Regulational Effect Of Activin A-Smads Pathway On The Local Estrogen Synthesis In Endometriosis

Background：Endometriosis, one of the most common gynecological disordersaffecting10%of reproductive-aged women, is defined by the presence of functionalendometrial tissue outside the uterine cavity, primarily on the pelvic peritoneum andovaries. Traditionally speaking, it has been considered as an estrogen-responsivedisease. A molecular link between inflammation and estrogen production inendometriotic tissue was recently uncovered. This is mediated by a positive feedbackcycle that favors overexpression of key steroidogenic genes, most notably aromatase,continuous local production of estradiol, and increased secretion of transforminggrowth factor-beta(TGF-βs) and prostaglandin E2(PGE2) in endometriotic tissue. Ourprevious studies assessed the different cytokine expression in peritoneal fluid betweenwomen with minimal/mild and moderate/severe endometriosis and those withoutendometriosis using the cytokine array. Activin A was one of the altered expressioncytokine, which up-regulated in the women with endometriosis. Activin A, a highlyconserved member of the TGF-β superfamily, which initiates signal transductionpathways critical for reproductive functions and development. Recent evidencesuggested that Activin A secreted by glandular and stromal endometrial cells wassevenfold and threefold higher in women with endometriosis compared to thosewithout endometriosis. In KGN cells, cytochrome P-450aromatase (P450arom)activity was up-regulated by activin stimulation through TGF-β type I receptor(ALK4). We hypothesized that Activin A stimulates P450arom expressed inendometrial stromal cells(ESCs)of endometriosis via ALK4-Smads pathway, thenpromote the synthesis of estradiol in ectopic stromal cells.Objective:(1) To investigate the biological activity of Activin A on endometrialstromal cells (ESC).(2) Up-or down-regulate the level of Activin A, Activin receptorand Smads by molecular and cellular methods, the level of estradiol, the expressionand the enzymatic activity of P450aromand the moleculars of Activin A-Smads signalpathway was been detected. The result will complete the estrogen–dependent theoryof endometriosis and provide the experimental basis to explore the new biologicaltarget of endometriotic treatment.Methods：1、In the invitro culture system of eutopic endometrium stromal cellsfrom EMs, we studied the effect of different dose of Activin A（0,2.5,25,50ng/mL）on estradiol secration, apoptosis, proliferation and cellcycle of ESCs for different time(2h,12h,24h,48h,72h). Estradiol concentrations in the culture media were measuredby means of electrochemiluminescence immunoassay. Cell proliferative activity wasmeasured by MTT. Cell apoptosis and cell cycle ratew cytom were determined byfloetery. The capacity of Activin A induce invasion was tested using TranswellMatrigel invasion chambers.2、Real-time PCR and Western blot were used todetermine the different expression of P450arom in endometrial and endometrioticcells.3、In the invitro culture system of eutopic endometrium stromal cells from EMs,ESCs were cultured in vitro and stimulated with various concentrations (0,2.5,25,50ng/mL)of Activin A for different time(0h，2h,12h,24h). Real-time PCR and Westernblot were used to determine the expression of P450arom. Addition testosterone to cellcultures containing Activin A, the concentrations of estradiol was determined.4、Todetermine whether Activin A-induced P450arom expression is via ALK4-smadssignaling pathway independent of PGE2, ESCs were treated with H89, an inhibitor of PKA，Smad4-siRNA, to down-regulate smad4expression, and SB431542, aninhibitor of ALK4. To observe the phosphorylation of Smad3and its subsequentnuclear translocation, the protein of the cytoplasmic and nuclear were extractedseparately. Estradiol concentrations in the culture media were measured by means ofelectrochemiluminescence immunoassay.The mRNA level of P450arom wasinvestigated by Real-time PCR. The protein of P450arom, p-samd3, smad3, smad4,and smad7were determined by Western blot and Immunofluorescence.Results:1、Compared with the control group,estradiol levels in cultured mediaincreased in a dose-and time-dependent manner by Activin A with the maximal effectobserved at the dose of25ng/ml for48h (p<0.05). Neither proliferative activity northe cell apoptosis and cell cycle were affected by the Activin A.Activin A induced theincrease in cell invasiveness and the expression of MMP-2and MMP-9inESCs(p<0.05).In addition, it induced a decreased mRNA expression ofN-cadherin(p<0.05).2、P450arom expressed in all kinds of endometriual stromallcells,and it was more abundant in eutopic and ectopic stromal cells from patients withendometriosis compared to women without endometriosis(p<0.01).3、Activin A caninduce the expression of P450arom mRNA and protein in ESCs from wemen withendometriosis(p<0.01). Either activin A or testosterone can induce a pronounced levelof estradiol, and they have synergistic effect(p<0.05). Neither estradiol production northe expression of P450arom mRNA and protein were affected by the Activin A in theESCs from women without endometriosis(p>0.05).4、The effect of Activin A onESCs of endometriosis was not affected by H89(p>0.05).Exposure of ESCs to ActivinA resulted in phosphorylation of Smad3, which was increased as early as5minutes,peaked at15minutes(p<0.01), slightly decreased by1hour. Similar results weredetected for Samd4and Smad7after Activin A exposure. In the absence of Activin Aexposure,p-Smad3was weakly both in the cytoplasm and in the nucleus. Upon ActivinA stimulation,most p-Smad3accumulated in the nucleus. The effect of ActivinA on estradiol induction,P450arom expression, and Smad3nuclear translocation was,in part, abrogated by the pretreatment of ESC with SB431542and Smad4-siRNA.Conclusions:（1）Activin A can stimulate the ESCs to secrete estradiol and increaseinvasiveness,which cues the increased invasiveness may be concerned with highconcentrations estradiol in ectopic endometrium.（2）P450arom is more abundant ineutopic and ectopic stromal cells from patients with endometriosis compared towomen without endometriosis,and its expression and activity can be enhanced byActivin A.These support that endometriosis is an estrogen-dependent disease,and Activin Amay be the initiation factor in progenesis of endometriosis.（3）Activin A stimulates ESCs ofendometriosis to express aromatase via ALK4-Smads pathway independent of PGE2.(4)Activin A promoted the secretion of estradiol of ESCs by increasing the expressionof P450arom via ALK4-Smads pathway, which might be an important reason forpromoting ectopic foci survival and development.