Mechanism of action of pituitary adenylate cyclase-activating polypeptide on human glycoprotein hormone alpha-subunit transcription in alphaT3-1 gonadotropes.
Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to increase glycoprotein hormone alpha-subunit synthesis and release from pituitary cells. We have used alphaT3-1 clonal gonadotropes to investigate the intracellular mechanisms involved in PACAP regulation of alpha-subunit gene transcription; and using deletion, mutation, and heterologous constructs of the alpha-promoter linked to a luciferase reporter gene, we have defined DNA sequences responsive to PACAP. Stimulation of alphaT3-1 cells for 24 h with PACAP, GnRH, or vasoactive intestinal peptide (VIP) resulted in a time- and concentration-dependent increase in alpha-promoter transcription at 100 nM for GnRH (17.5-fold, P < 0.001), PACAP (12.7-fold, P < 0.01), and VIP (4.1-fold, P < 0.05). Incubation of alphaT3-1 cells in calcium-depleted medium suggested that the transcriptional response to PACAP was less dependent on changes in intracellular calcium concentration, in contrast to the results seen with GnRH or VIP, where alpha-subunit transcription was significantly reduced. Transfection of an alpha-promoter construct containing a mutant cAMP response element (CRE) suggested that the CRE region is involved in PACAP and VIP responsiveness, with stimulatory effects on the mutant construct by PACAP (11.1-fold) and VIP (7.6-fold) being significantly (P < 0.001) reduced, compared with their stimulatory effects (PACAP: 25.6-fold, VIP: 23.1-fold) on the native alpha-promoter. In the same experiment, the transcriptional response of the mutant CRE construct and the native CRE construct to GnRH was not significantly different. Both PACAP and VIP enhanced GnRH-stimulated alpha-subunit gene transcription, but this additive effect was lost when their combined effects on the mutant CRE were examined. Deletion analysis indicated that sequences between -244 and -195 bp were involved in mediating the response to PACAP, with a dramatic reduction in fold-stimulation by PACAP (2.0-fold) of the -195-bp construct, compared with the -244-bp construct (15.8-fold). Constructs containing only upstream alpha-promoter sequences from -517 bp to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited a similar loss of responsiveness to PACAP below -298 bp. Thus, our studies show that, unlike GnRH, PACAP stimulation of alpha-subunit gene transcription in alphaT3-1 cells is less dependent on changes in intracellular calcium concentration; and full transcriptional activation of the alpha-subunit by PACAP requires an intact CRE. PACAP responsiveness involves sequences between -244 and -195 bp of the alpha-promoter. These sequences have been implicated also in GnRH-responsiveness and may thus provide a mechanism for coordinated regulation of the alpha-subunit gene by PACAP and GnRH in alphaT3-1 cells.
Burrin
J M
JM
Department of Clinical Biochemistry, St. Bartholomew's and the Royal London School of Medicine and Dentistry, United Kingdom. j.m.burrin@mds.qmw.ac.uk
Aylwin
S J
SJ
Holdstock
J G
JG
Sahye
U
U
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D017353:Gene Deletion; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D009479:Neuropeptides / Q000494:pharmacology*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects*; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
C495401:ADCYAP1 protein, human; C495403:Adcyap1 protein, rat; D015292:Glycoprotein Hormones, alpha Subunit; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D007987:Gonadotropin-Releasing Hormone; D014660:Vasoactive Intestinal Peptide; C010661:surfagon; D000242:Cyclic AMP; D002118:Calcium