title: HIF1A-dependent increase in endothelin 2 levels in granulosa cells: role of hypoxia, LH/cAMP, and reactive oxygen species.
abstract: Hypoxia-inducible factor 1 alpha (HIF1A) and endothelin 2 (EDN2) are transiently expressed during the same time window in the developing corpus luteum (CL). In this study, we sought to investigate the involvement of LH/cAMP, reactive oxygen species (ROS), and a hypoxia-mimetic compound (CoCl2) on HIF1A expression and how it affected EDN2 levels, using transformed human granulosa cells (thGCs) and primary bovine granulosa cells (GCs). CoCl2 elevated HIF1A protein levels in thGCs in a dose-dependent manner. Forskolin alone had no significant effect; however, forskolin and CoCl2 together further induced HIF1A protein and EDN2 mRNA expression in thGCs. Similarly, in primary GCs, LH with CoCl2 synergistically augmented HIF1A protein levels, which resulted in higher expression of EDN2 and another well-known hypoxia-inducible gene, VEGF (VEGFA). Importantly, LH alone elevated HIF1A mRNA but not its protein. The successful knockdown of HIF1A in thGCs using siRNA abolished hypoxia-induced EDN2 and also the additive effect of forskolin and CoCl2. We then examined the roles of ROS in thGCs: hydrogen peroxide (20 and 50 μM) elevated HIF1A protein as well as the expression of EDN2, implying that induction of HIF1A protein levels is sufficient to stimulate the expression of EDN2 (and VEGF) in normoxia. A broad-range ROS scavenger, butylated hydroxyanisole, inhibited CoCl2-induced HIF1A protein with a concomitant reduction in the mRNA expression of EDN2 and VEGF in thGCs. The results obtained in this study suggest that HIF1A, induced by various stimuli, is an essential mediator of EDN2 mRNA expression. The results may also explain the rise in the levels of HIF1A-dependent genes (EDN2 and VEGF) in the developing CL.
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Herzl Street, Rehovot 76100, IsraelIVF UnitDepartment of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Mount Scopus, Jerusalem, Israel.
authors.forename: Ronit
authors.initials: R
authors.lastname: Yalu
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Herzl Street, Rehovot 76100, IsraelIVF UnitDepartment of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Mount Scopus, Jerusalem, Israel.
authors.forename: Adepeju Esther
authors.initials: AE
authors.lastname: Oyesiji
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Herzl Street, Rehovot 76100, IsraelIVF UnitDepartment of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Mount Scopus, Jerusalem, Israel.
authors.forename: Iris
authors.initials: I
authors.lastname: Eisenberg
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Herzl Street, Rehovot 76100, IsraelIVF UnitDepartment of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Mount Scopus, Jerusalem, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Imbar
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Herzl Street, Rehovot 76100, IsraelIVF UnitDepartment of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Mount Scopus, Jerusalem, Israel rina.meidan@mail.huji.ac.il.
authors.forename: Rina
authors.initials: R
authors.lastname: Meidan
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D019333:Endothelin-2 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000494:pharmacology*; D010053:Ovary / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D019333:Endothelin-2; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D007986:Luteinizing Hormone; D000242:Cyclic AMP