Transcriptional regulation of the glycoprotein hormone α-subunit gene by pituitary adenylate cyclase-activating polypeptide (PACAP) in αT3-1 cells

Abstract

We showed previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases glycoprotein hormone α-subunit gene expression and secretion in αT3-1 cells. We have now used 5′-flanking deletion and clustered point mutations of the mouse α-subunit promoter fused to the luciferase (LUC) reporter gene in transient transfection assays to further characterize the cell signaling pathways and sequences involved in responsiveness to PACAP. PACAP stimulated LUC activity at a lower concentration than VIP, supporting the notion that PACAP acts through type 1 receptors. The effect of PACAP on LUC activity was observed by 2 h, peaked at 4–12 h, and persisted until at least 20 h. αT3-1 cells were transfected with mouse α-LUC constructs truncated at −507, −424, −288, −205, −146, and −133, and treated with PACAP, a cell-permeable cAMP analog (8Br-cAMP), phorbol myristate acetate (PMA), or control medium. Transcriptional activation by PACAP was highest with the −288 and −205 mouse α-LUC vectors (7–8-fold stimulation) and decreased significantly with truncation of the 5′-flanking region to −146 or −133. The pattern of α-subunit stimulation by cAMP closely paralleled that of PACAP. With PMA, stepwise decrements in LUC activity were observed between −507 and −424 and, especially, −424 and −288, and there was no further loss of activity with deletion to −205, −146, or −133. Clustered point mutations in the pituitary glycoprotein hormone basal element (−337 to −330) or the gonadotropin-releasing hormone response element (GnRH-RE) (−406 to −399) of the −507 to +46 mouse α-promoter significantly ( P<0.05) increased and decreased, respectively, PACAP's effect on transcriptional activity. These results indicate that there are several regions of the mouse α-subunit promoter that mediate responsiveness to PACAP. The co-localization of PACAP and cAMP responsiveness as well as the results of studies involving specific inhibitors of protein kinase A (H-89) or protein kinase C (PKC) (bisindolylmaleimide) suggests that the action of PACAP on α-subunit transcription is mediated primarily by the protein kinase A (PKA) pathway.