Estrogen receptor α36 mediates a bone‐sparing effect of 17β‐estrodiol in postmenopausal women

Recently, a membrane‐based estrogen receptor (ER), ER‐α36, was identified and cloned that transduces membrane‐initiated estrogen signaling such as activation of the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signaling pathway. Here we show that the postmenopausal level of estradiol (E2) induces mitogenic, antiapoptotic, and antiosteogenic effects and proapoptotic effects in postmenopausal osteoblasts and osteoclasts with high levels of ER‐α36 expression, respectively. We also found that ER‐α36 mediated the effects of postmenopausal‐level E₂ on proliferation, apoptosis, and differentiation of osteoblasts through transient activation of the MAPK/ERK pathway, whereas ER‐α36‐mediated postmenopausal‐level E₂ induces apoptosis of osteoclasts through prolonged activation of the MAPK/ERK pathway with the involvement of reactive oxygen species. We also show that the levels of ER‐α36 expression in bone are positively associated with bone mineral density but negatively associated with bone biochemical markers in postmenopausal women. Thus the higher levels of ER‐α36 expression are required for preserving bone mass in postmenopausal and menopausal women who become osteoporotic if ER‐α36‐mediated activities are dysregulated. © 2011 American Society for Bone and Mineral Research.     

Osteoclast TGF‐β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

Osteoblast‐mediated bone formation is coupled to osteoclast‐mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age‐related bone loss. Osteoclasts release and activate TGF‐β from the bone matrix. Here we show that osteoclast‐specific inhibition of TGF‐β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF‐β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF‐β receptor signaling. Osteoclasts in aged murine bones had lower TGF‐β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF‐β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF‐β availability with age. Therefore, osteoclast responses to TGF‐β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age‐related bone loss. © 2015 American Society for Bone and Mineral Research.     

Fibrillin‐1 Regulates Skeletal Stem Cell Differentiation by Modulating TGFβ Activity Within the Marrow Niche

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age‐related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin‐1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin‐1 (Fbn1^Prx1–/– mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1^Prx1–/– mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1^Prx1–/– mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1‐silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin‐1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1^Prx1–/– limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin‐1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. © 2015 American Society for Bone and Mineral Research.     

Regulation of RANKL‐induced osteoclastogenesis by TGF‐β through molecular interaction between Smad3 and Traf6

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling. © 2011 American Society for Bone and Mineral Research.     

β‐catenin promotes bone formation and suppresses bone resorption in postnatal growing mice

Genetic studies in the mouse have demonstrated multiple roles for β‐catenin in the skeleton. In the embryo, β‐catenin is critical for the early stages of osteoblast differentiation. Postnatally, β‐catenin in mature osteoblasts and osteocytes indirectly suppresses osteoclast differentiation. However, a direct role for β‐catenin in regulating osteoblast number and/or function specifically in the postnatal life has not been demonstrated. Addressing this knowledge gap is important because low‐density lipoprotein receptor‐related protein 5 (LRP5), a coreceptor for WNT signaling proposed to function through β‐catenin, controls osteoblast number and function in postnatal mice or humans. To overcome the neonatal lethality caused by embryonic deletion of β‐catenin in early‐stage osteoblast‐lineage cells, we use the Osx‐CreER^T2 mouse strain to remove β‐catenin in Osterix (Osx)‐expressing cells by administering tamoxifen (TM) temporarily to postnatal mice. Lineage‐tracing experiments in the long bones demonstrate that Osx‐CreER^T2 targets predominantly osteoblast‐lineage cells on the bone surface, but also transient progenitors that contribute to bone marrow stromal cells and adipocytes. Deletion of β‐catenin by this strategy greatly reduces the bone formation activity of the targeted osteoblasts. However, the targeted osteoblasts rapidly turn over and are replaced by an excessive number of non‐targeted osteoblasts, causing an unexpected increase in bone formation, but an even greater increase in osteoclast number and activity produces a net effect of severe osteopenia. With time, the mutant mice also exhibit a marked increase in bone marrow adiposity. Thus, β‐catenin in postnatal Osx‐lineage cells critically regulates bone homeostasis by promoting osteoblast activity and suppressing osteoblast turnover, while restraining osteoclast and marrow fat formation. © 2013 American Society for Bone and Mineral Research.     

Active TGF‐β1 Expression in kidney transplantation: a comparative study of Cyclosporin‐A (CyA) and tacrolimus (FK506)

Abstract Chronic rejection is a major cause of graft dysfunction following kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF‐β). Previous studies have suggested that cyclosporin‐A (CyA) might increase production of this growth factor. The current study was designed to measure the expression of TGF‐β in renal transplant biopsies from patients immunosuppressed with either CyA or tacrolimus. Paraffin‐embedded renal biopsies were sectioned, dewaxed and incubated with primary antibody against active TGF‐β₁ antibody. After washing, the sections were treated with secondary antibody conjugated with fluorescein isothiocyanate (FITC). In each case the sections were assessed by semi‐quantitative scanning laser confocal microscopy. Biopsies from patients receiving CyA expressed significantly more active TGF‐β₁ than biopsies from patients receiving tacrolimus (P < 0.0001, Mann‐Whitney test). The increased level of active TGF‐β₁ expression in renal biopsies of patients receiving CyA may indicate a mechanism of chronic rejection.     

Skeletal Colonization by Breast Cancer Cells Is Stimulated by an Osteoblast and β2AR‐Dependent Neo‐Angiogenic Switch

The skeleton is a common site for breast cancer metastasis. Although significant progress has been made to manage osteolytic bone lesions, the mechanisms driving the early steps of the bone metastatic process are still not sufficiently understood to design efficacious strategies needed to inhibit this process and offer preventative therapeutic options. Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to stimulate sympathetic nerve outflow. In this study, we show that stimulation of the beta 2‐adrenergic receptor (β2AR) by isoproterenol, used as a pharmacological surrogate of sympathetic nerve activation, led to increased blood vessel density and Vegf‐a expression in bone. It also raised levels of secreted Vegf‐a in osteoblast cultures, and accordingly, the conditioned media from isoproterenol‐treated osteoblast cultures promoted new vessel formation in two ex vivo models of angiogenesis. Blocking the interaction between Vegf‐a and its receptor, Vegfr2, blunted the increase in vessel density induced by isoproterenol. Genetic loss of the β2AR globally, or specifically in type 1 collagen‐expressing osteoblasts, diminished the increase in Vegf‐positive osteoblast number and bone vessel density induced by isoproterenol, and reduced the higher incidence of bone metastatic lesions induced by isoproterenol after intracardiac injection of an osteotropic variant of MDA‐MB‐231 breast cancer cells. Inhibition of the interaction between Vegf‐a and Vegfr2 with the blocking antibody mcr84 also prevented the increase in bone vascular density and bone metastasis triggered by isoproterenol. Together, these results indicate that stimulation of the β2AR in osteoblasts triggers a Vegf‐dependent neo‐angiogenic switch that promotes bone vascular density and the colonization of the bone microenvironment by metastatic breast cancer cells. © 2017 American Society for Bone and Mineral Research.     

Activation of mTORC1 in B Lymphocytes Promotes Osteoclast Formation via Regulation of β‐Catenin and RANKL/OPG

The cytokine receptor activator of nuclear factor‐κB ligand (RANKL) induces osteoclast formation from monocyte/macrophage lineage cells. However, the mechanisms by which RANKL expression is controlled in cells that support osteoclast differentiation are still unclear. We show that deletion of TSC1 (tuberous sclerosis complex 1) in murine B cells causes constitutive activation of mechanistic target of rapamycin complex 1 (mTORC1) and stimulates RANKL but represses osteoprotegerin (OPG) expression and subsequently promotes osteoclast formation and causes osteoporosis in mice. Furthermore, the regulation of RANKL/OPG and stimulation of osteoclastogenesis by mTORC1 was confirmed in a variety of RANKL‐expressing cells and in vivo. Mechanistically, mTORC1 controls RANKL/OPG expression through negative feedback inactivation of Akt, destabilization of β‐catenin mRNA, and downregulation of β‐catenin. Our findings demonstrate that mTORC1 activation‐stimulated RANKL expression in B cells is sufficient to induce bone loss and osteoporosis. The study also established a link between mTORC1 and the RANKL/OPG axis via negative regulation of β‐catenin. © 2016 American Society for Bone and Mineral Research.     

Hyperactive Transforming Growth Factor‐β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

Dysregulated transforming growth factor beta (TGF‐β) signaling is associated with a spectrum of osseous defects as seen in Loeys‐Dietz syndrome, Marfan syndrome, and Camurati‐Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF‐β1 signaling pivotally underpins osseous defects in Nf1^flox/?;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF‐β1 levels are fivefold to sixfold increased both in Nf1^flox/?;Col2.3Cre mice and in a cohort of NF1 patients. Nf1‐deficient osteoblasts, the principal source of TGF‐β1 in bone, overexpress TGF‐β1 in a gene dosage–dependent fashion. Moreover, Nf1‐deficient osteoblasts and osteoclasts are hyperresponsive to TGF‐β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21‐Ras–dependent hyperactivation of the canonical TGF‐β1–Smad pathway. Reexpression of the human, full‐length neurofibromin guanosine triphosphatase (GTPase)‐activating protein (GAP)‐related domain (NF1 GRD) in primary Nf1‐deficient osteoblast progenitors, attenuated TGF‐β1 expression levels and reduced Smad phosphorylation in response to TGF‐β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF‐β receptor 1 (TβRI) kinase inhibitor, SD‐208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1^flox/?;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF‐β1 signaling in the pathogenesis of NF1‐associated osteoporosis and pseudarthrosis, thus implicating the TGF‐β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies. © 2013 American Society for Bone and Mineral Research.     

Indirubin‐3′‐Oxime Reverses Bone Loss in Ovariectomized and Hindlimb‐Unloaded Mice Via Activation of the Wnt/β‐Catenin Signaling

Osteoporosis is a major global health issue in elderly people. Because Wnt/β‐catenin signaling plays a key role in bone homeostasis, we screened activators of this pathway through cell‐based screening, and investigated indirubin‐3′‐oxime (I3O), one of the positive compounds known to inhibit GSK3β, as a potential anti‐osteoporotic agent. Here, we show that I3O activated Wnt/β‐catenin signaling via inhibition of the interaction of GSK3β with β‐catenin, and induced osteoblast differentiation in vitro and increased calvarial bone thickness ex vivo. Intraperitoneal injection of I3O increased bone mass and improved microarchitecture in normal mice and reversed bone loss in an ovariectomized mouse model of age‐related osteoporosis. I3O also increased thickness and area of cortical bone, indicating improved bone strength. Enhanced bone mass and strength correlated with activated Wnt/β‐catenin signaling, as shown by histological analyses of both trabecular and cortical bones. I3O also restored mass and density of bone in hindlimb‐unloaded mice compared with control, suspended mice, demonstrating bone‐restoration effects of I3O in non‐aged–related osteoporosis as well. Overall, I3O, a pharmacologically active small molecule, could be a potential therapeutic agent for the treatment and prevention of osteoporosis. © 2014 American Society for Bone and Mineral Research.     

An O‐Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through α4β1 Integrin

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O‐deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O‐glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4β1 or α4β7 integrins, these integrins were evaluated. We show that integrin α4β1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4β1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O‐glycosylation in the variable region that results in decreased integrin‐mediated signaling. This deleterious effect can be thwarted by binding α4β1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model. © 2016 American Society for Bone and Mineral Research.     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

SH3BP2 Cherubism Mutation Potentiates TNF‐α–Induced Osteoclastogenesis via NFATc1 and TNF‐α–Mediated Inflammatory Bone Loss

Cherubism (OMIM# 118400) is a genetic disorder with excessive jawbone resorption caused by mutations in SH3 domain binding protein 2 (SH3BP2), a signaling adaptor protein. Studies on the mouse model for cherubism carrying a P416R knock‐in (KI) mutation have revealed that mutant SH3BP2 enhances tumor necrosis factor (TNF)‐α production and receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclast differentiation in myeloid cells. TNF‐α is expressed in human cherubism lesions, which contain a large number of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells, and TNF‐α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2^KI/KI). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF‐α–mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF‐α–mediated osteoclast formation and bone loss. Here, we show that bone marrow–derived M‐CSF–dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2^KI/+) mice are highly responsive to TNF‐α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves spleen tyrosine kinase (SYK) and phospholipase Cγ2 (PLCγ2) phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF‐α injection model as well as in a human TNF‐α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP‐positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF‐α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF‐α–induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders. © 2014 American Society for Bone and Mineral Research.     

Connective Tissue Growth Factor Promotes Fibrosis Downstream of TGFβ and IL‐6 in Chronic Cardiac Allograft Rejection

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long‐term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFβ, IL‐6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFβ transduction. Further, intragraft CTGF expression was inhibited by IL‐6 neutralization whereas TGFβ expression remained unchanged, indicating that IL‐6 effects may potentiate TGFβ‐mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFβ and IL‐6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.     

Dullard/Ctdnep1 Regulates Endochondral Ossification via Suppression of TGF‐β Signaling

Transforming growth factor (TGF)‐β signaling plays critical roles during skeletal development and its excessive signaling causes genetic diseases of connective tissues including Marfan syndrome and acromelic dysplasia. However, the mechanisms underlying prevention of excessive TGF‐β signaling in skeletogenesis remain unclear. We previously reported that Dullard/Ctdnep1 encoding a small phosphatase is required for nephron maintenance after birth through suppression of bone morphogenetic protein (BMP) signaling. Unexpectedly, we found that Dullard is involved in suppression of TGF‐β signaling during endochondral ossification. Conditional Dullard‐deficient mice in the limb and sternum mesenchyme by Prx1‐Cre displayed the impaired growth and ossification of skeletal elements leading to postnatal lethality. Dullard was expressed in early cartilage condensations and later in growth plate chondrocytes. The tibia growth plate of newborn Dullard mutant mice showed reduction of the proliferative and hypertrophic chondrocyte layers. The sternum showed deformity of cartilage primordia and delayed hypertrophy. Micromass culture experiments revealed that Dullard deficiency enhanced early cartilage condensation and differentiation, but suppressed mineralized hypertrophic chondrocyte differentiation, which was reversed by treatment with TGF‐β type I receptor kinase blocker LY‐364947. Dullard deficiency induced upregulation of protein levels of both phospho‐Smad2/3 and total Smad2/3 in micromass cultures without increase of Smad2/3 mRNA levels, suggesting that Dullard may affect Smad2/3 protein stability. The phospho‐Smad2/3 level was also upregulated in perichondrium and hypertrophic chondrocytes in Dullard‐deficient embryos. Response to TGF‐β signaling was enhanced in Dullard‐deficient primary chondrocyte cultures at late, but not early, time point. Moreover, perinatal administration of LY‐364947 ameliorated the sternum deformity in vivo. Thus, we identified Dullard as a new negative regulator of TGF‐β signaling in endochondral ossification. © 2014 American Society for Bone and Mineral Research.     

Role of TGF‐β in a Mouse Model of High Turnover Renal Osteodystrophy

Altered bone turnover is a key pathologic feature of chronic kidney disease‐mineral and bone disorder (CKD‐MBD). Expression of TGF‐β1, a known regulator of bone turnover, is increased in bone biopsies from individuals with CKD. Similarly, TGF‐β1 mRNA and downstream signaling is increased in bones from jck mice, a model of high‐turnover renal osteodystrophy. A neutralizing anti‐TGF‐β antibody (1D11) was used to explore TGF‐β's role in renal osteodystrophy. 1D11 administration to jck significantly attenuated elevated serum osteocalcin and type I collagen C‐telopeptides. Histomorphometric analysis indicated that 1D11 administration increased bone volume and suppressed the elevated bone turnover in a dose‐dependent manner. These effects were associated with reductions in osteoblast and osteoclast surface areas. Micro‐computed tomography (µCT) confirmed the observed increase in trabecular bone volume and demonstrated improvements in trabecular architecture and increased cortical thickness. 1D11 administration was associated with significant reductions in expression of osteoblast marker genes (Runx2, alkaline phosphatase, osteocalcin) and the osteoclast marker gene, Trap5. Importantly, in this model, 1D11 did not improve kidney function or reduce serum parathyroid hormone (PTH) levels, indicating that 1D11 effects on bone are independent of changes in renal or parathyroid function. 1D11 also significantly attenuated high‐turnover bone disease in the adenine‐induced uremic rat model. Antibody administration was associated with a reduction in pSMAD2/SMAD2 in bone but not bone marrow as assessed by quantitative immunoblot analysis. Immunostaining revealed pSMAD staining in osteoblasts and osteocytes but not osteoclasts, suggesting 1D11 effects on osteoclasts may be indirect. Immunoblot and whole genome mRNA expression analysis confirmed our previous observation that repression of Wnt/β‐catenin expression in bone is correlated with increased osteoclast activity in jck mice and bone biopsies from CKD patients. Furthermore, our data suggest that elevated TGF‐β may contribute to the pathogenesis of high‐turnover disease partially through inhibition of β‐catenin signaling. © 2014 American Society for Bone and Mineral Research.