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Isolation and Characterization of a Rat Nitric Oxide Synthase Type I Gene Promoter that Confers Expression and Regulation in Pituitary Gonadotrope Cells

Endocrinologie Cellulaire et Moléculaire de la Reproduction, Centre National de la Recherche Scientifique-ESA 7080, Université Pierre et Marie Curie, 75252 Paris, France

Address all correspondence and requests for reprints to: Dr. Raymond Counis, Endocrinologie Cellulaire et Moléculaire de la Reproduction, Centre National de la Recherche Scientifique-ESA 7080, Université Pierre et Marie Curie, 4, place Jussieu, Case 244, 75252 Paris cedex 05, France. E-mail: raymond.counis{at}snv.jussieu.fr

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 T3–1 and LßT2 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 LßT2 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.

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