The Role Of PEA3in Epithelial-mesenchymal Transition And The Raltive Mechanism

Along with the social economy development and people life style changes, thespectrum of human diseases are changing and chronic kidney disease (CKD) hasbecome popular. With high prevalence of CKD, large medical cost, easy to causehigh mortality and morbidity of cardiovascular disease, CKD has become the maindisease affecting the national health at present^1]. Renal tubular interstitial ifbrosis isthe common pathway by which a variety of causes of CKD develop into end-stagerenal failure. In recent years, studies have shown that the severity of the renal tubuleinterstitial injury affects the prognosis of CKD [2]. Therefore，how to prevent andreduce the renal tubular interstitial injury as soon as early is very important to delaythe progress of CKD. It has been conifrmed that epithelial cells and mesenchymalcells can be mutually transformated between each other[3]. In embryonic development,kidney mesenchymal cells can be transformed into epithelial cells，which was calledMET. In adulthood, renal tubular epithelial cell damage factors stimulation can occurafter phenotype transformation which differentiate from epithelial cells intomesenchymal cells and renal tubular interstitial damage, ultimately develop to renaltubular interstitial ifbrosis. The studies found that more than one-third of theinterstitial ifbroblasts derived from partial renal tubular epithelial cells in theoccurrence of EMT [4]，thus, EMT is central in varies processes to cause renal tubularinterstitial ifbrosis [5，6].Our group constructed subtractive library of kidneys of21days old andnewborn rat by suppression subtractive hybridization technique previously and found multiple copies of PEA3. PEA3belongs to the Ets family which codes protein Ccontains a very conservative Ets structure domain which is also a DNA bindingdomain structure. The Ets family protein regulates a variety of gene expression,including growth, transcription, and T cell activation and organ development, etc.PEA3mainly expresses in the epithelial tissues and organs ungergone the mutualtransformation between epithelial cells and mesnchymal cells[7]. Our group found thatPEA3expressed in embryonic kidney development by the induction of WT1expression and transformation from mesenchymal cells to normal renal tissuestructure to induce MET. It has been comifrmed that BMP-7could inhibit EMT andprotect renal ifbrosis.What’s more，we found there was two binding sites in BMP-7promoter, so we speculated PEA3might block EMT by upregulating BMP-7.In addtion, it has been reported that TGF-betal could also induce HK-2cellsROS release and enhance smad2phosphorylation which also can aggravate theoccurrence of EMT [6]. Our team also found that mitochondrial dysfunction couldaggravate HK-2cells EMT induced by aldosterone and coifrmed that PGC-1alphaoverexpression could attenuate mitochondrial dysfunction and then inhibited EMT.These will provide a new way to explore the mechanism of EMT and interventiontargets. PGC-1alpha plays an important role in blocking mitochondrial dysfunction,whose main function is to stimulate mitochondrial biology biosynthesis and enhancedmitochondrial metabolism ability by stimulating the expression of mitochondrialtranscription factor A and regulating mtDNA synthesis; promote the synthesis of ATPby the regulation of mitochondrial respiratory factor and mitochondrial oxidativephosphorylation process; prevent the damage caused by ROS by activating severalclassical antioxidant enzymes such as UCPs antioxidant defense system. Thus,weassumed whether PEA3worked by upregulating PGC-1alpha.However, the role of PEA3in renal EMT and ifbrosis is ot clear. Therefore，we tried to explore PEA3function in vitro and in vivo and the role of BMP-7andPGC-1alpha in this process.All trans-retinoic acid (ATRA) is an active metabolite of vitamin A，which has avariety of biological activities in the embryo and adult tissues, such as cellproliferation, apoptosis, differentiation, reproductivation, maintaining normal cellularfunction, immune regulating, etc. In recent years, the role of ATRA in kidney ifbrosisdisease has attracted the schol’arss attention. A number of research conifrmed that ATRAcould improve glomerular hypertrophy, delay the renal ifbrosis, protect renal function.But the mechanisms of ATRA in renal ifbrosis is still unclear. It ha benn found ATRAcould raise the expression of BMP-7at the transcriptional level, promote endogenousBMP-7expression, then binds with its receptors and so on to protect kidney ifbrosis.So we tried to observe the role of ATRA in epithelial-to mesychamal transition and itsmachanisms.Part1: PEA3expression in experimental nephropathyanimal modelsObjective: To observe PEA3expression in experimental nephropathy animalmodels.Methods: We established animal models of kidney disease as follows: cisplatinmodel，aldosterone model，adriamycin nephropathy animal models，UUO andgentamicin model，then observe PEA3expression in experimental nephropathymodels by Real-time PCR，western blot and immunohistochemistry..Results: Real-time PCR and western blot showed that PEA3expressionincreased signiifcantly in UUO group; immunohistochemical results showed PEA3 expression increased signiifcantly from the seventh day in UUO model and to themaximum at14days which mainly expressed in the glomerular and renal interstitialcells，little in renal tubule; PEA3expression increased signiifcantly in gentamicingroup, to the peak at5days, then gradually declined which mainly expressed in renaltubular; PEA3expression increased signiifcantly in adriamycin model，mainly inrenal tubular; PEA3is visible from the third day in aldosterone model which exsitedin glomerular, then gradually increased, more in renal tubular and glomerular; PEA3expression increased in cisplatin (20mg/kg) model,mainly in the renal tubules.Conclusion： With the aggravation of renal ifbrosis，PEA3expression graduallyincreased which implied that it might play a role in the process of renal ifbrosis and itmainly expressed in renal tubules and glomeruli which prompted us to suggest that itmay be involved in epithelial-to-mesenchymal transition.Part2: The role of PEA3in epithelial-to-mesenchymaltransition and the relative mechanismsObjective: The purpose of this study was to discuss the role of PEA3in renalepithelial-to-mesenchymal transition and its possible mechanisms from theobservation in the experimental nephropathy animal models.Methods:(1) We built PEA3eukaryotic expression vector and transfected itinto HK-2cell，then observed PEA3and BMP-7mRNA and protein expression byReal-time PCR and Western Blot;(2)We used TGF-betal to stimulate PEA3overexpression HK-2cells，or added TGF-betal after pretreatment of BMP-7siRNA,then detected smad-12，3，5，8，alpha-SMA, CTGF，Collagen, E-cadherin，ZO-1expression by Real-time PCR and Western Blot;(3) We built PEA3siRNA andtransfected it into the HK-2cell line to suppress PEA3gene expression, thenobserved PEA3and BMP-7mRNA and protein expression, smad-l，2，3，5，8，alpha-SMA, CTGF, Collagen, E-cadherin, ZO-1expression by Real-time PCR andWestern Blot.(4) In vitro，TGF-beta1(5ng/ml) stimulated HK-2cells, we tested mitochondrial dysfunction relative indexes at different time points;(5)PEA3plasmidand PGC-1alpha siRNA was transfected into HK-2cells，followed by addition ofTGF-betal, we detected ROS by2，7-dihydrogen fluorescein acetyl acetate(DCFDA); mitochondria membrane potential by JC-1; mitochondrial DNA copynumbers by real-timePCR; PEA3, PGC-1alpha, TFAM, E-cadherin, Vimentin, ILKand alpha SMA was assessed by Real-timePCR and Western blot after PGC-1alphawas added.Results:(1)PEA3mRNA and protein expression increased signiifcantly aftertransfection, and BMP-7mRNA and protein expression are also correspondinglyincreased;(2) Real-time PCR and Western Blot results showed that after TGF-betalstimulation of HK-2cells，alpha-SMA, CTGF，Coll and smad2/3phosphorylationincreased, E-cadherin，Collagen, ZO-1，smadl/5/8phosphorylation is reduced inTGF-betal alone group, and alpha-SMA, CTGF, smad2phosphorylation is reducedin PEA3overexpression group, E-cadherin, ZO-1, smadl/5/8phosphorylationincreased compared with TGF-betal group; Real-time PCR and Western Blot showedPEA3protection was attenuated after adding BMP-7siRNA;(3)Real-time PCR andWestern Blot showed that effective PEA3siRNA interfered almost50%of itsexpression. PEA3，BMP-7mRNA and protein expression were reduced inPEA3siRNA group, alpha-SMA, CTGF, Coll, smad2/3phosphorylation, E-cadherin,ZO-1, smadl/5/8phosphorylation was decreased signiifcantly in both PEA3siRNAand TGF-betal treament group.(4)TGF-beta1(5ng/ml) could induce mitochondrialdysfunction;(5) PEA3overexpression could raise PGC-1alpha and mitochondrialtranscription factor (TFAM) expression, and attenuate the TGF-betal inducedmitochondrial dysfunction; PGC-1-alpha siRNA weakened the protective effect ofPEA3on mitochondrial dysfunction and EMT induced by TGF-betal.Conclusion： PEA3could block TGF-betal induced epithelial-to-mesenchymaltransition through BMP-7and PGC-1-alpha. Part3： The role of PEA3in All trans-retinoic acidinhibiting epithelial-to-mesenchymal transitionObjective: To discuss the effect of all trans-retinoic acid in epithelial-to-mesench-ymal transitionand, its relative mechanisms and the role of PEA3in the process.Methods:(1) HK-2cells was cultured,30minutes after ATRA pretreatment,added TGF-betal to stimulate for48h, EMT related moleculars such as E-cadherin,ZO-1，alpha-SMA, Col I and CTGF mRNA and protein expression was detected byReal-time PCR and Western Blot;(2)In UUO with ATRA treatment, TGF-beta,E-cadherin, ZO-1, alpha SMA，Coll and CTGF mRNA and protein expression wasdetected by Real-time PCR and Western Blot;(3)We built BMP-7promoter andluciferase report gene recombinant plasmid and tranfected it into HK-2cell, thenstimulated it by ATRA and luciferase activity was tested. BMP-7and smadl/5/8activation was detected by Real-time PCR and Western Blot.(4) After transfection ofBMP-7siRNA, TGF-betal and ATRA were applicated, E-cadherin, ZO-1, alpha-SMA,Coll and CTGF mRNA and protein expression was detected by Real-time PCR andWestern Blot;(5)Transfected the recombinant plasmid transfection to HK-2cells，then overexpressed and interfered RAR alpha and RXR alpha, luciferase activity wasdetected after stimulated by ATRA for24h;(6) PEA3expression was tested byWestern Blot after different doses of ATRA stimulation, then the direct combinationof BMP-7and PEA3was obsreved by Chip; overexpressed and interfered PEA3inHK-2cells with BMP-7promoter and luciferase report gene recombinant plasmidtransfection, then stimulated cells with ATRA for24h to detect the luciferase activity;(7) Detected the direct combination between RAR alpha and PEA3by Chip anddetermined PEA3role during the process of BMP-7induction by ATRA.Results:(1) ATRA signiifcantly inhibited TGF-beta1-induced the increase ofalpha-SMA, Coll and CTGF expression and decrease of E-cadherin and ZO-1expression in dose dependent manner;(2)TGF-betal, alpha-SMA, Col I and CTGFexpression signiifcantly increased, whereas E-cadherin and ZO-1expression declinedsigniifcantly in UUO while was opposite after ATRA treatment;(3)ATRA increasedBMP-7promoter activity in dose dependent, also promoted BMP-7mRNA andprotein expression, at the same time, we also found that ATRA signiifcantly promoted αsmadl/5/8phosphorylation which implied ATRA might activate the BMP-7signalling pathways.(4) BMP-7siRNA blocked ATRA inhibition of EMT，BMP-7siRNA promoted the alpha-SMA, Coll and CTGF expression and inhibitedE-cadherin and ZO-1expression.(5) After overexpressing RAR alpha and RXR alpha,ATRA increased BMP-7luciferase activity which was increased more in both RARalpha and RXR alpha overexpression group, otherwise not;(6) After overexpressingPEA3, ATRA increased BMP-7luciferase activity, while not in PEA3siRNA group;PEA3could bind with BMP-7directly;(7)RAR alpha could be directly combinedwith PEA3，and RXR alpha and RAR alpha worked with PEA3together in theprocess of induction of BMP-7by ATRA.Conclusion: Studies in vivo and in vitro suggested that ATRA could blockepithelial-to-mesenchymal transition and might work through PEA3/BMP-7/smadl,5，8signaling pathways.