Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation.

Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGFbeta-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation.