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.

Abstract

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), can efficiently increase cAMP levels in pituitary cells and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH3 rat pituitary cells to PACAP results in increases in cellular cAMP levels, PRL promoter activity, and PRL messenger RNA levels. We have employed this system to further characterize PACAP regulation of PRL gene expression. RT-PCR analysis showed that GH3 cells express transcripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, we investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the intracellular Ca2+ concentration in GH3 cell cultures and that the optimal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphosphate-Ca2+ pathway is not significantly involved in PACAP action on the PRL promoter. Four kinase inhibitors exhibited similar profiles of inhibition of the activity on PRL-chloramphenicol acetyltransferase (PRL-CAT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The observations that coexpression of the dominant PKA inhibitor RAB completely blocked either FSK or PACAP action on PRL-CAT and that these actions of FSK and PACAP were completely nonadditive imply that the cAMP-PKA pathway plays a dominant role in PACAP regulation of PRL gene expression. Coexpression of low levels of KCREB, a cAMP response element (CRE)-binding protein (CREB) dominant inhibitor, partially blocked regulation of PRL-CAT activity by PACAP, but not TRH, implying that PACAP 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 CRE-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 independent pathways to regulate the PRL promoter, and that PACAP action is exerted virtually entirely via a cAMP/PKA-mediated pathway that is strongly dependent upon an intact CLE sequence and at least partially dependent upon the activity of a CREB-related protein.

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…Cytofluorimetric studies, conducted on dispersed rat pituitary cells, have shown that PACAP, acting through three different mechanisms, induces calcium elevation in all categories of endocrine cells Gracia-Navarro et al, 1992;Rawlings et al, 1993Rawlings et al, , 1994Rawlings and Hezareh, 1996;Alarcón and García-Sancho, 2000). Consistent with this observation, PACAP stimulates the release of GH, adrenocorticotropin, LH, follicle-stimulating hormone (FSH), PRL (Goth et al, 1992;Hart et al, 1992;Coleman and Bancroft, 1993;Koch and Lutz-Bucher, 1993;Perrin et al, 1993;Arbogast and Voogt, 1994;Hashizume et al, 1994;Velkeniers et al, 1994;Coleman et al, 1996;Martínez-Fuentes et al, 1998c;Ortmann et al, 1999) and somatolactin (Azuma et al, 2009). The effects of PACAP on the different pituitary cell types are summarized in Table 9.…”

Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-Activating Polypeptides Stimulate Mitogen-Activated Protein Kinase in the Pituitary Cell Line GH4C1 by a 3′,5′-Cyclic Adenosine Monophosphate Pathway

“…VIP is a recognized stimulator of PRL release, whereas the role of PACAP38 on PRL synthesis and release may be species dependent [49]. However, additivity of PACAP38 and TRH effects on the PRL promoter has already been described in the pituitary cell line GH3 [50]. In our study, concentrations of VIP and PACAP38 (1–2 n M ) able to elicit half-maximal activation of ERK in GH4C1 cells are compatible with a physiological role of this transduction pathway.…”

Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells

“…Although cAMP generated upon stimulation with VIP can activate the secretion of PRL (Guild and Drummond, 1983;Onali et al, 1983), no such relationship was found for TRH. The main action of TRH on PRL secretion was instead attributed to elevations in [Ca 2+ ] cyt , and the activation of PKC (Gershengorn, 1986 (Coleman et al, 1996;Hayakawa et al, 2002;Lin et al, 1996). Likewise, TRH does impact gene expression, because it directly stimulates the promoter of the gene encoding PRL via activation of MAPK (Wang and Maurer, 1999).…”