Gremlin-1 Induces Epithelial-mesenchymal Transition And Interferes With Redifferentiation In Fetal RPE Cells

Purpose: To investigate the role of Gremlin-1,which is an endogenous antagonist of the bone morphogenetic protein(BMP)signaling pathway,in inducing epithelium-mesenchymal transition(EMT)in fetal RPE cells after repeated wounds.Methods: Subconfluent repetitive passages in fetal RPE cells was regarded as a model of repeated wounds.A phase contrast microscope was used to observe the morphology and pigment formation in cells.The expression of GREM1 and EMT-or RPE-related genes in cells were evaluated by quantitative PCR(q-PCR).Recombinant human protein Gremlin-1(0.1?g/ml)was added every day to investigate the molecular effects of Gremlin-1 on fetal RPE cells.The cell migration rate was investigated using a cell wound scratch assay,and a western blot was used to analyze the representative proteins(P-cadherin,ZO-1,Vimentin,Smad4,and phosphorylated-Smads).In addition,transfection of si RNA was used to explore the rescue effects on EMT cells through the downregulation of GREM1.Finally,LDN193189,which is a type of pan-inhibitor to BMP receptors,was used to verify whether complete blocking of the BMP pathway would interfere with the redifferentiation in low-passage fetal cells,even if the cells were treated by TGF-?inhibitors.Results: In fetal RPE cells,the expression of GREM1 were gradually upregulated with repetitive passages,and at the same time,the function-specific genes in fetal RPE cells(TJP1,PMEL,BEST1,RPE65,MERTK)were downregulated while the EMT-specific genes were upregulated.In addition,GREM1 had a similar expression pattern with SNAI1,which is a key transcription factor to trigger EMT.Recombinant human Gremlin-1 promoted EMT with the upregulation of SNAI1 and elevated the cell migration rate in a cell scratch assay as well as,decreased the expression of two key transcription factors ofRPE embryonic development(MITF and OTX2)and the RPE marker,RPE65.Furthermore,the EMT marker,Vimentin and the TGF-?pathway downstream transcription factor phosphorylated-Smad2(p-Smad2)increased,but the epithelial marker,ZO-1,was reduced.Additionally,Smad4,which plays a role as a Snail1 cooperator by binding Smad3 was also increased.In contrast,GREM1 silencing increased the expression of MITF,OTX2 which means there was a better redifferentiation in subconfluent fetal RPE cells,but it had little influence on phosphorylated-Smad2 compared to the Negative Control group.Finally,by adding LDN193189,the BMP signaling pathway was blocked,and this block led to poor redifferentiation in low-passage cells,even though the cells were treated with TGF-?inhibitors.In addition,as positive feedback to block the BMP pathway,GREM1 was subsequently upregulated.Conclusions: In fetal RPE cells,Gremlin-1 induces EMT and inhibits redifferentiation by promoting the TGF-? pathway and inhibiting the BMP pathway.GREM1 silencing alleviates EMT and increases the redifferentiation of cells by relieving the blockade of the BMP pathway.However,GREM1 silencing has no effects on the TGF-? pathway.Thus,Gremlin-1 may serve as a novel target to treat proliferative vitreoretinopathy(PVR)and inhibit subretinal fibrosis,which is a risk factor for influencing the therapeutic effects of anti-vascular endothelial growth factor(anti-VEGF)on neovascular age-related macular degeneration(nAMD).