Integration of BMP and FGF signaling in calvarial osteoblasts

FGF and BMP signaling play critical roles in osteogenesis. However, whether these two osteogenic signaling pathways directly interact with each other in differentiating osteoblasts is not known. Objectives: This study was carried out to determine whether FGF signaling modulates activation of the BMP signaling pathway. Specifically, we determined if FGF9 treatment of osteoblasts would alter expression of BMP2 and noggin genes. We also studied whether FGF9 induces phosphorylation/activation and nuclear translocation of Smad1. Methods: Primary osteoblast cultures were prepared from postnatal day 10 mouse calvarial bones. The cultures were treated with FGF9 (10ng/ml) for 24 hours and examined for expression of BMP2 and noggin genes by Northern analysis. For Smad-1 studies, nuclear and cytoplasmic extracts were prepared from calvarial osteoblasts that had been treated with FGF9 for 24 hours. Phospho Smad-1 was detected by immunoblot analysis using antibodies specific for Ser463/465. To identify the FGF9 signaling pathway which induces phosphorylation of Smad1 at Ser463/465, the cells were pretreated with an Erk inhibitor (U0126) and PKC inhibitors (Calphostin C, Rottlerin). Results: Our data show that FGF9 treatment increases BMP2 and decreases noggin gene expression. FGF9 treatment also induces phosphorylation of Smad1 at Ser463/465, which was subsequently translocated into the nucleus. FGF9 failed to induce Smad1 phosphorylation in osteoblasts expressing dominant negative FGFR2. Inhibitor studies showed that the PKC inhibitors, but not the Erk inhibitor, block FGF9 induced Smad1 phosphorylation. Conclusion: This study shows for the first time that Ser 463/465 residue of Smad1 is phosphorylated by FGF9 and that this phosphorylation requires activation of PKC. (Supported by NIHR03HD40282)