Ubiquitin-specific Protease 2 Regulates Ang Ⅱ–induced Cardiac Fibrosis By Stabilizing β-catenin

【Background】cardiac fibrosis,is an important event in the process of cardiac remodeling and is involved in almost every type of heart disease,featuring abnormally elevated extracellular matrix accumulation,decreases tissue compliance,impairs cardiac function and accelerates heart failure.Mounting evidence suggests that the ubiquitin proteasome pathway is involved in cardiac fibrosis.As a DUB,USP2 can reverse target protein degradation via removal of the ubiquitin from the respective substrates and regulate many cell processes such as cell cycle regulation and many signaling pathways.In this study,we investigated the role of USP2 in cardiac fibrosis,demonstrating that inhibition of USP2 can promote the degradation of β-catenin and thus reduce cardiac fibrosis,providing a potential therapeutic target for cardiac fibrosis.【Methods】1.To investigate the role of USP2 in cardiac fibrosis,2μmol/L Ang Ⅱ was added into cardiac fibroblasts(CFs)to establish a cell model of cardiac fibrosis.The experiment was divided into two groups: Control group and Ang Ⅱ group.Western blot was used to detect the expressions of fibrosis related marker proteins(Collagen III,CTGF,Periostin)and target protein USP2 in CFs.2.In order to investigate the effect of USP2 on proliferation and migration of cardiac fibroblasts,we treated Ang Ⅱ-activated CFs with sh-USP2 and different concentrations of USP2 inhibitor ML364,and detected the cell viability of CFs by MTS assay.Appropriate concentrations of ML364 were selected for subsequent experiments,and the experimental groups were: CON group,Ang Ⅱ group,Ang Ⅱ+ML364 group,ML364 group,sh-NC group,Ang Ⅱ+ sh-NC group,Ang Ⅱ+sh-USP2 group,and sh-USP2 group.The proliferation and migration of CFs under the action of Ang Ⅱ and the reversal of USP2 inhibition were investigated by Ed U assay and would healing assay.3.In order to investigate the effect of USP2 on cardiac fibroblast cycle,western blot and flow cytometry were used in this study to investigate the expression of cyclin and the changes of cell cycle when USP2 was inhibited or not inhibited by ML364.The study was divided into four groups: Con group,Ang Ⅱ group,Ang Ⅱ+ML364 group,and ML364 group.4.In order to study the influence of USP2 on the formation of cardiac fibrosis,different treatments were used in this study to inhibit USP2(inhibitors ML364 and sh-USP2)in the cell model of cardiac fibrosis,and the expression of fibrosis related marker proteins was detected.5.To investigate whether USP2 regulates the expression of β-catenin in CFs,western blot was used to detect the protein expression of β-catenin in CFs before and after ML364 and sh-USP2 treatment.The expression of β-catenin in cells was observed by confocal laser microscopy after the use of ML364 to inhibit USP2.6.In order to investigate whether β-catenin protein levels are regulated by the ubiquitin-proteasome system,this study used proteasome inhibitor MG132 to performβ-catenin protein level reversal assay.7.In order to investigate the interaction between USP2 and β-catenin,the present study used Co-IP assay to detect CFs before and after Ang Ⅱ treatment,and Western blot to quantitatively detect β-catenin after ubiquitination.【Results】1.Western blot results showed that USP2 expression was increased in Ang Ⅱ-induced cardiac fibrosis.2.MTS results showed that ML364 could effectively reduce cell viability at appropriate concentrations,and sh-USP2 also inhibited the activity of CFs.The results of Ed U assay showed that inhibiting USP2 can inhibited the proliferation of CFs.The results of would healing assay showed that inhibition of USP2 could inhibit the migration of CFs.3.Flow cytometry results showed that ML364 blocked the progression of cell cycle in G0/G1 phase.Western blot results showed that the expression of Cyclin D1 was decreased and the expression of P27 was increased after ML364 treatment.4.Western blot results showed that Ang Ⅱ significantly increased the expression level of USP2,and the expression of fibrosis related proteins,including Collage III,CTGF and Periostin were also significantly up-regulated.However,the high expression of these proteins was inhibited by ML364 and sh-USP2.5.Western blot results showed that Ang Ⅱ activated the Wnt/β-catenin pathway,promoted the phosphorylation of GSK-3β,and significantly increased the level ofβ-catenin while activating CFs and upregulating USP2 protein level.However,ML364 can reverse these effects and inactivate Wnt/β-catenin signaling.Knocking down the USP2 results are the same as the ML364 results.The results of immunofluorescence were also consistent with those of western blot.6.Western blot results showed that inhibition of USP2 could reduce the protein expression level of β-catenin,while the addition of proteasome inhibitor MG132 could increase the protein expression level of β-catenin.7.The results of Co-IP showed that USP2 could interact with β-catenin,and the effect of Ang Ⅱ did not affect their interaction.Inhibition of USP2 significantly increased the ubiquitination level of β-catenin,supporting USP2 as a deubiquitination enzyme of β-catenin,reversing its ubiquitination and thus stabilizing β-catenin protein.【Conclusion】Ubiquitin-specific protease 2 regulates Ang Ⅱ –induced cardiac fibrosis by stabilizing β-catenin.