The Effects And Mechanisms Of LAMP-2A-Mediated Chaperon Autophagy In Fibrosis

IntroductionFibrosis is a common disease that seriously endangers human health.End-stage fibrosis occurs in important organs,not only brings great physical and economic burden to patients,but also leads to death due to organ failure.However,until now there are still limitations for the treatment of fibrosis,which are characterized by little available drugs and narrow application.Therefore,it is necessary to find new molecular targets and signaling pathways that regulate the process of fibrosis,and explore new methods for the treatment of fibrosis at the present stage.A growing number of studies have shown that chaperone-mediated autophagy(CMA)plays an important role in various diseases.Levels of CMA are controlled by lysosomeassociated membrane protein 2A(LAMP-2A).Our previous study found that the levels of LAMP-2 A were reduced in fibrosis that indicated CMA was likely to participate in the pathogenesis of fibrosis.Therefore,our research focus on the effects and mechanisms of LAMP-2A-mediated CMA in fibrosis based on the decreased level of LAMP-2A,which would be important for understanding the pathogenesis of fibrosis and finding new strategies for the treatment of fibrosis.ObjectiveThe purpose of our study is to explore the molecular mechanisms of LAMP-2Amediated chaperon autophagy in fibrosis and find new strategies for the treatment of fibrotic diseases.Contents and Methods:Our research involve three sections:(1)Indicate the expression level of LAMP-2A was down-regulated and its important roles during fibrosis.(2)Explore the mechanism of fibrosis regulated by LAMP-2A-mediated chaperon autophagy.(3)Identify the effects of sunitinib in fibrosis through up-regulating the level of LAMP-2A.Animal model of pulmonary fibrosis in mice was induced by intratracheal instillation bleomycin.High-dose bleomycin(5 mg/kg)was used to observe the survival time of mice,while low-dose bleomycin(3 mg/kg)was used to observe the pathological changes;HE and Masson staining were used to assess fibrosis.Animal model of liver fibrosis in mice were induced by intraperitoneal injection of carbon tetrachloride(CCl4)and the occurrence of liver fibrosis was investigated by blood biochemical indexes and pathological analysis.Western blot was used to detect protein levels in vitro and in vivo.Real-time PCR was used to investigate the mRNA levels in vitro and in vivo.Combined gene silence and overexpression techniques with western blot to investigate the correlation between different proteins.Effects of different protein degration pathways were investigated by using proteasome inhibitors and lysosomal inhibitors.LAMP-2A plasmid was packaged with adeno-associated virus serotype 9(AAV9)to detect the influence of LAMP-2A on pulmonary and liver fibrosis.Mass spectrometry was used to find proteins that could interact with HSC70(Heat-shock 70KD protein,HSC70).Immunoprecipitation was used to investigate the interaction of different proteins with HSC70.Results:(1)The protein and transcriptional level of LAMP-2 A reduced in the process of pulmonary and liver fibrosis.(2)The protein and transcriptional level of LAMP-2A in three cell lines(3T6,HFL1 and LX-2)reduced during TGF-β treatment.(3)LAMP-2A and HSC70 regulated the expression of TGF-β-induced type Ⅰ collagen in fibroblast.(4)AAV9-LAMP-2A could alleviate pulmonary and liver fibrosis.(5)Mass spectrometry showed that SMAD4 could interact with HSC70.(6)HSC70 could interact with SMAD4 and SMAD2,but not SMAD3.(8)The protein levels of SMAD4 and SMAD2 were upregulated after the inhibition of CMA.(9)Sunitinib could up-regulate the level of LAMP-2A and down-regulate the level of SMAD4 and SMAD2.(10)Sunitinib could protect fibrosis.Conclusion:In this study,we found that in the process of fibrosis,the reduced level of LAMP2A-mediated chaperone autophagy resulted in the abnormal accumulation of SMAD4 and SMAD2,thus promoting fibrosis by enhancing the generation and secretion of COL1A1.More importantly,we found that sunitinib could upregulate the transcriptional level of LAMP-2A and finally exert its anti-fibrosis effect at a low dose.Our study not only identified the importance and mechanisms of LAMP-2A-mediated chaperone autophagy in the process of fibrosis,but also provided sufficient experimental basis for LAMP-2A as a potential new target for the treatment of fibrosis.