Bcl-2 is a crucial regulator of cell survival and is frequently over expressed in epidermal cancers. Regulation of bcl-2 expression in epidermal keratinocytes is not well characterized. In the epidermis bcl-2 is expressed only in keratinocytes of the basal layer and the outer root sheath of hair follicles. Suprabasalar keratinocytes lose bcl-2 expression. The calcium gradient in the epidermis is able to regulate keratinocyte differentiation. Increasing calcium concentrations are associated with differentiation and resulted in the down regulation of a 2.9 kb bcl-2 promoter luciferase construct. The AP-1 family of transcription factors is differentially expressed in the various strata of the epidermis and, has been shown to be involved in the stage specific expression of various differentiation markers in the epidermis. In silico analysis of the bcl-2 promoter identified several potential AP-1 binding sites. Co-transfection of JUNB and JUND, but not other AP-1 dimers, caused a significant upregulation of the bcl-2 promoter in primary keratinocytes grown in low calcium concentrations. This suggests a novel role for JUNB and JUND in transcriptional regulation of bcl-2. Immunoelectrophoretic mobility shift assays identified JUNB and JUND as the factors that bind to the AP-1 element. Mutational analysis of this binding site on the bcl-2 promoter combined with luciferase assays identified it as the site involved in bcl-2 regulation. In vivo chromatin immunoprecipitation studies indicate JUNB/JUND binding to this site in keratinocytes grown in low calcium media and an abrogation of binding in differentiating keratinocytes. Using phosphorylation mutants of JUND, we determined that the activation of the bcl-2 promoter requires phosphorylation of JUND at the Ser90/Ser100 sites of JUND. We further identified the MAPK pathway mediates the phosphorylation of JUND. These studies show a role for AP-1 in bcl-2 activation and, elucidate a potential mechanism for the regulation of bcl-2 in primary keratinocytes.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA