Isolation and characterization of a rat nitric oxide synthase type I gene promoter that confers expression and regulation in pituitary gonadotrope cells.
Nitric oxide synthase type I (NOS I) is expressed and up-regulated in rat pituitary gonadotrophs. Using rapid amplification of cDNA ends-PCR, 2 major transcripts with 5' ends corresponding to exon 1a but truncated of its first 369 or 384 nucleotides, indicative of two pituitary-specific transcription start sites, were identified. By chromosome walking, we isolated 5'-upstream of this truncated exon termed 1p, a novel -1653/+384-bp genomic region. Transient transfections, using the gonadotrope-derived alpha T3-1 and L beta T2 cell lines and the full-length or 5'-deleted sequences fused to a luciferase reporter gene, demonstrated that cell-specific positive and negative regions were present especially within the -246/-73 region, whereas the +12/+384 region was crucial for transcription. Moreover, in L beta T2 cells, the luciferase activity was increased by GnRH, with the full-length sequence being the most efficient and the -73/+60 region corresponding to the essential zone. The latter region was also crucial for cholera toxin-induced activation. Interestingly, GnRH and cAMP effects were not additive, implying a convergent step in the transduction cascade. These data provide evidence for the presence of several elements controlling NOS I expression in gonadotrophs and demonstrate that GnRH, the prime regulator of gonadotrope function, and cAMP may induce the transcription of NOS I in these cells.
Bachir
L K
LK
Endocrinologie Cellulaire et Moléculaire de la Reproduction, Centre National de la Recherche Scientifique-ESA 7080, Université Pierre et Marie Curie, 75252 Paris, France.
Laverrière
J N
JN
Counis
R
R
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002460:Cell Line; D005091:Exons; D015870:Gene Expression / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006065:Gonadotropins, Pituitary / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052248:Nitric Oxide Synthase Type I; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D006065:Gonadotropins, Pituitary; D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat