Endothelin-2 (EDN2) expression in granulosa cells was previously shown to be highly dependent on the hypoxic mediator, hypoxia inducible factor 1 alpha (HIF1A). Here, we investigated whether sirtuin-1 (SIRT1), by deacetylating HIF1A and class III histones, modulates EDN2 in human granulosa-lutein cells (hGLCs). We found that HIF1A was markedly suppressed in the presence of resveratrol or a specific SIRT1 activator, SRT2104. In turn, hypoxia reduced SIRT1 levels, implying a mutually inhibitory interaction between hypoxia (HIF1A) and SIRT1. Consistent with reduced HIF1A transcriptional activity, SIRT1 activators, resveratrol, SRT2104, and metformin, each acting via different mechanisms, significantly inhibited EDN2. In support, knockdown of SIRT1 with siRNA markedly elevated EDN2, whereas adding SRT2104 to SIRT1-silenced cells abolished the stimulatory effect of siSIRT1 on EDN2 levels further demonstrating that EDN2 is negatively correlated with SIRT1. Next, we investigated whether SIRT1 can also mediate the repression of the EDN2 promoter via histone modification. Chromatin immunoprecipitation (ChIP) analysis revealed that SIRT1 is indeed bound to the EDN2 promoter and that elevated SIRT1 induced a 40% decrease in the acetylation of histone H3, suggesting that SIRT1 inhibits EDN2 promoter activity by inducing a repressive histone configuration. Importantly, SIRT1 activation, using SRT2104 or resveratrol, decreased the viable numbers of hGLC, and silencing SIRT1 enhanced hGLC viability. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. Taken together, these findings propose novel, physiologically relevant roles for SIRT1 in downregulating EDN2 and survival of hGLCs.

Summary sentence

Inhibition of EDN2 transcription by SIRT1 is mediated by two plausible mechanisms: lowering HIF1A protein levels and its transcriptional activity and via deacetylation of histone H3 at the EDN2 promoter, inducing a repressive histone configuration.

Graphical Abstract

Illustrative summary depicting the biological effects of SIRT1 in human granulosa cells. SIRT1 activation is achieved by either SRT2104, resveratrol or metformin. SIRT1 activation diminishes HIF1A levels that may reduce its transcriptional activity exerted via HRE. Hypoxia, on the other hand, suppresses SIRT1 activity, indicating a mutually inhibitory interaction between hypoxia and SIRT1. SIRT1-induced deacetylation of histone H3 produces repressive epigenetic modifications of the EDN2 promoter. SIRT1 activation, using SRT2104 or resveratrol also decreased the viable numbers of hGLC. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. For both these actions, EDN2 and cell viability, siRNA silencing of endogenous SIRT1 corroborated the effects observed with SIRT1 activators.