Receptor Tyrosine Kinases Inhibit Bone Morphogenetic Protein‐Smad Responsive Promoter Activity and Differentiation of Murine MC3T3‐E1 Osteoblast‐like Cells

Growth factors such as fibroblast growth factor‐2 (FGF‐2) and epidermal growth factor (EGF) that activate extracellular signal‐regulated kinases (ERKs) through receptor tyrosine kinases (RTKs) stimulate proliferation but suppress differentiation of osteoblasts. To study the mechanism of this inhibitory action of these growth factors on osteoblastic differentiation, we evaluated Smad1 transactivity in MC3T3‐E1 osteoblast‐like cells by reporters of promoter activity of mouse Smad6, an early response gene to bone morphogenetic proteins (BMPs). FGF‐2 and EGF inhibited alkaline phosphatase activity and Smad6 promoter activity stimulated by BMP‐2. Overexpression of constitutively active MEK by adenovirus mimicked, but that of dominant negative Ras or treatment with a MEK1 inhibitor, PD098059, reversed, the inhibitory effects of these growth factors on both activities. These effects are mediated by BMP‐responsive elements (BMPREs) on Smad6 promoter, because an artificial reporter driven by three tandem BMPREs gave similar results, and these effects were all abolished when the BMPREs were mutated. RTK‐ERK activation inhibited the promoter activity even when BMP signal was mediated by a mutant Smad1, which lacks phosphorylation sites by ERKs, or by a Smad1 fused to Gal4 DNA binding domain, which constitutively localizes in the nucleus. These results show that the RTK‐Ras‐ERK pathway suppresses BMP signal by interfering with Smad1 transactivity. Because direct phosphorylation of Smad1 by ERKs is not required for the inhibition, other transcriptional factors that are phosphorylated by ERKs might be involved in the regulation of osteoblastic differentiation by ERKs.