The rat pituitary promoter of the neuronal nitric oxide synthase gene contains an Sp1-, LIM homeodomain-dependent enhancer and a distinct bipartite gonadotropin-releasing hormone-responsive region.
The neuronal nitric oxide synthase (NOS I) is expressed and hormonally regulated in rat anterior pituitary gonadotropes. In the present study, we investigated the mechanisms that underlie the constitutive and GnRH up-regulated activity of the pituitary exon 1p promoter of the NOS I gene in these cells. Through the use of 5'-deletions and transient transfections in L beta T2, a gonadotrope-derived cell line, we delineated a NOS I cell-specific (NCS) enhancer region (-73/-59) that is required for constitutive activity. Independently of the NCS enhancer, GnRH responsiveness is supported by a bipartite regulatory domain referred to as the GnRH response element I and II located between -33/-10 and -4/+4, the latter consisting of a cAMP-like response element. By combining transient transfections, gel shift, and supershift assays, we demonstrate that Sp1 and LIM-homeodomain-related protein bind the NCS enhancer, whereas cAMP response element binding protein and cAMP regulatory element modulator-like factors bind the GnRH response element II motif. We further show that factors involved in GnRH regulation are also implicated in constitutive activity, suggesting intimate links between constitutive and regulated promoter activity. We speculate that specific expression of the NOS I gene in gonadotropes together with its regulation by GnRH is suggestive of a critical participation of NOS I in gonadotrope function.
Bachir
Lydia K
LK
Signalisation Cellulaire, Régulation de Gènes et Physiologie de l'Axe Gonadotrope, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 7079, Physiologie et Physiopathologie, Université Pierre et Marie Curie, 75252 Paris, France.
Garrel
Ghislaine
G
Lozach
Anne
A
Laverrière
Jean-Noël
JN
Counis
Raymond
R
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D051786:Cyclic AMP Response Element Modulator; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052248:Nitric Oxide Synthase Type I; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C499522:Lhx1 protein, rat; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051786:Cyclic AMP Response Element Modulator; D007987:Gonadotropin-Releasing Hormone; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases