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J Biol Chem, Vol. 273, Issue 28, 17318-17325, July 10, 1998

Three Activator Protein-1-binding Sites Bound by the Fra-2·JunD Complex Cooperate for the Regulation of Murine Laminin 3A (lama3A) Promoter Activity by Transforming Growth Factor-

Thierry Virolle, Marie-Noëlle Monthouel, Zied Djabari, Jean-Paul Ortonne^¶, Guerrino Meneguzzi, and Daniel Aberdam

From  INSERM U385, Faculté de Médecine, 06107 Nice Cedex 2 and the ^¶ Service de Dermatologie, Hôpital L'Archet, 06002 Nice Cedex 1, France

Several lines of evidence suggest a role for laminin-5 in skin wound healing. We report here that transforming growth factor- (TGF-), which elicits various responses during cutaneous healing, stimulates transcription of the mouse laminin 3A (lama3A) gene. To identify the TGF--responsive elements (TGF-REs) on the lama3A promoter, we have generated a series of 5'-deletions of the promoter upstream of the -galactosidase reporter gene. Transient cell transfection assays using mouse PAM212 keratinocytes revealed that TGF-REs lie between nucleotides 297 and 54 relative to the transcription start site. Insertion of the TGF-RE in front of the unresponsive minimal SV40 promoter conferred TGF- 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- responsiveness by 50%. Simultaneous mutation of these two AP-1 sites resulted in a 65% decline in the response to TGF-, 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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