一类新的BMP信号传递因子在成骨细胞分化中的作用

TGF-b/BMP family ligands interact with specific membrane receptor complexes that have serine/threonine kinase activities. The receptor phosphorylation and activation induced by the ligands lead to phosphorylation of the Smads proteins that translocate to the nucleus, controlling gene expression. Thus, regulation of Smad proteins is a key step in TGF-b/BMP-induced signal transduction. Here we report a novel mechanism of regulation of SMAD-mediated signaling, by which Smad protein levels are controlled through expression of the CHIP protein. CHIP is a U-box-dependent E3 ubiquitin ligase, and has been previously identified as a co-chaperon protein. However, we have isolated CHIP as a Smad-interacting protein in a yeast two-hybrid screen using Smad1. Furthermore we have observed the CHIP interacted with Smad3 using the GST-CHIP protein and the overexpressed Flag tagged Smad3 in 293T cells by an in vitro GST-pull-down assay. Furthermore, the CHIP/Smad3 interaction has been confirmed in vivo in mammalian cells through co-immunoprecipitation. We also showed that the interaction of the endogenous CHIP/Smad3, which were labeled by 35S-methionine, could be observed in the Mv1Lu cells. The TPR domain in CHIP protein and MH2 domain in Smad3 protein were confirmed to be important for the interaction of the two proteins. Interestingly, we demonstrated that the co-expression of Smad3 with the CHIP protein results in the degradation of Smad3 through a ubiquitin-mediated process. Consistent with the observation that CHIP induces Smad degradation, we further show that the expression of CHIP can inhibit the transcription activities of Smad3/4 that is the complex induced by TGF-b signal by luciferase reporter assays. Using RNAi, we successfully knocked down the expression of CHIP protein and we observed that CHIP RNAi facilitated the TGF-b signal. Finally, in both Mv1Lu and BaF3 cell lines stably expressing CHIP, we observed that over-expression of CHIP protein abolished the cell growth inhibition to TGF-b response. At the same time, over-expression of CHIP blocked the JUN-B gene expression, which was a direct target gene for TGF-b signal. These results suggest that CHIP can interact with Smad3, and possibly block the TGF-b signal transduction through the ubiquitin-mediated degradation of Smad3.

成骨细胞分化减少是骨质疏松产生的根本原因之一。BMP可以诱导成骨细胞生成。Smad蛋白在BMP TGF－β信号传递过程中起着关键作用。虽然发现Smad2,3可以与核蛋白结合捶⑾諷mad1下游的结合蛋白。本项目研究由酵母双杂所得的11个强作用候选新基因在BMP信号传递和成骨细胞分化中的功能，对于防止和治疗骨质疏松具有非常重要的意义。..