Yap Stimulates Fibroblast Activation Depending On Extracellular Matrix Stiffness Via A Feed-forward Manner

Objective 1.To investigate the expression of Yap in the model of renal fibrosis induced by unilateral renal ischemia-reperfusion injury(UIRI);2.To investigate the effects of Yap on the activation of renal fibroblasts and the expression of extracellular matrix(ECM)protein;3.To investigate the expression of Yap and myofibroblast labelled protein SMA-α and ECM proteins of renal fibroblasts in different stiffness matrix hydrogels.4.To investigate the effect of CTGF on the activation of fibroblasts and the expression of Yap and ECM protein,so that to determine whether Yap/CTGF pathway regulates the activation of renal fibroblasts.Methods 1.Clean grade male wild type C57/B6 mice were randomly divided into three groups,six in each group.In the operation group,the left renal blood flow was blocked by microvascular clamp for 45 minutes,and the kidneys were collected at 1 week and 4 weeks after operation respectively,while in the sham group,the kidneys were collected at 4 weeks.The renal tissue structure was detected by PAS and HE staining,and collagen was detected by PASM and Masson staining.The expression levels of Yap,CTGF,SMA-α,Fn and Col 1 were detected by immunohistochemistry and WB(Western blot).2.The expression of Yap,CTGF,SMA-α,Fn,Col 1 protein was detected by WB after TGF-β1-induced fibroblast activation.The lentiviral overexpression vector Yap(5SA)and the knockout vector sg-Yap were respectively transfected into fibroblasts,WB test detected the expression of Yap,CTGF,SMA-α,Fn,Col 1.Inhibited Yap transcription with different verteporfin concentrations and the expression levels of CTGF,SMA-α,Fn,Col-1,caspase-3 and cleared caspase were detected by WB.The binding of Yap and TEAD was blocked by transfection of TEAD(Δ289)-myc,then TGF-β1(4ng/ml)induced fibroblasts for 24 hours,the expression levels of CTGF,SMA-α and Col 1 were detected by WB.3.Adjusting the modulus of hydrogel by changing the concentration of Bis-acrylamide and acrylamide to make different stiffness matrix hydrogel to culture fibroblasts.WB test detected the expression of Yap,CTGF,SMA-α,Fn,Col 1,and immunofluorescence staining detected the cell location of F-actin,SMA-α and Yap.Fibroblasts cultured on different stiffness matrix,and inhibited the expression of Yap by sg-Yap,TEAD(Δ 289)-Myc and verteporfin,and the expression of Yap,CTGF,SMA-α,Fn and Col 1 were detected by WB test.4.The fibroblasts cultured on different stiffness matrix were incubated with exogenous CTGF(50μg/ml),and the growth morphology was observed under microscope;the fibroblasts were incubated with different CTGF concentrations and transfected with lentivirus knockout vector sg-CTGF,and the protein levels of Yap,SMA-α and Col 1 were detected by WB.Result 1.Yap expression in the model of unilateral renal ischemia-reperfusion injury in mice The results of renal pathology in UIRI operation group after 4 weeks showed that the renal structure was disordered,the number of renal tubules was decreased,the lumen was dilated and deformed,the epithelial cells of tubules were atrophic,the renal interstitium was widened,a large number of cells were infiltrated and collagen was deposited.WB detection showed that the expression of Yap and its target protein CTGF,myofibroblast marker protein SMA-α,and ECM protein Fn,Col 1 in UIRI renal tissue significantly increased,particularly in 4 weeks UIRI group.Compared with sham group,the difference of protein expression was statistically significant(P<0.05).Immunohistochemical staining showed that in the renal interstitium of UIRI 4 weeks group,myofibroblasts with SMA-α and FSP-1 positive infiltrated,Fn and Col-1 were positive,Yap was strongly positive in the renal interstitial cells and renal tubular epithelial cells,that location in the nucleus.2.The effect of Yap on the activation of renal fibroblasts and the expression of ECM protein The protein expression of SMA-α,Fn,Col 1,Yap and CTGF increased significantly in TGF-β1-induced renal fibroblast activation,and had a concentration gradient dependence.Yap(5SA)overexpression induced fibroblast activation and increased the expression of SMA-α,CTGF and Col 1 protein,which was significantly different from the control group(P<0.05).Knockout fibroblasts Yap gene by sg-Yap,the expression of SMA-α,CTGF,Fn,Col-1 was significantly lower than that of TGF-β1 stimulation group(P<0.05).Inhibited Yap signal respectively by TEAD(Δ 289)-Myc plasmid and drug verteporfin,suppressed the protein expression of SMA-α,CTGF,Fn,Col 1 even induced with TGF-β1,which was significantly different from that of TGF-β1 group(P<0.05).3.The effect of extracellular matrix stiffness on fibroblast activation and Yap expression The results showed that the stiff matrix(50k Pa)promoted the rapid growth and proliferation of fibroblasts,and cells were star shaped with the pseudopodia,while on the soft matrix(0.5k Pa)fibroblasts were round,cluster and easy to fall off,the growth and proliferation were limited.The results of immunofluorescence showed that SMA-α of fibroblasts was negative in soft matrix,no filamentous stress fibers were produced and Yap was expressed in cytoplasm;while in hard matrix SMA-α was positive,Yap expressed in nucleus.The result of WB test showed that with the increase of gel matrix stiffness,the activation of fibroblasts was more obvious,especially at 50 k Pa stiff matrix,and the expression of Yap,CTGF,Col 1,SMA-α increased.Knockout of Yap gene blocked the activation of fibroblasts induced by stiff matrix.The expression of Col 1,CTGF,SMA-α decreased significantly compared with the stiff matrix control group(P<0.05).Similarly,the inhibition of Yap by TEAD(Δ289)-Myc and verteporfin blocked the activation of fibroblasts even on the stiff matrix,and the expression of SMA-α,CTGF,Fn decreased significantly compared with the stiff matrix control group(P<0.05).High stiffness matrix activated Yap expression,promoted fibroblasts activation and ECM protein expression,but inhibition of Yap signal blocked the fibroblasts activation on high stiffness matrix.4.The effect of CTGF on the activation of renal fibroblasts and the expression of ECM protein Exogenous CTGF significantly promoted the fibroblasts proliferation and growth even cultured on soft matrix;CTGF induced the expression of Yap,SMA-α,Col 1 in fibroblasts,and enhanced with the increase of CTGF concentration;knockout of CTGF gene(sg-CTGF)blocked the effect of TGF-β1 on fibroblasts,and the the expression of protein Yap,SMA-α,Fn,Col 1 decreased significantly,compared with TGF-β1 group,(P<0.05).CTGF regulates the activation of fibroblasts.Conclusion Yap overexpression promoted the activation of fibroblasts and the synthesis of ECM protein;decreasing the expression of Yap inhibited the activation of fibroblasts mediated by extracellular matrix mechanics;CTGF knockout inhibited the activation of fibroblasts that was induced by TGF-β1.Therefore,intervening Yap signal to regulate the activation,proliferation and synthesis of fibroblasts will provide a new idea for the treatment of renal fibrosis.