TGF-?1 Mediates Aromatase Expression In Human Granulosa Cells And The Molecular Mechanism

The TGF-? superfamily is a conserved but functionalized protein that regulates many important physiological activities.TGF-? superfamily plays an important role in the regulation of ovarian function,including the recruitment of antral follicles,steroidogenesis,follicles development,ovulation and the subsequent luteinization.TGF-?1 is one of the most important members of the TGF-? subfamily.Studies have shown that TGF-?1 is expressed in theca cells,oocytes and granulosa cells.In the meantime,TGF-?1 plays an important role in the regulation of reproductive activity,like follicular development and steroid production..Polycystic ovarian syndrome(PCOS)is a common complex abnormal endocrine disorders in women of reproductive age,which could seriously affect women's reproductive capacity.The clinical manifestations are: oligomenorrhea or amenorrhea,hirsutism,acne,obesity and so on.The most important endocrine characteristic is hyperandrogenism,androgen metabolic disorders can lead to increased androgen,which could be caused by abnormal synthesis and metabolism of androgen.The synthesis of androgen is regulated by a variety of factors on molecular levels such as insulin growth factor,renin and so on.P450 aromatase cytochrome(P450arom)is encoded by the CYP19 gene.During the ovarian steroidogenesis process,P450 arom is expressed in a variety of tissues,and it is a key rate-limiting enzyme in the process of conversion of androgen to estrogen.P450 arom is a rate-limiting enzyme in the conversion of testosterone to estradiol,therefore the abnormal expression of P450 arom is often considered to be one of the important causes of hyperandrogenism in PCOS patients.Studies have shown that P450 arom expression is regulated by a variety of regulatory factors,such as gonadotropin,steroid hormone receptors and so on.In addition,the mutation,loss of function and single nucleotide polymorphisms of CYP19 gene could lead to altered expression of P450 arom.At present,it is still not clear that the level of P450 arom in PCOS patients and its specific regulatory mechanism.Studies have shown that the expression of T?R I and T?R?mRNA in the ovary of PCOS rat is significantly higher than that in normal group,which may directly affect the regulation of follicular development by TGF-?1,which is the main reason for the hyperandrogenism in PCOS rats.In human beings,the serum TGF-?1 concentration of PCOS patients was higher than that of normal people,but TGF-? receptors were inhibited,which suggested that TGF-?1 may be related to the pathogenesis of PCOS.Some studies have shown that the serum estrogen concentration of PCOS was lower than that of normal,and the expression of P450 arom in granulosa cells was decreased,which was considered as an important reason for the hyperandrogenism and anovulation of PCOS patients.TGF-? promotes the transcription of P450 arom gene in osteoblasts,THP-1 cells and so on.However,TGF-?1 inhibits the transcription of P450 arom gene in fetal liver cells,skin fibroblasts,human trophoblast cells and bovine granulocytes.In human granulosa cells,the effect of TGF-?1 on P450 arom expression and its specific regulatory mechanism are still unclear.Therefore,the aim of this study is to investigate the role of TGF-?1 in the expression of P450 arom in human granulosa cells and its molecular mechanism.ObjectiveTo investigate the effects of TGF-?1 on P450 arom expression and its molecular mechanism in human primary granulosa cells.MethodsHuman primary granulosa cells were used as the research model to explore the regulation of TGF-?1 to the expression of P450 arom and its molecular mechanism.Firstly,investigate whether TGF-?1 is involved in the regulation of P450 arom expression.We tested different concentrations of TGF-?1 on P450 arom expression,and treatment for 3h,6h,12 h,24h,respectively.We found that treatment with 10ng/ml TGF-?1 for 24 h exhibited comparable effects on the upregulation of P450 arom mRNA and protein levels.The real-time quantitative PCR(RT-qPCR)and western blotting were used to detect the expression of RNA and protein in granulosa cells,respectively.In order to explore whether TGF-? receptor was involved in the regulation of TGF-? on P450 arom expression,Treatment with 10 nM SB431542,which is a specific and potent inhibitor of T?RI,could abolish the TGF-?–induced upregulation of P450 arom mRNA and protein levels.In order to confirm the involvement of canonical Smad2/3 signaling pathway in the regulation of TGF-?1 on P450 arom expression,we examined the effect of TGF-?1 on phosphorylation levels of Smad2 and Smad 3 in granulosa cells.In order to further confirm the involvement of Smads signaling pathway in the regulation of TGF-?1 on P450 arom expression,SiRNA-mediated knockdown of Smad4 was used.ResultsIn human granulosa cells,TGF-?1 could upregulate the expression of P450 arom,and treatment with 5ng / ml of TGF-?1 for 24 h exhibited comparable effects on upregulation of P450 arom.Treatment with 10 nM SB431542 could abolish the TGF-?1-induced upregulation of P450 arom expression,indicating that T?RI is required for the TGF-?1-mediated upregulation of P450 arom expression.we examined the effect of TGF-?1 on phosphorylation levels of Smad2 and Smad3 in granulosa cells.Western blotting analyses showed that treatment with TGF-?1 for 30 min and 60 min induced significant phosphorylation of Smad2 and Smad3,indicating that Smad2/3 downstream signaling pathway was involved in TGF-?1-induced-upregulation of P450 arom expression.Smad4 siRNA was used to knocdown endogenous Smad4 mRNA levels.We found that the TGF-?-induced upregulation of P450 arom expression could abolish by the Smad4 knockdown,indicating that Smads signaling pathway was required for the TGF-?1-induced upregulation of P450 arom expression.Conclusion 1.In human primary granulosa cells,treatment with 10 ng / ml TGF-?1 for 24 h could promote the expression of P450 arom mRNA and protein and promote the production of estrogen.2.In human granulosa cells,TGF-?1 promotes the expression of P450 arom mRNA and protein by binding to T?RI.3.In human granulocytes,TGF-?1 promotes the expression of P450 arom mRNA and protein levels by activating Smads signaling pathway.