Three activator protein-1-binding sites bound by the Fra-2.JunD complex cooperate for the regulation of murine laminin alpha3A (lama3A) promoter activity by transforming growth factor-beta.
Several lines of evidence suggest a role for laminin-5 in skin wound healing. We report here that transforming growth factor-beta (TGF-beta), which elicits various responses during cutaneous healing, stimulates transcription of the mouse laminin alpha3A (lama3A) gene. To identify the TGF-beta-responsive elements (TGFbeta-REs) on the lama3A promoter, we have generated a series of 5'-deletions of the promoter upstream of the beta-galactosidase reporter gene. Transient cell transfection assays using mouse PAM212 keratinocytes revealed that TGFbeta-REs lie between nucleotides -297 and -54 relative to the transcription start site. Insertion of the TGFbeta-RE in front of the unresponsive minimal SV40 promoter conferred TGF-beta inducibility. Computer analysis of the promoter sequence identified three canonical activator protein-1 (AP-1) sites located at nucleotides -277 (AP-1A), -125 (AP-1B), and -69 (AP-1C). Site-directed mutagenesis of either the AP-1A or AP-1C site did not drastically alter the basal activity of the lama3A promoter, but reduced TGF-beta responsiveness by 50%. Simultaneous mutation of these two AP-1 sites resulted in a 65% decline in the response to TGF-beta, suggesting a cooperative contribution of each site to the overall promoter activity. In contrast, mutation of the AP-1B site markedly reduced the basal activity of the lama3A promoter, indicating that this AP-1 site is essential for gene expression. Mobility shift assays demonstrated specific binding of Fra-2 and JunD to the AP-1 sites, suggesting for the first time a possible regulatory function for the Fra-2.JunD AP-1 complex in a basal keratinocyte-specific gene.
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INSERM U385, Faculté de Médecine, 06107 Nice Cedex 2, France.
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Djabari
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Ortonne
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Meneguzzi
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Aberdam
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051921:Fos-Related Antigen-2; D005786:Gene Expression Regulation / Q000187:drug effects*; D007797:Laminin / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D051921:Fos-Related Antigen-2; C497873:Fosl2 protein, mouse; D007797:Laminin; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C095567:laminin alpha 3; D004247:DNA