Abstract
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 oPACAP38 which was blocked by PACAP antagonist but not the VIP antagonist. The stimulatory effect of oPACAP38 was mimicked by activation of AC/cAMP and voltage-sensitive Ca2+ channel (VSCC) signaling, or induction of Ca2+ entry. In parallel, PACAP-induced PRL promoter activity was negated or inhibited by suppressing cAMP production, inhibiting PKA activity, removal of extracellular Ca2+, 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 oPACAP38 as the stimulant for PRL promoter activity. Moreover, PACAP-induced PRL promoter activity was also blocked by inhibition of PLC signaling, attenuation of [Ca2+]i immobilization via IP3 receptors, and blockade of PI3K/P70S6K 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 Ca2+/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. Apparently, other signaling pathways, including PLC/IP3 and PI3K/P70S6K cascades, may also be involved in PACAP induction of PRL gene transcription.
Highlights
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
PRL secretion and PRL mRNA expression were up-regulated by PACAP but not vasoactive intestinal peptide (VIP) treatment via functional coupling of Ca2+/ calmodulin (CaM)- and c-Jun N-terminal kinase (JNK)-dependent cascades with the cAMP/PKA pathways[21]
These findings suggest that PACAP but not VIP can serve as a stimulator for PRL synthesis in the carp species and this stimulatory action is mediated through activation of PAC1 but not VPAC receptors at the pituitary level
Summary
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. 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 Ca2+/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. PRL secretion and PRL mRNA expression were up-regulated by PACAP but not VIP treatment via functional coupling of Ca2+/ calmodulin (CaM)- and c-Jun N-terminal kinase (JNK)-dependent cascades with the cAMP/PKA pathways[21] These findings suggest that PACAP but not VIP can serve as a stimulator for PRL synthesis in the carp species and this stimulatory action is mediated through activation of PAC1 but not VPAC receptors at the pituitary level. To further elucidate the mechanisms for PACAP regulation of grass carp PRL gene expression, the effect of PACAP treatment on PRL promoter activity and the post-receptor signaling cascades involved were examined in αT3-1 cells. To shed light on the transcription factors mediating the genomic action of PACAP in terms of PRL gene transcription, the effect of over-expression of cAMP response element binding protein (CREB) on basal PRL promoter activity and siRNA/ domain negative (DN) mutant of CREB on PACAP-induced PRL promoter activity were investigated in αT3-1 cells
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