The Role And Mechanism Of SPOCK1 In Hepatic Stellate Cell Activation And Liver Fibrosis

Background & aims: The long-term development of a variety of chronic liver diseases can lead to liver fibrosis and then develop into liver cirrhosis.There are approximately 1.2million deaths due to liver cirrhosis worldwide each year,and China accounts for about11% of them.Liver cirrhosis is one of the most common disease burdens in China,however,the therapeutic strategies are limited.The most distinctive feature of liver fibrosis is abnormal deposition and distribution of extracellular matrix(ECM),and its core event is the activation of hepatic stellate cells(HSCs).Thus,elucidating the molecular mechanism of liver fibrosis is of great significance for blocking or even reversing the occurrence and progression of fibrosis.ECM is a key element in regulating HSCs activation and liver fibrosis,and matricellular protein is one of the main components of ECM.Sparc/osteonectin,cwcv,and kazal-like domain proteoglycan 1(SPOCK1)belongs to the secreted protein acidic and rich in cysteine(SPARC)family,which is reported to exert crucial roles in growth,metastasis and survival of various tumor cells.In the present study,we are aimed to explore the role and mechanism of SPOCK1 in HSCs activation and liver fibrosis,in order to provide new potential targets for the intervention of liver fibrosis.Methods: The correlation of SPOCK1 and α-smooth muscle actin(α-SMA)messenger RNA(mRNA)expression in normal and cirrhotic liver tissues was analyzed in Gene Expression Omnibus(GEO)dataset(GSE25097).Immunohistochemistry(IHC)staining,real time quantitative polymerase chain reaction(RT-qPCR),Western blot,and double immunofluorescence(IF)staining were performed to assess the expression and localization of SPOCK1 in human and rat fibrotic and normal liver tissues,and in rat primary HSCs(R-HSCs).Mechanistically,RT-qPCR,Western blot,luciferase reporter assay,forkhead box M1(Fox M1)small interfering RNA(si RNA),inhibitors of phosphatidylinositol3-kinase(PI3K)and extracellular signal-regulated kinase(ERK),chromatin immunoprecipitation(Ch IP)were used to investigate the regulation of PDGF-BB on SPOCK1 expression and the underlying mechanism.Then,RT-qPCR,Western blot,cell counting kit-8(CCK-8)and Transwell assays were performed to investigate the role of SPOCK1 in PDGF-BB-induced HSCs activation,proliferation and migration.Subsequently,Western blot analysis was performed to investigate the signaling pathway regulated by SPOCK1 downregulation/overexpression or recombinant SPOCK1(r SPOCK1)treatment,and the PI3 K inhibitor LY294002 was utilized to verify whether SPOCK1 regulated HSCs activation,proliferation and migration through activating the PI3K/Akt pathway.Co-immunoprecipitation(Co-IP)and double IF were performed to investigate the interaction between SPOCK1 and integrin α5β1.Specific neutralizing antibodies were used to investigate whether blocking integrin α5 or/and integrin β1 reduced r SPOCK1-induced HSCs activation,proliferation and migration and Akt phosphorylation.Finally,the HSCs-specific-targeted lentivirus(LV-SMA-sh SPOCK1-Flag)was injected via tail vein to rats to knockdown SPOCK1 in HSCs,and liver fibrotic model was established by intraperitoneal thioacetamide(TAA)administration.The successful delivery of the lentivirus to HSCs was verified by IHC,IF and Western blot.Then,the degree of liver fibrosis was assessed by hematoxylin & esoin(H&E),Masson’s trichrome,α-SMA staining,and measurement of hydroxyproline,alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Then,RT-qPCR and Western blot analyses were performed to assess the expression of markers of HSCs activation,proliferation and migration in liver tissues and R-HSCs.Results:Part Ⅰ: SPOCK1 mRNA expression was positively correlated with α-SMA in normal and cirrhotic liver tissues in GSE25097.The results of IHC,RT-qPCR and Western blot showed that SPOCK1 expression was upregulated in human cirrhotic/fibrotic liver tissues than in normal individuals.The results of IHC showed that SPOCK1 was upregulated in TAA-induced rat fibrotic liver tissues.The results of RT-qPCR and Western blot analyses showed SPOCK1 was upregulated in activated R-HSCs when compared with the quiescent R-HSCs.The results of double IF staining showed that SPOCK1 was expressed in HSCs,hepatocytes and in the ECM of human cirrhotic liver tissues,and SPOCK1 was co-localized with α-SMA in the cytoplasm in R-HSCs.Taken together,SPOCK1 is positively correlated with liver fibrosis and HSCs activation and might be involved in the pathogenesis of hepatic fibrogenesis.Part Ⅱ: RT-qPCR and Western blot showed that both transforming growth factor-β1(TGF-β1)and platelet derived growth factor-BB(PDGF-BB)upregulated SPOCK1 expression in LX-2 and R-HSCs,and PDGF-BB exerted stronger effect,however,the effects of connective tissue growth factor(CTGF)and vascular endothelial growth factor(VEGF)were insignificant.Luciferase reporter assay indicated that PDGF-BB upregulated the promoter activity of SPOCK1 gene.Serially truncated mutation and site-directed mutation indicated that the first Fox M1 binding site located in-1356 ～-741 bp of SPOCK1 promoter was responsible for PDGF-BB-induced SPOCK1 promoter activity.Furthermore,knockdown of Fox M1 by si RNA transfection reduced PDGF-BB-induced promoter activity of SPOCK1 gene and its mRNA and protein expression.Then,Western blot showed that PDGF-BB upregulated SPOCK1 expression by activating PI3K/Akt pathway rather than the ERK pathway.Finally,the results of Ch IP indicated that PDGF-BB activated the PI3K/Akt pathway,and thus regulating the binding of Fox M1 to SPOCK1 promoter.Taken together,PDGF-BB promotes SPOCK1 expression through activating the PI3K/Akt/Fox M1 pathway.Part Ⅲ: The upregulation and downregulation of lentivirus transfection were verified by RT-qPCR and Western blot analyses.The results of RT-qPCR and Western blot indicated that SPOCK1 knockdown reduced the expression of α-SMA and collagen type I alpha 1(COL1A1),while PDGF-BB upregulated the expression of α-SMA and COL1A1,however,knockdown of SPOCK1 reduced the effects of PDGF-BB.The results of CCK-8 and Transwell assays showed that knockdown of SPOCK1 reduced the proliferative and migratory abilities of LX-2 and R-HSCs,while PDGF-BB promoted proliferation and migration of LX-2 and R-HSCs,however,knockdown of SPOCK1 reduced the effects of PDGF-BB on HSCs proliferation and migration.The results of RT-qPCR and Western blot indicated that knockdown of SPOCK1 reduced expression of cyclin B1,cyclin D1,MMP2 and MMP9,while PDGF-BB promoted expression of these markers,however,knockdown of SPOCK1 reduced the promotion of PDGF-BB on these markers.Taken together,SPOCK1 is involved in PDGF-BB-induced HSCs activation,proliferation and migration.Part Ⅳ: The results of Western blot indicated that knockdown of SPOCK1 reduced the level of phosphorylated Akt(p-Akt),while SPOCK1 overexpression or r SPOCK1 treatment upregulated the level of p-Akt,however,no significant changes were observed on Akt expression,and phosphorylation and expression of ERK,JNK and p38.These results indicated that SPOCK1 activated PI3K/Akt pathway in HSCs.Subsequently,the results of RT-qPCR,Western blot,CCK-8 and Transwell assays showed that SPOCK1 overexpression or r SPOCK1 treatment enhanced α-SMA and COL1A1 expression,HSCs proliferation and migration,however,blocking PI3 K by LY294002 reduced the profibrotic roles of SPOCK1 overexpression and r SPOCk1 treatment.Furthermore,SPOCK1 overexpression and r SPOCK1 treatment enhanced expression of cyclin B1,cyclin D1,MMP2 and MMP9,while these effects were reduced by LY294002 administration.Taken together,SPOCK1 promotes HSCs activation,proliferation and migration through activating the PI3K/Akt pathway.Part Ⅴ: The results of Co-IP showed that SPOCK1 interacted integrin α5β1 in LX-2 cells,and the results of double IF staining further confirmed that SPOCK1 colocalized with integrin β1.RT-qPCR and Western blot showed that specific neutralizing antibodies against integrin α5(anti-α5)or integrin β1(anti-β1)attenuated r SPOCK1-induced α-SMA and COL1A1 expression.The results of CCK-8 and Transwell assays showed that anti-α5and anti-β1 dramatically abolished r SPOCK1-induced proliferation and migration of HSCs.Furthermore,a combination of anti-α5 and anti-β1 exerted a synergistic inhibitory effects on r SPOCK1-induced HSCs activation,proliferation and migration.RT-qPCR and Western blot showed that blocking integrin α5β1 reduced r SPOCK1-induced expression of cyclin B1,cyclin D1,MMP2,MMP9 and p-Akt.Taken together,SPOCK1 interacts with integrinα5β1,activates PI3K/Akt pathway,and thus promoting HSCs activation,proliferation and migration.Part Ⅵ: The results of IHC showed that Flag was selectively expressed in the portal and perisinusoidal area,but not in the parenchymal cells.The double IF staining of Flag and Desmin in liver samples showed that Flag only expressed in the Desmin-positive cells.The results of Western blot showed that Flag only expressed in the R-HSCs of LV-SMA-sh SPOCK1-Flag-treated rats,but not in the LV-MOCK-treated rats.These results confirmed the successfully specific delivery of lentivirus to HSCs.The results of H&E,Masson’s Trichrome,α-SMA staining,and hydroxyproline measurement showed that HSCs-specific knockdown of SPOCK1 alleviated TAA-induced collagen deposition.HSCs-specific knockdown of SPOCK1 reduced the serum level of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)which were enhanced by TAA administration.Furthermore,HSCs-specific knockdown of SPOCK1 reduced the mRNA and protein levels of α-SMA,COL1A1,cyclin B1,cyclin D1,MMP2 and MMP9,both in the liver tissues and in R-HSCs.Taken together,HSCs-specific knockdown of SPOCK1 ameliorated TAA-induced liver fibrosis in rats.Conclusion: SPOCK1 expression was significantly increased in liver fibrosis and HSCs activation.PDGF-BB upregulated SPOCK1 expression in HSCs through activating the PI3K/Akt/Fox M1 pathway.Furthermore,SPOCK1 was involved in PDGF-BB-induced profibrotic responses,and SPOCK1 interacted with integrin α5β1,activated the PI3K/Akt pathway,and thus promoting HSCs activation,proliferation and migration.In vivo,HSCs-specific knockdown of SPOCK1 ameliorated TAA-induced liver fibrosis in rats.Collectively,this study elucidated a novel mechanism for liver fibrogenesis,and SPOCK1 might serve as a potential therapeutic target for liver fibrosis.