Role And Mechanism Of RELM-β In TGF-β1-Induced Endothelial-to-Mesenchymal Transition In Human Pulmonary Artery Endothelial Cells

Objective: To study the role and mechanism of RELM-β in TGF-β1-induced endothelial-to-mesenchymal transition in human pulmonary artery endothelial cells(HPAECs)and human umbilical vein endothelial cells(HUVECs).Methods:(1).We induced TGF-β1-induced EndMT cell model and detected the expression of RELM-β for different concentrations of TGF-β1(0,5,10,15,20 ng/ml),to Select the Proper concentration of TGF-β1.(2).Cells had been given lentivirus intratracheally to knockdown RELM-β,used Western-blot to Detect the interference efficiency and the phenotypes of CD31,VE cadherin and α-SMA,detected cell proliferation by MTT assays and cell migration by Transwell assays.(3).SB432542,an inhibitor of SMADs,used to reverse TGF-β1/SMADs,detected RELM-β/SMAD2/p-SMAD2/SMAD3/p-SMAD3/SMAD4/CD3 1/VE cadherin/α-SMA by Western blot.(4).Plasmid RELM-βLuc and pRK5-βGal,sub-cloned the wt-RELM-β or mut-binding RELM-β element and co-transfected the constructs with pRK5/SMAD2+3 or pRK5/ SMAD2+3+4 luciferase reporter gene vectors into 293 T cells,to detected the luciferase activity.Results:(1).TGF-β1 increased the protein level of RELM-β in a concentration-dependent manner;RELM-β protein level was remarkably induced by ≥ 10 ng/ml of TGF-β1 [(1.484±0.038)compared to(1.055±0.068),(1.691±0.124)compared to(0.99±0.103),P<0.01].(2).RELM-β knockdown suppressed HUVECs and HPAECs proliferation [(1.141±0342)compared to(1.190±0.069),(0.775±0.036)compared to(0.919±0.048),P < 0.05],migration [(55.580±2.108)compared to(104.28±3.103),(46.407±4.727)compared to(106.624±8.446),P<0.01] and EndMT,in other words,decreased the protein level of.VE-cadherin [(0.802±0.054)compared to(0.356±0.033),(0.707±0.067)compared to(0.404±0.025),P<0.05] or decreased the protein level of.VE-cadherin CD31 [(0.715±0.047)compared to(0.414±0.044),(0.737±0.075)compared to(0.363±0.021),P<0.05] or increased the protein level of a-SMA [(1.789±0.064)compared to(2.273±0.264),(1.682±0.140)compared to(2.271±0.459),P<0.05].(3).TGF-β1 dramatically induced the protein levels of RELM-β,p-SMAD2,p-SMAD3 and SMAD4 [(2.207±0.290)compared to(0.356±0.033),(1.893±0.148)compared to(1.009±0.031),(2.624±0.095)compared to(0.997±0.016),(1.724±0.120)compared to(1.103±0.089),P<0.05],On the contrary,SB432542 reduced the protein levels than TGF-β1 group [(1.260±0.055)than(1.951±0.080),(1.481±0.093)than(3.478±0.333),(1.344±0.016)than(2.794±0.169),(1.019±0.157)than(1.774±0.153),P<0.05].(4).The luciferase activity of wt-RELM-β was significantly up-regulated by SMAD2+3 or SMAD2+3+4 co-transfection [(4.752±1.986)than(2.077±0.437),(9.519±0.406)than(2.077±0.437),P < 0.01];after mutation in the predicted binding sites,the changes of the luciferase activity were eliminated.Conclusions: TGF-β1-induced SMAD2/3/4 activation promotes RELM-β transcription to modulate the endothelium-mesenchymal transition in HPAECs.