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Article: Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Role of protein kinase C and activating protein 1

TitleTranscriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Role of protein kinase C and activating protein 1
Authors
Issue Date2000
PublisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.org
Citation
Endocrinology, 2000, v. 141 n. 10, p. 3611-3622 How to Cite?
AbstractClinical 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 (αT3-1) 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 μM 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(C)/(G)T(A)/(C)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 αT3-1 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 GnRHR 5'-flanking region has been functionally identified to be involved in mediating this down-regulatory effect.
Persistent Identifierhttp://hdl.handle.net/10722/84867
ISSN
2015 Impact Factor: 4.159
2015 SCImago Journal Rankings: 2.363
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorCheng, KWen_HK
dc.contributor.authorNgan, ESWen_HK
dc.contributor.authorKang, SKen_HK
dc.contributor.authorChow, BKCen_HK
dc.contributor.authorLeung, PCKen_HK
dc.date.accessioned2010-09-06T08:58:03Z-
dc.date.available2010-09-06T08:58:03Z-
dc.date.issued2000en_HK
dc.identifier.citationEndocrinology, 2000, v. 141 n. 10, p. 3611-3622en_HK
dc.identifier.issn0013-7227en_HK
dc.identifier.urihttp://hdl.handle.net/10722/84867-
dc.description.abstractClinical 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 (αT3-1) 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 μM 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(C)/(G)T(A)/(C)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 αT3-1 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 GnRHR 5'-flanking region has been functionally identified to be involved in mediating this down-regulatory effect.en_HK
dc.languageengen_HK
dc.publisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.orgen_HK
dc.relation.ispartofEndocrinologyen_HK
dc.rightsEndocrinology. Copyright © The Endocrine Society.en_HK
dc.titleTranscriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Role of protein kinase C and activating protein 1en_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0013-7227&volume=141&spage=3611&epage=3622&date=2000&atitle=Transcriptional+Down-Regulation+of+Human+Gonadotropin-Releasing+Hormone+(GnRH)+Receptor+Gene+by+GnRH:+Role+of+Protein+Kinase+C+and+Activating+Protein+1en_HK
dc.identifier.emailNgan, ESW: engan@hku.hken_HK
dc.identifier.emailChow, BKC: bkcc@hku.hken_HK
dc.identifier.authorityNgan, ESW=rp00422en_HK
dc.identifier.authorityChow, BKC=rp00681en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.pmid11014215-
dc.identifier.scopuseid_2-s2.0-0033752089en_HK
dc.identifier.hkuros56561en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0033752089&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume141en_HK
dc.identifier.issue10en_HK
dc.identifier.spage3611en_HK
dc.identifier.epage3622en_HK
dc.identifier.isiWOS:000089394800012-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridCheng, KW=35081802000en_HK
dc.identifier.scopusauthoridNgan, ESW=22234827500en_HK
dc.identifier.scopusauthoridKang, SK=25623598700en_HK
dc.identifier.scopusauthoridChow, BKC=7102826193en_HK
dc.identifier.scopusauthoridLeung, PCK=55085135300en_HK

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