Transcription of the vasoactive intestinal peptide gene in response to glucocorticoids: differential regulation of alternative transcripts is modulated by a labile protein in rat anterior pituitary.
Expression of the vasoactive intestinal peptide (VIP) gene is controlled by glucocorticoids in a tissue- and endocrine status-specific manner. We have investigated the molecular mechanisms that determine glucocorticoid regulation of VIP gene expression in the rat pituitary. In initial experiments, using explant cultures of rat pituitary glands, we have demonstrated that treatment with the glucocorticoid agonist dexamethasone leads to a marked increase in VIP mRNA levels. This effect was found to be selective for the larger of two alternatively polyadenylated VIP transcripts, and in addition, protein synthesis inhibitors markedly enhanced the magnitude of this response indicating that a labile pituitary protein acts to attenuate the transcript-selective response to glucocorticoids. Nuclear run-on analysis of transcription demonstrated that the effects of dexamethasone in vitro are mediated largely, if not completely, at the level of transcription. In order to investigate the role of VIP promoter sequence in the glucocorticoid response, we then demonstrated that the activity of rat VIP gene promoter/reporter constructs in GH3 pituitary cells are up-regulated by dexamethasone. This up-regulation is virtually abolished following removal of promoter sequence between -162 and -89 of the start of transcription. Using an in vitro electrophoretic mobility shift assay, we have also demonstrated that this region of the promoter binds recombinant glucocorticoid receptor protein. The results of our study therefore indicate a direct mechanism of action for the modulation of VIP gene expression by glucocorticoids, and furthermore provide evidence of a mechanism that permits selective glucocorticoid regulation of alternative VIP transcripts.
Chew
L J
LJ
Institute of Molecular and Cell Biology, National University of Singapore, Singapore.
Burke
Z D
ZD
Morgan
H
H
Gozes
I
I
Murphy
D
D
Carter
D A
DA
D000818:Animals; D002460:Cell Line; D046508:Culture Techniques; D003513:Cycloheximide / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D005938:Glucocorticoids / Q000494:pharmacology*; D008297:Male; D010903:Pituitary Gland, Anterior / Q000187:drug effects* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014660:Vasoactive Intestinal Peptide / Q000235:genetics*
D005938:Glucocorticoids; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide; D003907:Dexamethasone; D003513:Cycloheximide