Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.     

The orphan receptor tyrosine kinase Ror2 modulates canonical Wnt signaling in osteoblastic cells

Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.     

Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling

The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass. In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293T cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by overexpression of Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transduction of canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the high bone mass phenotype associated with the loss of sclerostin may be attributed, at least in part, to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.     

Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells

Multipotential adult mesenchymal stem cells (MSCs) are able to differentiate along several known lineages, and lineage commitment is tightly regulated through specific cellular mediators and interactions. Recent observations of a low/high bone-mass phenotype in patients expressing a loss-/gain-of-function mutation in LRP5, a coreceptor of the Wnt family of signaling molecules, suggest the importance of Wnt signaling in bone formation, possibly involving MSCs. To analyze the role of Wnt signaling in mesenchymal osteogenesis, we have profiled the expression of WNTs and their receptors, FRIZZLEDs (FZDs), and several secreted Wnt inhibitors, such as SFRPs, and examined the effect of Wnt 3a, as a representative canonical Wnt member, during MSC osteogenesis in vitro. WNT11, FZD6, SFRP2, and SFRP3 are upregulated during MSC osteogenesis, while WNT9A and FZD7 are downregulated. MSCs also respond to exogenous Wnt 3a, based on increased beta-catenin nuclearization and activation of a Wnt-responsive promoter, and the magnitude of this response depends on the MSC differentiation state. Wnt 3a exposure inhibits MSC osteogenic differentiation, with decreased matrix mineralization and reduced alkaline phosphatase mRNA and activity. Wnt 3a treatment of fully osteogenically differentiated MSCs also suppresses osteoblastic marker gene expression. The Wnt 3a effect is accompanied by increased cell number, resulting from both increased proliferation and decreased apoptosis, particularly during expansion of undifferentiated MSCs. The osteo-suppressive effects of Wnt 3a are fully reversible, i.e., treatment prior to osteogenic induction does not compromise subsequent MSC osteogenesis. The results also showed that sFRP3 treatment attenuates some of the observed Wnt 3a effects on MSCs, and that inhibition of canonical Wnt signaling using a dominant negative TCF1 enhances MSC osteogenesis. Interestingly, expression of Wnt 5a, a non-canonical Wnt member, appeared to promote osteogenesis. Taken together, these findings suggest that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor MSC population, whereas non-canonical Wnts facilitate osteogenic differentiation. Release from canonical Wnt regulation is a prerequisite for MSC differentiation. Thus, loss-/gain-of-function mutations of LRP5 would perturb Wnt signaling and depress/promote bone formation by affecting the progenitor cell pool. Elucidating Wnt regulation of MSC differentiation is important for their potential application in tissue regeneration.     

Porphyromonas gingivalis lipopolysaccharide inhibits the osteoblastic differentiation of preosteoblasts by activating Notch1 signaling

Although Porphyromonas gingivalis lipopolysaccharide (P‐LPS) is known to inhibit osteoblast differentiation, the exact molecular mechanisms underlying this phenomenon remain unclear. Here, we investigated the role of Notch signaling in the osteoblastic differentiation of both MC3T3E‐1 cells and primary mouse bone marrow stromal cells (BMSCs). P‐LPS stimulation activated the Notch1 signaling cascade and increased expression of the Notch target genes HES1 and HEY1. P‐LPS can also act as an inhibitor because it is capable of suppressing Wnt/β‐catenin signaling in preosteoblasts by decreasing both glycogen synthase kinase‐3β (GSK‐3β) phosphorylation and the expression of nuclear β‐catenin. These effects were rescued, however, by inhibiting Notch1 signaling. Furthermore, P‐LPS treatment inhibited osteoblast differentiation in preosteoblasts as demonstrated by reductions in alkaline phosphatase activity, osteoblast gene expression, and mineralization, all of which were rescued by suppression of Notch1 signaling. Moreover, inhibition of GSK‐3β, HES1, or HEY1 partially reversed the P‐LPS‐induced inhibition of osteoblast differentiation. Together, these findings suggest that P‐LPS inhibits osteoblast differentiation by promoting the expression of Notch target genes and suppressing canonical Wnt/β‐catenin signaling. J. Cell. Physiol. 225: 106–114, 2010. © 2010 Wiley‐Liss, Inc.     

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP_M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.     

Control of osteogenesis by the canonical Wnt and BMP pathways in vivo

Although many regulators of skeletogenesis have been functionally characterized, one current challenge is to integrate this information into regulatory networks. Here, we discuss how the canonical Wnt and Smad‐dependent BMP pathways interact together and play antagonistic or cooperative roles at different steps of osteogenesis, in the context of the developing vertebrate embryo. Early on, BMP signaling specifies multipotent mesenchymal cells into osteochondroprogenitors. In turn, the function of Wnt signaling is to drive these osteochondroprogenitors towards an osteoblastic fate. Subsequently, both pathways promote osteoblast differentiation, albeit with notable mechanistic differences. In osteocytes, the ultimate stage of osteogenic differentiation, the Wnt and BMP pathways exert opposite effects on the control of bone resorption by osteoclasts. We describe how the dynamic molecular wiring of the canonical Wnt and Smad‐dependent BMP signaling into the skeletal cell genetic programme is critical for the generation of bone‐specific cell types during development.     

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.     

Cross-talk between Wnt signaling pathways in human mesenchymal stem cells leads to functional antagonism during osteogenic differentiation

Wnt signaling is involved in developmental processes and in adult stem cell homeostasis. This study analyzes the role(s) of key Wnt signaling mediators in the maintenance and osteogenesis of mesenchymal stem cells (MSCs). We focus specifically on the involvement of low-density lipoprotein-related protein 5 (LRP5), T-cell factor 1 (TCF1), and Frizzled (Fz) receptors, in the presence or absence of exogenous, prototypical canonical (Wnt3a), and non-canonical (Wnt5a) Wnts. In undifferentiated MSCs, LRP5 and TCF1 mediate canonical Wnt signal transduction, leading to increased proliferation, enhanced synergistically by Wnt3a. However, LRP5 overexpression inhibits osteogenic differentiation, further suppressed by Wnt3a. Wnt5a does not affect cell proliferation but enhances osteogenesis of MSCs. Interestingly, Wnt5a inhibits Wnt3a effects on MSCs, while Wnt3a suppresses Wnt5a-mediated enhancement of osteogenesis. Flow cytometry revealed that LRP5 expression elicits differential changes in Fz receptor profiles in undifferentiated versus osteogenic MSCs. Taken together, these results suggest that Wnt signaling crosstalk and functional antagonism with the LRP5 co-receptor are key signaling regulators of MSC maintenance and differentiation.     

Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations.     

Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2

Wnts are lipid-modified secreted glycoproteins that regulate diverse biological processes. We report that Wnt5a, which functions in noncanonical Wnt signaling, has activity on endothelial cells. Wnt5a is endogenously expressed in human primary endothelial cells and is expressed in murine vasculature at several sites in mouse embryos and tissues. Expression of exogenous Wnt5a in human endothelial cells promoted angiogenesis. Wnt5a induced noncanonical Wnt signaling in endothelial cells, as measured by Dishevelled and ERK1/2 phosphorylation, and inhibition of canonical Wnt signaling, a known property of Wnt5a. Wnt5a induced endothelial cell proliferation and enhanced cell survival under serum-deprived conditions. The Wnt5a-mediated proliferation was blocked by Frizzled-4 extracellular domain. Wnt5a expression enhanced capillary-like network formation, whereas reduction of Wnt5a expression decreased network formation. Reduced Wnt5a expression inhibited endothelial cell migration. Screening for Wnt5a-regulated genes in cultured endothelial cells identified several encoding angiogenic regulators, including matrix metalloproteinase-1, an interstitial collagenase, and Tie-2, a receptor for angiopoietins. Thus, Wnt5a acts through noncanonical Wnt signaling to promote angiogenesis.     

R-spondin1 synergizes with Wnt3A in inducing osteoblast differentiation and osteoprotegerin expression

R-spondins are a new group of Wnt/beta-catenin signaling agonists, however, the role of these proteins in bone remains unclear. We reported herein that R-sponin1 (Rspo1) acted synergistically with Wnt3A to activate Wnt/beta-catenin signaling in the uncommitted mesenchymal C2C12 cells. Furthermore, we found that Rspo1 at concentrations as low as 10 ng/ml synergized strongly with Wnt3A to induce C2C12 osteoblastic differentiation and osteoprotegerin expression. These events were blocked by Wnt/beta-catenin signaling antagonist Dickkopf-1. Finally, we demonstrated that Rspo1 synergized with Wnt3A to induce primary mouse osteoblast differentiation. Together, these findings suggest that Rpos1 may play an important role in bone remodeling.     

Wdr5, a WD-40 protein, regulates osteoblast differentiation during embryonic bone development

Wdr5 accelerates osteoblast and chondrocyte differentiation in vitro, and is developmentally expressed in osteoblasts as well as in proliferating and hypertrophic chondrocytes. To investigate the role of Wdr5 during endochondral bone development, transgenic mice overexpressing Wdr5 under the control of the 2.3-kb fragment of the mouse alpha(1) I collagen promoter were generated. The transgene was specifically expressed in the osteoblasts of transgene positive mice and was absent in the growth plate. Histological analyses at embryonic day 14.5 demonstrated that the humeri of transgene positive embryos were longer than those isolated from wild-type littermates largely due to an expansion of the hypertrophic chondrocyte layer. Acceleration of osteoblast differentiation was observed with greater and more extensive expression of type I collagen and more extensive mineral deposition in the bone collar of transgene positive embryos. Acceleration of vascular invasion was also observed in transgene positive mice. Postnatal analyses of transgenic mice confirmed persistent acceleration of osteoblast differentiation. Targeted expression of Wdr5 to osteoblasts resulted in earlier activation of the canonical Wnt signaling pathway in the bone collar as well as in primary calvarial osteoblast cultures. In addition, overexpression of Wdr5 increased the expression of OPG, a target of the canonical Wnt signaling pathway. Overall, our findings suggest that Wdr5 accelerates osteoblast differentiation in association with activation of the canonical Wnt pathway.     

BK Channel Deficiency in Osteoblasts Reduces Bone Formation via the Wnt/β-Catenin Pathway

Global knockout of the BK channel has been proven to affect bone formation; however, whether it directly affects osteoblast differentiation and the mechanism are elusive. In the current study, we further investigated the role of BK channels in bone development and explored whether BK channels impacted the differentiation and proliferation of osteoblasts via the canonical Wnt signaling pathway. Our findings demonstrated that knockout of Kcnma1 disrupted the osteogenesis of osteoblasts and inhibited the stabilization of β-catenin. Western blot analysis showed that the protein levels of Axin1 and USP7 increased when Kcnma1 was deficient. Together, this study confirmed that BK ablation decreased bone mass via the Wnt/β-catenin signaling pathway. Our findings also showed that USP7 might have the ability to stabilize the activity of Axin1, which would increase the degradation of β-catenin in osteoblasts.     

Collagen, type V, alpha1 (COL5A1) is regulated by TGF-beta in osteoblasts.

Bone matrix contains high concentrations of growth factors that are known to play important regulatory roles during osteogenesis, particularly transforming growth factor-beta (TGF-beta). Divergent effects of TGF-beta on bone formation have been reported both in vitro and in vivo depending upon experimental conditions, cells employed and their stage of maturation. In this study, we have used a clonal osteoblastic cell line MC3T3-E1, derived from newborn mouse calvaria, as an in vitro model of bone development. These cells undergo an ordered, time-dependent developmental sequence characterized by three stages (proliferation, differentiation and mineralization), over a 30-35-day period. In this study, cDNA microarray technology was used to study the expression profile of 8470 genes, in the presence of TGF-beta1 during osteoblast development. Microarray analysis revealed 120 cDNAs to be differentially expressed in MC3T3-E1 osteoblasts that had been treated with TGF-beta1. From the 120 differentially expressed genes, we selected Collagen, type V, alpha1 (COL5A1) {differential expression=+4.9} for further studies since it represents a previously uncharacterized component of the bone matrix. Using Northern blotting, we found that, when MC3T3-E1 cells were treated with TGF-beta1, COL5A1 was up-regulated during the proliferation and differentiation phases of osteogenesis. Furthermore, by a combination of RNA in situ hybridization and Northern blotting, we found COL5A1 mRNA to be expressed in the calvaria and developing bone of the E17.5 mouse embryos. Lastly, significant COL5A1 protein expression was observed by immunohistochemistry in the developing bone of the E17.5 mouse embryos. In conclusion, by the use of in vitro and in vivo approaches, we have discovered that the COL5A1 gene is a target of TGF-beta during osteogenesis.     

VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells

Osteoblasts and adipocytes are thought to derive from a common bone marrow stromal cell (BMSC) precursor. Activation of the canonical Wnt signaling pathway plays a pivotal role in the differentiation of BMSCs along either of these two lineages, promoting osteogenesis and inhibiting adipogenesis. Liganded nuclear receptors, including the vitamin D receptor (VDR) and peroxisomal proliferator-activated receptor gamma (PPARgamma), can also affect BMSCs differentiation. To address whether VDR ablation modulates the differentiation of BMSCs into the osteoblast or adipogenic lineages, BMSCs were isolated from VDR null mice and from their wild-type littermates. VDR ablation did not alter osteoblastic differentiation. However, when cultured under adipogenic conditions, BMSCs from the VDR null mice expressed higher mRNA levels of PPARgamma and of markers of adipogenic differentiation. An increase in the size and number of mature adipocyte foci was also observed in cultures isolated from VDR null mice relative to those isolated from wild-type mice. To address whether the increased adipogenesis observed in the VDR null cultures was associated with inhibition of the canonical Wnt signaling pathway, mRNA levels for DKK1 and SFRP2 were examined. Cultures from the VDR null mice expressed higher levels of mRNA encoding DKK1 and SFRP2 than did the wild-type cultures. This difference is, at least in part, due to ligand-dependent actions of the VDR, since 1,25-dihydroxyvitamin D3 suppressed DKK1 and SFRP2 expression in wild-type cultures. Thus, the VDR inhibits adipogenesis of BMSCs at least in part by suppressing the expression of inhibitors of the canonical Wnt signaling pathway.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.