The authors state that they have no conflicts of interest.
IGF-I Receptor Is Required for the Anabolic Actions of Parathyroid Hormone on Bone†
Article first published online: 29 MAY 2007
Copyright © 2007 ASBMR
Journal of Bone and Mineral Research
Volume 22, Issue 9, pages 1329–1337, September 2007
How to Cite
Wang, Y., Nishida, S., Boudignon, B. M., Burghardt, A., Elalieh, H. Z., Hamilton, M. M., Majumdar, S., Halloran, B. P., Clemens, T. L. and Bikle, D. D. (2007), IGF-I Receptor Is Required for the Anabolic Actions of Parathyroid Hormone on Bone. J Bone Miner Res, 22: 1329–1337. doi: 10.1359/jbmr.070517
- Issue published online: 4 DEC 2009
- Article first published online: 29 MAY 2007
- Manuscript Accepted: 22 MAY 2007
- Manuscript Revised: 27 APR 2007
- Manuscript Received: 6 JUL 2006
- IGF-I signaling;
- bone formation rate
We showed that the IGF-IR–null mutation in mature osteoblasts leads to less bone and decreased periosteal bone formation and impaired the stimulatory effects of PTH on osteoprogenitor cell proliferation and differentiation.
Introduction: This study was carried out to examine the role of IGF-I signaling in mediating the actions of PTH on bone.
Materials and Methods: Three-month-old mice with an osteoblast-specific IGF-I receptor null mutation (IGF-IR OBKO) and their normal littermates were treated with vehicle or PTH (80 μg/kg body weight/d for 2 wk). Structural measurements of the proximal and midshaft of the tibia were made by μCT. Trabecular and cortical bone formation was measured by bone histomorphometry. Bone marrow stromal cells (BMSCs) were obtained to assess the effects of PTH on osteoprogenitor number and differentiation.
Results: The fat-free weight of bone normalized to body weight (FFW/BW), bone volume (BV/TV), and cortical thickness (C.Th) in both proximal tibia and shaft were all less in the IGF-IR OBKO mice compared with controls. PTH decreased FFW/BW of the proximal tibia more substantially in controls than in IGF-IR OBKO mice. The increase in C.Th after PTH in the proximal tibia was comparable in both control and IGF-IR OBKO mice. Although trabecular and periosteal bone formation was markedly lower in the IGF-IR OBKO mice than in the control mice, endosteal bone formation was comparable in control and IGF-IR OBKO mice. PTH stimulated endosteal bone formation only in the control animals. Compared with BMSCs from control mice, BMSCs from IGF-IR OBKO mice showed equal alkaline phosphatase (ALP)+ colonies on day 14, but fewer mineralized nodules on day 28. Administration of PTH increased the number of ALP+ colonies and mineralized nodules on days 14 and 28 in BMSCs from control mice, but not in BMSCs from IGF-IR OBKO mice.
Conclusions: Our results indicate that the IGF-IR null mutation in mature osteoblasts leads to less bone and decreased bone formation, in part because of the requirement for the IGF-IR in mature osteoblasts to enable PTH to stimulate osteoprogenitor cell proliferation and differentiation.
PTH is a major regulator of calcium and phosphate homeostasis. Although it has been well established that PTH exerts both anabolic(1,2) and catabolic(3,4) actions on bone in animals and humans, the mechanism is unclear. The bone marrow stromal cells (BMSCs)/osteoblasts have been shown to be the primary target cells for actions of PTH on bone. PTH binds to a seven membrane-spanning G protein–coupled receptor (PTH/PTHrP receptor) on the surface of stromal cells/osteoblasts and activates both the cAMP protein kinase A (PKA) and phosphoinositide protein kinase C (PKC) pathways. When given intermittently, PTH increases bone mass and strength by increasing the number(5) or function of osteoblasts(6) and by inhibiting osteoblast apoptosis(7,8) (anabolic effect). Continuous infusion of PTH leads to bone loss by stimulating a net increase in bone resorption(3,4) through an increase in the gene expression ratio of RANKL/osteoprotegerin (OPG) (catabolic effect). These effects are both direct actions of PTH on osteoblast lineage cells and indirect through modulation of local growth factor production. In particular, IGF-I has been shown to be an important mediator for the anabolic actions of PTH on bone.(9,10)
Previous studies indicated that the IGF-I system plays a key role in bone metabolism. It acts in diverse patterns, through a combination of endocrine, autocrine, and paracrine modes of action, to regulate the functions of both osteoblasts and osteoclasts.(11–13) Signaling through the IGF-I receptor (IGF-IR), which is expressed in bone cells, not only promotes osteoblast proliferation and/or differentiation but also mediates anti-apoptotic actions.(14,15) PTH stimulates IGF-I production by bone cells, and antibodies to IGF-I can block the actions of PTH on bone. When PTH was administered to global IGF-I knockout mice, anabolic actions of PTH on bone were suppressed.(9,10) Although all of these previous studies indicated the importance of IGF-I in mediating the anabolic actions of PTH, the mechanisms by which IGF-I mediates the actions of PTH on bone and the function of IGF-IR in mediating these actions remain poorly understood. Because of complex relationships in the signaling processes of IGF-I regulation,(16,17) the complex effects of PTH on bone, and the poor survival rate of global IGF-I–null mice,(18) it has been difficult to define individual in vivo aspects of the skeletal actions of IGF-I as a mediator of bone actions of PTH. Compared with mice with global IGF-I–null mutations(18) and global IGF-I receptor (IGF-IR)–null mutations,(19) mice with IGF-IR–null mutations specifically in osteoblasts(14) have a normal survival. As such, these mice provide us with a new tool to study the role of IGF-I signaling in mediating the skeletal actions of PTH.
MATERIALS AND METHODS
IGF-IR OBKO mice were generated by crossing homozygous conditional mutants carrying modified IGF-IR alleles (with loxP sites flanking exon3)(20) with mice carrying the cre recombinase transgene under the control of an osteocalcin promoter.(14) These mice were normal in survival rate, body size, and weight. Twelve-week-old control mice and mice with an osteoblast-specific IGF-IR–null mutation were treated with either PTH [lsqb]PTH(1-34), rat; Bachem[rsqb] 80 μg/kg body weight (BW) or vehicle (150 mM NaCl, 1 mM 1 N HCl, and 2% heat-inactivated FBS dissolved in distilled water) everyday by subcutaneous injection for 2 wk. Each group contained equal numbers of male and female mice. To determine both trabecular and cortical bone formation during the period of PTH administration, the mice were injected subcutaneously with fluorochrome (each 15 mg/kg) on day 0 (the day PTH administration was started) (calcein), day 6 (demeclocycline), and day 12 (calcein) of PTH administration to label the mineralization fronts of the bone. Two days later, the mice were weighed and killed. The right tibias were obtained for fat-free weight, μCT, cortical bone mineral appositional rate (MAR), and bone formation rate (BFR) at the tibiofibular junction (TFJ). The right distal femurs were obtained for trabecular bone histomorphometry. To determine the effect of PTH on BMSC differentiation and mRNA levels of bone markers, the mice were killed 1 h after the last PTH treatment. Left tibias and femurs were obtained for mRNA determinations. BMSCs from left tibias and femurs were collected individually for cell culture. These studies were approved by the Animal Use Committee of the San Francisco Veterans Affairs Medical Center where the animals were raised and studied.
The tibias were cleaned of adherent tissue, extracted in ethanol and diethyl ether using a Soxhlet apparatus (Fisher Scientific, Pittsburgh, PA, USA), dried at 100°C overnight, and weighed.
The tibias were analyzed with a Scanco Medical AG μCT apparatus (Scanco Medical, Basserdorf, Switzerland) as previously described.(9,21,22) 3D information was obtained by stacking successively measured slices on top of each other. The voxel size was 9 μm in all three spatial dimensions. One hundred twenty-eight slices were measured in each sample, covering a total of 1.15 mm of the metaphysis. To analyze only cancellous fractions of the tibias, the compact part of the bone was masked out in the following way: with a 3D box-shaped low-pass filter applied to the original gray-scale CT images, an artificial partial volume effect was created, which blurs out the individual trabeculae but leaves the dense compact shell intact. The cortex mask was extracted with a simple thresholding operation with a fixed threshold of 20.0% of the maximal grayscale value. To analyze the cortex, a low-pass Gaussian filter (s = 3.0) was applied to the original images, with a threshold of 10.0%. The axial position and extent are identical to the trabecular volume. The trabecular region was masked out, and a 3D component labeling of the cortex was performed additionally to extract only the main connected component. The cortex was evaluated with the direct distance transformation method to calculate its thickness. Bone volume (BV) and bone surface (BS) were calculated using a tetrahedron meshing technique generated with the marching cubes method. Total volume (TV) was calculated from the volume of the conforming voxels of interest (VOIs).
Trabecular bone: Right distal femurs were dehydrated in graded ethanol solutions and xylene and embedded undecalcified in modified methyl methacrylate.(23) These bone samples were sectioned longitudinally with a Leica RM 2165 microtome at indicated thicknesses of 8–10 μm. The 8-μm sections were stained with modified Goldner trichrome protocol to determine trabecular bone volume, whereas the 10-μm sections remained unstained for dynamic measurements of bone formation by assessing the labeling by demeclocycline and the second calcein injection (6-day interval).
Histomorphometric data were collected using the Bioquant Bone Morphometry System (Nashville, TN, USA) and reported according to standard bone histomorphometry nomenclature.(24)
Cortical bone: Diaphyseal segments of the right tibias were dehydrated, defatted in acetone followed by ether, and embedded in bioplastic (Tap Plastics, Dublin, CA, USA). After polymerizing overnight, the blocks were sectioned at a thickness of 60 μm using a Leica SP 1600 circular bone saw (Leica, Deerfield, IL, USA). The section containing the TFJ was digitized with a Hamamatsu video camera (Carl Zeiss, Thornwood, NY, USA) coupled to a Leica DMR microscope, and periosteal and endosteal MAR and BFR were determined using the NIH Image program (BFR and MAR were measured by assessing the two calcein labels (12-day interval).
The left tibial and femoral BMSCs were harvested using techniques previously described.(25) Briefly, the tibias and femurs were collected, the soft tissue, and the epiphyses of each bone were removed. The bone marrow was flushed from the diaphysis with a syringe and a 26-gauge needle. The marrow from each individual mouse was collected in primary culture medium (α-MEM; containing l-glutamine and nucleosides; Mediatech, Herndon, VA, USA), supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA, USA), 100 U/ml penicillin/streptomycin (Mediatech), and 0.25 μg/ml fungizone (Life Technologies, Rockville, MD, USA). A single-cell suspension was obtained by repeated passage through an 18-gauge needle. A pool of BMSCs was made from the tibia and femur of each mouse. The cells were plated at 10 × 106 cells/dish in 10-cm dishes. Nonadherent cells were removed by aspiration, and the primary medium was replenished on day 5. On day 7, the cells were provided with secondary medium (the primary medium with 3 mμ β-glycerophosphate and 50 μg/ml ascorbic acid; Sigma, St Louis, MO, USA). Subsequent medium changes were performed every 2 days for up to 28 days.
Determination of alkaline phosphatase–positive colony number and calcium nodule formation
BMSCs were grown in 10-cm dishes for up to 28 days. At day 14 of culture, cells were assessed using a commercial kit (diagnostics kit, procedure no. 86; Sigma) to determine alkaline phosphatase (ALP)+ colony-forming units (CFUs) following the manufacturer's instructions. At day 28 of culture, calcium nodules were stained with Alizarin red (AR; Sigma) for 10 min, the stain was aspirated, and the dishes were rinsed five times by distilled water to remove loosely bound stain. The number of mineralized nodules was determined, and the specifically bound stain was eluted with a solution of 0.5 N HCl/5% SDS and quantitated in a spectrophotometer at 415 nm.
Quantitative real-time PCR
RNA was extracted from the long bones (bone marrow flushed out) of control and IGF-IR OBKO mice. mRNA levels of osteoblast differentiation markers were determined by quantitative real-time PCR as previously described.(9) Primers and probes are as follows: GAPDH (forward: 5′-TGCACCACCAACTGCTTAG-3′; reverse: 5′- GGATGCAGGGATGATGTTC-3′; probe: 5′-CAGAAGACTGTGGATGGCCCCTC-3′), RUNX2 (forward:5′- GGCTCTGGCGTTTAAATGGTT-3′; reverse: 5′-GTGCCCTCTGTTGTAAATACTGCTT-3′; probe: 5′-CCACCGAGACCAACCGAGTCATTTAAGG-3′), ALP (forward: 5′-TCCTGACCAAAAACCTCAAAGG-3′; reverse: 5′-TGCTTCATGCAGAGCCTGC-3′; probe: 5′- CTGGTGGAAGGAGGCAGGATTGACC-3′), osteocalcin (forward: 5′-CTCACAGATGCCAAGCCCA-3; reverse: 5′-CAAGGTAGCGCCGGAGTCT-3′; probe: 5′- CCCTGAGTCTCTGACAAAGCCTTCATGTCCA-3′), and RANKL (forward: 5′- GGCCACAGCGCTTCTCAG-3′; reverse: 5′-GAGTGACTTTATGGGAACCCGAT-3′; probe: 5′-CAGCTATGATGGAAGGCTCATGGTTGGA-3′).
Data are presented as mean ± SD. All data were analyzed using two-factor (genotype and treatment) ANOVAs followed by a posthoc Fisher's protected least significant difference (PLSD) test with a SuperANOVA program. Statistical significance was stated for p < 0.05.
The fat-free weights of tibias (normalized by body weight, FFW/BW) are shown in Fig. 1. By one-way ANOVA analysis, in vehicle-treated mice, FFW/BW of IGF-IR OBKO mice (1.21 ± 0.11 mg/g) was significantly lower than FFW/BW of control mice (1.44 ± 0.12 mg/g; p = 0.001). PTH significantly decreased FFW/BW by 10% in control mice (1.32 ± 0.13 mg/g; p = 0.02) but had less effect in IGF-IR OBKO mice (1.12 ± 0.09 mg/g; p = 0.06). Two-way ANOVA revealed a significant PTH treatment-IGF-IR gene mutation interaction (p < 0.05).
Bone structure: proximal tibia
μCT was used to evaluate the effects of PTH on bone structure of control and IGF-IR OBKO. The field of interest was the secondary spongiosa. The results are shown in Fig. 2. In IGF-IR OBKO mice, cortical bone thickness (C.Th) was significantly less than in control mice (Fig. 2B). PTH significantly decreased bone volume (BV, 30%; Fig. 2A) and increased C.Th (Fig. 2B) in control mice, but induced no significant changes in IGF-IR OBKO mice, although the trend was comparable.
Trabecular bone at primary and secondary spongiosa: We first determined whether the IGF-IR is required for PTH stimulation of bone formation in trabecular bone. As shown in Fig. 3, trabecular bone volume (BV/TV) was significantly decreased (33%, p < 0.05) in the primary spongiosa of the IGF-IR OBKO mice (Figs. 3A3 and 3B) than in the control mice (Figs. 3A1 and 3B). PTH stimulated BV/TV (50%, p < 0.05) in the primary spongiosa of the control mice (Figs. 3A2 and 3B), but had less effect in the IGF-IR OBKO mice (Figs. 3A4 and 3B). On the other hand, at the secondary spongiosa, PTH showed no effects on BV/TV (Fig. 3C) in either control or IGF-IR OBKO mice. In the secondary spongiosa, both single-labeled surfaces (sLS) and double-labeled surfaces (dLS) tended to be lower in the IGF-IR OBKO (sLS: 653.4 ± 70.6 μm; dLS: 326.7 ± 35.3 μm) compared with the control mice (sLS: 962.0 ± 102.2 μm; dLS: 960.1 ± 202.6 μm). In the control mice, PTH showed no significant effects on sLS (973.6 ± 184.6 μm) or dLS (1313.6 ± 254.3 μm). In IGF-IR OBKO mice, PTH increased dLS (1112.1 ± 190.2 μm), but induced no changes on sLS (983.6 ± 209.8 μm). Trabecular bone formation rate/bone surface (BFR/BS) in the IGF-IR OBKO mice was 48% (p < 0.05) lower than that in the control mice (Fig. 3D). PTH stimulated BFR/BS by 114% (p < 0.05) in control mice and 85% (p < 0.05) in IGF-IR OBKO mice (Fig. 3D), although the absolute increase in BFR/BS in the IGF-IR KO mice was substantially less than controls owing to the lower basal levels of BFR/BS. There were no differences in the MAR between the control mice and the IGF-IR OBKO mice. PTH increased MAR by 52% (p < 0.05) in control mice but had no effect in the IGF-IR OBKO mice (Fig. 3E).
Cortical bone at tibiofibular junctions: We determined whether the IGF-I receptor is needed for PTH stimulation of bone formation at the TFJ by bone histomorphometry. The results are shown in Fig. 4. Periosteal BFR (Fig. 4A) and MAR (Fig. 4B) at the TFJ were markedly lower in the IGF-IR OBKO mice than in the control mice. PTH treatment increased periosteal BFR (40%, p = 0.058; Fig. 4A) and MAR (40%, p = 0.058; Fig. 4B) at the TFJ in control mice, but not in IGF-IR OBKO mice. Endosteal BFR (Fig. 4C) and MAR (Fig. 4D) were comparable in control and IGF-IR OBKO mice, but PTH stimulated endosteal BFR (76%) and MAR (76%) only in the control animals (p < 0.05; Figs. 4C and 4D).
Osteoblast progenitor proliferation and mineralization
On day 14 of BMSC culture (Fig. 5A), the numbers of ALP+ colonies formed in the cultures from the IGF-IR OBKO mice (122 ± 12) and the control mice (124 ± 21) were equivalent. However, PTH administration in vivo significantly increased the number of ALP+ colonies (160 ± 24) in BMSC cultures subsequently obtained from control mice (129% of vehicle treated control mice, p < 0.05; Fig. 5A), but induced no changes in that from IGF-IR OBKO mice. On day 28 of BMSC culture (Fig. 5B), the number of mineralized nodules in the cultures from the IGF-IR OBKO mice (100 ± 9) was significantly less (p < 0.05) than that observed in the cultures from the control mice (164 ± 19; Fig. 5B). This progressive loss of differentiation capacity in the IGF-IR OBKO was accompanied by a progressive loss of IGF-IR expression in vitro. On day 14 of BMSC culture, the mRNA level of IGF-IR in the IGF-IR OBKO cells was 75%, whereas on day 28 of BMSC culture, it decreased to 38% of the control cells (data not shown). The mRNA levels of IGF-IR in the control cells remained constant between days 14 and 28 (data not shown). Administration of PTH in vivo increased the number of mineralized nodules 1.5-fold in BMSC cultures subsequently obtained from control mice (from 164 ± 19 to 246 ± 8, p < 0.05), but not in BMSC cultures from IGF-IR OBKO mice (Fig. 5B; from 100 ± 9 to 122 ± 12). Two-way ANOVA showed a significant interaction between PTH and IGF-IR genotype on the number of mineralized nodules (p = 0.03).
mRNA levels of bone markers
The mRNA levels of selected bone markers in extracts of intact bone were determined by quantitative real-time PCR. The results are shown in Table 1. These values are expressed as a percentage of GAPDH expression. The mRNA levels of IGF-I were significantly higher in the IGF-IR OBKO mice than in the control mice. PTH significantly increased the mRNA levels of IGF-I (1.5-fold) in the control mice but induced no changes in the IGF-IRKO mice. The mRNA levels of RUNX2, ALP, and RANKL were equivalent in the control mice and the IGF-IR OBKO mice, but the mRNA levels of osteocalcin were significantly lower in the IGF-IR OBKO mice than in control mice. PTH significantly increased the mRNA levels of RUNX2, ALP, osteocalcin, and RANKL in the control mice (4.9-, 5.5-, 5.7-, and 23.7-fold, respectively), but not in IGF-IR OBKO mice. The interactions between PTH and IGF-IR genotype on the expression of these genes were significant (two-way ANOVA).Table 1.
PTH has complex effects on bone, not all of which are direct. IGF-I is an attractive candidate as a mediator for at least some of these actions. In this study, we determined the effects of PTH on a mouse model in which the floxed type I IGF-IR gene was selectively deleted in mature osteoblasts using an osteocalcin promoter driven cre-recombinase (IGF-IR OBKO). Previous studies(14) showed that the IGF-IR OBKO mice were normal in size and body weight but had lower trabecular bone volume, connectivity, trabecular number, mineralization rate, and an increased trabecular spacing, findings that we confirmed in this study. In this study, we further showed that the IGF-IR–null mutation in mature osteoblasts, not only affected trabecular bone but also cortical bone formation and mineralization. The lower periosteal bone formation rate and mineralization at the TFJ resulted in decreased cortical bone thickness, which together with the abnormalities in trabecular bone, resulted in a lower FFW/BW. At the cellular level, we observed that the IGF-IR–null mutation in mature osteoblasts did not affect the osteoprogenitor numbers, as indicated by ALP+ CFUs, but markedly reduced their differentiation into mineralized nodules. These results are consistent with the loss of IGF-IR as the osteoblasts differentiate in vitro, turning on the osteocalcin driven cre recombinase that inactivates the floxed IGF-IR gene in late stage cultures. At the molecular level, the IGF-IR–null mutation in mature osteoblasts did not affect the mRNA levels of early osteoblast differentiation markers RUNX2 and ALP, but decreased the mRNA levels of the late osteoblast differentiation marker osteocalcin, consistent with the failure of the osteoprogenitor cells from the IGF-IR OBKO mice to fully differentiate.
The major question we addressed in this study was whether the actions of PTH on bone were altered in the IGF-IR OBKO mice. Consistent with the results from earlier studies,(26,27) PTH increased trabecular bone, but in this study, this anabolic action was confined to the primary spongiosa. That PTH had a greater anabolic effect in this region may be caused by the greater expression of IGF-I in the osteoblasts of the primary spongiosa.(28) Deletion of IGF-IR in the mature osteoblasts blunted the anabolic effects of PTH on BV/TV, indicating that IGF-IR in mature osteoblasts was involved in mediating the anabolic actions of PTH on trabecular bone at the primary spongiosa. On the other hand, in the secondary spongiosa, as shown by other investigators,(29) PTH stimulated BFR and MAR, but induced no changes in trabecular bone volume in either control or IGF-IR OBKO mice. This may be because of differences in the balance between anabolic and catabolic actions of PTH in the primary and secondary spongiosa, perhaps because of reduced IGF-I expression or differences in structure between primary and secondary spongiosa. For example, there are fewer bone surfaces on which new bone formation can occur in the secondary spongiosa. It also may be that, in our study, 2 wk of PTH administration was not long enough to maximize the anabolic effects of PTH in the secondary spongiosa. The deletion of IGF-IR in mature osteoblasts did not completely block the stimulatory effects of PTH on BFR in trabecular bone as it did in cortical bone. Unlike bone histomorphometry, but consistent with our earlier studies,(9,30) μCT measurements of the secondary spongiosa indicated that PTH had catabolic effects on trabecular bone. Because FFW/BW showed decreased mineral content in the PTH-treated groups, this may have led to an underestimation of BV by μCT. Our data, together with that from other studies, suggest that μCT measurements should also be performed of the primary spongiosa. In the diaphysis, the periosteal and endosteal osteoblasts also express high levels of IGF-I.(28,31) As analyzed by BFR, MAR, and bone structural changes, PTH increased endosteal and periosteal bone formation and mineralization and increased cortical bone thickness. As seen in trabecular bone, the anabolic effects of PTH on cortical were blunted in the IGF-IR OBKO mice. Here the lack of anabolic effects of PTH in the IGF-IR OBKO was more pronounced, showing the importance of IGF-I signaling in the mature osteoblast for mediating the anabolic actions of PTH on bone.
BMSCs/osteoblasts play central roles in directing the anabolic and catabolic effects of PTH on bone.(6,32) At the cellular level, the anabolic effects of PTH may be attributed to several different mechanisms such as increasing the total number of osteoprogenitor cells(5) or inhibiting apoptosis.(7,33) In this study, we analyzed the effects of PTH given in vivo on BMSC proliferation and differentiation in vitro, and found that PTH increased both ALP+ colony number and mineralization in the cultures from the control mice. These findings support the concept that PTH acts by increasing the number of osteoprogenitor cells. However, our observation that deletion of the IGF-IR from the mature osteoblast blocks the ability of PTH to increase the numbers of ALP+ colonies suggests that this action of PTH is indirect (i.e., mediated by the mature osteoblast). Depending on the different protocols(5,7,33) and animal models used, previous studies have shown inconsistent results in the effect of PTH on cell proliferation. Using a rat model, Nishida et al.(5) showed that 3 wk of PTH administration (30 μg/kg body weight) increased ALP+ colony number at day 13 of BMSC cultures. In contrast, using mouse models, Knopp et al.(33) and Jilka et al.(7) showed that 4 wk of PTH administration at a dose of 95 or 400 μg/kg body weight, respectively, did not increase the number of ALP+ colonies at day 21(33) or day 28(7) of BMSC cultures, but extended the life span of osteoblasts, resulting in an increase in bone deposition with time. Our data support the concept that the anabolic effects of PTH are caused by increased osteoprogenitors that differentiate into osteoblasts to form more bone. However, our data do not exclude an effect on PTH on enhanced osteoblast survival. At the molecular level, PTH stimulated the expression of IGF-I in the control mice, which would act as an autocrine/paracrine factor to stimulate osteoblast proliferation and differentiation. PTH also stimulated the expression of RUNX2 and ALP, two early stage osteoblast differentiation markers, and osteocalcin, a late stage osteoblast differentiation marker, consistent with its effects on BMSC differentiation. PTH also stimulated the expression of RANKL. RANKL binds to its receptor RANK on the surface of osteoclast precursors, initiates osteoclastogenesis,(34–36) leading to the catabolic actions of PTH on bone.(3,37) In the BMSC cultures from the IGF-IR OBKO mice, as indicated by the measurement of ALP+ colony number, the loss of IGF-IR in mature osteoblasts blunted the stimulatory effects of PTH given in vivo on osteoprogenitor cell proliferation measured in vitro. Similarly, loss of IGF-IR in mature osteoblasts blocked the stimulatory effects of PTH given in vivo on the gene expression of early (RUNX2, ALP) and late (osteocalcin) osteoblast differentiation markers in bone, and osteoblast differentiation and mineral matrix production in vitro. Finally, the loss of IGF-IR in mature osteoblasts blocked the stimulatory effects of PTH given in vivo on RANKL expression in bone, thus blunting the catabolic effects of PTH on mineral content. We conclude that IGF-I signaling in mature osteoblasts mediates both the anabolic effects of PTH on bone by inducing osteoprogenitor cell proliferation and differentiation and the catabolic effects of PTH by inducing RANKL expression. The IGF-IR in the mature osteoblast is needed for these PTH stimulated events to occur. The mechanism for this is unclear but is under active study in our laboratory.
In summary, our results indicate that the IGF-IR in mature osteoblasts is needed both for normal bone remodeling and for PTH responsiveness of both mature osteoblasts and osteoblast progenitors. The later may be affected secondarily perhaps by PTH-stimulated IGF-I production from mature osteoblasts, but the precise role of the IGF-IR in the mature osteoblast in mediating these actions of PTH remains for future study.
This work was supported by grants from the National Institutes of Health RO1 DK 54793 and the National Aeronautic and Space Administrations NN04CC67G.