PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE REGULATES PROLACTIN PROMOTER ACTIVITY VIA A PROTEIN-KINASE A-MEDIATED PATHWAY THAT IS INDEPENDENT OF THE TRANSCRIPTIONAL PATHWAY EMPLOYED BY THYROTROPIN-RELEASING-HORMONE

The hypothalamic peptide, pituitary adenylate cyclase-activating polyp eptide (PACAP), can efficiently increase cAMP levels in pituitary cell s and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH(3) rat pituitary cells to PACAP results in increases in cellular c AMP levels, PRL promoter activity, and PRL messenger RNA levels. We ha ve employed this system to further characterize PACAP regulation of PR L gene expression. RT-PCR analysis showed that GH(3) cells express tra nscripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, w e investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the int racellular Ca2+ concentration in GH(3) cell cultures and that the opti mal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphospha te-Ca2+ pathway is not significantly involved in PACAP action on the P RL promoter. Four kinase inhibitors exhibited similar profiles of inhi bition of the activity on PRL-chloramphenicol acetyltransferase (PRL-C AT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The obs ervations that coexpression of the dominant PKA inhibitor R(AB) comple tely blocked either FSK or PACAP action on PRL-CAT and that these acti ons of FSK and PACAP were completely nonadditive imply that the cAMP-P KA pathway plays a dominant role in PACAP regulation of PRL gene expre ssion. Coexpression of low levels of KCREB, a cAMP response element (C RE)-binding protein (CREB) dominant inhibitor, partially blocked regul ation of PRL-CAT activity by PACAP, but not by TRH, implying that PACA P action is mediated at least in part by a CREB family member that can dimerize with CREB. The PRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CR E-like element (CLE). Mutation of either the left or right 4 bp of the CLE yielded a strong decrease in the response to either FSK or PACAP, but not to TRH. These data imply that PACAP and TRH employ independen t pathways to regulate the PRL promoter, and that PACAP action is exer ted virtually entirely via a cAMP/PKA-mediated pathway that is strongl y dependent upon an intact CLE sequence and at least partially depende nt upon the activity of a CREB-related protein.