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HIF1A-dependent increase in endothelin 2 levels in granulosa cells: role of hypoxia, LH/cAMP, and reactive oxygen species.
- Source :
-
Reproduction (Cambridge, England) [Reproduction] 2015 Jan; Vol. 149 (1), pp. 11-20. - Publication Year :
- 2015
-
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.<br /> (© 2015 Society for Reproduction and Fertility.)
- Subjects :
- Animals
Blotting, Western
Cattle
Cells, Cultured
Endothelin-2 genetics
Female
Granulosa Cells cytology
Humans
Hypoxia drug therapy
Hypoxia pathology
Hypoxia-Inducible Factor 1, alpha Subunit antagonists & inhibitors
Hypoxia-Inducible Factor 1, alpha Subunit genetics
Ovary cytology
Ovary drug effects
Ovary metabolism
RNA, Messenger genetics
RNA, Small Interfering genetics
Reactive Oxygen Species
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Cyclic AMP metabolism
Endothelin-2 metabolism
Granulosa Cells drug effects
Granulosa Cells metabolism
Hypoxia metabolism
Hypoxia-Inducible Factor 1, alpha Subunit metabolism
Luteinizing Hormone pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 1741-7899
- Volume :
- 149
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Reproduction (Cambridge, England)
- Publication Type :
- Academic Journal
- Accession number :
- 25433027
- Full Text :
- https://doi.org/10.1530/REP-14-0409