| Background A normal level of estrogens synthesized in the testis plays a vital role in regulating the development of male reproduction and maintenance of spermatogenesis.Aromatase,encoded by Cyp19 gene,is the crucial enzyme for estradiol(E2)synthesis in the testis and is mainly derived fron Leydig cells(LCs)of adult males.The transcriptional activity of Cyp19 gene is controlled by several transcriptional factors,including SF-1(steroidogenic factor-1),LRH-1(liver receptor homolog-1),CREB(cyclic AMP response element-binding protein)and CREM(cyclic AMP response element modulator).Transforming growth factor beta 1(TGF-?1)regulates a range of biological processes via the Smad dependent signalling pathway.T?RI(TGF-? type I receptor)is also known as ALK5(activin receptor-like kinase 5).After identifying the TGF-?1 ligand,activated ALK5 phosphorylates Smad2/3 proteins,which transduct the signals into the nucleus where the TGF-?1 signalling pathway exerts regulatory effects.Our recent study showed that TGF-?1 inhibits estradiol(E2)secretion via down-regulating Cyp19 gene expression in mature rat LCs.In order to explore the mechanism by which TGF-?1 regulated E2 secretion and aromatase expression in Leydig cells,we firsly investigated the effect of TGF-?1 signalling pathway on the expression levels of Cyp19 related transcriptional factors both in vitro and in vivo.Secondly,we observed the effect of TGF-?1 signalling pathway on the transcriptional activity of Cyp19 gene.At last,we investigated the possible involvement of mi RNA in the regulatory effect of TGF-?1.Methods 1.The primary LCs and R2 C cells were incubated with TGF-?1(5 ng/m L)for 20 h,and the rat testicular interstitium was injected with 200 ?l TGF-?1(1 ?M)under a stereoscopic microscope.Western blot and immunofluorescence(IF)staining were performed to investigate the effect of TGF-?1 on the expression levels of Cyp19 related transcriptional factors,including SF-1,LRH-1,CREB and CREM.2.To observe the involvement of TGF-?1 receptors in the regulation of SF-1/LRH-1 expression,the ALK5 inhibitor SB431542 was added to the primary LCs cells or injected into the rat testicular interstitium.Western blot was performed to examine the effect of SB431542 on the protein levels of SF-1,LRH-1 and CYP19.Electrochemiluminescence immunoassay(ECLIA)was used to test the effect of TGF-?1 and ALK5 on estradiol(E2)production in testicular interstitial fluid(TIF).3.In order to explore the special Smad involved in TGF-?1 signalling pathway in LCs,R2 C cells were treated with 5 ng/m L of TGF-?1 for 20 h.Western blot was applied to detect the phosphorylation levels of Smad2 and Smad3.4.The 293 T cells were transfected with Cyp19 promoter luciferase reporter gene vector and SF-1/LRH-1 expression vectors,then the cells were treated with TGF-?1.Dual-luciferase reporter gene assays were performed to investigate the effect of TGF-?1 on SF-1/LRH-1 induced Cyp19 transcription.5.The 293 T cells were transfected with Cyp19 promoter luciferase reporter gene vector and SF-1/LRH-1 expression vectors,then the cells were co-transfected with Smad2 expression vector.Dual-luciferase reporter gene assays were performed to investigate the effect of Smad2 on SF-1/LRH-1 induced Cyp19 transcription.6.R2 C cells were treated with 5 ng/m L of TGF-?1 for 20 h,after which the total RNA was extracted.High-throughput sequencing and q RT-PCR(quantitative reverse transcription-polymerase chain reaction)were conducted to identify the mi RNA that were involved in TGF-?1 induced down-regulation of SF-1 and LRH-1.Results 1.TGF-?1 down-regulated the protein levels of SF-1/LRH-1 rather than CREB/CREM in primary LCs,R2 C cells and the rat testis.2.SB431542 attenuated the TGF-?1 induced down-regulation of SF-1 and LRH-1 expression in primary LCs and in vivo,and alleviated the TGF-?1 induced down-regulation of CYP19 expression and E2 production in TIF in vivo.3.Under TGF-?1 stimulation,the phosphorylation level of Smad2 was markedly increased,but the phosphorylation level of Smad3 was not obvious.4.Dual-luciferase reporter gene assays showed that both TGF-?1 and Smad2 overexpression significantly inhibited the transcriptional activity of Cyp19 promoter induced by SF-1 or LRH-1.5.High-throughput sequencing showed that,amoung the identified mi RNA,17 kinds of mi RNA were up-regualted and 16 kinds were down-regulated under TGF-?1 stimulation in R2 C cells.QRT-PCR showed that mi R-21-3p,mi R-532-3p and mi R-107,which might target SF-1,as well as mi R-339-5p,which might target LRH-1,were up-regulated under TGF-?1 stimulation.Conclusions 1.Both in vitro and in vivo,TGF-?1 suppressed the expression of Cyp19 related transcriptional factors(SF-1 and LRH-1)in rat LCs,which demonstrated that TGF-?1 inhibited the expression of CYP19 via down-regulating the accumulation of SF-1 and LRH-1.2.The inhibition of TGF-?1 on SF-1/LRH-1 expression was attenuated by ALK5 inhibitor SB431542,which indicated that the TGF-?1 acted through the canonical signaling pathway involving ALK5 to inhibit the expression of SF-1 and LRH-1,and eventually attenuated the expression of CYP19 and the production of E2.3.TGF-?1 significantly induced the phosphorylation of Smad2 in LCs,indicating that Smad2 was involved in TGF-?1 induced down-regulation of SF-1 and LRH-1expression.4.Both TGF-?1 and Smad2 overexpression significantly inhibited the transcriptional activity of Cyp19 promoter induced by SF-1 or LRH-1,which indicated that TGF-?1 acted through Smad2 signalling pathway to inhibit the expression of Cyp19 at transcriptional level.5.Mi R-21-3p,mi R-532-3p,mi R-107 and mi R-339-5p,which might target SF-1 and LRH-1,were up-regulated under TGF-?1 stimulation,indicating that they might play important roles in TGF-?1 induced down-regulation of SF-1 and LRH-1. |
PDF Full Text Request