Diverse actions of sirtuin-1 on ovulatory genes and cell death pathways in human granulosa cells

Abstract

Background Human granulosa-lutein cells (hGLCs) amply express sirtuin-1 (SIRT1), a NAD + -dependent deacetylase that is associated with various cellular functions. SIRT1 was shown to elevate cAMP on its own and additively with human chorionic gonadotropin (hCG), it is therefore interesting to examine if SIRT1 affects other essential hGLC functions. Methods Primary hGLCs, obtained from the follicular aspirates of women undergoing IVF and SV40-transfected, immortalized hGLCs (SVOG cells), were used. Primary cells were treated with SIRT1 specific activator SRT2104, as well as hCG or their combination. Additionally, siRNA-targeting SIRT1 construct was used to silence endogenous SIRT1 in SVOG cells. PTGS2, EREG, VEGFA and FGF2 expression was determined using quantitative polymerase chain reaction (qPCR). Apoptotic and necroptotic proteins were determined by specific antibodies in western blotting. Cell viability/apoptosis was determined by the XTT and flow cytometry analyses. Data were analyzed using student t-test or Mann–Whitney U test or one-way ANOVA followed by Tukey HSD post hoc test. Results In primary and immortalized hGLCs, SRT2104 significantly upregulated key ovulatory and angiogenic genes: PTGS2, EREG, FGF2 and VEGFA, these effects tended to be further augmented in the presence of hCG. Additionally, SRT2104 dose and time-dependently decreased viable cell numbers. Flow cytometry of Annexin V stained cells confirmed that SIRT1 reduced live cell numbers and increased late apoptotic and necrotic cells. Moreover, we found that SIRT1 markedly reduced anti-apoptotic BCL-XL and MCL1 protein levels and increased cleaved forms of pro-apoptotic proteins caspase-3 and PARP. SIRT1 also significantly induced necroptotic proteins RIPK1 and MLKL. RIPK1 inhibitor, necrostatin-1 mitigated SIRT1 actions on RIPK1 and MLKL but also on cleaved caspase-3 and PARP and in accordance on live and apoptotic cells, implying a role for RIPK1 in SIRT1-induced cell death. SIRT1 silencing produced inverse effects on sorted cell populations, anti-apoptotic, pro-apoptotic and necroptotic proteins, corroborating SIRT1 activation. Conclusions These findings reveal that in hGLCs, SIRT1 enhances the expression of ovulatory and angiogenic genes while eventually advancing cell death pathways. Interestingly, these seemingly contradictory events may have occurred in a cAMP-dependent manner.

Divergent roles of sirtuin 1 in human granulosa-lutein cells: similarities to human chorionic gonadotropin

“…Controversial results were reported on SIRT1's impact on GC viability from various species, which will be described below [51][52][53][55][56][57]. In a recent study aimed to reveal the effect of SIRT1 on hGLC viability, SRT2104 was incubated with intact primary hGLCs and SVOG cells or with SIRT1-silenced cells [51,53]. It was found that SRT2104 dose-and time-dependently decreased viable cell numbers.…”

“…Studies with SVOG cells have produced results comparable to those obtained in primary cells, demonstrating that they indeed serve as a reliable model for hGLCs [9, 51, 53, 85, 86]. For example, as will be detailed below, both cell types responded similarly to different SIRT1 activators in terms of SIRT1 induction, various GCs' gene expression as well as cell viability [21, 51, 53].…”

“…A number of in vitro studies also suggested that SIRT1 is involved in diverse ovarian functions including ovarian cell proliferation, apoptosis, folliculogenesis, oogenesis, and secretory activity [30,[47][48][49][50][51][52][53][54][55][56][57][58]. This review aims to provide state-of-the-art information on the expression and divergent roles of SIRT1 in GC function.…”

“…The two main sources for nonprimary human GCs available for in vitro studies are those derived from GC tumors, KGN cells, which exhibit a follicular phenotype [77–79], or immortalized nontumorigenic cells such as GC1a, HGL5, HO-23, SVOG, and HGP53, established from luteinized GCs obtained in IVF cycles [80–84]. Studies with SVOG cells have produced results comparable to those obtained in primary cells, demonstrating that they indeed serve as a reliable model for hGLCs [9, 51, 53, 85, 86]. For example, as will be detailed below, both cell types responded similarly to different SIRT1 activators in terms of SIRT1 induction, various GCs' gene expression as well as cell viability [21, 51, 53].…”

“…Ablation of endogenous SIRT1 with siRNA produces opposing actions, thereby corroborating the effects observed with SIRT1 activation. Adapted from [51].

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