Induction of proenkephalin gene expression in cultured bovine chromaffin cells is dependent on protein synthesis of AP-1 proteins.

In bovine chromaffin cells forskolin, phorbol ester, or high potassium levels induce a rapid increase of c-fos, c-jun, and junB mRNA levels, which precede an induction of proenkephalin gene expression. Preincubation of the cells with cycloheximide inhibited induction of proenkephalin mRNA levels by each of these agents, indicating that newly synthesized transcription factors are involved. Transient transfection of reporter genes showed that the ENKCRE-2 element of the proenkephalin promoter was sufficient for basal and second messenger-induced expression. Gel mobility shift assays revealed that stimulation increased the binding of nuclear proteins to ENKCRE-2 and AP-1 oligonucleotides but not to CRE oligonucleotides. Western analysis showed that the induction of AP-1 binding activity was associated with Fos protein synthesis. Moreover, cotransfection of c-fos, but not of c-jun or CREB, expression plasmids transactivated the expression of the PENKCAT reporter genes. These results suggest that Fos and/or other components of AP-1 transcription factors, rather than CREB or other preexisting proteins, play a specific role in the induction of the proenkephalin gene in bovine chromaffin cells.