Requirement of an AP-1 site in the calcium response region of the involucrin promoter

Involucrin is a major protein of the cornified envelope of keratinocytes th at provides much of the structural integrity of the skin. The gene expressi on of this differentiation marker is induced by elevated extracellular calc ium in cultured human keratinocytes. A 3.7-kilobase fragment of this gene c ontains the necessary elements to drive a luciferase reporter in a calcium- dependent manner. We have sequenced the upstream region of the involucrin p romoter and localized a calcium response element that contains an activatin g protein-1 (AP-1) site (TGAGTCA), Mutation of this site abolished the prom oter activation by calcium. Compared with cells grown in 0.03 mar calcium, the binding activity of factors within nuclear extracts from keratinocytes for this AP-1 site was enhanced 3-fold in cells grown in 1.2 mM calcium. Im munoelectrophoretic mobility shift (supershift) assays identified JunD, Fra 1, and Fra2 as the major factors that bind to the AP-1 element. Western ana lysis of the proteins in the nuclear extracts showed that the levels of c-J un, JunB, JunD, FosB, and Fra2 increased and the levels of c-Fos and Fra1 d ecreased slightly with calcium treatment. The effect of calcium on the invo lucrin promoter was enhanced synergistically by phorbol 12-myristate: 13-ac etate (PMA) in a protein kinase-dependent manner. In conclusion, calcium-re gulated involucrin gene expression is mediated at least in part by AP-1 tra nscription factors.