Reduced bone turnover in mice lacking the P2Y(13) receptor of ADP

Osteoporosis is a condition of excessive and uncoupled bone turnover, in which osteoclastic resorption exceeds osteoblastic bone formation, resulting in an overall net bone loss, bone fragility, and morbidity. Although numerous treatments have been developed to inhibit bone loss by blocking osteoclastic bone resorption, understanding of the mechanisms behind bone loss is incomplete. The purinergic signaling system is emerging to be a pivotal regulator of bone homeostasis, and extracellular ADP has previously been shown to be a powerful osteolytic agent in vitro. We report here that deletion of the P2Y(13) receptor, a G protein-coupled receptor for extracellular ADP, leads to a 40% reduction in trabecular bone mass, 50% reduction in osteoblast and osteoclast numbers in vivo, as well as activity in vitro, and an overall 50% reduction in the rate of bone remodeling in mice in vivo. Down-regulation of RhoA/ROCK I signaling and a reduced ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin observed in osteoblasts from P2Y(13)R(-/-) mice might explain this bone phenotype. Furthermore, because one of the main causes of osteoporosis in older women is lack of estrogen, we examined the effect of ovariectomy of the P2Y(13)R(-/-) mice and found them to be protected from ovariectomy-induced bone loss by up to 65%. These data confirm a role of purinergic ADP signaling in the skeleton, whereby deletion of the P2Y(13) receptor leads to reduced bone turnover rates, which provide a protective advantage in conditions of accelerated bone turnover such as oestrogen deficiency-induced osteoporosis.     

Inhibition of osteoblastic metalloproteinases in mice prevents bone loss induced by oestrogen deficiency

Matrix metalloproteinases (MMPs) are key mediators in extra-cellular matrix remodelling and implicated primarily in bone growth, and particularly in osteoclastic bone resorption. We hypothesise that MMPs have a role in the increased bone remodelling resulting from oestrogen deficiency. Transgenic (TG) mice overexpressing TIMP-1 in their osteoblastic cells and their wild-type (WT) littermates were ovariectomised. One month after surgery, bone mineral density (BMD) and bone microarchitecture were assessed. Primary cells from WT and TG mice were used to determine how TIMP-1 affects osteoclast and osteoblastic cells. The reduction of BMD induced by ovariectomy in WT mice was not observed in the transgenic mice. The transgene overexpression also dampened the post-ovariectomy increase in bone resorption in contrast to the WT mice. In vivo, osteoclastic surfaces and D-pyridinoline were not increased in TG mice, and ex vivo, the differentiation of osteoclasts from TG bone marrow precursor cells were unaffected by in vivo oestrogen deficiency or treatment. We showed also that TIMP-1 overexpression reduces and delays the osteoblastic proliferation and differentiation respectively, and reduced the generation of the active form of TGFbeta1 in the supernatant of TG osteoblasts. Our findings support the hypothesis that in vivo inhibition of osteoblastic MMPs prevented the bone loss induced by oestrogen deficiency, with a significant decrease in bone resorption. This effect was presumably resulting from (1) a direct inhibition of osteoclastic resorption activity by the TIMP-1 and (2) the modification in the local activation of extra-cellular signalling factors such as TGFbeta1 and the OPG/RANKL ratio.     

Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass.

Transforming growth factor-beta (TGF-beta) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. To explore the role of endogenous TGF-(beta) in osteoblast function in vivo, we have inhibited osteoblastic responsiveness to TGF-beta in transgenic mice by expressing a cytoplasmically truncated type II TGF-beta receptor from the osteocalcin promoter. These transgenic mice develop an age-dependent increase in trabecular bone mass, which progresses up to the age of 6 months, due to an imbalance between bone formation and resorption during bone remodeling. Since the rate of osteoblastic bone formation was not altered, their increased trabecular bone mass is likely due to decreased bone resorption by osteoclasts. Accordingly, direct evidence of reduced osteoclast activity was found in transgenic mouse skulls, which had less cavitation and fewer mature osteoclasts relative to skulls of wild-type mice. These bone remodeling defects resulted in altered biomechanical properties. The femurs of transgenic mice were tougher, and their vertebral bodies were stiffer and stronger than those of wild-type mice. Lastly, osteocyte density was decreased in transgenic mice, suggesting that TGF-beta signaling in osteoblasts is required for normal osteoblast differentiation in vivo. Our results demonstrate that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties.     

Over-expression of fibroblast growth factor-2 causes defective bone mineralization and osteopenia in transgenic mice

Over-expression of human FGF-2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1-6 month-old wild (NTg) and TgFGF2 mice were studied. FGF-2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro-CT of trabecular bone of the distal femurs from 6-month-old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double-labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non-targeted over-expression of FGF-2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF-2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.     

Attenuation of bone mass and increase of osteoclast formation in decoy receptor 3 transgenic mice

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT, and TL1A, induces osteoclast formation from monocyte, macrophage, and bone stromal marrow cells. However, the function of DcR3 on bone formation remains largely unknown. To understand the function of DcR3 in bone formation in vivo, transgenic mice overexpressing DcR3 were generated. Bone mineral density (BMD) and bone mineral content (BMC) of total body were significantly lower in DcR3 transgenic mice as compared with wild-type controls. The difference in BMD and BMC between DcR3 transgenic and control mice was confirmed by histomorphometric analysis, which showed a 35.7% decrease in trabecular bone volume in DcR3 transgenic mice in comparison with wild-type controls. The number of osteoclasts increased in DcR3 transgenic mice. In addition, local administration of DcR3 (30 microg/ml, 10 microl, once/day) into the metaphysis of the tibia via the implantation of a needle cannula significantly decreased the BMD, BMC, and bone volume of secondary spongiosa in tibia. Local injection of DcR3 also increased osteoclast numbers around trabecular bone in tibia. Furthermore, coadminstration of soluble tumor necrosis factor receptor inhibitor/Fc chimera (TNFRSF1A) but not osteoprotegerin inhibited the action of DcR3. In addition, in an assay of osteoclast activity on substrate plates, DcR3 significantly increased the resorption activity of mature osteoclasts. Treatment with higher concentrations of DcR3 slightly increased nodule formation and alkaline phosphatase activity of primary cultured osteoblasts. These results indicate that DcR3 may play an important role in osteoporosis or other bone diseases.     

Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.     

Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling

Bone morphogenetic proteins (BMPs) function during various aspects of embryonic development including skeletogenesis. However, their biological functions after birth are less understood. To investigate the role of BMPs during bone remodeling, we generated a postnatal osteoblast-specific disruption of Bmpr1a that encodes the type IA receptor for BMPs in mice. Mutant mice were smaller than controls up to 6 months after birth. Irregular calcification and low bone mass were observed, but there were normal numbers of osteoblasts. The ability of the mutant osteoblasts to form mineralized nodules in culture was severely reduced. Interestingly, bone mass was increased in aged mutant mice due to reduced bone resorption evidenced by reduced bone turnover. The mutant mice lost more bone after ovariectomy likely resulting from decreased osteoblast function which could not overcome ovariectomy-induced bone resorption. In organ culture of bones from aged mice, ablation of the Bmpr1a gene by adenoviral Cre recombinase abolished the stimulatory effects of BMP4 on the expression of lysosomal enzymes essential for osteoclastic bone resorption. These results demonstrate essential and age-dependent roles for BMP signaling mediated by BMPRIA (a type IA receptor for BMP) in osteoblasts for bone remodeling.     

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.     

G蛋白偶联受体激酶的调控

G蛋白偶联受体激酶(G protein-coupled receptor kinase,GRK)特异地使活化的G蛋白偶联受体(G protein-coupled receptor,GPCR)发生磷酸化及脱敏化,从而终止后者介导的信号转导通路。研究表明,GRK的功能被高度调控,并具有下行调节GPCR的能力。调控GRK功能的机制包括两个层次：(1)多种途径调控激酶的亚细胞定位及活性,包括GPCR介导、G蛋白偶联、磷脂作用、Ca^2 结合蛋白调控、蛋白激酶C活化、MAPK反馈抑制、小窝蛋白抑制等;(2)调控GRK表达水平,主要体现在其与某些疾病的联系。     

EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation

G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.     

In vivo impact of a 4 bp deletion mutation in the DLX3 gene on bone development

Distal-less 3 (DLX3) gene mutations are etiologic for Tricho-Dento-Osseous syndrome. To investigate the in vivo impact of mutant DLX3 on bone development, we established transgenic (TG) mice expressing the c.571_574delGGGG DLX-3 gene mutation (MT-DLX3) driven by a mouse 2.3 Col1A1 promoter. Microcomputed tomographic analyses demonstrated markedly increased trabecular bone volume and bone mineral density in femora from TG mice. In ex vivo experiments, TG mice showed enhanced differentiation of bone marrow stromal cells to osteoblasts and increased expression levels of bone formation markers. However, TG mice did not show enhanced dynamic bone formation rates in in vivo fluorochrome double labeling experiments. Osteoclastic differentiation capacities of bone marrow monocytes were reduced in TG mice in the presence of osteoclastogenic factors and the numbers of TRAP(+) osteoclasts on distal metaphyseal trabecular bone surfaces were significantly decreased. TRACP 5b and CTX serum levels were significantly decreased in TG mice, while IFN-γ levels were significantly increased. These data demonstrate that increased levels of IFN-γ decrease osteoclast bone resorption activities, contributing to the enhanced trabecular bone volume and mineral density in these TG mice. These data suggest a novel role for this DLX-3 mutation in osteoclast differentiation and bone resorption.     

Adenosine A2B receptors play an important role in bone homeostasis

Bone homeostasis is a finely regulated mechanism involving different molecular pathways including adenosine signaling. The aim of this study is to determine the bone phenotype of adenosine A2B receptor knockout (A2BRKO) mice and to measure their ability to form new bone. Moreover, we analyzed the functionality of osteoclasts and osteoblasts from A2BRKO mice. Microcomputed tomography (μCT) analysis revealed a decrease of bone substance, bone mineral density, and trabecular number in A2BRKO mice compared to the WT mice at the same age. We measured the new bone formation by injecting fluorescent markers: it was reduced in femur and tibia of A2BRKO mice compare to the WT. A2BRKO young mice have fewer osteoblasts and an increase of osteoclasts was measured in the hind limbs of young and adult mice. A2BRKO osteoclasts are also more active in vitro, showing an increase of pit formation in dentin discs. Surprisingly in mature osteoblasts from A2BRKO mice, we measured an increase of calcified matrix production, collagen deposition, and alkaline phosphatase activity. These results demonstrate that A2BR on osteoblasts and osteoclasts regulate bone homeostasis.     

Ultrastructural changes in bones of the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mice, was developed as an animal model for senile osteoporosis. In the present study, we investigated the bone morphology, together with serum calcium and bone mineral density (BMD) in SAMP6 and age-matched normal mice SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 month of age with regard to the serum compositions and bone morphology. As compared with SAMR1, BMD, the femoral weight, femoral calcium and phosphorus levels were significantly reduced in SAMP6 at 2 and 5 months of age. The number of osteoblasts in trabecular bones was also significantly reduced. Swollen mitochondria and myelin-like structures were found in osteoblasts and osteocytes of SAMP6 mice at 2 and 5 months of age. There was a greater proportion of resting surface and less forming surface in the femoral endosteal surfaces of SAMP6 mice. The amount of trabecular bone in the lumbar vertebra and the distal metaphysis of the femur was reduced. The number of the mast cells in bone marrow of the tibia significantly increased in SAMP6 mice. These findings indicate that the lower bone mass in SAMP6 was due to the reduction in osteoblast formation and suggested that mast cells in bone marrows play a role in the pathogenesis of senile osteoporosis.     

Deletion of the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption

The P2X7 nucleotide receptor is an ATP-gated ion channel expressed widely in cells of hematopoietic origin. Our purpose was to explore the involvement of the P2X7 receptor in bone development and remodeling by characterizing the phenotype of mice genetically modified to disrupt the P2X7 receptor [knockout (KO)]. Femoral length did not differ between KO and wild-type (WT) littermates at 2 or 9 months of age, indicating that the P2X7 receptor does not regulate longitudinal bone growth. However, KO mice displayed significant reduction in total and cortical bone content and periosteal circumference in femurs, and reduced periosteal bone formation and increased trabecular bone resorption in tibias. Patch clamp recording confirmed expression of functional P2X7 receptors in osteoclasts from WT but not KO mice. Osteoclasts were present in vivo and formed in cultures of bone marrow from KO mice, indicating that this receptor is not essential for fusion of osteoclast precursors. Functional P2X7 receptors were also found in osteoblasts from WT but not KO mice, suggesting a direct role in bone formation. P2X7 receptor KO mice demonstrate a unique skeletal phenotype that involves deficient periosteal bone formation together with excessive trabecular bone resorption. Thus, the P2X7 receptor represents a novel therapeutic target for the management of skeletal disorders such as osteoporosis.     

G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis

Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1-125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2(+/-) mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2(+/-) mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2(+/-) animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2(+/-) mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2(+/-) animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.     

New insights into the epigenetics of osteoporosis

Osteoporosis is characterized by reduced bone formation and accumulation of adipocytes in the bone marrow compartment. The decrease in bone mass results from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The deficiency of bone cells to replace the resorpted bone can be due to a preferential differentiation of bone marrow stromal cells into adipocytes at the expense of osteoblasts. Consequently, the processes that control the differentiation of osteoclasts, osteoblasts and adipocytes play a crucial role in bone metabolism. It is known that epigenetic mechanisms are critical regulator of the differentiation programs for cell fate and moreover are subject to changes during aging. Here, we summarize recent findings on the role of epigenetics in the modulation of mechanisms that may be associated with osteoporosis. In particular, we focus on disturbances in the bone remodeling process described in human studies that address the epigenetic regulation of the osteoblast/adipocyte balance.     

Hydrogen peroxide impairs GRK2 translation via a calpain-dependent and cdk1-mediated pathway

Oxidative mechanisms of injury are involved in many neurodegenerative diseases such as stroke, ischemia-reperfusion injury and multiple sclerosis. G protein-coupled receptor kinase 2 (GRK2) plays a key role in G protein-coupled receptor (GPCR) signaling modulation, and its expression levels are decreased after brain hypoxia/ischemia and reperfusion as well as in several inflammatory conditions. We report here that hydrogen peroxide downregulates GRK2 expression in C6 rat glioma cells. The hydrogen peroxide-induced decrease in GRK2 is prevented by a calpain protease inhibitor, but does not involve increased GRK2 degradation or changes in GRK2 mRNA level. Instead we show that hydrogen peroxide treatment impairs GRK2 translation in a process that requires Cdk1 activation and involves the mTOR pathway. This novel mechanism for the control of GRK2 expression in glial cells upon oxidative stress challenge may contribute to the modulation of GPCR signaling in different pathological conditions.     

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.     

Osteoblastic Responses to TGF-β during Bone Remodeling

Bone remodeling depends on the spatial and temporal coupling of bone formation by osteoblasts and bone resorption by osteoclasts; however, the molecular basis of these inductive interactions is unknown. We have previously shown that osteoblastic overexpression of TGF-β2 in transgenic mice deregulates bone remodeling and leads to an age-dependent loss of bone mass that resembles high-turnover osteoporosis in humans. This phenotype implicates TGF-β2 as a physiological regulator of bone remodeling and raises the question of how this single secreted factor regulates the functions of osteoblasts and osteoclasts and coordinates their opposing activities in vivo. To gain insight into the physiological role of TGF-β in bone remodeling, we have now characterized the responses of osteoblasts to TGF-β in these transgenic mice. We took advantage of the ability of alendronate to specifically inhibit bone resorption, the lack of osteoclast activity in c-fos^−/− mice, and a new transgenic mouse line that expresses a dominant-negative form of the type II TGF-β receptor in osteoblasts. Our results show that TGF-β directly increases the steady-state rate of osteoblastic differentiation from osteoprogenitor cell to terminally differentiated osteocyte and thereby increases the final density of osteocytes embedded within bone matrix. Mice overexpressing TGF-β2 also have increased rates of bone matrix formation; however, this activity does not result from a direct effect of TGF-β on osteoblasts, but is more likely a homeostatic response to the increase in bone resorption caused by TGF-β. Lastly, we find that osteoclastic activity contributes to the TGF-β–induced increase in osteoblast differentiation at sites of bone resorption. These results suggest that TGF-β is a physiological regulator of osteoblast differentiation and acts as a central component of the coupling of bone formation to resorption during bone remodeling.