Endocrine Abstracts

Bone is not only a skeletal scaffold but also an endocrine organ. Its formation and remodeling initiate from the recruitment and subsequent differentiation of mesenchymal stem cells (MSCs), in which the signals are known to be dominated by SMAD2/3 signaling driven by TGF-βs and SMAD1/5/8 signaling driven by BMPs, respectively. Of interest, distinct from the above concepts, we found that TGF-β1 induces not only SMAD2/3 phosphorylation but also SMAD1/5/8 phosphorylation in either primary MSCs or C3H10T1/2 MSC line. To further investigate the underlying mechanism, individual Smad knockdown, receptor profiling and signaling inhibitors were applied. Our results indicated that TGF-β1 mainly induces SMAD1 phosphorylation via ALK5-containing receptor complexes in MSCs. In terms of functional characterization, we demonstrated that TGF-β1-induced phosphorylated SMAD1 complexes can be translocated into the nucleus; this then increases the transcription of BMP-responsive genes, such as Id1 and Timp3. However, in contrast to SMAD2/3 activation, we found that SMAD1/5/8 phosphorylation driven by TGF-β1 retained transiently and this may in turn dampen the BMP4-mediated activities in C3H10T1/2 MSC line. Indeed, TGF-β1 co-treatment significantly inhibited BMP4-activated reporter activity as well as expression of osteogenesis-related genes, such as Runx2. Using co-immunoprecipitation assay, we proposed that the aforementioned inhibitory effect may involve the formation of SMAD1/5-SMAD2/3 mix complex, which then recruits yet unknown co-repressors in MSCs. Taken together, our findings unveil a previously uncharacterized TGF-β-stimulated SMAD mechanism in MSCs and would further draw interests in exploring whether this signaling participates in balancing the BMP activity during MSC specification.