Transcription factor AP-2 regulates human insulin-like growth factor binding protein-5 gene expression.
Insulin-like growth factor binding protein-5 (IG-FBP-5) is an important modulator of IGF actions. IG-FBP-5 mRNA is abundant in human fibroblasts and is regulated by cAMP. To understand the molecular mechanism underlying this cell type-specific expression and regulation, we isolated the 5'-flanking region of the human IGFBP-5 gene and fused it to a promoter-less reporter plasmid encoding luciferase. Transient transfection of the construct into fibroblasts displayed both constitutive and cAMP-induced promoter activity in an orientation-specific manner. Sequence analysis revealed the existence of distal and proximal consensus AP-2 recognition sites located 5' from the TATA box. Both sequences bound specifically to human AP-2 in vitro by gel shift mobility assay. The possible role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with the IGFBP-5 promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated IGFBP-5 promoter activity. This trans-activation was IGFBP-5 promoter and AP-2 specific. In AP-2 abundant fibroblasts, expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed IGFBP-5 promoter activity. In HepG2 cells, AP-2B alone had no significant effect, but the AP-2-induced activation of promoter activity was inhibited by AP-2B in a dose-dependent manner. The relative functional importance of the putative AP-2 binding sites was examined using a number of deletion mutants and point mutations. When the first two distal CCCCACCC-like putative AP-2 sites were deleted or mutated, there was no change in AP-2-induced trans-activation. Deletion or mutation of the proximal GCCNNNGGC-like sequences, however, abolished the AP-2-induced activation. These results suggest that AP-2 regulates the IGFBP-5 gene expression through the proximal GCCNNNGGC-like sequences. This AP-2-mediated trans-activation contributes at least in part to the constitutively high expression of IGFBP-5 in fibroblasts and to the cAMP responsiveness of this gene.
Duan
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Department of Medicine, University of North Carolina at Chapel Hill 27599, USA.
Clemmons
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DR
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics*; D008969:Molecular Sequence Data; D013002:Somatomedins / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D018975:Insulin-Like Growth Factor Binding Protein 5; D013002:Somatomedins; D050656:Transcription Factor AP-2; D014157:Transcription Factors