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Endocrinology Vol. 139, No. 4 1731-1737
Copyright © 1998 by The Endocrine Society

ARTICLES

Mechanism of Action of Pituitary Adenylate Cyclase-Activating Polypeptide on Human Glycoprotein Hormone {alpha}-Subunit Transcription in {alpha}T3–1 Gonadotropes1

J. M. Burrin, S. J. B. Aylwin, J. G. Holdstock and U. Sahye

Department of Clinical Biochemistry, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London E1 2AD, United Kingdom

Address all correspondence and requests for reprints to: J. M. Burrin, Department of Clinical Biochemistry, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Turner Street, London E1 2AD, United Kingdom. E-mail: j.m.burrin{at}mds.qmw.ac.uk

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to increase glycoprotein hormone {alpha}-subunit synthesis and release from pituitary cells. We have used {alpha}T3–1 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 {alpha}T3–1 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 {alpha}T3–1 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 {alpha}T3–1 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 {alpha}T3–1 cells.




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