Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes

Mesenchymal cells can differentiate into osteoblasts, adipocytes, myoblasts, or chondroblasts. Whether mesenchymal cells that have initiated differentiation along one lineage can transdifferentiate into another is largely unknown. Using 3T3-F442A preadipocytes, we explored whether extracellular signals could redirect their differentiation from adipocyte into osteoblast. 3T3-F442A cells expressed receptors and Smads required for bone morphogenetic protein (BMP) signaling. BMP-2 increased proliferation and induced the early osteoblast differentiation marker alkaline phosphatase, yet only mildly affected adipogenic differentiation. Retinoic acid inhibited adipose conversion and cooperated with BMP-2 to enhance proliferation, inhibit adipogenesis, and promote early osteoblastic differentiation. Expression of BMP-RII together with BMP-RIA or BMP-RIB suppressed adipogenesis of 3T3-F442A cells and promoted full osteoblastic differentiation in response to retinoic acid. Osteoblastic differentiation was characterized by induction of cbfa1, osteocalcin, and collagen I expression, and extracellular matrix calcification. These results indicate that 3T3-F442A preadipocytes can be converted into fully differentiated osteoblasts in response to extracellular signaling cues. Furthermore, BMP and retinoic acid signaling cooperate to stimulate cell proliferation, repress adipogenesis, and promote osteoblast differentiation. Finally, BMP-RIA and BMP-RIB induced osteoblast differentiation and repressed adipocytic differentiation to a similar extent.     

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.     

Differential effects of secreted frizzled-related proteins (sFRPs) on osteoblastic differentiation of mouse mesenchymal cells and apoptosis of osteoblasts

Secreted frizzled-related proteins (sFRPs) are modulators of Wnt signaling. This study was undertaken for definitive assessment of contribution of different sFRPs in osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. Treatment of C3H10T1/2 cells with sFRP-2 at concentrations of 10, 50, and 100 nM and sFRP-4 at low concentrations (5 nM) significantly increased Wnt-3A-induced alkaline phosphatase (ALP) activities, whereas sFRP-1 or 3 did not. Retroviral transduction of the sFRP-2 but not other sFRPs also significantly enhanced ALP activity induced by β-glycerophosphate and ascorbic acid. Furthermore, transfection of all the sFRP expression vectors significantly increased β-catenin/TCF reporter activity and the effects were most prominent with sFRP-2 and -4. In osteoblast apoptosis assay, only sFRP-3 increased etoposide-induced apoptosis in MC3T3-E1 mouse osteoblasts. In conclusion, we found that different repertoires of sFRPs exert differential effects on osteoblastic differentiation of mouse mesenchymal cells and cellular apoptosis of mouse osteoblasts in vitro.     

The Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation induces PKC-independent downregulation of FGFR-2 associated with premature calvaria osteoblast differentiation

We recently showed that the Apert Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation causes premature osteoblast differentiation and increased subperiosteal calvaria bone matrix formation. To gain further insight into the cellular mechanisms involved in these effects, we examined the effects of the mutation on the expression of FGFRs in relation to cell proliferation and differentiation markers in vivo and in vitro, and we analyzed the underlying signaling pathways in mutant cells. Immunohistochemical analysis of the Apert calvaria suture showed that the Ser252Trp FGFR-2 mutation increased type 1 collagen, osteocalcin, and osteopontin expression in preosteoblasts compared to normal, whereas cell growth was not affected. The premature osteoblast differentiation induced by the mutation was associated with lower than normal FGFR-2 immunolabeling, whereas FGFR-1 and FGFR-3 levels were not decreased. Immunocytochemical analysis in osteoblasts isolated from Apert coronal suture showed that the Ser252Trp mutation induced constitutive downregulation of FGFR-2 in mutant cells. Western blot analysis of FGFRs in immortalized mutant osteoblastic cells confirmed that the mutation induced FGFR-2 downregulation. FGFR-2 mRNA levels were not altered in mutant cells, indicating that FGFR-2 downregulation resulted from receptor internalization rather than from changes in receptor mRNA. The signaling pathway involved in FGFR-2 downregulation was studied using specific inhibitors of FGF signaling molecules. The selective PKC inhibitor calphostin C markedly reduced FGFR-2 protein levels in mutant cells, in contrast to the p38 MAP kinase inhibitor SB 203580 or the Erk 1,2 MAP kinase inhibitor PD-98059, showing that PKC is involved in FGFR-2 regulation, but not in FGFR-2 downregulation in mutant cells. The results indicate that the premature osteoblast differentiation induced by the FGFR-2 Ser252Trp mutation is associated with a PKC-independent downregulation of FGFR-2 in human calvaria cells.     

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.     

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.     

Akt Activation is Required for TGF-β1-Induced Osteoblast Differentiation of MC3T3-E1 Pre-Osteoblasts

Background

We have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.

Methodology/Principal Findings

MC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.

Conclusions

TGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.     

Lentiviral-mediated RNAi targeting p38MAPK ameliorates high glucose-induced apoptosis in osteoblast MC3T3-E1 cell line

The p38 mitogen activated protein kinase (p38MAPK) pathway is an important signaling cascade involved in cell growth, differentiation and apoptosis. High glucose activates p38MAPK pathway in different cells, including osteoblasts. In the present study, role of p38MAPK in high glucose induced osteoblast apoptosis and potential of RNA interference (RNAi) targeting p38MAPK as a therapy strategy have been reported. Lentiviral-mediated RNAi effectively reduced p38MAPK and p-p38MAPK expressions in osteoblastic cell line (MC3T3-E1) following high glucose (22 mM) induction. Inhibition of p38MAPK activity significantly suppressed high glucose induced apoptosis of MC3T3-E1 cell and was confirmed by flow cytometry and ultra-structural examination by transmission electronic microscope. Inhibition of p38MAPK also significantly attenuates caspase-3 and bax protein expressions, but increased significantly bcl-2 expression as determined by Western blot analysis. The results suggested that p38MAPK mediates high glucose induced osteoblast apoptosis, partly through modulating the expressions of caspase-3, bax and bcl-2. Inhibition of p38MAPK with lentiviral-mediated RNAi or its specific inhibitor provides a new strategy to treat high glucose induced osteoblast apoptosis.     

Adiponectin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the AMPK/mTOR pathway

Vascular calcification is common in patients with peripheral artery diseases and coronary artery diseases. The osteoblastic differentiation of vascular smooth muscle cells (VSMCs) contributes significantly to vascular calcification. Adiponectin has been demonstrated to exert a protective effect in osteoblastic differentiation of VSMCs through regulating mTOR activity. However, the upstream and downstream signaling molecules of adiponectin-regulated mTOR signaling have not been identified in VSMCs with osteoblastic differentiation. In this study, the VSMC differentiation model was established by beta-glycerophosphate (β-GP) induction. The mineralization was identified by Alizarin Red S staining. Protein expression and phosphorylation were detected by Western blot or immunofluorescence. Adiponectin attenuated osteoblastic differentiation and mineralization of β-GP-treated VSMCs. Adiponectin inhibited osteoblastic differentiation of VSMCs through increasing the level of p-AMPKα. Pretreatment of VSMCs with AMPK inhibitor blocked while AMPK activator enhanced the effect of adiponectin on osteoblastic differentiation of VSMCs. Adiponectin upregulated TSC2 expression and downregulated mTOR and S6K1 phosphorylation in β-GP-treated VSMCs. Adiponectin treatment significantly attenuates the osteoblastic differentiation and calcification of VSMCs through modulation of AMPK–TSC2–mTOR–S6K1 signal pathway.