Although BMP2 is well known to induce adipogenesis of mesenchymal cells (Ahrens et al., 1993
; Ji et al., 2000
; Sottile and Seuwen, 2000
), to date, the molecular mechanisms by which BMP2 promotes adipocytic differentiation are elusive. In this study, we have shown that BMP2 induces the expression of PPARγ in association with adipocytic differentiation of C3H10T1/2 cells. We also found that dominant-negative PPARγ markedly blocked BMP2-induced adipocytic differentiation of C3H10T1/2 cells. Furthermore, introduction of PPARγ is enough to induce adipogenesis of C3H10T1/2 cells in the absence of BMP2. Collectively, it is most likely that BMP2 exhibits its adipogenic effects by inducing PPARγ. This conclusion is supported by the results that pretreatment of C3H10T1/2 cells with BMP2 was able to bring out the adipogenic effects of TZD. Moreover, overexpression of Smad6, which blocked activation of Smad1 in response to BMP2 stimulation, abolished BMP2-induced adipogenesis of C3H10T1/2 cells as well as the induction of PPARγ expression. Thus, activation of Smad family signaling is a prerequisite for the induction of PPARγ expression, thereby inducing differentiation of C3H10T1/2 cell into adipocytes.
Recently, the data that Smad regulates the function of transcriptional factors, nuclear receptors, or coactivator through its physical association are accumulated (Massague and Wotton, 2000
). We have shown herein that BMP2 increased the transcriptional activity of PPARγ. BMP2 also enhanced adipogenesis caused by overexpression of PPARγ. It is, therefore, possible that Smad signaling promoted the adipogenesis in cooperation with PPARγ. However, Smad6 failed to inhibit the transcriptional activity of PPARγ enhanced by BMP2. In addition, overexpression of Smad1 or Smad5 together with Smad4 had no effects on transcriptional activity of PPARγ (our unpublished data). Furthermore, BMP2 enhanced PPARγ-promoted adipogenesis of C3H10T1/2 cells in which Smad signaling is blockaded by overexpression of Smad6. These data suggest that Smad signaling is implicated in the regulation of the PPARγ expression but not in activation of its function. We found that specific inhibitors for p38 kinase, SB203580, PD169316, and FR167653, decreased the transcriptional activity of PPARγ enhanced by BMP2 treatment to the basal level and markedly inhibited BMP2-induced adipocyte differentiation of C3H10T1/2 cells. Consistently, treatment with SB203580 or overexpression of dominant-negative MKK3 inhibited BMP2-induced adipogenesis of C3H10T1/2 cells. Furthermore, overexpression of TAK1 and TAB1, which markedly induced activation of p38 kinase, was sufficient to up-regulate the transcriptional activity of PPARγ. This effect of TAK1 and TAB1 was also suppressed by treatment with SB203580. These results indicate that p38 kinase but not Smad signaling accounts for up-regulation of PPARγ activity, which leads to further promotion of adipogenesis. The potential schematic model in which BMP2 regulates adipogenesis through activation of Smad1 and p38 kinase is delineated in Figure .
Figure 8 Schematic model of BMP2-induced adipogenesis. Activation of Smad1 is essential for induction of PPARγ expression, which is critical for adipogenic effects of BMP2. In contrast, activation of p38 kinase, presumably through MKK3 and MKK6, plays (more ...)
The mechanism for the up-regulation of PPARγ by p38 kinase remains unknown. The direct up-regulation of PPARγ by p38 kinase through phosphorylation is unlikely because PPARγ possesses only one consensus phosphorylation site by MAP kinases at serine112, which is shown to be phosphorylated by extracellular signal-regulated kinase kinase, thereby inhibiting the transcriptional activity of PPARγ (Hu et al., 1996
). Consistent with this report, we confirm that a specific inhibitor for mitogen-activated protein kinase kinase, PD98059, enhanced the function of PPARγ- and BMP2-induced adipogenesis in C3H10T1/2 cells (our unpublished data). Another possibility is that PPARγ might be up-regulated by forming the complex with a certain transcription factor. ATF-2 is known to be regulated by p38 kinase and to control transcription through cross-talk with other transcriptional factors or coactivators (Davis, 2000
). We observed that BMP2 phosphorylates ATF-2 in C3H10T1/2 cells (our unpublished data). It is, therefore, possible that interaction of ATF-2 with PPARγ might account for the up-regulation of PPARγ by p38 kinase, although the interaction of ATF-2 and PPARγ, and the biological relevance of this interaction are needed to be addressed.
Recently, Sano et al. (1999)
have reported the direct cooperation between Smad3 and p38 kinase signaling. Similarly, it has been shown that p38 pathway is involved in transforming growth factor-β–induced gene expression by interacting with Smad signaling (Hanafusa et al., 1999
, Watanabe et al., 2001
). In contrast, our results with SB203580 indicated that p38 kinase pathway is not involved in the regulation of PPARγ expression, which is controlled by Smad signaling. Kimura et al. (2000)
and Yanagisawa et al. (2001)
reported that activation of p38 signaling was blocked by Smad6 or Smad7 in mouse hybridoma or PC12 cells. On the other hand, we showed herein that Smad6 failed to inhibit the transactivation of PPARγ by BMP2 and that BMP2 increased adipogenic function of PPARγ even in the presence of Smad 6, suggesting that Smad6 has little effect on the up-regulation of PPARγ by p38 kinase. Because we observed that SB203580 had no effect on phosphorylation of Smad1, and that the inhibitory effect of Smad6 on p38 activation was modest in C3H10T1/2 cells, the extent of interaction of both Smad pathway and p38 signaling might depend on types of cells or tissues. Alternatively, it is also possible that experimental models and/or levels of Smad6 expression may account for these differences. Further dissection of relationship of both pathways would solve this issue.
Because we have shown that BMP2 induced expression of PPARγ in C3H10T1/2 cells and that Smad6 blocked the effects of BMP2, we were concerned that these effects of BMP2 or Smad6 might affect our transcriptional assay for PPARγ. To eliminate this possibility, we exogenously introduced the appropriate amount of PPARγ by transfection when we examined the effects of BMP2 on the transcriptional activity of PPARγ. As we expected, the amount of PPARγ introduced by transfection was much larger than that induced by BMP2 (Figure E). Moreover, when we performed the reporter assay, the amounts of PPARγ were not affected by treatment with BMP2 or Smad6 (Figure A). It is, therefore, likely that our transcriptional assay for PPARγ was independent of levels of PPARγ induced by BMP2 treatment or effect of Smad6.
It is known that adipocytes share their origin in bone marrow with osteoblasts (Prockop, 1997
; Pittenger et al., 1999
). We and others have demonstrated that BMP2 also differentiates pluripotent mesenchymal cells toward osteoblasts by activating Smad signaling (Yamamoto et al., 1997
; Nishimura et al., 1998
). In this study, we showed that BMP2 regulated PPARγ expression via Smad signaling. Interestingly, we also observed that overexpression of PPARγ in bone marrow stromal cells or primary osteoblasts inhibited the osteoblastic differentiation process (Hata and Nishimura, unpublished data). Abnormally accelerated adipogenesis in bone marrow, also known as fatty marrow, is often observed in the patients with osteoporosis, which is a common metabolic bone disease characterized by the impaired function and differentiation of osteoblasts (Ducy et al., 2000
). Therefore, the identification of molecular mechanisms that regulate the direction between adipogenesis and osteoblastogenesis may contribute to the further understanding of pathogenesis of metabolic bone disease such as osteoporosis.
In conclusion, our data suggest that BMP2 induces the differentiation of undifferentiated mesenchymal cells into adipocytes by induction of PPARγ expression through activation of Smad1 and up-regulation of its transcriptional activity via activation of p38 kinase. We believe that these results further our understanding of the molecular mechanism underlying the BMP2-induced adipogenesis.