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

• Published in: Journal of cellular biochemistry 2001-03, Vol.80 (4), p.550-559
• Main Authors: Fang, Meika A., Glackin, Carlotta A., Sadhu, Archana, McDougall, Skye
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Binding, Competitive; Blotting, Northern; Cell Differentiation; Cell Division; Cell Nucleus - metabolism; Collagen - biosynthesis; Collagen Type I; DNA-Binding Proteins - metabolism; Dose-Response Relationship, Drug; Down-Regulation; Early Growth Response Protein 1; Egr-1; FGF-2; Fibroblast Growth Factor 2 - biosynthesis; Fibroblast Growth Factor 2 - metabolism; Fibroblast Growth Factor 2 - pharmacology; Gene Expression Regulation; Genes, Reporter; helix-loop-helix; Humans; Immediate-Early Proteins; Luciferases - metabolism; Mice; Nuclear Proteins - metabolism; osteoblast; Osteoblasts - metabolism; Promoter Regions, Genetic; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Time Factors; transcription factor; Transcription Factors - metabolism; Transcription, Genetic; Transfection; Twist-Related Protein 1
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/1097-4644(20010315)80:4<550::AID-JCB1009>3.0.CO;2-1

Fibroblast growth factor‐2 (FGF‐2) stimulates proliferation and inhibits differentiated function of osteoblasts by suppressing synthesis of type I collagen and other proteins. However, little is known regarding the molecular mechanisms regulating the suppressive effects of FGF‐2 on type I collagen synthesis in osteoblasts. The zinc finger transcription factor Egr‐1 and the basic helix‐loop‐helix (bHLH) family of proteins have been implicated in the regulation of genes crucial to mesodermal cell growth and differentiation. 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 α2(I) collagen expression in MC3T3‐E1 osteoblasts which undergo a developmental sequence in vitro. Upon treatment of undifferentiated MC3T3‐E1 cells 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 analyzed the transcriptional control of α2(I) collagen gene expression by FGF‐2 by transient transfection of an α2(I) collagen‐luciferase construct (pH5) into undifferentiated MC3T3‐E1 cells. The activity of the pH5 luciferase promoter decreased in a dose‐dependent manner following treatment with.01 and 1 nM FGF‐2. We identified putative Egr‐1 and TWIST recognition sequences in the proximal region of the promoter for the murine α2(I) collagen gene and a putative Egr‐1 site in the 5′ region of the murine TWIST promoter. In gel mobility shift assays, potential Egr‐1 response elements in the 5′ region of the murine TWIST and α2(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 expressed 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 α2(I) collagen gene expression. J. Cell. Biochem. 80:550–559, 2001. Published 2001 Wiley‐Liss, Inc.