The authors have no conflict of interest.
Perturbations in Bone Formation and Resorption in Insulin-Like Growth Factor Binding Protein-3 Transgenic Mice†
Article first published online: 1 OCT 2003
Copyright © 2003 ASBMR
Journal of Bone and Mineral Research
Volume 18, Issue 10, pages 1834–1841, October 2003
How to Cite
Silha, J. V., Mishra, S., Rosen, C. J., Beamer, W. G., Turner, R. T., Powell, D. R. and Murphy, L. J. (2003), Perturbations in Bone Formation and Resorption in Insulin-Like Growth Factor Binding Protein-3 Transgenic Mice. J Bone Miner Res, 18: 1834–1841. doi: 10.1359/jbmr.2003.18.10.1834
- Issue published online: 2 DEC 2009
- Article first published online: 1 OCT 2003
- Manuscript Accepted: 5 MAY 2003
- Manuscript Revised: 10 MAR 2003
- Manuscript Received: 9 DEC 2002
- insulin-like growth binding protein-3;
- transgenic mice
IGF-I and their binding proteins are important in bone health. Examination of BMD, osteoblast proliferation, and markers of bone resorption in transgenic mice that constitutively overexpress IGFBP-3 indicates that overexpression of IGFBP-3 increases osteoclast number and bone resorption, impairs osteoblast proliferation, and has a significant negative effect on bone formation.
Introduction: Low serum insulin-like growth factor I (IGF-I) levels correlate with an increased risk of osteoporotic fractures. Serum IGF-I is largely bound to IGF-binding protein-3 (IGFBP-3), which can inhibit IGF-I action and enhance delivery of IGF-I to tissues. Its role in bone biology is unclear.
Methods: Bone mineral density (BMD), osteoblast proliferation, and markers of bone resorption were examined in transgenic (Tg) mice that constitutively overexpressed human IGFBP-3 cDNA driven by either the cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter.
Results: Cultured calvarial osteoblasts from Tg mice expressed the transgene and grew more slowly than cells from wild-type (Wt) mice, and the mitogenic response to IGF-I was attenuated in osteoblasts from Tg mice. Total volumetric BMD and cortical BMD, measured in the femur using peripheral quantitative computed tomography (pQCT) were significantly reduced in both Tg mouse strains compared with Wt mice. PGKBP-3 Tg mice showed the most marked reduction in bone density. Osteocalcin levels were similar in Wt and CMVBP-3 Tg mice but were significantly reduced in PGKBP-3 Tg mice. Urinary deoxypyridinoline and osteoclast perimeter, markers of bone resorption, were significantly increased in both Tg mouse strains compared with Wt mice. Using double labeling with tetracycline, we demonstrated that pericortical and endocortical mineral apposition rate was significantly reduced in PGKBP-3 Tg mice compared with Wt mice.
Conclusions: These data show that overexpression of IGFBP-3 increases osteoclast number and bone resorption, impairs osteoblast proliferation, and has a significant negative effect on bone formation.
Insulin-like growth factors (IGF)-I and -II are present in plasma and most biological fluids as a complex with IGF binding proteins (IGFBPs). Of the six IGFBPs so far identified, IGFBP-3 is the most abundant in plasma(1) and is abundantly expressed in skeletal tissue.(2) However, the function of IGFBP-3 in vivo is not clear. In vitro experiments examining the effects of IGFBP-3 on various cell cultures have provided conflicting data. Both enhancement and inhibition of IGF-I actions by IGFBP-3 is observable depending on cell types and culture conditions used.(3–5) For example, in human skin fibroblasts, IGFBP-3 either inhibits or potentates IGF-I-induced DNA synthesis.(3) In bovine fibroblasts, IGFBP-3 increased IGF-I stimulated amino acid uptake through enhanced sensitivity of the protein kinase B/AKT pathway.(4) Similarly, in vivo experiments with IGF-I/IGFBP-3 complex have demonstrated enhanced biological activity of this complex compared with IGF-I alone.(5)
In rodents, IGF-I enhances osteoblast proliferation, differentiation, and function.(6–8) In human subjects, low serum IGF-I levels correlate with a higher risk of osteoporotic fractures.(8) On the other hand, systemic administration of IGFBP-4 and -5 increases bone formation as does the administration of IGF-I/IGFBP-3 complex in animals and humans.(9–12) Because we had previously reported that global overexpression of IGFBP-3 resulted in greatly increased serum IGF-I concentrations,(13) we postulated that overexpression of IGFBP-3 might result in a pronounced skeletal phenotype.
MATERIALS AND METHODS
CD-1 mice were used to generate transgenic mice overexpressing human IGFBP-3 as described elsewhere.(13) To ensure that the effects observed were related to overexpression of IGFBP-3 rather than the transgene promoter, we examined two independent transgenic lineages, overexpressing IGF BP-3 ubiquitously under control of the murine PGK promoter or the CMV promoter. Wild-type (Wt), nontransgenic littermates provided control mice of the same genetic background in all experiments.
Male Tg and Wt mice were used for all experiments. The animals were housed in a controlled environment with 11-h light/13-h dark cycles at 22°C with food and water ad libitum. All experiments were performed in accordance with protocols approved by the Animal Care Committee of the Faculty of Medicine, University of Manitoba.
One-day-old mice were killed by decapitation. After removal of the scalp, muscle, and other connective tissue, calvariae were excised from skull and placed into dishes containing serum-free culture media. Calvariae were digested in 2 mg/ml collagenase A (Worthington, NJ, USA) in serum-free DMEM containing 0.1% trypsin and antibiotic-antimycotic mixture (Gibco BRL, Grand Island, NY, USA). Five successive digestions were performed. Each digestion was done in a 15-ml centrifuge tube in 8–10 ml of solution at 37°C with shaking. The first two digestions were discarded, and third, fourth, and fifth digestions were collected and pooled together. Subsequently, cells were washed twice in enzyme-free media and grown in DMEM with 10% FBS and 100 μg/ml ascorbic acid. More than 90% of the cells were positively stained with antibody to osteocalcin.
Cells were seeded at a plating density of 4 × 103/well and cultured for 24 h to allow them to adhere to the plate. After preincubation, culture media were changed with serum-free experimental media supplemented with different concentrations of IGF-I and allowed to grow for 6 days. In between, serum-free-supplemented media were changed every 48 h. On day 6, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 10 μg/100 μl of media) was added, and the cells were incubated for 3 h at 37°C. MTT is a water-soluble tetrazolium salt that is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by mitochondrial dehydrogenases that are only functional in viable cells. At the end of the incubation, the colored tetrazolium product was solubilized in acidic isopropanol, and the OD was read at 540 nm in a spectrophotometer. Data are presented as percentage of controls.
Cell growth curves
Cells were seeded at a density of 2 × 105 cells/35-mm dish and kept in humidified atmosphere of 95% air and 5% CO2 for 24 h to allow adherence to the plate. After 24 h, cells were cultured in DMEM containing 5% FBS, and medium was changed every other day. Cells were trypsinized on days 2, 4, and 6 and counted by hemocytometer.
Serum and conditioned medium (CM) collected from osteoblasts derived from Wt and Tg mice was analyzed on a 11% SDS-PAGE gel. After electrophoretic transfer of the resolved proteins to nitrocellulose, the membranes were blocked in 5% milk, washed in TBST (0.5% Tween-20), and incubated with anti-mouse IGFBP-3 or anti-human IGFBP-3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted to 1:300, for 1 h at room temperature. After washing in TBST, membranes were incubated with secondary antibody-horseradish peroxidase conjugate diluted 1:3000 (Life Technologies Inc.) for 1 h at room temperature. After washing, membranes were analyzed using enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Baie d'Urfé, QC, Canada) and Kodak BioMax X-ray film (Eastman Kodak Co., Rochester, NY, USA).
Bone mineral density measurements
Isolated femurs were prepared from 16-week-old mice, an age at which peak adult bone mineral density (BMD) is achieved.(14) Bone were stored in 75% ethanol before analysis. Peripheral quantitative computed tomography (pQCT; Stratec XTC 960M; Norland Medical Systems, Ft. Atkinson, WI, USA) was used to assess BMD as previously described.(14) Briefly, thresholds of 2000 differentiated high-density cortical bone from bone of lower density. Isolated femurs were scanned at 2-mm intervals over their entire length. Total vBMD was calculated by dividing the total mineral content by the total bone volume. Femoral cortical bone density volume and thickness were compared at three consecutive mid-diaphyseal cross-sections. μCT imaging was performed on the distal femur as previously described.(15)
Histomorphometric procedures were performed using a semiautomated image analysis system (OsteoMetrics, Inc., Atlanta, GA, USA) in 7- to 9-week-old mice during the period of maximal bone formation.(14) Tetracycline and demeclocycline were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). Tetracycline (0.2 mg in 0.2 ml of sterile saline) was injected intraperitoneally to 49-day-old mice. Demeclocycline (0.2 mg in 0.2 ml of sterile saline) was injected intraperitoneally to 62-day-old mice. Tibias were collected 24 h later, fixed overnight in neutral buffered formalin at 4°C, and stored in 70% ethanol. Isolated bones were dehydrated in a series of increasing concentrations of ethanol, embedded without demineralization in a mixture of methylmethacrylate/2-hydroxymethylmethacrylate (12.5:1) to retain fluorochrome labeling. Cortical and cancellous bone measurements were performed as previously described.(14) The cancellous bone perimeter lined by osteoclasts was measured and expressed as a percent. Histochemical demonstration of acid phosphatase activity in tissue sections was performed using the method of Liu et al.(16) Osteoclasts were identified as acid phosphatase positive, multinucleated cells lining bone surface. These cells usually had other characteristics of osteoclasts, including a foamy cytoplasm and location in a pronounced lacuna.
Nonfasted, 9-week-old mice were anesthetized with 2.5% Avertin, and blood was collected from the retro-orbital sinus using heparinized capillaries between 9:00 and 11:00 a.m. Samples were stored at −70°C until analysis. Plasma osteocalcin was measured using a mouse osteocalcin RIA kit manufactured by Biomedical Technologies Inc. (Stoughton, MA, USA). The sensitivity of the assay was 4 ng/ml, and the intra-assay CV was <10%.
For measurement of urinary free deoxypyridinoline (Dpd), random urine samples were collected from 9-week-old mice and stored at −70°C. All samples were analyzed in a single assay in duplicate using an enzyme immunoassay supplied by Metra Biosystems, Inc. (Mountain View, CA, USA). The intra-assay CV and the sensitivity of the assay were 4.8% and 1.1 nM, respectively. Results are expressed as the ratio of the Dpd (nM) to urine creatinine (mM) to correct for variations in urine concentration. Creatinine in urine was measured in duplicates with colorimetric kit purchased from Sigma-Aldrich Canada Ltd.
Data are expressed as the mean ± SE. Student's t-test was used for single comparisons between Tg and Wt mice. For determining statistical differences between multiple groups, an ANOVA with repeated measures followed by Dunnett's t-test was used.
Cultured calvarial osteoblasts from Tg mice grew less rapidly than cells from Wt mice (Fig. 1). Osteoblasts from the PGKBP-3 Tg mice were more slowly growing than osteoblasts from CMVBP-3 Tg mice. In addition, the proliferative response of osteoblasts from Tg mice to exogenous IGF-I was attenuated compared with osteoblasts from Wt mice (Fig. 2). Cells from PGKBP-3 Tg mice were less responsive than cells from CMVBP-3 Tg mice.
Western blots of CM using antibody against human IGFBP-3 indicated that osteoblasts from both PGKBP-3 and CMVBP-3 Tg mice expressed the transgene. Slightly higher levels of transgene expression were consistently observed in CM from PGKBP-3 cells (Fig. 3). This antibody did not detect mouse IGFBP-3 present in serum from Wt mice but detected the transgene in serum from PGKBP-3 Tg mice. Using antibody against mouse IGFBP-3, no IGFBP-3 was detectable in CM from Wt mice. A small amount of IGFBP-3 was detectable in CM from CMVBP-3 and PGKBP-3 osteoblasts, possibly as a result of cross-reaction with human IGFBP-3 present in CM from osteoblasts derived from the Tg mice.
Consistent with our previously reported growth curves for the PGKBP-3 and CMVBP-3 Tg mice,(13) body weight in 16-week-old PGKBP-3 Tg mice was significantly less that Wt mice (35.0 ± 0.9 versus 38.6 ± 0.4, n = 13, p = 0.02). CMVBP-3 Tg mice that have reduced birth weight and growth retardation in early life but later show catch-up growth(13) were slightly heavier than Wt mice at 16 weeks (40.7 ± 1.6 versus 38.6 ± 0.9, n = 13, p = not significant).
Femoral length was measured using hand calipers in 16-week-old male mice from each of the groups of mice. Femoral length was similar in PGKBP-3 Tg and Wt mice (16.29 ± 0.07 versus 16.47 ± 0.13 mm, n = 8, p = 0.26) but was significantly increased in CMVBP-3 Tg mice (17.51 ± 0.11 versus 16.47 ± 0.13 mm, n = 8, p < 0.001). Total and cortical bone density was measured at three points in the mid-femoral diaphysis. Both parameters were significantly reduced in both Tg mouse strains compared with Wt mice. PGKBP-3 Tg mice showed the most marked reduction (Fig. 4). Cortical bone volume was also significantly reduced in PGKBP-3 Tg mice compared with Wt mice (1.55 ± 0.5 versus 1.71 ± 0.5 mm3, p = 0.019). In CMVBP-3 Tg mice, the reduction in cortical bone volume did not achieve statistical significance. Mid-diaphysis cortical bone thickness was also significantly reduced in PGKBP-3 Tg mice compared with Wt mice (p = 0.021). Whereas cortical bone from PGKBP-3 Tg mice was less dense and thinner, trabecular architecture, as measured by μCT in the distal part of the femur, were similar in Tg and PGKBP-3 Wt mice (Table 1). In CMVBP-3 Tg mice, relative cancellous bone volume (BV/TV) and trabecular thickness were significantly reduced compared with Wt mice.
Bone histomorphometry combined with double tetracycline labeling was undertaken to determine the bone formation rate in the tibia of Tg and Wt mice. Cortical area in the tibia was significantly reduced in PGKBP-3 Tg mice (0.616 ± 0.013 versus 0.698 ± 0.015 μm, n = 8, p = 0.002) but similar in CMVBP-3 and WT mice. Both periosteal and endocortical bone formation rate were reduced in PGKBP-3 Tg mice compared with Wt mice, but this difference only achieved statistical significance for periosteal bone formation rate (Fig. 5). In CMVBP-3 Tg mice, the periosteal bone formation rate was similar to that observed in Wt mice, and endocortical bone formation rate was modestly increased. There was no significant difference between Wt and CMVBP-3 Tg mice in terms of periosteal or endocortical mineral apposition rates (Fig. 5). However, both periosteal and endocortical mineral apposition rates were significantly reduced in PGKBP-3 Tg mice.
Cancellous bone formation was examined using bone histomorphometry in the proximal tibia at ∼9 weeks of age at the time of peak bone formation (Table 2). Cancellous bone volume was reduced in both PGKBP-3 and CMVBP-3 Tg mice; however, the differences between the Tg and Wt mice did not achieve significance (p = 0.066 and p = 0.089, respectively). Trabecular number and thickness was reduced in PGKBP-3 Tg mice compared with Wt mice, and trabecular separation was increased (Fig. 6). A similar trend but of lesser magnitude was seen in CMVBP-3 Tg mice (Table 2). Cancellous bone formation rate expressed in terms of trabecular surface and bone mineral apposition rate was not statistically different between Tg and Wt mice. Osteoclast perimeter was dramatically increased in both PGKBP-3 and CMVBP-3 transgenic mice (Table 2).
Serum osteocalcin was measured by radioimmunoassay (RIA) in 9-week-old male mice. Osteocalcin levels were similar in Wt and CMVBP-3 Tg mice but were significantly reduced in PGKBP-3 Tg mice (Table 3). Urinary deoxypyridinoline, a marker of bone resorption, was significantly increased in both PGKBP-3 and CMVBP-3 Tg mice compared with Wt mice.
We have demonstrated that overexpression of IGFBP-3 results in a reduction in BMD using two different transgenic mouse strains. Significant effects were observed in both strains of IGFBP-3 mice but were less marked in CMVBP-3 mice. We attribute the reduction in bone mass in IGFBP-3 Tg mice directly to inhibition of IGF-I action in skeletal tissue. This is supported by our in vitro findings of a reduced mitogenic response of osteoblasts from the Tg mice to IGF-I and reduced proliferation rate of osteoblasts from Tg mice in culture medium containing fetal calf serum. This inhibition was more marked in osteoblasts from PGKBP-3 mice than those from CMVBP-3 mice and paralleled the increased abundance of IGFBP-3 in the CM from PGKBP-3 cells. These data are complementary to an earlier report that targeted overexpression of IGF-I to osteoblasts increases bone volume and mass.(15)
The deleterious effect on femoral BMD was apparent in both Tg strains, although it was most marked in the PGKBP-3 Tg mouse. In the proximal tibia, periosteal bone formation rate but not trabecular bone formation rate was reduced in PGKBP-3 Tg mice. Neither periosteal bone formation rate nor trabecular bone formation rate was reduced in the tibia of CMVBP-3 Tg mice. Interestingly, although both Tg strains are smaller at birth than Wt mice, CMVBP-3 mice show catch-up growth, and by 6–12 weeks of age, there was no significant difference in body length between CMVBP-3 Tg and Wt mice.(13) In contrast, PGKBP-3 Tg mice remained lighter and shorter than Wt mice. In addition to the catch-up growth, CMVBP-3 mice consumed more food and were more obese than PGFBP-3 mice.
Several lines of evidence suggest that accelerated or compensatory growth may lead to some unique skeletal features in CMVBP-3 mice. First, CMVBP-3 mice had longer femur lengths than Wt or PGFBP-3 mice. Second, we found an increase in endocortical bone formation and mineral apposition rate in the CMVBP-3 mice compared with Wt, while periosteal bone formation rates did not vary among strains. Third, serum osteocalcin levels were similar in CMVBP-3 and Wt compared with suppressed values in the PGKBP-3 animals. These findings imply that there is a marked increase in endocortical bone turnover in the CMVBP-3 Tg strain compared with PGKBP-3 mice. However, it appears that this acceleration in remodeling is somewhat unbalanced such that formation is only increased on the endocortical surface, and the overall rate of resorption exceeds that of formation. The cause for this catch-up phenomena in skeletal remodeling is not evident from this study, although osteoblast expression of the transgene is lower in CMVBP-3 mice than PGKBP-3. In fact, this response may be important and represent a critical compensatory mechanism related to excess skeletal IGFBP-3. However, irrespective of the underlying cause, uncoupling, if persistent, could in part explain the reduced BMD in light of increased bone turnover.
IGF-I has been shown to enhance BMD in ovariectomized rats.(15,17–19) An anabolic effect of IGF-I in bone has also been demonstrated in transgenic mice that overexpress IGF-I in osteoblasts under the human osteocalcin promoter.(17) Interestingly in this animal model, the increase in the rate of bone formation was not caused by enhanced osteoblast proliferation,(17) although other reports indicate that IGF-I enhances both osteoblast growth and differentiation in other circumstances.(6)
The observed increase in urinary deoxypyridinoline and osteoclast perimeter in both Tg strains indicate that overexpression of IGFBP-3 results in increased bone resorption. Bone formation is generally coupled to the prevailing level of bone resorption. Failure to observe a corresponding increase in bone formation suggests that IGFBP-3/IGF-I complex plays a role in coupling of bone resorption and formation.
While IGF-I seems to have stimulatory effects on bone mass, the role of the binding proteins is unclear. Human osteoblasts express all six of the IGFBPs, with IGFBP-3, -4, and -5 being the most abundant.(20) We were, however, unable to confirm using immunoblotting that osteoblasts from Wt mice express IGFBP-3. This may reflect the relative lack of sensitivity of this technique in our hands rather than a species difference in IGFBP-3 expression in osteoblasts. Factors such as transforming growth factor-beta and 1,25-dihydroxyvitamin D3, which regulate proliferation and differentiation of osteoblast precursors, enhance the expression of IGFBP-3.(21,22)
The systemic administration of the IGFBP-3/IGF-I complex increases bone formation in ovariectomized rats.(11,12) Similarly, in human osteoporotic subjects, the IGFBP-3/IGF-I complex increased bone mineral mass.(23) In mice, systemic administration of IGFBP-4 and IGFBP-5 increased bone formation as assessed by serum markers.(9,10) In the case of IGFBP-4 administration, the effect on bone formation was not seen with a protease-resistant IGFBP-4 mutant, and thus, the effect was considered to be caused by enhanced bioavailability of IGF-I to skeletal tissue. Interestingly, local administration of IGFBP-4 actually inhibited IGF-I-induced bone formation.(24)
Epidemiological studies have demonstrated a strong association between low serum IGF-I levels and osteoporotic fractures in healthy postmenopausal women.(8) Only a weak association was evident between serum IGF-I levels and BMD, suggesting that the association between low IGF-I and fracture was independent of BMD and nutritional status.(8) In the same study, no association was found with plasma IGFBP-3 levels and osteoporotic fractures. However, an association between femoral neck BMD and circulating IGFBP-3 concentrations has recently been reported in Swedish men.(25)
IGFBP-3 is the most abundant of the IGF binding proteins in plasma(1) and is thought to be important in the transport and modulation of the biological actions of the IGFs. Both inhibition and potentiation of IGF-I actions are demonstrable in vitro and in vivo depending on the experimental conditions and whether it is administered with IGF-I. In the IGFBP-3 Tg mice reported here, there is ubiquitous expression of the transgene and the predominant effect seems to be inhibition of IGF action as manifested by the growth retardation apparent at birth in both strains of Tg mice.(13) As noted previously, postnatal growth retardation was less marked in CMVBP-3 Tg mice than PGKBP-3 mice despite similar circulating IGFBP-3 levels and tissue IGFBP-3 mRNA abundance.(13) And indeed, by 9 weeks of age, growth rates accelerated in the CMVBP-3 mice. Although the mechanism for this catch-up remains unclear, factors such as hypernutrition, increased body fat, or higher serum leptin might contribute to this response.
It is of interest that the reduction in BMD was observed in IGFBP-3 Tg mice despite enhanced circulating IGF-I levels.(13) Because systemic administration of the IGF-I/IGFBP-3 complex has been previously shown to stimulate bone formation,(11,12,23) our observations in IGFBP-3 Tg mice suggests that enhanced levels of circulating levels of IGF-I/IGFBP-3 complex are unable to stimulate bone formation in the presence of increased osteoblast IGFBP-3 expression.
In summary, the data reported here provide compelling evidence that IGFBP-3 overexpression reduces bone formation and increases bone resorption in the mouse. These results point to a major role for the IGF-I regulatory system in murine bone acquisition and are consistent with other transgenic and conditional mutagenic studies of IGF-I and the IGF type 1 receptor.
This research was supported by grants from CIHR (LJM), US PHS NIAMS (ARA45433 to CJR), and National Institutes of Health (DR38773 to DRP, AA11140 to RTT). JVS is a recipient of a Canadian Diabetes Association Postdoctoral Fellowship. LJM is a recipient of a CIHR Senior Scientist award and an endowed Research Professorship in Metabolic Diseases.
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