Bone morphogenetic protein activities are enhanced by 3′,5′-cyclic adenosine monophosphate through suppression of Smad6 expression in osteoprogenitor cells

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF)-β superfamily, and some display potent osteogenic activity both in vivo and in vitro. The BMP signaling cascade involving BMP receptors at the cell membrane and intracellular messengers (Smads) has been elucidated, but the regulatory mechanisms of BMP signaling have not been clarified. We previously found that pentoxifyline (PeTx), a nonspecific inhibitor of phosphodiesterase (PDE), and rolipram, a PDE-4-specific inhibitor, enhance BMP-4-induced osteogenic differentiation of mesenchymal cells, probably through the elevation of intracellular cyclic adenosine monophosphate (cAMP) accumulation and modulation of BMP signaling pathways as enhanced BMP-4 action was reproduced by addition of dibutylyl-cAMP (dbcAMP). However, the precise mechanisms underlying the enhancing effects of those agents on BMP signaling were not completely revealed. As already reported, BMPs utilize a specific intracellular signaling cascade to target genes via R-Smads (Smad1,5,8), Co-Smad (Smad4) and I-Smads (Smad6,7). One possibility for cAMP-mediated effects on BMP signaling might be suppression of I-Smads expression since these proteins form a negative feedback loop in BMP signaling. To examine this possibility, changes in I-Smad (Smad6) expression on addition of dbcAMP or PeTx were examined in a bone-marrow-derived osteogenic cell line (ST2). Alkaline phosphatase activity in ST2 cells was consistently induced by BMP-4 treatment (300 ng/ml), and Smad6 mRNA expression was also induced by BMP-4 treatment. Although concurrent treatment of ST2 cells with BMP-4 and dbcAMP elicited further activation of alkaline phosphatase, addition of dbcAMP reduced BMP-4-induced Smad6 expression in a dose-dependent manner. Furthermore, detection of phosphorylated Smad1/5/8 on Western blotting analysis was prolonged, suggesting prolonged kinase activity of BMP receptors through suppressed expression of Smad6. Elevated intracellular cAMP might thus enhance BMP signaling by suppressing Smad6 induction and prolonging intracellular BMP signaling.