Transcriptional regulation of alpha 2(1) collagen gene expression by fibroblast growth factor-2 in MC3T3-E1 osteoblast-like cells

Fibroblast growth factor-2 (FGF-2) stimulates proliferation and inhibits di fferentiated function of osteoblasts by suppressing synthesis of type I col lagen and other proteins. However, little is known regarding the molecular mechanisms regulating the suppressive effects of FGF-2 on type I collagen s ynthesis in osteoblasts. The zinc finger transcription factor Egr-1: and th e basic helix-loop-helix (bHLH) family of proteins have been implicated in the regulation of genes crucial to mesodermal cell growth and differentiati on. The aim of this study was to determine whether Egr-1 and TWIST might be potential transcriptional regulators of the inhibitory effects of FGF-2 on alpha2(I) collagen expression in MC3T3-E1 osteoblasts which undergo a deve lopmental sequence in vitro. Upon treatment of undifferentiated MC3T3-E1 ce lls with 1 nM FGF-2, Egr-1 mRNA increased with the effect maximal after 30- 60 min. TWIST mRNA also increased with the effect maximal at 2 h. We analyz ed the transcriptional control of alpha2(I) collagen gene expression by FGF -2 by transient transfection of an alpha2(I) collagen-luciferase construct (pH5) into undifferentiated MC3T3-E1 cells. The activity of the pH5 lucifer ase promoter decreased in a dose-dependent manner following treatment with. 01 and 1 nM FGF-2. We identified putative Egr-1: and TWIST recognition sequ ences in the proximal region of the promoter for the murine alpha2(I) colla gen gene and a putative Egr-1 site in the 5' region of the murine TWIST pro moter. In gel mobility shift assays, potential Egr-1 response elements in t he 5' region of the murine TWIST and alpha2(I) collagen genes demonstrated specific Egr-1 binding activity with bFGF-treated nuclear extracts obtained from MC3T3-E1 cells. These results indicate that Egr-1 and TWIST are expre ssed in undifferentiated MC3T3-E1 osteoblast-like cells following treatment with FGF-2 and they may be potential transcriptional regulators of FGF-2s negative effects on alpha2(I) collagen gene expression. J. Cell. Biochem. 8 0:550-559, 2001. Published 2001 Wiley-Liss, Inc.dagger