The Angiogenic Effects And Mechanisms Of 17?-estradiol-dependent Smad1 Protein Activation In Endometriosis

BackgroundEndometriosis(EMs)is one of the most common benign diseases in gynecology,which seriously affects both women's physical and mental health.Recent epidemiology studies revealed that the prevalence of EMs has approached 6-10%in the general female population.Alarmingly,in women suffering from pain,or infertility,or both,the prevalence frequency of EMs is 35-50%.It is understood that EMs is a complex syndrome characterized by an estrogen-dependent chronic inflammatory process.Current effective therapeutic options for EMs management,are composed of treatments using medicinal drug compounds and surgical interventions.Although anti-hormonal therapy for EMs is effective,it is not favored as treatment option in for patient who trying to get pregnant.While surgical is an effective treatment for EMs with or without symptoms of pelvic pain and infertility.It has high rates of recurrences because of its limitations in rooting out the underlying cause of EMs.Therefore,complete understanding on the pathogenesis of EMs is of great significance in order to identify novel EMs therapeutic targets as well as to develop improved clinical and targeted drug treatments.Previous studies have revealed that EMs formation is closely associated with the initiation of angiogenesis,in which it is suggested that estrogen can induce endometriosis formation by mediating the production of angiogenic factors.Recently,a study by Benn A et al.has identified that Smadl protein,which is highly expressed in the endothelium,can play important biological roles such as but not limited to proliferation,migration and tube formation in both physiological and pathological settings.Besides,it is reported that Smadl genetically knockout mice is embryonic lethal,which is due to the pathological development of vascular dysplasia.ObjectivesTo investigate the underlying effects and mechanisms of 17?-estradiol(E2)in endometriosis mediated through Smadl protein activation dependent angiogenesis.Methods and ResultsA total of 10 clinical ectopic endometrial specimens and a total of 10 normal endometrial specimens were collected.Collected samples were then subjected for cryosectioning and fixed on slides.The distribution of phosphorylated-Smad1(p-Smad1)and vascular endothelial marker CD31 were observed by immunofluorescence after frozen section.Our results showed that the expression of vascular endothelial p-Smad1 in ectopic endometrium is higher than the normal endometrium.We next used primary cultured human umbilical vein endothelial cells(HUVECs)to perform our cellular studies.Firs1 we used western blot and immunofluorescence to explore the effect of E2 on Smad1 protein expression in HUVECs.Our results suggested that E2 stimulation can activate Smad1 protein expression in HUVECs by promoting the translocation of p-Smad1 protein into the nucleus to regulate target genes expression.Besides,cell-impermeable estradiol-bovine serum albumin(E2-BSA)treatment on HUVECs also promoted Smad1 protein phosphorylation,which suggest that the activation of p-Smad is modulated through E2 nongenomic pathway.Additional experiments using translation inhibitor cycloheximide(CHX)or the transcription inhibitor actinomycin D(Act D)also did not have significant effect on E2 dependent Smad1 protein activation.In order to identify the downstream effectors modulated by E2-dependent Smad1 protein activation,we further performed western blot using ERa selective agonist propylpyrazole(PPT),ER? selective agonist diarylpropionitrate(DPN),ERa selective antagonist(MPP),ER? selective antagonist(PHTPP)and ER antagonist ICI 182,780(ICI).Our results indicated that E2 activates Smad1 protein primarily through membrane ER?,which further supported our observation on E2 nongenomic pathway activation.Subsequently,we used western blot to validate the effect,of PI3K inhibitor wortmannin(WM),mTOR inhibitor rapamycin(Rapa),Gi protein inhibitor pertussis toxin(PTX),NF-kB inhibitor pyrollidine dithiocarbamate(PDTC),extracellular signal-regulated kinase(ERK)inhibitor PD98059(PD),and cellular Src(c-Src)inhibitor PP2 on E2 dependent Smad1 protein activation.We observed that only treatment with ERK inhibitor PD and c-Src inhibitor PP2 have significant inhibitory effects on Smad1 protein activation.In order to further validate the roles of ERK and c-Src on E2-dependent Smad1 activation,we transfected HUVECs with either c-Src siRNA,ERK2 siRNA,ERK2 plasmid and pcDNA3-c-Src plasmid.We found that E2-induced Smad1 activation requires both c-Src and ERK1/2.Since E2 nongenomic mechanism is activated at the plasma membrane level,we further tested if caveolin-1,another plasma membrane protein which has been found associated with the activation of E2 non-genomic signaling,is required.Our results revealed that treatment with either the caveolae disruptor ?-cyclodextrin(MCD)or Cav-1 siRNA tranfection can significantly inhibit E2 from activating Smad1 protein.Furthermore,our immunoprecipitation results using caveolae extracts from HUVECs revealed that caveolin-1(Cav-1),ER?,and c-Src interacts with each other,which further suggest that E2 stimulation may promote the formation of complex consists of caveolin-1(Cav-1),ER?,and c-Src.On the other hand,our primary cell functional studies revealed that E2 can promote tube formation in HUVECs,in which the effect is abolished in the presence of ?-MCD,PD,PP2 treatments and Smad1 siRNA transfection.We also performed HUVECs proliferation and migration studies using xCELLigence RTCA DP instrument,in which the HUVECs were monitored real-time.Our data analysis of cell index curves also showed that Smad1 is required to promote HUVECs proliferation and migration,and this effect is dependent on E2 stimulation.ConclusionsE2 activates Smad1 protein through E2-dependent nongenomic signaling pathway in order to promote HUVECs tube formation,proliferation,and migration.The activation of this nongenomic signaling pathway also requires the formation of ER?/c-Src/Cav-1 complex and downstream activation of ERK1/2 signaling.In a nutshell,the completion of our research may help us to better understand the molecular mechanisms of E2 in promoting endometriosis progression.