title: Molecular characterization and transcriptional regulation of a disintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1) in bovine preovulatory follicles.
abstract: The ovulatory process involves a complex remodeling of the extracellular matrix during which a desintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1) is thought to play a key role, but its transcriptional regulation in bovine follicles remains largely unknown. The objectives of this study were to characterize the regulation of ADAMTS1 in bovine follicles before ovulation and to determine its transcriptional control in bovine granulosa cells. Regulation of ADAMTS1 was assessed using total RNA isolated from bovine preovulatory follicles obtained at various times after human chorionic gonadotropin treatment. Results from RT-PCR analyses showed that levels of ADAMTS1 mRNA were very low at 0 hours but increased at 6 to 24 hours after human chorionic gonadotropin in granulosa cells. To determine the regulatory mechanisms controlling ADAMTS1 gene expression in vitro, primary cultures of bovine granulosa cells were established, and treatment with forskolin up-regulated ADAMTS1 mRNA levels. Promoter activity assays, 5'-deletion, and site-directed mutagenesis identified a minimal region conferring full-length basal and forskolin-stimulated ADAMTS1 promoter activities, with both being dependent on Ebox cis-acting elements. EMSAs revealed upstream stimulating factor (USF) proteins as key trans-activating factors interacting with Ebox. Chromatin immunoprecipitation assays confirmed such interactions between USF and Ebox in vivo, and USF binding to Ebox elements was increased by forskolin treatment. ADAMTS1 promoter activity and mRNA expression were increased by forskolin and overexpression of the catalytic subunit of protein kinase A, but not by cotreatment with inhibitors of protein kinase A, ERK1/2, and epidermal growth factor receptor signaling pathways. Furthermore, treatment with a soluble epidermal growth factor induced ADAMTS1 mRNA expression in granulosa cells. Collectively, results from this study describe the gonadotropin/forskolin-dependent up-regulation of ADAMTS1 mRNA in granulosa cells of bovine preovulatory follicles in vivo and in vitro and identify for the first time some of the molecular mechanisms responsible for ADAMTS1 promoter activation in follicular cells of a large monoovulatory species.
authors.affiliation: Centre de Recherche en Reproduction Animale and the Département de Biomédecine, Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada. k.sayasith@umontreal.ca
authors.forename: Khampoun
authors.initials: K
authors.lastname: Sayasith
authors.forename: Jacques
authors.initials: J
authors.lastname: Lussier
authors.forename: Jean
authors.initials: J
authors.lastname: Sirois
mesh_terms: D051722:ADAM Proteins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D006080:Ovarian Follicle / Q000378:metabolism*; D010060:Ovulation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects; D051720:Upstream Stimulatory Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D006063:Chorionic Gonadotropin; D051720:Upstream Stimulatory Factors; D005576:Colforsin; D008156:Luciferases; D051722:ADAM Proteins