Absence of pituitary adenylate cyclase-activating polypeptide-stimulated transcription of the human glycoprotein α-subunit gene in LβT2 gonadotrophs reveals disrupted cAMP-mediated gene transcription

Abstract

Hormone regulation of anterior pituitary expression of the common glycoprotein hormone α-subunit (αGSU) is mediated by multiple response elements residing in the first-435 bp of the human promoter. In rat pituitary cells and mouse αT3-1 precursor gonadotrophs, the human αGSU promoter is strongly responsive to activators of the adenylyl cyclase/cAMP pathway, such as the hypothalamic releasing hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) and forskolin (an adenylyl cyclase activator). However, the role of PACAP and cAMP in regulating αGSU transcription in the more differentiated LβT2 gonadotroph in unclear. Here, we investigate the regulation of the human αGSU promoter by PACAP and forskolin in LβT2 and αT3-1 gonadotrophs. PACAP failed to stimulate αGSU promoter activity or cAMP production in LβT2 cells, in marked contrast to αT3-1 cells. LβT2 gonadotrophs expressed extremely low levels of any PACAP type 1 receptors (PAC1-R) Isoform by RT-PCR and lacked PAC1-R by radioligand binding. Forskolin stimulated the αGSU promoter in LβT2 cells, but by less than 30% of the response seen in αT3-1 gonadotrophs. This blunted cAMP transcriptional effect was not due to different levels of cAMP generation, or altered expression of the cAMP target proteins CREB, Akt, CBP or ICER. However, only LβT2 cells showed detectable expression of the protein kinase A type IIα regulatory subunit. Binding of activating transcription factor-2 and phosphorylated CREB to the consensus CRE was observed in both LβT2 and αT3-1 gonadotrophs, yet forskolin failed to stimulate either CRE- or CREB-mediated transcription in LβT2 cells. Collectively, these data demonstrate the lack of functional PACAP receptors in LβT2 gonadotrophs, and a pronounced attenuation in the responsiveness of this differentiated gonadotroph cell line to cAMP stimulus.