Fig. 6

The TGF-β1-induced mixed SMADs further form a transcriptional suppression complex. A Illustration of the correlations of SMAD2-interacting transcriptional regulators with osteogenesis and adipogenesis in the IPA database. Factors related to [Repression of RNA] were highlighted in purple. Factors related to [Differentiation of osteogenesis] process only were marked with a blue frame, while factors related to [Differentiation of adipogenesis] process only were marked with an orange frame. Co-repressors and co-activators involved in both processes are listed in the purple box and the gray box, respectively. B Hierarchical clustering of the temporal expression patterns of selected SMAD2-binding co-repressors in TGF-β1-treated hBM-MSCs. C and D STRING diagrams depicted the PPI networks between the seven TGF-β1-induced co-repressors in human (C) and mouse (D) models. Interaction confidence scores were shown, with 1 being the highest score. Significance of the PPI enrichment p-value suggests that these proteins are more interconnected than expected by chance. E-I Quantification of the BRE-driven luciferase reporter activity in C3H10T1/2 cells infected with lentivirus-packaged shLuc (E), shHdac1 (F), shTgif1 (G), shAtf3 (H), or shNcor2 (I). The cells were treated with TGF-β1 (0.1 nM) and/or BMP4 (0.1 nM) as indicated. Data are means ± SD. ***P < 0.001 compared to the indicated sample, Student’s t test. n = 3 independent experiments. J The nuclear extracts from C3H10T1/2 cells with indicated treatments for 24 h were subjected to DNA pull-down assays using the BRE-containing or mutant probes. The presence of corresponding co-repressors was confirmed by Western blot. Blots are representative of three independent experiments. K Schematic diagram of the TGF-β1 signaling in controlling MSC differentiation. TGF-β1 activates two distinct branches of SMAD signals in MSCs via the receptor complex formed by TGFBR2 and ALK5. The signaling of canonical SMAD2/3 branch counteracts the BMP-driven adipogenesis. Parts of the induced co-repressors, such as HDAC1, TGIF1 and ATF3, can couple with the novel branch of SMAD1-SMAD2 complex to suppress BMP-driven osteogenesis. Functional disruption of this novel branch is predicted to tune the osteogenic suppression of TGF-β1 down specifically