Pituitary Adenylate Cyclase-Activating Polypeptide Stimulates Proenkephalin Gene Transcription through API- and CREB-Dependent Mechanisms

The effects of the pituitary adenylase cyclate-activating peptides (PACAP) 27 and 38 on proenkephalin (PENK) gene transcription were examined in PC 12 (rat pheochromocytoma) cells using transient transfection assays. Both ligands stimulated PENK gene transcription in a dose-dependent manner, with an apparent ED₅₀ close to 5 X 10^-11 M. Inactivation of cAMP dependent-protein kinase (PKA) with a dominant inhibitory mutant strongly reduced PACAP-stimulated PENK transcription. Using reporter genes driven by either the minimal TPA-responsive element (TRE: TGACTCA) or cAMP-responsive element (CRE: TGACGTCA), we showed that the two PACAPs activate transcription through both regulatory sequences. These effects could result from direct post-translational activation of Jun and CREB, as shown using GAL4-Jun or GAL4-CREB fusion proteins. Expression of a dominant inhibitory mutant of CREB decreased by 60% the response to PACAP, suggesting that CREB is implicated in PENK transactivation. Similarly, expression of c-fos antisense RNA reduced by 80% the stimulatory effects of PACAP. Taken together, these results indicate that PACAP stimulates PENK transcription by members of both the API and the CREB families. However, API by itself is not sufficient to increase PENK transcription, as insulin-like growth factor 1 (IGF1), which stimulates API activity but not cAMP production, is unable to stimulate PENK transcription. These results indicate a cooperative effect of API and CREB on PENK transcription.