co-factor AEBP1. Additionally, bone marrow expression of PPARγ2 and CEBPα was also quantified. Our results showed that CR induced a significant reduction in weight, body mass index and serum leptin levels in both casein and soy protein fed groups as compared with the ad libitium groups (p < 0.001). We found that in the CR rats fed with soy protein there is a significantly higher expression of both Sirtl and AEBP1 detected by both in-situ hybridization and immunohistochemistry as compared with casein and ad libitum fed rats (p < 0.01). Additionally, levels of both PPARγ2 and CEBPα expression were significantly reduced in the soy protein group (p < 0.01). Our results indicate that the combination of CR and soy protein has an inhibitory effect on bone marrow adipogenesis through the stimulation of Sirtuins (Sirtl and co-factor AEBP1) thus suppressing the expression of PPARγ2. These findings provide the basis for further investigation on the role of Sirtuins in age-related bone marrow adipogenesis.
Disclosures: G. Duque. None.
This study received funding from: Canadian Institutes for Health Research.
See Sunday Plenary Number S049
Bespoke Bioceramic Scaffolds Support Mesenchymal Stem Cell Growth and Osteogenic Differentiation.
J. Dyson*1, P. Genever*2, K. Dalgarno*3, D. Wood*1. 1Leeds Dental Institute, University of Leeds, Leeds, United Kingdom, 2Department of Biology, University of York, York, United Kingdom, 3School of Mechanical and Systems Engineering, University of Newcastle, Newcastle, United Kingdom.
There is a clinical need for new bone replacement materials which combine long implant life with complete integration and appropriate mechanical properties. Apatite-wollastonite (A-W) is a bioactive glass-ceramic that shows excellent biocompatibility in vivo and similar mechanical properties to bone. Selective laser sintering (SLS) is a rapid prototyping technology used to produce intricate porous three-dimensional scaffolds to match the exact geometry of a patient's graft site, and can be applied to A-W powder. Mesenchymal stem cells (MSCs) can be induced to undergo osteogenic differentiation to mature bone-forming osteoblasts making MSCs attractive candidates for orthopaedic tissue engineering applications. We have used human MSCs to populate porous A-W scaffolds produced by SLS to create bespoke bone replacements.
MSCs were cultured on A-W scaffolds for up to 21 days. Confocal and scanning electron microscopy (SEM) were used to determine optimal seeding densities, to demonstrate that MSCs adhered and retained viability on the surface and penetrated into the pores of A-W scaffolds over 21 days culture. We identified a significant increase (p < 0.01) in the number of MSCs growing on the scaffolds over 7 days. Using BrdU incorporation we demonstrated that 28.4% ± 2.6 of MSCs proliferated over a period of 24 hours. Using realtime PCR we analyzed the expression of osteogenic markers by MSCs cultured in the absence of osteogenic supplements on A-W scaffolds compared to tissue culture plastic. We identified up to a 3-fold increase in cbfa1, 4-fold increase in alkaline phosphatase, 8-fold increase in type-I collagen, 170-fold increase in osteopontin, 1-fold increase in osteocalcin and a 14-fold increase in osteonectin expression on A-W compared to plastic at different time points over 21 days (p < 0.05). At all time points the expression of osteogenic markers was equivalent to or significantly greater on A-W than plastic. We also identified significantly higher (p < 0.01) alkaline phosphatase activity at days 7 and 14 compared to a calcium phosphate scaffold. Bespoke A-W scaffolds were engineered with a central channel to increase cell penetration in dynamic culture. Confocal microscopy and SEM were used to demonstrate that MSCs adhered to the scaffolds and retained viability for 21 days culture and cell in-growth was facilitated. These results indicate for the first time the biocompatibility and osteo-supportive capacity of SLS-generated A-W scaffolds and their potential as a bone replacement material.
Disclosures: J. Dyson. None.
This study received funding from: White Rose.
Enhanced Mesenchymal Stem Cell Properties in Dynamic Three-Dimensional Cultures.
J. E. Frith*1, B. Thompson*2, P. G. Genever*1. 1Department of Biology, University of York, York, United Kingdom, 2Smith and Nephew Research Centre, York, United Kingdom.
Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to self renew and differentiate along the osteogenic, adipogenic and chondrogenic lineages. This makes them good candidates for a range of therapeutic applications. MSCs are routinely cultured as monolayers attached to tissue culture plastic but there are indications that they lose their capacity for proliferation and differentiation after prolonged culture periods. We have grown MSCs as three-dimensional (3D) spheroids under dynamic conditions in both spinner flasks and a NASA rotating wall vessel (RWV). We hypothesised that increased cell-cell interactions, potential for communication and conditions of shear-stress and mass transfer may more closely mimic the in vivo environment to improve retention of stem cell properties.
We have shown that human MSCs spontaneously aggregate when prevented from adhering to culture plastic. These aggregates compact over a period of seven days into dense spheroids with decreased cell size and altered cell surface antigen expression compared to parallel monolayer MSCs, as shown by SEM and flow cytometric analysis. Live/dead staining confirmed that the cells remained viable in both spinner flasks and RWV culture after 7 days of dynamic culture. MSCs were cultured as monolayers and as spheroids in spinner flasks and the RWV in osteogenic medium for a period of 21 days. Staining showed the presence of alkaline phosphatase in all cultures from 7 days onwards with von Kossa-positive discrete staining for mineralised deposits in both dynamic 3D cultures, but not monolayers, at 14 and 21 days. RNA was extracted at 7, 14 and 21 days for analysis of osteogenic marker expression by qRT-PCR. As compared to monolayers, expression of Cbfal and osteocalcin was increased in 3D culture across all time points and expression of osteonectin and osteopontin was increased after 21 days. Increases in alkaline phosphatase and type-I collagen were also present in RWV cultures after 14 and 21 days. A comparison of adipogenic differentiation showed lipid droplet accumulation, by Oil red O staining, in dynamic 3D cultures after just seven days with accumulation in monolayers not occurring until 21 days. qRT-PCR analysis of adipogenic marker expression showed increased expression of C/EBP alpha and FABP4 in spinner flasks and of PPAR gamma, LPL, C/EBP alpha and FABP4 in RWV cultures. Together these results show that dynamic 3D methods are viable for MSC culture and that both osteogenic and adipogenic differentiation potential are enhanced as compared to standard monolayer cultures.
Disclosures: J.E. Frith, None.
This study received funding from: BBSRC and Smith and Nephew.
See Sunday Plenary Number S052
Increasing Osteoblast Number Is Not Sufficient to Enhance the Function of the Hematopoietic Stem Cell Niche.
S. Lymperi, A. Cope*, F. Dazzi*, N. Horwood. Kennedy Institute of Rheumatology, London, United Kingdom.
The regulation of haemopoietic stem cell (HSC) fate requires a specialized microenvironment in the bone marrow (BM) cavity called the HSC niche. Accumulating evidence indicates that osteoblasts (OB) are a key component of this niche, regulating HSC number and differentiation state. In this study the role of OB in the niche was further investigated by manipulating their activity with strontium (Sr). Since Sr increases osteoblast activity whilst inhibiting osteoclast differentiation and function, we hypothesized that Sr would improve the HSC supporting capacity of the niche. Sr, like PTH, promoted bone nodule formation (alizarin red S and Von Kossa staining) and increased production of runx2 mRNA by RT-PCR in primary OB cultures. Administration of Sr to mice resulted in elevated levels of serum osteocalcin as measured by ELISA. Sr-treated mice showed increased bone volume and trabecular thickness defined by micro-CT analysis. In terms of haemopoiesis, Sr-treated mice exhibited increased numbers of haemopoietic progenitor cells compared to untreated control mice as assessed by colony forming unit-cells assay. However, no difference in primitive HSC numbers was detected between the two groups as evaluated by long term culture-initiating cells assay and FACS analysis for lin-, sca1+, c-kit+ cells. When Sr-treated mice were used as donors in HSC transplantation experiments, no difference in HSC engraftment ability was observed. However, when treated mice were used as recipients a delayed recovery was observed. Therefore despite an augmenting effect on OB function, the administration of Sr had no influence on primitive HSC, although the number of haemopoietic progenitors was higher than untreated controls.
It was recently shown that osteoclasts are important in stress-induced mobilization of haemopoietic stem and progenitors cells. The impaired engraftment of HSC observed in Sr-treated recipients, together with the fact that strontium inhibits osteoclastic resorption, implies that BM space could be intimately linked to the effect of bone remodeling whereby factors released from the bone matrix or produced by the osteoclasts themselves are required to provide signals that maintain the stem cell niche. In conclusion, we have shown that, although osteoblasts are indeed key functional component of the niche, an increase in their activity is not enough to expand the stem cell pool size. Therefore, we propose that active osteoclasts and balanced bone remodeling are required for the generation of the optimal HSC niche.
Disclosures: S. Lymperi, None.
This study received funding from: Leukemia Research Fund.
Alteration of Endothelin-1 Effects on Calvarial Osteoblastic Cells from Connexin-43-deficient Mice.
G. Geneau*1, C. Niger*2, N. Defamie*1, S. Rodriguez*1, M. Mesnil*1, L. Cronier*1. 1UMR 6187, Institute of Physiology and Cellular Biology, CNRS, University of Poitiers, Poitiers, France, 2Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA.
Gap junctional intercellular communication (GJIC) is known to permit coordinated cellular activities during development and differentiation processes. Accordingly GJIC dysfunction or mutations of gap junction protein (connexin, Cx) genes have been implicated in human pathologies. In bone, in vitro and in vivo studies have demonstrated the implication of Cx43 expression and GJIC in osteoblastic differentiation and in the mineralization process (i.e. Schiller et al., 2001; Chung et al., 2006). Cumulated data support the fact that Cx43 expression is very important for normal bone formation and regulation of cellular responses to hormones/growth factors stimulation and to mechanical load. In this context, since Endothelin-1 (ET1) has been also implicated in the control of osteoblast proliferation/differentiation and in some bone pathologies like prostatic metastasis, the possible cross talk between Cx43 and ET1 was investigated in cultured calvarial osteoblastic cells (OB) isolated from Cx43+/− and Cx43+/+ mice.
Interestingly, microcomputed tomographic analysis of 3days-old Cx43+/− mice confirmed the hypomineralization observed in Cx43−/− embryos by Civitelli's group. However, this delayed ossification was restricted to calvaria and in vitro characterization demonstrated that Cx43 protein expression, GJIC, alkaline phosphatase (ALP) activity, osteocalcin mRNA level and mineral deposits were significantly reduced in Cx43+/− OB compared to Cx43+/+ OB. Pharmacological approaches using specific antagonists and agonist and quantitative RT-PCR using specific primers for ET1 receptor subtypes (ETA and ETB) revealed that the partial deletion of Cx43 gene was not related to a decrease or a modification in the expression of both receptor subtypes in OB. ET1 (10−8M) induced an inhibitory effect on OB differentiation in both genotypes whereas this peptide has a mitogenic effect only in Cx43+/+ OB. Moreover, the ET1 inhibitory effect on cell differentiation was correlated to a reduced Cx43 expression (-22%) and GJIC (-46%) in Cx43+/+ OB. Surprisingly, this correlation was not demonstrated in Cx43+/− OB, suggesting an alternative regulatory pathway. The link between ET1 effect and GJIC was confirmed in the presence of a well-known uncoupler (18-AGA; 30 μM) in the culture medium of Cx43+/+ OB cells. In conclusion, these data strongly suggest that the level of Cx43 expression influences the osteoblastic response to ET1.
Disclosures: G. Geneau, None.
This study received funding from: Ligue Régionale contre le cancer.
Modulation of P2X7 Receptors in Human Osteoblasts by Oestrogen and Corticosteroids.
M. Al-qallaf*1, E. J. Kidd*1, B. A. J. Evans2. 1Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom, 2Child Health, Cardiff University, Cardiff, United Kingdom.
The P2X, receptor (P2X,R) is usually identified by the greater affinity of dibenzoyl ATP (DBzATP) compared to ATP. Prolonged activation of the P2X7R results in the formation of a non-selective pore in the cell membrane permeable to molecules as large as 900 Da. Oestrogen has been shown to inhibit osteoblast apoptosis, and hence increase their activity and lifespan. Additionally, oestrogen has been shown to inhibit osteoblast proliferation, an action believed to be through the oestrogen receptor-α. Interestingly, oestradiol has been found to exert non-genomic inhibition of the human P2X7R in transfected CV-1 monkey kidney cells. The aims of this project were to investigate the modulation of P2X7R expression and function by oestrogen and dexamethasone in human osteoblasts.
We have studied the effects of pre-incubating (30 minutes or 2 days) human osteoblasts with oestradiol or dexamethasone on P2X7R expression and function using a cell line representing a late stage of osteoblast differentiation (SaOS2). To investigate the effect of oestradiol and dexamethasone on P2X7R expression. Western blotting was performed. P2X7R function was studied by measuring pore formation upon activation with ATP using the YO-PRO1 (a dye that fluoresces on binding to nucleic acids) uptake method.
Dexamethasone (5 μM) and oestradiol (10 μM) were found to have no effect on P2X7R protein expression. Concentration-effect curves (CEC) for ATP using the YO-PRO 1 assay gave EC50 values of 0.36 mM ± 0.12. Oestradiol and dexamethasone were found to have no effect on the ATP EC50 values, but they decreased the maximum YO-PRO 1 uptake induced by the agonist. Oestradiol significantly decreased maximum YO-PRO 1 uptake after pre-incubation for 30 minutes and 2 days by 22 and 38%, respectively. Dexamethasone significantly decreased maximum YO-PRO 1 uptake after 30 minutes incubation by 19%, but had no effect following a 2 day pre-incubation.
The results show that the P2X7R expression at the protein level is not modulated by oestradiol or dexamethasone. However, oestradiol was found to have an inhibitory action on P2X7R function after 30 minutes and 2 days pre-incubation. A non-classical, non-genomic mechanism must be responsible for the action of dexamethasone and oestradiol after 30 minutes incubation. The effect of the 2 day incubation with oestradiol could be genomic or non-genomic. These effects of oestradiol and dexamethasone on the P2X7R might be important mechanisms for their modulation of osteoblast function. Further experiments are required to elucidate the function of the P2X7R in these cells.
Disclosures: M. Al-qallaf, None.
See Sunday Plenary Number S056
Silencing Dkk1 Expression Rescues Dexamethasone-induced Bone Loss In Vitro.
J. S. Butler1, C. J. Hurson*1, R. T. Moon*2, J. M. O'Byrne*3, P. P. Doran*1. 1UCD School of Medicine & Medical Science, Mater Misericordiae University Hospital, Dublin, Ireland, 2Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, WA, USA, 3Dept of Trauma & Orthopaedic Surgery, Royal College of Surgeons in Ireland, Dublin, Ireland.
Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive micro-architectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. The Wnt/β-catenin pathway is a major signaling cascade in bone biology, playing a key role in regulating bone development and remodeling, with aberrations in signalling resulting in disturbances in bone mass.
Our objectives were to assess the gene expression profile of primary human osteoblasts (HOBs) exposed to dexamethasone with a view to identifying key genes driving bone mass regulation and to assess the effects of the Wnt antagonist Dickkopf-1 (Dkk1) on the bone profile of primary human osteoblasts exposed in vitro to dexamethasone.
HOBs were cultured in vitro and exposed to 10–8M dexamethasone over a time course of 4hr, 12hr and 24hr. RNA isolation, cDNA synthesis, in vitro transcription and microarray analysis were performed. Microarray data was validated by quantitative real time RT-PCR. Dkk1 expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Dex-treated HOBs were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition, osteocalcin expression, along with cell proliferation and cellular apoptosis.
Global changes in HOB gene expression were elicited by dexamethasone. Development associated gene pathways were co-ordinately dysregulated with the expression profile of key genes of the Wnt Pathway significantly altered. Dkk1 expression in HOBs was increased in response to dexamethasone exposure with an associated reduction in alkaline phosphatase activity, calcium deposition and osteocalcin expression. Silencing of Dkk1 expression, as confirmed by quantitative RT-PCR, was associated with an increase in alkaline phosphatase activity and calcium deposition, along with increased cell proliferation and reduced cellular apoptosis.
Dkk1 is an antagonist of Wnt/β-catenin signalling and plays a key role in regulating bone development and remodeling. Silencing the expression of Dkk1 in primary human osteoblasts has been shown to rescue the effects of dexamethasone-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass.
Disclosures: J.S. Butler, None.
See Sunday Plenary Number S058
Glucocoiticoid Suppresses the Differentiation of Osteoblasts by Enhancing the Expressions of BMP Antagonists, Follistatin and Dan, and Pretreatments with Alendronate and PTH Abolish this Process.
K. Havashi*, T. Yamaguchi, S. Yano, M. Yamauchi, M. Yamamoto, T. Sugimoto. Internal Medicine 1, Shimane University Faculty of Medicine, Izumo, Japan.
Glucocorticoid-induced osteoporosis (GIO) is known to be caused by the suppression of osteoblast-mediated osteogenesis, and to be effectively treated by bisphosphonate and parathyroid hormone (PTH). However, the exact mechanisms by which glucocorticoid(GC) suppresses osteoblast functions, or alendronate and PTH cure GIO are still unclear. The impaired BMP-Runx2 signal is known to be one of candidates for the suppression of osteogenesis by GC. The present study was performed by using osteoblastic MC3T3-E1 cells in order to clarity the mechanisms by which GC modulated this signal pathway and whether or not bisphosphonate as well as PTH antagonized GC-induced effect on this process. Dex (10−7M) significantly inhibited the proliferation of the cells through Day 7 by cell counting and BrdU incorporation. Dex (10−9 — 10−7 M) also strongly and dose-dependently suppressed the mineralization of the cells at Days 21 and 28 by Alizarin red stain and von Kossa stain. Realtime PCR revealed that Dex (10−7 M) reduced the mRNA expressions of type I collagen, osteocalcin, and Runx2. In contrast, mRNA expressions of BMP antagonists, Follistatin and Dan, were increased by Dex (10−7 M). Western blotting also revealed that protein expressions of Dan and follistatin were increased by Dex (10−7 M). The addition of a neutralizing antibody for Dan partially reversed the Dex-induced suppression of mineralization of the cells. Moreover, pretreatments of the cells with alendronate (10−8 M) or human PTH-(1–34) (10−8 M) also reversed the Dex-induced enhancement in mRNA expression of follistatin and Dan. The present findings suggest that Dex may inhibit the differentiation of osteoblasts by suppressing the BMP signal pathway through enhanced expressions of its antagonists, follistatin and Dan, and that the effectiveness of bisphosphonate and PTH in treating GIO may be partly explained by the abolishment of this process.
Disclosures: K. Hayashi, Merk & Co., Inc. 2.
This study received funding from: Merck & Co., Inc.
See Sunday Plenary Number S060
ChIP-on-chip Analysis Reveals Novel Glucocorticoid Response Genes Adjacent to Genomic Binding Sites for Steroid Hormone Receptor in Osteoblastic Cells.
K. Horie-Inoue*1, K. Takavama*2, S. Inoue2. 1Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan, 2Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Glucocorticoid causes secondary osteoporosis by increasing bone resorption and impairing bone formation. Investigation of the glucocorticoid-dependent gene regulatory network in osteoblasts will provide useful information to elucidate the pathophysiology of glucocorticoid-induced osteoporosis. Glucocorticoid receptor (GR) belongs to the nuclear receptor superfamily, and functions as a ligand-induced transcriptional factor that regulates the expression of its target genes by recognizing and binding to the steroid hormone response elements (SHREs) in the gene regulatory regions. The prototypic consensus SHRE is a palindromic sequence that consists of two hexameric half-sites arranged as inverted repeats with a 3-bp spacer. Besides GR, other steroid hormone receptors for androgen, mineralocorticoid, and progesterone can also recognize and bind to the identical consensus SHREs. In the present study, we investigate whether novel glucocorticoid response genes can be identified in the vicinity of SHREs that have been previously validated as androgen response elements. Using chromatin immunoprecipitation (ChIP) assay combined with DNA microarray technique (ChlP-on-chip), we identified 10 bona-fide androgen-dependent SHREs in human prostate cancer LNCaP cells in the genomic regions that are being investigated by the ENCyclopedia Of DNA Elements (ENCODE) project. We examined whether these SHREs in the ENCODE regions could also recruit GR ligand-dependently and the expression of their adjacent genes could be modulated by glucocorticoid in osteoblastic Saos2 cells. Among the genes in the vicinity of the SHREs, pepsinogen C (PGC) and UDP glucuronosyltransferase 1A1 (UGT1A1) mRNAs were up-regulated by >2-fold in Saos2 cells (4.6-fold and 2.9-fold, respectively) in response to dexamethasone (10 nM) treatment for 6 h. The SHREs in the proximal upstream region of PGC and the 17-kb upstream region of UGT1A1 were shown to recruit GR ligand-dependently by ∼ 2-fold in response to dexamethasone treatment for 1 h. The present data suggest that the systemic investigation of functional SHREs by ChIP-on-chip analysis is useful to identify novel glucocorticoid response genes in osteoblastic cells, which may play roles in the pathogenesis of glucocorticoid-induced osteoporosis as well as in the physiological hormone regulation.
Disclosures: K. Horie-Inoue, None.
See Sunday Plenary Number S062
Acid-induced Stimulation of COX-2 and RANKL in Osteoblasts Is Mediated by IP3.
N. S. Krieger, A. C. Michalenka*, K. LaPlante Strutz*, D. A. Bushinsky. Medicine, University of Rochester, Rochester, NY, USA.
Chronic metabolic acidosis stimulates net calcium (Ca) efflux from bone by regulation of osteoblastic activity leading to stimulation of osteoclastic resorption. This acid-induced bone resorption is mediated primarily by stimulation of osteoblastic cyclooxygenase 2 (COX2) mRNA and subsequent prostaglandin E2-mediated increase in RANKL. We hypothesize that extracellular [H+] transduces its intracellular signal through a G protein-coupled proton sensing receptor, OGR1, recently identified in osteoblasts and coupled to inositol trisphosphate (IP3)-mediated Ca release. We have shown previously that acid-induced bone Ca efflux from neonatal mouse calvariae is inhibited by TMB-8 and 2-APB, both of which inhibit IP3 mediated Ca signaling. To further define the signaling pathway that responds to H+, we now demonstrate that these inhibitors also block osteoblastic COX2 and RANKL production. Neonatal mouse calvariae were incubated for 48h in Met (pH ∼7.11) or neutral (Ntl, pH ∼7.40) medium in the absence or presence of 100 μM TMB-8 or 50 μM 2-APB. Medium was changed at 24 h. As before, incubation in Met induced a marked increase in net Ca efflux and this bone resorption was significantly blocked by each inhibitor. In parallel, immunoblot analysis shows a significant increase in COX2 protein at 48h in calvariae in response to Met compared with Ntl (Ntl = 0.37 ± 0.04, Met = 1.18 ± 0.16, normalized to actin, p < 0.05). This increase in COX2 protein was also significantly inhibited by treatment with TMB-8 (Met + TMB-8 = 0.13 ± 0.05) or 2-APB (met + 2-APB = 0.20 ± 0.10). Our previous studies have shown that the increase in COX2 in response to Met occurs within 6h in primary osteoblastic cells isolated from the calvariae. We now demonstrate that incubation of cells for 6h in the presence of TMB-8 also significantly inhibits the acid-induced increase in COX2 mRNA, as measured by realtime PCR (Ntl = 2.49 ± 0.47, Met = 5.70 ± 1.19, Met + TMB-8 = 1.54 ± 0.27 normalized to actin). Acid stimulation of osteoblastic COX2 protein, measured by immunoblot analysis, was also inhibited by TMB-8 and 2-APB (Ntl = 0.84 ± 0.11, Met = 1.18 ± 0.11, Met + TMB-8 = 0.43 ± 0.05, Met + APB = 0.61 ± 0.08, normalized to actin). Increases in RANKL in response to Met occur downstream of stimulation of COX2. In primary cells Met increased RANKL mRNA compared to Ntl after 48h and this stimulation of RANKL mRNA was significantly inhibited in the presence of TMB-8 (Ntl = 3.27 ± 0.59, Met = 7.81 ± 2.02, Met + TMB-8 = 1.87 ± 0.56, normalized to actin). Thus, our results are consistent with a requirement for a H+-induced increase in osteoblastic IP3-stimulated Ca release from intracellular stores which would stimulate production of COX2 and then RANKL, leading to the subsequent increase in osteoclastic bone resorption.
Disclosures: N.S. Krieger, None.
This study received funding from: NIH. RR1.
See Sunday Plenary Number S064
ATP Is Released From Human Osteoblasts By Multiple Mechanisms.
J. A. Gallagher1, J. P. Dillon*1, P. M. Wilson*1, H. E. Burrell*1, M. J. Havton*1, W. D. Fraser2, A. Gartland3. 1Human Anatomy and Cell Biology, University of Liverpool, Liverpool, United Kingdom, 2Clinical Chemistry, University of Liverpool, Liverpool, United Kingdom, 3Bone Biology, University of Sheffield, Sheffield, United Kingdom.
There is a growing recognition that extracellular ATP is one of the most important regulators of bone cell function. Multiple P2 receptor subtypes are expressed by bone cells and these regulate many functions including osteoblast proliferation and gene expression, osteoclast formation and resorptive capacity, and apoptosis of both osteoblasts and osteoclasts. Recent evidence indicates that extracellular ATP plays a pivotal role in mechanotransduction in bone, signalling in part through the P2×7 receptor. Less is known about how ATP is released into the bone microenvironment. We used a combination of end-point and real-time assays, utilising luciferin/luciferase bioluminescence, to monitor release of ATP into the extracellular environment from osteoblast cell lines, SaOS-2, MG63 and Te85, as well as primary human osteoblasts. Real-time assays revealed that ATP is released in to the medium constitutively and that this release is exquisitely sensitive to changes in fluid flow. In response to increased flow, there was a rapid rise in ATP concentration in medium bathing the cells, followed by a slower decline back to resting levels. At steady state, the concentration of ATP in the medium of cultures of osteosarcoma cells lines was usually less than 5 nM ATP. In contrast, the ATP concentration in the medium of cultures of primary osteoblasts was up to six fold higher, depending on the donor. The concentration of ATP in medium is a function of ATP release from cells and degradation by ectonucleotidases. End-point assays revealed that fluid flow in the form of medium displacement resulted in a rise in the concentration of ATP which correlated with the number of displacements. After six displacements there was an approximate 10-fold increase in control cultures and 16-fold in cultures treated with ionomycin. Treatment with N-ethylmaleimide inhibited constitutive and fluid displacement-stimulated release, probably by blocking N-ethylmaleimide sensitive factor, a protein that regulates membrane fusion events in exocytosis. Addition of a monoclonal antibody that blocks the action of the P2×7 receptor had variable effects on extracellular concentration of ATP, both in control and fluid displacement cultures, but it was never completely effective in blocking ATP release. These data demonstrate that ATP release varies between osteoblastic cell lines, can be stimulated by fluid flow and is mediated by more that one mechanism. The effect of fluid flow on ATP release could be a key factor in mechanotranduction in bone.
Disclosures: J. A. Gallagher, None.
Microstructural Variation in the Bone Matrix and Cell Attachments Modify Strain Amplification on the Osteocyte.
A. R. Bonivtch1, L. F. Bonewald2, D. P. Nicolella1. 1Mechanical and Materials Engineering, Southwest Research Institute, San Antonio, TX, USA, 2School of Dentistry, University of Missouri-Kansas City, Kansas City, MO, USA.
Osteocytes make up over 90% of all bone cells and are hypothesized to be the mechanosensors in bone that mediate the effects of loading. Measurements of bone strain in humans using strain gages have been used for strain applications in in vitro bone cell experiments. The limitation of applying this strain magnitude to cells in vitro is that the in vivo strain gage measurements represent continuum measures of bone deformation. Clearly, bone is not a continuum, but heterogeneous, especially at the spatial level of bone cells. Microstructural inhomogeneities result in inhomogeneous strain fields in which local tissue strains are magnified. The objective of this study was to investigate how microstructural bone tissue strains may be transmitted to an embedded osteocyte and its cell processes and to determine how changes in the perilacunar bone tissue structure and properties affect osteocyte strain and deformation. Previously we had created a parametric finite element model composed of an osteocyte lacuna, canaliculi, perilacunar tissue, and surrounding bone tissue. This model showed that a decrease in perilacunar tissue elastic modulus amplifies the perilacunar tissue strain and canalicular deformation. As the perilacunar elastic modulus decreases with glucocorticoid treatment, this may change the level of strain perceived by the osteocyte. In the present model, an osteocyte, cell processes, and glycocalyx attachments to the cell and cell processes were added. A displacement was applied to the model resulting in a global strain of 2000 microstrain and 27 simulations were performed with varying osteocyte and cell process moduli, perilacunar tissue moduli, and glycocalyx and cell process attachment stiffness. Maximum strain levels in the osteocyte reached over 10,000 microstrain, 5 times the applied macroscopic strain of 2000 microstrain. Similar strain levels had been observed in lacunae in bone slices previously. Maximum strain within the osteocyte was found to occur on the cell membrane, parallel to the plane of applied strain. Maximum osteocyte strain was found to increase with a decrease in perilacunar tissue modulus and decrease with an increase in perilacunar tissue modulus consistent with perilacunar tissue strains in the earlier model. A decrease in the osteocyte, glycocalyx, and cell process stiffness led to a decrease in the maximum strain transmitted to the osteocyte. In summary, this model can predict the influence of each component of the osteocyte structure and microenvironment on location and magnitude of cellular and dendrite deformation.
Disclosures: A.R. Bonivtch, None.
This study received funding from: NIH/NIAMS.
Bone Microarchitecture of Irradiated and Bone Marrow Transplanted Mice.
A. Dumas*1, M. Brigitte*2, M. F. Moreau*1, M. F. Baslé*1, R. K. Ghérardi*2, D. Chappard1. 1Faculté de Médecine, INSERM, EMI 0335, Angers, France, 2Faculté de Médecine, Université Paris XII, INSERM, EMI 0011, Créteil, France.
Osteopenia and osteoporosis are complications of renal, hepatic, cardiac and bone marrow transplants. Bone loss is attributed to transplant-related treatments (ciclosporine, glucocorticoids.) whether they act directly on bone cells or they deregulate the hormonal system. Total body irradiation provokes hypogonadism and increases osteoclast number and activity without increase in bone formation.
We investigated the effects of total body irradiation and bone marrow transplantation (BMT), without additional treatment, on bone mass and microarchitecture in a murine model. C57B1/6 (B6) mice were lethally irradiated and within 1 day received syngeneic bone marrow cells expressing the green fluorescent protein. Transplanted mice (T-B6) and age-control B6 mice were euthanized 1, 3 and 6 months after BMT. Bone mass (BV/TV), mean cortical thickness (Ct.Th), trabecular characteristics (Tb.Th, Tb.N, Tb.Sp, Tb.Pf and SMI) were analyzed by microCT at the distal femur.
One month after irradiation and BMT, T-B6 presented a 56.9% bone loss compared to B6 at the same age. T-B6 did not restore their bone mass with time whereas BV/TV of B6 increased. The difference in bone mass between both groups reached 80.8% six months after BMT. Bone microarchitecture was altered in T-B6 mice: Tb.N dramatically decreased 1 month after BMT, Tb.Sp increased whereas Tb.Th was reduced after 6 months. The trabecular bone was less connected with fewer plates. Ct.Th increased significantly with time and, 6 month after BMT, T-B6 had cortical bone of the femoral diaphysis significantly thicker than age-matched B6 (+16%).
Our findings suggest that transplanted bone cells do not restore of the irradiation-induced trabecular bone loss despite an increase in cortical thickness. Effects of irradiation on cortical or cancellous bone remodeling can be considered in the different envelopes.
Disclosures: A. Dumas. None.
See Sunday Plenary Number S068
Lacunocanalicular Fluid Flow and Regulation of Basic Multicellular Unit Activity.
G. C. Goulet*1, D. M. L. Cooper*2, D. Coombe*3, D. L. Thomas*4, J. G. Clement*4, R. F. Zernicke*5. 1Schulich School of Engineering, University of Calgary, Calgary, AB, Canada, 2Department of Orthopaedics, University of British Columbia, Vancouver, BC, Canada, 3Computer Modelling Group, Ltd., Calgary, AB, Canada, 4School of Dental Science, University of Melbourne, Melbourne, Australia, 5Schulich School of Engineering; Departments of Kinesiology and Medicine, University of Calgary, Calgary, AB, Canada.
Human bone is continuously regenerated through remodeling. This process is carried out by a complex mediator mechanism called the Basic Multicellular Unit (BMUs). Cortical BMUs proceed by osteoclastic excavation of a resorption space (cutting cone) followed by new bone formation by osteoblasts (closing cone). Current theories suggest that the BMU is strain and/or fluid-flow regulated [1,2,3]. While these regulatory models have focused on idealized forms, BMU morphology is variable, including unidirectional, bidirectional, and branched forms. The purpose of this study was to examine strain and fluid flow in relation to realistic BMU forms directly observed in human cortical bone by 3D micro-CT imaging. Computational models were developed in STARS, a coupled finite difference fluid flow and finite element mechanics simulator (Computer Modelling Group, Ltd., Calgary, AB, Canada). Models of a unidirectional and branched BMU were generated. These were subjected to compressive loads, which induced fluid flow through the simulated lacunocanalicular porosity. Upon loading, fluid flowed from the high-pressure bone matrix into the low-pressure porosity of the BMU. Consistent with previous models, the unidirectional canal model displayed decreased strain in front of the cutting cone, and increased strain behind it. Similarly, the branched model demonstrated strain reduction in front of both cutting cones and increased strain behind them. However, the branched morphology also displayed a similar reduction in strain at the fork where it divided. Fluid stasis has been implicated as a regulating mechanism in BMU activity, whereby decreased osteocytic stimulation results in reduced production of nitric oxide, inducing apoptosis, which subsequently signals the recruitment of osteoclasts. The inter-branch strain shielding and related reduction in fluid flow observed in the branched BMU poses a challenge to this regulatory model since the region between resorption spaces would also be prone to osteocyte apoptosis and osteoclastic activity, effectively eliminating the branched structure. Therefore, strain and related fluid flow alone may not be sufficient to explain BMU regulation.
 Smit and Burger (2000). J Bone Miner Res, 15, 301–307.
 Burger et al. (2003). J Biomech, 36, 1453–1459.
 Smit et al. (2002). J Bone Miner Res, 17, 2021–2029.
Disclosures: G.C. Goulet. None.
This study received finding from: Natural Sciences and Engineering Research Council; Alberta Heritage Foundation for Medical Research: Wood Professorship in Joint Injury Research.
Increased Expression of Anti-adhesive PODXL in TIEG Knock-Out Osteoclast Precursors Delays Osteoclast Formation and Reduces Osteoclast Size.
M. Cicek1, A. Vrabel*1, M. Subramaniam2, T. C. Spelsgerg2, M. J. Oursler1. 1Endocrine Research Unit, Mayo Clinic, Rochester, MN, USA, 2Department of Molecular Biology and Biochemistry, Mayo Clinic, Rochester, MN, USA.
TIEG is a Krüppel-like transcription factor gene which was originally cloned from human osteoblasts as an early response gene to TGF-β treatment. As reported previously, TIEG−/− mice have decreased cortical bone thickness and lower vertebral volume, increased spacing between the trabeculae in the femoral head, and a lower breaking strength compared to wild-type mice. Therefore we investigated the role of TIEG in the osteoclasts that may be responsible for bone phenotype in TIEG−/− mice. In in vitro cultures of mouse marrow treated with RANKL and M-CSF, we found that osteoclasts differentiated more slowly and formed reduced numbers of osteoclasts compared to wild type precursors. Moreover, TIEG−/− osteoclasts were significantly smaller than wildtype osteoclasts. Flow cytometry analyses showed that there was no difference in CD11b+ and/or RANK+ and/or c-fms+ in osteoclast precursors in freshly harvested marrow cells between the genotypes. These data led us to hypothesize that loss of TIEG reduced the capacity of TIEG−/− precursors to differentiate into mature osteoclasts. We therefore investigated the role of the anti-adhesive glycosylated cell-membrane protein podocalyxin (PODXL) in osteoclastogenesis in vitro in TIEG−/− and wildtype precursor differentiation. We found that TIEG−/− precursors have increased expression of PODXL relative to the wildtype. Adding RANKL and M-CSF to the in vitro model significantly reduced the expression of PODXL in TIEG−/− precursors, suppressing expression relative to that of wildtype cells. Parallel treatment of wildtype precursors did not reveal any effect on PODXL expression in these cells. These data suggest that the reduced ability of TIEG−/− osteoclast differentiation may be due to an increased expression of anti-adhesive PODXL in these cells. We conclude that TIEG expression may be critical in controlling the rate of osteoclast differentiation and that increased PODXL expression in TIEG−/− may be a key factor in the reduced differentiation and/or the size of TIEG−/− osteoclasts.
Disclosures: M. Cicek. None.
Regulation of Integrin Expression by NFATc1 and PU.1 in Osteoclasts.
T. N. Crotti1, M. R. Flannery*1, J. D. Fleming*1, S. R. Goldring2, K. P. McHugh1. 1Rheumatology and Orthopaedics, Beth Israel Deaconess Medical Center, Boston, MA, USA, 2Hospital for Special Surgery, New York, NY, USA.
Beta-3 and beta-5 integrins play important and contrasting roles in osteoclast (Oc) differentiation and are accordingly reciprocally regulated. The integrin beta-5 is expressed by pre-Ocs and has an inhibitory role in Oc differentiation, as beta-5 knockout mice display enhanced Oc formation. The beta-3 integrin is expressed by mature Ocs and is required for normal Oc function.
NFATc1 is a RANKL-induced and Ca++-regulated transcription factor critical for Oc differentiation. NFATc1 with PU.1, an ETS family transcription factor, regulate expression of distinct target genes in Oc differentiation. We provide here direct evidence that NFATc1 and PU.1 are involved in the reciprocal regulation of integrin expression during Oc differentiation.
To test the involvement of NFATc1 in beta-3 integrin expression, we generated mouse beta-3 promoter/reporter constructs (-1350 to + 34bp; mB3–1350) which include a composite NFAT and PU.1 binding site conserved from mouse and man. The mB3–1350 construct was trans-activated >60X by co-transfected NFATc1 in RAW264.7 cells, consistent with our previous report on regulation of the human beta-3 promoter. Trans-activation was abolished by deletion to −1084, and further deletions were similarly unresponsive. Direct binding of NFATc1 and PU.1 to the mouse beta-3 promoter was demonstrated by EMSA. Consensus NFAT and PU.1 oligos competed, while oligos with mutant NFAT or ETS sites failed to compete. The identity of the protein moieties was verified by supershift with anti-NFATc1 and PU.1 antibodies. Mutation of either the NFAT or PU.1 binding site, in the context of mB3–1350, abrogated NFATc1 trans-activation. The specificity of PU.1, among ETS family members, to induce mB3–1350 was demonstrated by co-transfection in HEK293 cells. NFATc1 trans-activation of the mB3–1350 promoter was specifically facilitated by PU.1. NFATc1 had no effect when co-transfected with Ets-1, ESE1, or Elf, however Ets-1 and Elf mediated the highest basal expression.
We previously reported that TAT-mediated transduction with a dominant-negative (dn)NFATc1 construct dose-dependently blocked spreading of mouse osteoclasts and inhibited expression of the endogenous beta-3 integrin gene. In addition to this, we find reciprocal dose-dependent induction of the endogenous beta-5 integrin by TAT-dnNFATc1. The mouse beta-3 integrin gene is therefore a specific target of NFATc1 during RANKL-induced Oc differentiation through direct binding of NFATc1 and PU.1 to specific sites in the beta-3 promoter. Furthermore, NFATc1 function is coupled with, and required for, down-regulation of the beta-5 integrin gene Oc cells.
Disclosures: T.N. Crotti, None.
This study received funding from: The author of this paper holds a National Health and Medical Research Council (Aust) CJ Martin Fellowship (I.D. 200078).
This work was supported in part by National Institute of Health Grants N/AMS R01 AR45472 (to SRG), NIAMS R01 AR47229 (to KPM).
The Decisive Moment of Osteoclast Fusion and Apoptosis Captured Under a Microscope Connected with Time-Lapse Mosion Video Picture.
A. Itabashi1, T. Ohnuma*2, Y. Takada*1. 1Saitama Center for Bone Research, Kumagaya, Japan, 2Yone Production, Tokyo, Japan.
Osteoclasts are multinucleated bone cells that are formed by multiple cell fusions. Mature osteoclasts undergo several cycles of activation and inactivation, where bone is resorbed in the active state and osteoclasts migrate in the resting state, eventually, the cells die apoptotically. However, there are few pictures or videos that captured the moment of osteoclast fusion or apoptosis. In this study, we tried to capture those pictures in collaboration with a science movie company.
We first observed mouse calvarial bone cells under a microscope connected with time-lapse motion video picture in organ culture conditions continuously for more than 96 hours. We could observe osteoblast devision and bone matrix formation. We could also capture the decisive moment of osteoclast fusion using isolated rabbit osteoclasts with stromal cells. Osteoclasts had many cell processes folding in and sticking out from their cell surfaces, and moved around in a culture dish actively. When the cell process made contact with that of the other osteoclast, rapid movements between those processes were observed. Often they separated again after some conversation between them. But some cells started to make continuous contact with each other, with busy communication through cell membranes. And then one part of cell membrane overlapped with other cell membrane, and cell contents started to mingle and multiple nuclei from each osteoclast assembled to form a giant multinucleated osteoclast. The huge osteoclast moved around actively as one cell and it could be maintained in the plastic dish under culture conditions for several hours, then rapidly decreased movement. After that, spontaneous apoptosis occurred in that cell with cell shrinkage and fragmentation of nuclei.
Using time-lapse video picture system under regular culture conditions for certain amount of time, we can observe the way the osteoclasts fuse and die. This can contribute to understand the mechanism of osteoclast fusion and apoptosis.
Disclosures: A. Itabashi, None.
See Sunday Plenary Number S073
Tropomyosin-2/3 Regulate the Intracellular Scaffold of Osteoclasts.
P. Kotadiya*, B. K. McMichael, T. Singh*, B. S. Lee. Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA.
Tropomyosins (Tms) are actin-binding proteins that stabilize microfilaments and inhibit or recruit binding of other act in-associated proteins. In nonmuscle cells, over twenty alternately spliced isoforms are expressed from four distinct genes, namely the alpha gene (Tm-2, −3, −5a/b, −6), the beta gene (Tm-1), the gamma/5NM gene (Tms NM-1 to NM-11), and the delta/Tm-4 gene (Tm-4). Recent studies have indicated that individual Tm isoforms are distributed to different intracellular locations where they regulate specific actin pools. Therefore, the goal of this work is to determine the roles of tropomyosin isoforms in mature osteoclast activity.
Our previous studies showed that at least eight Tm isoforms are expressed in osteoclasts and that some of these, including Tm-2 and 3, are not present in monocyte-macrophage precursors but are upregulated during osteoclastogenesis. These isoforms differ by only a few amino acids; therefore, reagents that distinguish between them are limited. However, Tm-2 is present in osteoclasts at levels about four-fold higher than Tm-3. Immunocytochemical analysis showed that they are dispersed throughout the interior of osteoclasts and are only loosely intercalated in the podosomes and actin rings. To analyze the role of Tm-2/3 in osteoclasts, the effects of both Tm-2/3 suppression and overexpression have been studied in these cells. It was found that cells treated with Tm-2/3-specific siRNAs covered a greater surface area, but contained the same number of nuclei, as control cells. These results suggest that loss of Tm-2/3 results in increased cell spreading. This hypothesis is supported by data showing that the siRNA-treated cells are flattened relative to controls. These cells also are greatly diminished in their motility in Transwell assays. Additionally, resorption assays illustrated that the total resorption area was similar in Tm-2/3-suppressed cells and controls, but the siRNA-treated cells generated fewer, but larger, resorbed areas. On the other hand, increasing Tm-2 levels by overexpression also distorted cell shape. On glass, these cells were distinctly more spherical than controls and lacked podosomes. Instead, these overexpressing cells contained patches of actin and sometimes stress fibers at their base. This in turn led to decreased motility by these cells. In summary, these findings indicate that Tm-2/3 are involved in regulating cell shape and motility, likely due to stabilization of actin microfilaments that create a cellular scaffold.
Disclosures: P. Kotadiya, None.
Effect of Pore Size and Surface Modification of the Circular Poly-Ethylene Glycol(PEG) Polymer Scaffold on Adhesion of Cultured Human Ligamentum Flavum Cells.
C. Lee*1, H. Kim*1, J. Lee*1, M. Nan*1, J. Park*2, E. Moon*2, S. Park*3, H. Kim*2, J. Jahng*2, S. Moon2, H. Lee*2, H. Chun*4, H. Kim*2. 1Orthopaedic Surgery, Brain Korea 21 Medical Science Graduate School of Yonsei University, Seoul, Republic of Korea, 2Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea, 3Orthopaedic Surgery, Korea University College of Medicine, Seoul, Republic of Korea, 4Mechanical Engineering, School of Mechanical Engineering of Yonsei University, Seoul, Republic of Korea.
Tissue engineering requires viable cells and cellular-compatible scaffold. Poly-Ethylene Glycol(PEG) polymer is a probable option of scaffold with its unique advantages. Nevertheless, it is still needed to be characterized that the effect of surface modification with making pore with various size and coating with collagen. Hence, we report the effect of surface modification by circular pore and type I collagen coated to cellular attachment and viability of ligamentum flavum (LF) cells. Human LF tissue was harvested, digested. and cultured. Poly-Ethylene Glycol(PEG) hydrogel micropatterns were fabricated from PEG by using a photomask illuminated with UV light. Collagen-coated PEGs were made by immersion in type I collagen solution. Then LF cells (1 × 105)were seeded on PEG with various pore size (50, 100, 200 μm) with or without collagen coating. After 2 days' incubation, adherent cells were imaged in SEM. Cellular viability was assessed by live/dead cell viability kit. Most LF cells were found at the base of pore, however, some patches of cells were found on the surface of PEG and inner surface of the PEG pore. No visible cellular adhesion was noted in PEG with the pore size of 50 μm. However, in PEG with the pore size of 100 and 200 μm there is marked cellular adhesion to the surface of PEG and even inner surface of the PEG pore. Surface modification of PEG with type I collagen renders more efficient adhesion of LF cells on the modified surface of PEG Even with collagen coating, PEG with pore size of 50 μm failed to show any significant cellular adhesion. Adhered cells on the surface of PEG were all viable in all experiment group. In summary, PEG scaffold with the pore size of 100 and 200 μm was non-toxic to the LF cell and provided suitable surface for LF cell adhesion. Surface modification with type I collagen facilitated process of cellular adhesion to PEG surface. New modification such as one sided sealing of the pore is need for more efficient cellular containment within PEG pore.
Disclosures: C. Lee. None.
Myosin × Regulates Osteoclast Adhesion Through Linkage of Podosomes and Microtubules.
B. K. McMichael1, R. E. Cheney*2, B.S. Lee1. 1Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA, 2Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Osteoclasts generate two distinct actin-based adhesion structures–podosomes for migration, and the actin ring of the sealing zone for resorption. Although podosomes are actin-based structures, their patterning is microtubule dependent. In this study, the role of myosin × (myo10) in osteoclast adhesion was assessed. Myo10 is an unconventional myosin that consists of a motor domain, three light chain binding domains, and a unique tail consisting of pleckstrin homology domains (PH), a myosin tail homology 4 domain (MyTH4), and a FERM domain. The multifaceted myo10 has been shown to bind many osteoclast regulatory proteins including microtubules, β1/β3/β5 integrins, products of PI3 kinase, and VASP, along with binding actin via the motor domain.
Initial immunocytochemical analysis of osteoclasts showed myo10 to be localized immediately adjacent to, but not overlapping, podosome belts and the actin ring, suggesting a potential role in positioning of these structures. To analyze its role, myo10 expression was inhibited in both RAW264.7 and mouse marrow derived osteoclasts via RNA interference. Myo10 suppression resulted in diminished spreading of osteoclasts cultured on glass. Suppression also led to a decrease in the size of the actin ring with an even greater decrease in bone resorption and motility. Further, inhibition of myo10 levels resulted in a microtubule-based podosome patterning defect. When microtubules of osteoclasts were depolymerized by cold treatment, both siRNA-treated and control cells showed redistribution of podosomes away from the cell periphery. However, when allowed to recover, the siRNA-treated cells, unlike the control cells, could not distribute their podosomes back to the periphery even though they reformed the microtubule network. These results suggest that myo10 may act as a link between podosomes and microtubules through its tail domains. Indeed, detailed immunocytochemical analysis of osteoclasts during podosome positioning shows myo10 localized between podosomes and the surrounding microtubule system. Over-expression of sections of the tail, either PH, MyTH4, or FERM alone or in combination, caused formation of aberrant podosomes while over-expression of all three domains had a dominant negative effect similar to the phenotype manifested in the siRNA-treated cells. These results taken together clearly suggest a role for myo10 in linking the microtubule network to podosomes through its tail domain.
Disclosures: B.K. McMichael. None.
Expression and Possible Role of PVR/CD 155/ Necl-5 in Osteoclastogenesis.
I. Morita, S. Kakehi*, K. Nakahama. Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo, Japan.
Osteoclast, the bone-resorbing cell, is differentiated from hematopoietic precursors via two-step cell-cell interactions; interaction between osteoclast precursor and stromal cell, and interaction among osteoclast precursors. We have previously reported that some adhesion molecules have a crucial role in these interactions. Recently, poliovirus receptor (PVR, CD155, Necl-5) was reported to play important roles in cell adhesion and migration. However, there is no report of PVR in osteoclastogenesis. Here, we examined the expression of PVR and its ligand, DNAX accessory molecule-1 (DNAM-1, CD226) in osteoclast precursor, mature osteoclast and stromal cell. We found that the PVR was constitutively expressed in both osteoclast cells and stromal cells. The expression of PVR was not changed at various stages of osteoclast formation. In contrast, the expression of DNAM-1 was observed in mononuclear cells and was down-regulated during osteoclastogenesis. Moreover, multinucleated osteoclast formation was inhibited by treatment with extracellular domain of DNAM-1 (ED-DNAM-1) as a soluble ligand for PVR, but mononuclear preosteoclast formation was not affected. Especially, during the 7-days cultivation, osteoclast formation was suppressed by the treatment with ED-DNAM-1 on days 6 and 7, when mononuclear preosteoclast fused into multinucleated osteoclasts. This suppression was abrogated partially by small interfering RNA specific for PVR. These results indicated that the binding of PVR with DNAM-1 negatively regulates osteoclast formation via inhibition of cellular fusion caused by PVR mediated signaling. Our report may suggest the possibility of a new therapy for bone loss diseases by activation of PVR signaling.
Disclosures: I. Morita, None.
This study received funding from: JSPS COE Program.
Spacio-Temporal Analysis of Osteoclastogenesis Using Co-culture System.
K. Nakahama1, S. Ichinose*2, I. Morita1. 1Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo, Japan, 2Instrumental Analysis Research Center, Tokyo Medical and Dental University, Tokyo, Japan.
The mechanism of osteoclast differentiation has been well studied in vivo and in vitro. But, in our knowledge, little is known about the spacio-temporal aspects of preosteoclast differentiation into multinuclear osteoclast. To examine the spacio-temporal aspects of osteoclastogenesis, the stromal cells and spleen-derived mononuclear cells were labeled with calcein-AM and Cell Tracker Orange CMRA respectively. The spleen-derived mononuclear cells were co-cultured on the confluent stromal cells. In this co-culture system, tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts were observed under the stromal cells after 5-days culture without stimulation. Conforcal microscopic observation revealed that mononuclear cells migrated under stromal cells after 6-hrs co-culture. Surprisingly, in addition to the paracellular migration (migration via cell junction), transcellular migration (migration via stromal cell body) of spleen-derived mononuclear cell was detected. These two types of migrations were confirmed using scanning electron microscopy. Immunocytochemical study showed strong expression of intercellular adhesion molecule-1 (ICAM-1) and its counter receptor macrophage antigen-1 (Mac-1) on spleen-derived mononuclear cell, but not on stromal cells at 24-hrs after co-culture. The polarized localization of ICAM-1 indicated that ICAM-1 plays some roles in the migration of spleen-derived mononuclear cells. Two-days after co-culture, some aggregated mononuclear cells expressed ICAM-1 at cell-cell adhesion sites. After 5-days co-culture of the cells, multinucleated TRAP-positive osteoclasts were formed. Interestingly, we also detected the localization of ICAM-1 at cell-cell association site among osteoclasts. ICAM-1 localization in some osteoclasts was observed at apical surface of the membrane. Collectively, these results suggest that preosteoclasts will migrate under the stromal cells paracellularly and transcellularly, thereafter they will fuse each other. There may be suitable conditions at basolateral side of stromal cells. Polarized localization of ICAM-1 on osteoclast would indicate some important roles in migration and fusion of preosteoclasts.
Disclosures: K. Nakahama, None.
This study received funding from: JSPS COE Program.
HMGB1 Regulates Actin Cytoskeleton Organization and RANKL-induced Osteoclastogenesis in a Manner Dependent on RAGE.
Z, Zhou1, D. Ferguson*2, J. Xie*1, C. Xi*1, D. Stern*2, L. Mei*1, W. Xiong*1. 1Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA, 2University of Cincinnati, Cincinnati, OH, USA.
High-mobility group box 1 (HMGB1), a non-histone nuclear protein, is released by macrophages into the extracellular milieu consequent to cellular activation. Extracellular HMGB1 has properties of a pro-inflammatory cytokine via its interaction with receptor for advanced glycation endproducts (RAGE) and/or toll-like receptors (TLR2 and TLR4). Although HMGB1 is highly expressed in macrophages and differentiating osteoclasts, its role in osteoclastogenesis remains largely unknown. Here we present evidence for a function of HMGB1 in this event. HMGB1 is released from macrophages in response to RANKL stimulation, and is required for RANKL-induced osteoclastogenesis in vitro and in vivo. In addition, HMGB1, like other osteoclastogenic cytokines (e.g., TNFalpha), enhances RANKL-induced osteoclastogenesis in vivo and in vitro at sub-threshold concentrations of RANKL, which alone would be insufficient. The role of HMGB1 in osteoclastogenesis is mediated largely by its interaction with RAGE, and regulation of alpha v beta 3 integrin signaling and osteoclastic actin ring formation. Together, HMGB1-RAGE signaling appears to be important in regulating osteoclastic actin cytoskeleton reorganization, thereby participating in RANKL-induced osteoclastogenesis. These observations provide a new link between inflammatory mechanisms and bone resorption.
Disclosures: Z. Zhou. None.
Precursor Analyses of CD11c+Dendritic Cells-derived Osteoclasts.
M. Alnaeeli*, D. Mahamed*, J. Park*, Y. Teng*. Department of Microbiology & Immunology and Eastman Department of Dentistry, University of Rochester, Rochester, NY, USA.
Inflammation-induced osteoclastogenesis is a well established osteoimmunological phenomenon, whereby osteoclasts (OC) frequency and activity become elevated under inflammatory conditions. Dendritic cells (DC) are professional antigen presenting cells that not only infiltrate bone tissues during inflammation, but are also suggested to share common precursors with OC. Previously we reported and characterized the in vitro development of TRAP+ CT-R+ cathepsin-K+ CD11c+ MHC-II+ multinucleated functional OC from murine CD11c+DC (called; DDOC), in response to RANKL and bacterial or protein antigens during immune interactions [J. Immunol. 177(5): 3314–26 & JBMR, in press, 06/2007]. Here, we aimed to study the clonal characteristics of DDOC by determining their OC precursor frequency and proliferative capability. We employed colony forming unit assay and limiting dilution analysis, using purified murine bone marrow-derived CD11c+DC (purity ≥98%) in the presence of 100 ng/ml RANKL & 10μg/ml sonicated antigens of Aggregatibacter (Actinobacillus) actinomycetemcomitans; an anaerobic human pathogen. After 5 days culture and when compared to the controls, the purified CD11c+DC: i) did not manifest any proliferative capability (p = 0.003), and ii) had an estimated precursor frequency of 8.4 ± 0.06%, based on the number of TRAP+ multinucleated (≥3 nuclei) OC yielded. To investigate the physiological relevance of the DDOC phenomenon, we studied the osteoclastogenic potential of murine CD11c+DC in vivo. Interestingly, after the injection of carboxyfluoroscein succinimidyl ester-labelled freshly purified DC onto mouse calvarias, we found CFSE+TRAP+ multinucleated OC-like cells associated with higher local bone loss, compared to the control groups, suggesting that CD11c+DC can become TRAP+ multinucleated OC under inflammatory conditions in vivo. Furthermore, our immunohistochemical analysis detected the presence of CD11c+ multinucleated OC-like cells on the subchondral bone surfaces of the arthritic joints in DBA mice but not healthy controls. Collectively, these findings support the possible development of DDOC at the osteo-immune interface, which may be an integral part of the mechanisms underlying inflammation-induced osteoclastogenesis in inflammatory bone disorders such as rheumatoid arthritis and periodontal disease.
Disclosures: M. Alnaeeli, None.
This study received funding from: CIHR Grant MOP-37960 and NIH Grant DE 15786 to Y-T A Teng.
In Vivo Activation of Toll-Like Receptor 9 Modulates Oteoclastogenesis.
A. Amcheslavsky*, Z. Bar-Shavit. Experimental Medicine and Cancer Research, Hebrew University, Jerusalem, Israel.
Toll-like receptor (TLR) ligands are pathogen-derived molecules that activate the innate immune response. Many studies have shown that activation of these receptors in osteoclast lineage cells accelerates osteoclast formation and activity. In recent years, several groups have shown that pathogen-derived molecules such as peptidoglycan, viral RNA (mimicked by poly-IC), lipopolysaccharides and bacterial DNA (mimicked by CpG-oligodeoxynucleotides [CpG-ODNs], the ligands of TLR2, 3, 4 and 9, respectively, inhibit RANKL-induced osteoclastogenesis under certain conditions. In vitro studies led to the hypothesis that activation of TLRs in early osteoclast precursor cells inhibits their differentiation, while in cells that have been primed with RANKL and have already begun their osteoclastic differentiation, TLR ligands are potent stimulators of osteoclastogenesis. In the present study, we examine how in vivo administration of the TLR9 ligand CpG-ODN impacts osteoclastogenesis. Mice were injected, either i.p. or i.v., with the TLR9 ligand CpG-ODN. Bone marrow cells were harvested 1–7 days later and an osteoclastogenesis assay was performed. No effect was observed 1 day after injection, but in all other treatments the injection of CpG-ODN increased the number of osteoclasts formed in response to RANKL. Consistent with previous in vitro findings, osteoclast differentiation was greater in cells harvested from BALB/c mice injected with CpG-ODN than in cells from C57BL/6-injected mice. CpG-ODN did not exert any effect when injected to TLR9 knockout mice. The failure to identify conditions under which CpG-ODN inhibits osteoclast differentiation can be explained in two ways. Either, this effect occurs only in vitro, or both inhibition and stimulation of osteoclastogenesis take place in vivo, but the stimulatory effect is more pronounced and, therefore, a net inhibitory effect is not observed. Since the inhibitory effect of CpG-ODN is exerted on early precursors, we injected mice with 5-fluorouracil (5FU), which is known to enrich the bone marrow with early stem cells. Indeed, a reduction in osteoclast differentiation was observed when CpG-ODN was injected into 5FU-treated mice. Thus, TLR9 activation in vivo confirms the effect observed in vitro. The inhibition of osteoclast differentiation in early precursor cells may play a role in reducing the excessive bone loss caused by pathogenic infection and shifting the balance between the bone and immune systems during infection to recruit the immune system.
Disclosures: A. Amcheslavsky, None.
See Sunday Plenary Number S082
CreatinE Kinase Brain Type (CK-B) Involves in the Bone Resorptive Activity of Osteoclast Activity through by Regulating the V-ATPase and Rho Activity.
E. Chang*1, J. Ha*1, F. Oerlemans*2, H. Kim*1, B. Wieringa*2, Z. Lee1, H. Kim1. 1Department of Cell and Developmental Biology, College of Dentistry, BK21 and DR1, Seoul National University, Seoul, Republic of Korea, 2Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, Netherlands Antilles.
Osteoclasts, responsible for bone resorption, differentiate from the hematopoietic precursor cells of the monocyte/macrophage lineage. Receptor activator of nuclear factor-κB ligand (RANKL), in the presence of M-CSF, mediates osteoclast formation and activation through binding to its receptor RANK on osteoclast precursor cells. Some proteins highly expressed in osteoclasts have been suggested as molecular targets for development of anti-bone resorptive drugs. Previously, we identified through a proteomics approach that the brain type cytoplasmic creatine kinase (CK-B) is greatly increased in osteoclasts. In this study, we found that inhibition of CK-B blocked V-ATPase and Rho activity, resulting in the decrease of in vitro bone-resorbing activity of osteoclasts. In vivo experiments with a CK inhibitor also showed a reduced bone resorption in ovariectomized rat model. Using CK-B (-/-) KO and wild-type (WT) mice, we investigated the V-ATPase and Rho activity and in vitro bone-resorbing activity on dentine slice. Compared with that in WT mice, both the V-ATPase and Rho activity was decreased in KO osteoclasts. In aged mice, BMD in femur was significantly more increased in KO than in WT mice. These data reveal that CK-B, up-regulated in osteoclasts, plays an important role for bone resorbing activity of osteoclasts. Therefore, CK-B might be a good target for anti-resorptive drug development.
Disclosures: E. Chang, None.
See Sunday Plenary Number S084
Jagged1-Notch2 Signaling Promotes Osteoclast Differentiation In Vitro.
H. Fukushima1, A. Nakao*1, F. Okamoto1, H. Kajiya1, E. Jimi2, K. Okabe1. 1Physiological Science & Molecular Biology, Fukuoka Dental College, Fukuoka, Japan, 2Molecular Signalling & Biochemistry, Kyusyu Dental College, Fukuoka, Japan.
Notch related genes play a key role in various cell differentiation processes. Expression of Notch receptors in hematopoietic cells and expression of their ligands in bone marrow stromal cells suggests a possible role for Notch signaling in the regulation of osteoclastgenesis. However, the role of this Notch signaling pathway is unclear. Expression of Notch2 is upregulated during osteoclast differentiation, on the other hand, expression of Jagged1 of the Notch ligand family increases in osteoclasts and primary osteoblasts stimulated with osteoclast differentiation factors. To assess the involvement of Notch signaling in osteoclastogenesis, we generated RAW264.7 cells to permit the conditional induction of Notch2 specific shRNA using tetracycline-induced expression. Induction of Notch2 shRNA resulted in suppression of RANKL-induced osteoclast differentiation. Transient transfection of constitutively active intracellular domain of Notch2 in bone marrow macrophages increased NFATc1 activity and osteoclast differentiation. Treatment with Jagged1 activated Notch2 signaling and osteoclastgenesis. The selective γ-secretase inhibitor, L685,458 suppressed Notch signaling activity and RANKL-induced osteoclast differentiation from bone marrow macrophages. Furthermore, the intracellular domain of Notch2 was found to interact with p65 and p50 in NFκB signaling but not c-Fos. RBPJκ, a transcription factor in the downstream Notch signaling pathway, possesses a consensus sequence near the NFκB binding site of NFATc1/P1 promoter. Activation of Notch 2 increased the promoter activity of RANKL-induced NFATc1/P1 in RAW264.7 cells. Co-activation of Notch2 and p65 also activated NFATc1/P1 promoter. These data suggest that Notch2 interacts with Jagged1 and promotes osteoclastogenesis via activation of NFATc1 and NFκB signaling in osteoclast precursor cells.
Disclosures: H. Fukushima, None.
See Sunday Plenary Number S086
Extracellular Matrix Proteins Affect both Osteoclast Formation and Resorptive Activity.
A. Gramoun*1, D. P. Trebec2, N. Azizi*1, J. Sodek*1, M. F. Manolson1. 1Faculty of Dentistry, University of Toronto, Toronto, ON, Canada, 2Department of Biochemistry, University of Toronto, Toronto, ON, Canada.
Tissue destruction in arthritis is associated with bone loss and elevated levels of the extracellular matrix (ECM) proteins in affected joints. Osteoclasts (OCs), the bone resorbing multinucleated cells, are formed by fusion of mononuclear precursors. The ECM proteins, vitronectin (VN), fibronectin (FN) and osteopontin (OPN), all implicated in arthritis, interact with OCs through the integrin αvβ3. To determine the effects of VN, FN and OPN on OC formation and resorptive activity RAW 264.7 (RAW) cells were plated and differentiated (using 100 ng/ml RANKL) on dishes or Osteologic™ slides precoated with 0.01–20 μg/ml FN, VN, and OPN; concentrations shown not to affect cell proliferation. After 96 hours of differentiation, dose response experiments showed that OC number on VN and FN remained significantly lower than those on OPN and the uncoated controls. Measuring tartrate-resistant acid phosphatase (TRAP) activity levels revealed that only VN and OPN attenuated differentiation up to 72 hours. Furthermore, counting the number of mononuclear TRAP+ cells (preOCs) at 48 hours and large OCs (> 10 nuclei) at 96 hours showed that while FN did not affect pre OC number, it significantly decreased OC formation and multinucleation compared to the uncoated control. OPN on the other hand, had the opposite effect. Resorption studies revealed that both FN and OPN increased the total area of resorption and resorptive activity/OC by 40% compared to VN and the uncoated control groups. Further, FN increased the released TRAP/OC in cultures. Nitric oxide levels (as measured by Griess reagent) were significantly elevated by FN but not in any of the other groups. When cells were differentiated for 3 days then incubated with 1 μg/ml of the anti-rat αvβ3 antibody, VNR149, (or its IgG control) for 24 hours, VNR149 significantly increased the total OC number on FN compared to its control by 37% while VN, OPN and the control groups showed a significant decrease in OC numbers. We also observed that OCs on FN had a 1.5 fold increase in αvβ3 cell surface expression compared to uncoated group, as determined by flow cytometry. In conclusion, despite its inhibitory effect on preOC fusion and OC formation, FN significantly enhanced resorption/OC. FN's induction of nitric oxide and the increased αvβ3 expression, could both explain the increased OC activity. VN attenuates both differentiation and preOC fusion thus decreasing OC formation. This results in an overall reduction in total resorption and resorption per OC. Early effects of OPN on osteoclastogenesis are similar to VN. Beyond 48 hours, OPN promotes both the formation and fusion of preOCs resulting in an elevation of resorption.
Disclosures: A. Gramoun. None.
Suppression of Osteoclastogenesis by N,N-dimethyl-D-erythrosphingosine: A Sphingosine Kinase Inhibition-Independent Action.
H. Kim*, Y. Lee*, E. Chang*, H. Kim*, S. Hong*, Z. Lee*, J. Ryu*, J. Ko, H. Kim. Cell and Developmental Biology, College of Dentistry, BK21 and DRI, Seoul National University, Seoul, Republic of Korea.
N,N-dimethyl-D-erythro-sphingosine (DMS) competitively inhibits sphingosine kinase (SPHK) and has been widely used to assess the role of SPHK during cellular events including motility, proliferation, and differentiation. In the present study, the effect of DMS on the differentiation of bone marrow macrophages (BMMs) to osteoclasts was investigated. When the osteoclast precursor cells were treated with DMS, the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis was completely blocked. Surprisingly, however, knock-down of SPHK by siRNA in BMMs did not reduce osteoclastogenesis. Furthermore, both overexpression of SPHK and exogenous addition of sphingosine-1-phosphate, the product of SPHK activity, failed to overcome the anti-osteoclastogenic effect of DMS. These results suggest that DMS inhibited osteoclastogenesis independently of SPHK. Subsequent characterization of the DMS-mediated suppression of osteoclastogenesis revealed that DMS did not affect RANKL-induced activation of JNK, p38, NF-κB, and Ca2+ oscillation. On the other hand, DMS strongly inhibited two separate signaling pathways, the RANKL-induced activation of ERK and Akt, which eventually converged on the transcription factors c-Fos and NFATc1. There was significant increase in the osteoclast formation in the presence of DMS when BMMs were overexpressed with c-Fos, suggesting that c-Fos was a critical downstream target of DMS for the inhibition of osteoclastogenesis. Taken together, our data demonstrate that DMS has an anti-osteoclastogenic function independently of its SPHK inhibitory activity. Considering previously reported anti-cancer properties of DMS, our study may also propose that DMS is an ideal drug candidate for bone metastases, for which osteoclastic bone-resorption is crucial.
Disclosures: H. Kim. None.
PIAS3 Negatively Regulates RANKL-induced Osteoclastogenesis.
T. Hikata1, H. Takaishi*1, M. Matsumoto*2, H. Takavanagi*3, A. Yoshimura*4, H. Asahara*5, Y. Toyama*1. 1Department of Orthopaedic Surgery, School of Medicine, Keio, University, Tokyo, Japan, 2Department of Molecular Biology, Saitama Medical School, Saitama, Japan, 3Department of Cellular Physiological Chemistry, Tokyo Medical Dental University, Tokyo, Japan, 4Devision of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyusyu University, Kyusyu, Japan, 5Department of Innovative Surgery, National Center for Child Health and Development, Tokyo, Japan.
It is known that protein inhibitor of activated STAT3 (PIAS3) not only inhibits the DNA binding activity of STAT3 in the JAK/STAT signaling pathway, but also interacts with MITF that is an important transcription factor for the osteoclast differentiation, and to regulate the gene expression in the downstream. We found that PIAS3 was widely expressed in RAW 264.7, bone marrow-derived monocyte/macrophage lineage cells (BMMs) and mouse primary osteoblasts (POB). To clarify the physiological role of PIAS3 in the bone resorption, we examined the biological effect of PIAS3 on RANKL-induced osteoclastogenesis in the culture systems using hematopoietic osteoclast precursors. Retroviral gene transduction of PIAS3 in RAW264.7 and BMMs drastically inhibited the formation of the multinucleated TRAP-positive osteoclasts. PIAS3 also suppressed the expression of osteoclast differentiation markers such as TRAP and Cathepsin K, and attenuated the protein expression of c-Fos and NFATcl during RANKL-induced osteoclastogenesis. In PIAS3-transduced RAW264.7, the suppression of c-fos and NFATcl DNA-binding activity was observed using ELISA-based screening system for the several transcription factors regulating osteoclast differentiation. To confirm further the functional roles of PIAS3 in osteoclast-specific gene regulation, we performed reporter gene assays using 293T and Cos7 cells. PIAS3 suppressed NFATc1, PU.1, and MITF-induced promoter activity of the TRAP and CathepsinK in a dose dependent manner. PIAS3 also produced dose-dependent inhibition of MITF-induced c-Fos promoter activity. ChIP assays showed that overexpression of PIAS3 in RAW264.7 inhibited MITF binding to the c-Fos promoter region mediated by RANKL treatment as compared to the mock-transduced control. Furthermore, siRNA knockdown of PIAS3 led to accelerated expression of RANKL in the POB stimulated with IL-6 and sIL-6R. Taken together, our results demonstrate that a possible role of PIAS3 as a negative transcriptional regulator in osteoclastogenesis directly and indirectly. These results may offer molecular approaches for the treatment of inflammatory bone destruction.
Disclosures: T. Hikata, None.
See Sunday Plenary Number S090
Osteoclast Differentiation During Medullary Bone Formation Period in Japanese Quail Bone Marrow Cell Culture.
S. Hiyama, E. Yamanishi*, M. Watanabe*, T. Uchida*. Department of Oral Biology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan.
Medullary bone is a unique tissue of female birds, that is formed in the long bone marrow cavity to reserve and provide for calcium of egg-shell. This bone is remodeling during the reproductive period. On the other hand, when male birds are once injected estrogen (E2) into male birds, medullary bone is formed by osteoblasts that are derived by bone lining cells and immediately resorbed by osteoclasts. However, it is not clear why osteoclasts can differentiate rapidly, and can resorb to medullary bone. To address this question, we isolated to bone marrow cells (BMC) from femurs of male Japanese quails that injected E2 at 3 (Day3), 2 (Day2), 1 (Day1), and 0 days (Day0) before slaughter, and cultured with or without RANKL & M-CSF for 4 and 8 days. At culture 4 days, in either of the presence or the absence of RANKL & M-CSF, TRAP-positive mononuclear cells were existed. TRAP-positive multinucleated cells were slightly found in RANKL & M-CSF treated BMCs. TRAP-positive multinucleated cells were increased from culture 4 days to 8 days at BMCs of each Day. However, these cells in the absence of RANKL & M-CSF were small compared with these cells in the presence of RANKL & M-CSF. In Day3 RANKL & M-CSF treated BMCs, there was much number of TRAP-positive multinucleated cells in comparison with other Days BMCs, and these cells were very large. To confirm that these BMCs were expressed the transcription factors, NFATc1 and c-Fos, RT-PCR analysis was carried out. Although c-Fos mRNA was expressed all Days BMCs, non-RANKL & M-CSF treated BMCs were very low expressed and RANKL & M-CSF treated BMCs were high expressed. The expression of NFATc1 mRNA was increased in RANKL & M-CSF treated BMCs during culture, but not expressed in non-treated BMCs. Furthermore, to evaluate the ability to resorb of bone in these cells, we cultured with these cells on the calcium-phosphate coated culture dishes. In the absence of RANKL & M-CSF, resorption area was almost observed to neither BMCs of each Day. However, the area of resorption was recognized in RANKL & M-CSF treated BMCs, especially Day3 BMCs. In addition, bone resorption activity was inhibited by osteoprotegerin, as a decoy receptor of RANKL. Our results suggest that osteoclast precursor cells increase in the bone marrow cavity during medullary bone formation. Moreover osteoclast differentiation may be regulated by osteoblasts that formed medullary bone.
Disclosures: S. Hiyama, None.