Grass Carp Prolactin Gene: Structural Characterization and Signal Transduction for PACAP-induced Prolactin Promoter Activity

In this study, structural analysis of grass carp prolactin (PRL) gene was performed and the signaling mechanisms for pituitary adenylate cyclase-activating peptide (PACAP) regulation of PRL promoter activity were investigated. In αT3-1 cells, PRL promoter activity could be induced by oPACAP(38) which was blocked by PACAP antagonist but not the VIP antagonist. The stimulatory effect of oPACAP(38) was mimicked by activation of AC/cAMP and voltage-sensitive Ca(2+) channel (VSCC) signaling, or induction of Ca(2+) entry. In parallel, PACAP-induced PRL promoter activity was negated or inhibited by suppressing cAMP production, inhibiting PKA activity, removal of extracellular Ca(2+), VSCC blockade, calmodulin (CaM) antagonism, and inactivation of CaM kinase II. Similar sensitivity to L-type VSCC, CaM and CaM kinase II inhibition were also observed by substituting cAMP analog for oPACAP(38) as the stimulant for PRL promoter activity. Moreover, PACAP-induced PRL promoter activity was also blocked by inhibition of PLC signaling, attenuation of [Ca(2+)]i immobilization via IP3 receptors, and blockade of PI3K/P(70)(S6K) pathway. The PACAP-induced PRL promoter activation may involve transactivation of the transcription factor CREB. These results suggest that PACAP can stimulate PRL promoter activation by PAC1 mediated functional coupling of the Ca(2+)/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. Apparently, other signaling pathways, including PLC/IP3 and PI3K/P(70)(S6K) cascades, may also be involved in PACAP induction of PRL gene transcription.