Document Detail


Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1.
MedLine Citation:
PMID:  11014215     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Clinical applications of GnRH agonists (GnRHa) are based primarily on the decrease in gonadotropin release after down-regulation of the GnRH receptor (GnRHR) by continuous GnRHa administration. However, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR gene after prolonged GnRH treatment remain poorly understood. In the present study GnRHa-mediated regulation of human GnRHR gene transcription was studied by transiently transfecting the mouse gonadotrope-derived (alphaT3-1) cells with a 2297-bp human GnRHR promoter-luciferase construct (p2300-LucF). A dose- and time-dependent decrease in human GnRHR promoter activity was observed after GnRHa treatment. An average 71% decrease in promoter activity was observed after 24-h treatment with 0.1 microM GnRHa, which was blocked by cotreatment of the GnRH antagonist, antide. This effect was mimicked by phorbol 12-myristate 13-acetate (TPA) administration. In addition, the GnRHa- and TPA-mediated decrease in the human GnRHR promoter activity was reversed by a specific protein kinase C (PKC) inhibitor, GF109203X, or depletion of PKC by TPA pretreatment. These findings indicate that the activation of the PKC pathway is important in regulating the human GnRHR gene expression. By progressive 5'-deletion studies, we have identified a 248-bp DNA fragment (-1018 to -771, relative to the translation start site) at the 5'-flanking region of the human GnRHR gene that is responsible for the GnRHa-mediated down-regulation of human GnRHR promoter activity. Analysis of this sequence reveals the existence of two putative activating protein-1 (AP-1) sites with 87% homology to the consensus sequence (5'-TGA(G/C)T(C/A)A-3'), located at -1000 to -994 (5'-TTAGACA-3', in complementary orientation) and -943 to 937 (5'-TGAATAA-3'). Using competitive gel mobility shift assays, AP-1 binding was observed within this 248-bp region. Site-directed mutation of the putative AP-1-binding site located at -1000 to -994 abolished the GnRHa-induced inhibition. Further competitive GMSA and supershift experiments confirmed the identity of AP-1 binding in this region. By the use of Western blot analysis, a significant increase in c-Jun (100%; P < 0.05) and c-Fos (50%; P < 0.05) protein levels was observed after GnRHa treatment in alphaT3-1 cells. In addition, our data suggested that a change in AP-1 composition, particularly c-Fos, was important in mediating GnRHa-induced inhibition of human GnRHR gene expression. We conclude that activation of the PKC pathway by GnRH is important in controlling human GnRHR gene expression. In addition, the putative AP-1-binding site located at -1000 to -994 of the human GnRHR5'-flanking region has been functionally identified to be involved in mediating this down-regulatory effect.
Authors:
K W Cheng; E S Ngan; S K Kang; B K Chow; P C Leung
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Endocrinology     Volume:  141     ISSN:  0013-7227     ISO Abbreviation:  Endocrinology     Publication Date:  2000 Oct 
Date Detail:
Created Date:  2000-10-02     Completed Date:  2000-10-31     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0375040     Medline TA:  Endocrinology     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  3611-22     Citation Subset:  AIM; IM    
Affiliation:
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Line
Down-Regulation*
Gonadotropin-Releasing Hormone / agonists,  physiology*
Hormone Antagonists / pharmacology
Humans
Luciferases / metabolism
Mice
Oligopeptides / pharmacology
Promoter Regions, Genetic / drug effects,  genetics,  physiology
Protein Kinase C / physiology*
Proto-Oncogene Proteins c-fos / metabolism
Proto-Oncogene Proteins c-jun / metabolism
Receptors, LHRH / genetics*
Response Elements / physiology
Tetradecanoylphorbol Acetate / pharmacology
Transcription Factor AP-1 / physiology*
Transcription, Genetic / physiology*
Chemical
Reg. No./Substance:
0/Hormone Antagonists; 0/Oligopeptides; 0/Proto-Oncogene Proteins c-fos; 0/Proto-Oncogene Proteins c-jun; 0/Receptors, LHRH; 0/Transcription Factor AP-1; 112568-12-4/iturelix; 16561-29-8/Tetradecanoylphorbol Acetate; 33515-09-2/Gonadotropin-Releasing Hormone; EC 1.13.12.-/Luciferases; EC 2.7.11.13/Protein Kinase C

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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