Transcriptional regulation of alpha 2(I) collagen gene expression by fibroblast growth factor-2 in MC3T3-E1 osteoblast-like cells.
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 alpha2(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 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 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 alpha2(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 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 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 alpha2(I) collagen gene expression. J. Cell. Biochem. 80:550-559, 2001. Published 2001 Wiley-Liss, Inc.
Fang
M A
MA
Geriatric Research, Education and Clinical Center, GRECC 11G, VA Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA.
Glackin
C A
CA
Sadhu
A
A
McDougall
S
S
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D002454:Cell Differentiation; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis*; D024042:Collagen Type I; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D051766:Early Growth Response Protein 1; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D017874:Immediate-Early Proteins; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D051879:Twist-Related Protein 1
D024042:Collagen Type I; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C103173:TWIST1 protein, human; D014157:Transcription Factors; D051879:Twist-Related Protein 1; D016222:Fibroblast Growth Factor 2; D003094:Collagen; D008156:Luciferases