ATF4 Mediates The Expression Of COX2/PGE2,essential For Ovulation: A Potential Role Of ATF4 In PCOS

Objective:The objective of the study was to investigate the effects of ATF4 in ovarian ovulatory function and the potential anovulation role of ATF4 in PCOS.Main Outcome Measures:1.human granulosa cells(h GCs)from non-PCOS patients were cultured in vitro for three days,the localization and expression of ATF4 in h GCs were determined by immunofluorescence assay.2.h GCs from 59 newly diagnosed women with PCOS and 37 controls were used to determine ATF4 expression among which PCOS patients were diagnosed according to Rotterdam criteria.The expression of ATF4 m RNA in PCOS group and non-PCOS group was detected by real-time PCR.3.h GCs were transfected with si ATF4.The m RNA expression of ovulation-related genes was detected by real-time PCR.4.h GCs were treated with hCG(0,0.01,0.1,1,10,100IU/ml).The expression of ATF4 was detected by real-time PCR and western blot.5.Pharmacological inhibitors(CREB inhibitor: SQ22536 and H89、MEK inhibitor:PD980595、PI3K inhibitor: LY294002)were used to block the activation of signaling pathways,respectively.The expression of ATF4 was detected by real-time PCR and western blot.6.Ch IP-q PCR assay was performed to confirm the that ATF4 could directly bind to COX2 promoter.7.shAtf4 was injected in rats to establish an in vivo gene knockdown model for further confirming the studies above.Results:1.Immunofluorescence staining showed strong ATF4 density in h GCs.2.PCOS patients and non-PCOS patients(96 subjects)were recruited into our study.Statistical results showed that 59 subjects were PCOS patients,while the other 37 were non-PCOS.ATF4 m RNA expression was lower in h GCs from patients with PCOS than that from non-PCOS women.3.The m RNA abundance of genes including COX2,PTX3,CD44,TNFAIP6,and HAS2,associated with cumulus expansion in h GCs,were decreased when ATF4 was deficient.Other several genes which play a pivotal role in extracellular matrix(ECM)remodeling,including MMP2,MMP9,and ADAMTS1 in h GCs was impeded when ATF4 knockdown was achieved with small interfering RNA4.Results obtained from q RT-PCR and western blot analysis showed that hCG positively regulated the m RNA and protein abundance of ATF4 in h GCs in a dose-dependent manner.Especially,hCG at the concentration higher than 10IU/m L significantly induced ATF4 expression in h GCs.5.The phosphorylation levels of CREB,ERK1/2,and AKT were strongly increased in h GCs.hCG-induced ATF4 up-regulation was related with activation of PI3K/AKT pathway rather than PKA/CREB and ERK1/2 signaling pathways.6.Ch IP-q PCR assay extracted that ATF4 could directly bind to COX2 promoter and ATF4 knockdown could attenuate human chorionic gonadotropin(hCG)-induced COX2 expression and PGE2 production.7.The in vivo study showed that knockdown of Atf4 in rat ovaries via lentiviral infection obviously reduced the number of retrieved oocytes,and this might be attributed to the abnormal cumulus expansion.Conclusions:Our results demonstrated that decreased ATF4 expression in h GCs was correlated with the formation and development of PCOS-related anovulation.hCG could activate PI3K/AKT signaling pathway and then stimulated the expression of ATF4 in h GCs.Increased ATF4 directly bound to the promoter of COX2,which enhanced the transcription of COX2 and synthesis of PGE2 and consequently facilitated ovulation.Moreover,in vivo study,we confirmed that knockdown of ATFtf4 disrupted the potentiality of ovulation.Our study first reported that AKT signaling was required for hCG-induced ATF4 expression in h GCs.Increased ATF4 directly bound to the promoter of COX2,which enhanced the transcription of COX2 and synthesis of PGE2 and consequently facilitated ovulation.Our study provided a novel insight into the mechanisms underlying the formation and development of PCOS-related anovulation.In this case,ATF4 could probably be designed as a potential target for the treatment of PCOS in the future.