Three activator protein-1-binding sites bound by the Fra-2·JunD complex cooperate for the regulation of murine laminin α3A (lama3A) promoter activity by transforming growth factor-β

Abstract

Several lines of evidence suggest a role for laminin-5 in skin wound healing. We report here that transforming growth factor-β (TGF-β), which elicits various responses during cutaneous healing, stimulates transcription of the mouse laminin α3A (lama3A) gene. To identify the TGF-β-responsive elements (TGFβ-REs) on the lama3A promoter, we have generated a series of 5'-deletions of the promoter upstream of the β-galactosidase reporter gene. Transient cell transfection assays using mouse PAM212 keratinocytes revealed that TGFβ-REs lie between nucleotides -297 and -54 relative to the transcription start site. Insertion of the TGFβ-RE in front of the unresponsive minimal SV40 promoter conferred TGF-β inducibility. Computer analysis of the promoter sequence identified three canonical activator protein-1 (AP.1) sites located at nucleotides -277 (AP1A), -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-β responsiveness by 50%. Simultaneous mutation of these two AP-1 sites resulted in a 65% decline in the response to TGF-β, 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.