Regulation of α-subunit mRNA transcripts by pituitary adenylate cyclase-activating polypeptide (PACAP) in pituitary cell cultures and α T3-1 cells

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Pituitary adenylate cyclase activating polypeptide (PACAP) increases glycoprotein hormone α-subunit mRNA levels suggesting a role for PACAP in maintaining the high levels of α-subunit protein characteristic of the pituitary. The present study used primary pituitary cell cultures and the α T3-1 pituitary cell line to investigate how PACAP affects α-subunit mRNA transcripts. Stimulation of cultured pituitary cells with 10 nM PACAP38, 10 nM GnRH, or the combination, for 24 h increased α-subunit mRNA levels 1.5-fold, whereas GnRH more effectively ( P < 0.01) stimulated α-subunit protein release than did PACAP38 (3.2- vs. 2.0-fold). α-Subunit mRNA levels in α T3-1 cells were also increased by PACAP38 and by GnRH to maximum values at 12 h ( P < 0.05), and α-subunit protein secretion rose proportionately and in parallel with α-subunit MRNA levels. PACAP38 was a 100-fold more potent stimulator of α-subunit mRNA than was VIP, and a VIP-antagonist failed to block the stimulatory effect of PACAP38, suggesting an effect via type PACAP 1 receptors. Type I receptor mRNA transcripts were identified by Northern analysis in α T3-1 cells. Depletion of PKC activity by PMA failed to block the stimulatory effect of PACAP38, but prevented GnRH from increasing α-subunit mRNA levels and α-subunit secretion. PACAP38, like 8Br-cAMP and forskolin, stimulated ( P < 0.05) luciferase (LUC) activity in α T3-1 cells transfected with a plasmid containing the first 846 or 180 base pairs of the 5′-flanking region of the human α-subunit gene linked upstream to a LUC reporter gene. Finally, experiments using the transcription inhibitor DRB reveal that PACAP does not appreciably change α-subunit mRNA half-life. These findings are consistent with the proposal that PACAP contributes to the high levels of α-subunit protein characteristic of the pituitary by activating Type I receptors and stimulating α-subunit gene transcription in part by the cAMP/PKA pathway.