KRT8 Phosphorylation Regulates Retinal Pigment Epithelial-mesenchymal Transition Via Autophagy In The Pathogenesis Of Proliferative Vitreoretinopathy

BackgroundProliferative vitreoretinopathy(PVR)is a serious complication related to a number of intraocular diseases,including rhegmatogenous retinal detachment(RRD)and ocular trauma.Moreover,PVR is the most common cause of sxurgical failure of RRD.Retinal pigment epithelial(RPE)cells are considered to play a critical role in the development of PVR,as they undergo the epithelial-mesenchymal transition(EMT)process.In this process,RPE cells lose their polarity and cell-cell contact,trans-differentiate into fibroblast-like cells and produce extracellular matrix(ECM)components.Therefore,the EMT of RPE cells contributes to the formation of epiretinal fibrotic membrane.Due to the poor understanding of the molecular mechanisms underlying EMT,there is no effective prophylaxis or treatment of PVR have been achieved.Thus,it is of great significance to further explore the key molecular mechanisms and regulatory factors of EMT in RPE cells.Autophagy is an evolutionary conserved process by which lysosomes degrade and recycle cytoplasmic materials including long-lived proteins and old or damaged organelles.Autophagy plays a fundamental role in maintaining the homeostasis of RPE cells,and impairment of autophagy is likely to induce the dysfunction of RPE cells.Recent studies have demonstrated that autophagy may play a role in EMT process and participate in the fibrotic process of several human tissues and organs.However,the relation between autophagy and EMT in RPE cells is poorly understood.KRT8(keratin 8)is one of the most important keratin proteins and considered as a marker of mature RPE cells.Except for maintaining the mechanical integrity of cells like other cytoskeletal proteins,accumulating evidences have shown that keratins are involved in the regulation of cell growth,cell migration and cellular response to stress-related stimuli.These non-mechanical functions are based on the post-translational modifications(PTMs)of KRT8S especially phosphorylation.To date,there is no report on the effect of KRT8 and its phosphorylation on the EMT process of RPE cells,and the interaction mechanism between it and autophagy needs to be elucidated.Therefore,in this study,we have studied the role of autophagy and KRT8 phosphorylation in the EMT of RPE cells,and explored the relationship between them.Our study will further shed light on the pathogenesis of PVR and lay the foundation for clinical prevention and treatment of PVR.Methods1.To induce the EMT process,ARPE-19 cells were treated with TGF-?2(10ng/ml)for various time periods.The morphological change of ARPE-19 cells was observed under bright field microscopy and protein expression levels of EMT markers such as?-SMA,fibronectin and collagen ? were detected by western-blot assay.Based on this EMT model,the protein expression levels of autophagy markers such as LC3-?,Atg5-12,Beclin 1 and p62 were detected by western-blot assay,and the fluorescence of GFP-LC3 and mRFP-GFP-LC3 were observed under confocal microscope.ARPE-19 cells were pretreated with autophagy inhibitor(Baf-A1)and then subjected to TGF-?2 treatment,the change of LC3-? conversion was measured by western-blot assay.Moreover,ARPE-19 cells were pretreated with phannacological inhibitors of autophagy(3-MA or Baf-A1)or transfected with specific siRNA(si-ATG5 or si-BECN1)before exposed to TGF-p2.Then the migration ability of cells was examined by wound healing assay,and protein expression levels of EMT markers were detected by western-blot assay.2.Based on EMT model in vitro,the protein expression levels of KRT8 and p-KRT8 in ARPE-19 cells were detected by western-blot assay.Moreover,ARPE-19 cells were transfected with si-KRT8 before exposed to TGF-?2,then protein expression levels of EMT markers were measiured by western-blot assay.To investigate the relationship between KRT8 and autophagy,ARPE-19 cells were pretreated with autophagy inhibitors(3-MA or Baf-A1)or transfected with specific siRNA(si-ATG5 or si-KRT8).After stimulated by TGF-?2,the protein expression levels of autophagy markers(LC3-? and p62),KRT8 and p-KRT8 were detected by western-blot assay,and the expression of p-KRT8 in cells was further examined by immunofluorescence assay.Moreover,ARPE-19 cells were transfected with wild-type KRT8 or KRT8-S74A mutant and then exposed to TGF-?2,the protein expression levels of autophagy markers were detected by western-blot assay,mRFP-GFP-LC3 fluorescence was observed under confocal microscope,and the co-localization of LC3B and LAMP2 was.examined by immunofluorescence assay.3.The PVR membrane specimens were collected from patients undergoing standard vitreoretinal surgery for the treatment of retinal detachment complicated with PVR.The expression of autophagy marker(LC3B)and KLRT8 in the PVR membranes was examined by immunofluorescence assay.Results1.The expression levels of EMT markers such as ?-SMA,fibronectin and collagen IV showed a time-dependent upregulation in TGF-?2-stimulated ARPE-19 cells.Moreover,the morphology of cells changed significantly and transitioned into a fibroblast-like phenotype.Meantime,TGF-?2 significantly increased the expression of LC3-?,Atg5-12 and Beclin 1,while induced the gradual degradation of p62.TGF-?2 also increased the average number of GFP-LC3B puncta per cell.Pre-treatment of ARPE-19 cells with Baf-A1 enhanced the accumulation of LC3-? induced by TGF-?2.This result was futher confirmed by autophagosome fusion assay,in which TGF-?2 significantly increased both yellow and red fluorescence puncta.Besides,suppression of autophagy by autophagy inhibitor(3-MA or Baf-A1)or siRNA(si-ATG5 or si-BECN1)significantly attenuated the TGF-?2 induced cell migration,as well as the increased expression levels of mesenchymal specific proteins.2.TGF-?2 did not affect significantly the expression of KRT8,but enhanced the expression of its phosphorylated form(p-KRT8).Moreover,under TGF-?2 stimulation,the KRT8 knockdown resulted in decreased expression of EMT markers.While ARPE-19 cells showed decreased expression of p-KRT8 after the initial stage of autophagy has been blocked by 3-MA or si-ATGS,inhibition of the late stage of autophagy by Baf-A1 further enhanced the upregulation of p-KRT8 induced by TGF-?2.Moreover,under TGF-?2 stimulation,the KRT8 knockdown resulted in increased LC3-? expression and decreased p62 degradation.Besides,compared to wild-type KRT8,ARPE-19 cells expressing KRT8-S74A showed increased LC3-? and p62 levels after TGF-?2 treatment.They also showed increased number of yellow puncta in autophagosome fusion assay,and reduced co-localization of LC3B and LAMP-2.3.Immunofluorescent staining showed that dense KRT8 and LC3B fluorescence were present within the subretinal and epiretinal membranes.Moreover,the co-localization of KRT8 and LC3B was also observed.Conclusions1.TGF-?2 simultaneously induces EMT process and autophagy in ARPE-19 cells,and suppression of autophagy could inhibit the TGF-?2 mediated EMT process.2.TGF-?2 induces phosphorylation of KRT8 in ARPE-19 cells,and KRT8 phosphorylation is likely to be involved in the autophagosome-lysosome fusion,which will influence the process of EMT indirectly.3.Direct proof that the high expression of KRT8 and LC3B within the PVR subretinal and epiretinal membranes,as well as their co-localization.