Opposing activities of c-Fos and Fra-2 on AP-1 regulated transcriptional activity in mouse keratinocytes induced to differentiate by calcium and phorbol esters

The major differentiation products of maturing keratinocytes contain AP-I regulatory motifs, and AP-1 DNA binding activity increases in cultured keratinocytes induced to differentiate by calcium. Here, we have analysed AP-I transcriptional activity in mouse keratinocytes treated with calcium and 12-O-tetradecanoyl phorbol-13-acetate (TPA), two agents that induce terminal differentiation of keratinocytes with different phenotypic consequences. Reporter constructs representing multimers of AP-I sequences found in keratinocyte marker genes demonstrated that the calcium-induced AP-I DNA binding activity does not correlate with transcriptional activation. Moreover, expression from active subunits of the profilaggrin and spr 1 promoters increased in calcium-treated keratinocytes when the AP-1 sites were disrupted, indicating that AP-I may negatively regulate certain promoters in these cells. In contrast, AP-1 reporter activity was increased in keratinocytes treated with TPA. This induction was dependent upon the expression of c-Fos since AP-I transcriptional activity was not increased in TPA-treated keratinocytes derived from c-fos null mice. Analysis of AP-1 protein expression in calcium-and TPA-treated keratinocytes demonstrated that only TPA increased the expression of c-Jun, while Jun B and Jun D were induced by both of these agents. c-Fos was expressed only in TPA treated keratinocytes, Fra-2 was expressed only in calcium-treated cells, and Fra-1 was expressed in both. Exogenous expression of Fra-2 repressed AP-1 transcriptional activity in TPA-treated keratinocytes, while c-Fos expression activated the AP-I sequence in calcium-treated keratinocytes. These data indicate that Fra-2 and c-Fos play opposing roles in regulating AP-1 activity in keratinocytes and that multiple inducer-dependent regulatory pathways may exist for the expression of keratinocyte differentiation markers.