FOXA2 Protects The Liver Against Fibrogenesis By Inhibiting CHOP-and ROS-induced Hepatocyte Apoptosis

【Background and Objective】Hepatic fibrosis,an early stage of cirrhosis,is a consequence of pathological deposition of extracellular matrix（ECM）in the liver.The various chronic liver injuries,resulting from tissue damage induced by infections with hepatitis viruses,persistent heavy intake of alcoholic drinks,chemical insults（aflatoxin B1）,metabolic or autoimmune reactions,could give rise to fibrosis.China is a country with a high incidence of hepatitis B.Chronic hepatitis,liver fibrosis,cirrhosis and liver cancer,is the common pathological process of most chronic liver disease.If effective intervention of liver fibrosis can be taken as soon as possible,we could not only reverse the pathological process but also block the process of chronic liver diseases to cirrhosis.However,the pathogenesis of liver fibrosis is yet not completely clear,and there is no recognized clinical effective treatment for fibrosis.So it is significant to further explore the mechanism of hepatic fibrosis and find effective prevention and treatment programs.The hepatocyte nuclear factor（HNF）family,including HNF1,HNF3,HNF4,HNF6and CCAAT/enhancer binding protein,is predominantly expressed in hepatocytes and is crucial for liver differentiation as well as the intact of hepatocyte function.Forkhead box transcription factor A2（FOXA2）,previously known as HNF3β,is one of transcriptional regulators of HNF3 family and is mainly expressed in liver,pancreas,lung,etc.FOXA1and FOXA2 cooperate to establish competence in foregut endoderm and are required for the initiation of liver development.In adult liver,FOXA2 is critical for glucose,lipid and bile acid homeostasis.Besides,FOXA2 can regulate many hepatocyte-specific genes involved in maintenance of hepatic function.Our previous studies have revealed a significant inhibitory effect of HNF1αand HNF4αon liver fibrogenesis.We supposed that FOXA2 may also serve as a new treatment target for hepatic fibrosis.Previously,we collected the clinical liver tissue specimens of patients with hepatic fibrosis or cirrhosis and the thick liver sections from carbon tetrachloride（CCl4）-treated mice model of hepatic fibrosis,and then detected the FOXA2 expression.We found that compared with normal liver samples,both patients and mice fibrotic livers showed that the expression of FOXA2 was obviously decreased.The primary hepatocytes and HSCs were isolated from mice with CCl4 administration.Interestingly,during the process of hepatic fibrosis,the expression of FOXA2 was down-regulated in the primary hepatocytes while up-regulated in the HSCs.To further explore the role of FOXA2 in liver fibrosis,we constructed four different kinds of models of mice with CCl4.The results indicated that hepatocyte-specific ablation FOXA2 gene promoted liver fibrosis;Contrarily,enhanced expression of FOXA2 specifically in hepatocytes or non-specifically in liver could ameliorate liver fibrosis;Surprisingly,HSCs-specific overexpression of FOXA2 played a non-significant role in liver fibrosis.In conclusion,regulating the expression of FOXA2affects the fibrogenesis.Taken together,we hypothesize that FOXA2 inhibits hepatic chronic fibrosis via protection of hepatocyte rather than inhibition of activated hepatic stellate cells.However,the specific mechanism is not yet clear.Based on the anti-fibrosis action of FOXA2,the purpose of this article is to further investigate the underling mechanism of the FOXA2 effect on liver fibrogenesis.【Methods】1.Hepatic fibrosis model in mice induced by CCl₄ administrationMale mice（8 weeks of age,from Shanghai Experimental Center of Chinese Academy of Sciences）were injected intraperitoneally with 0.25 ml/kg CCl4（diluted 1:3（v/v）in olive oil）twice a week for 4 or 5 weeks to establish the hepatic fibrosis mouse model.2.Three different types of virus were constructed to regulate the expression of FOXA2 in the CCl₄-induced mice fibrotic livers（1）Hepatocyte-specific deletion of FOXA2 in CCl4-induced liver fibrosis model in mice:FOXA2^loxP/loxP mice were obtained from the Jackson Laboratory.A single dose of adeno-associated virus AAV8-TBG-Cre at 2×10¹¹ genome copies was injected into FOXA2^loxP/loxP mouse model（6 weeks of age）through tail vein to specifically knockout the expression of FOXA2 in hepatocytes(FOXA2^H-KO),2 weeks prior to CCl4 administration.（2）Hepatocyte-specific overexpression of FOXA2 in CCl4-induced liver fibrosis model in mice:For hepatic specific overexpression of FOXA2,vector pENN-AVV-TBG-PI-RBG（the Penn Vector Core,p1015-R）was used to construct AAV8-TBG-FOXA2 virus.To perform FOXA2 overexpression in hepatocytes,the mice were infused with AAV8-TBG-FOXA2 through tail vein 1 week after the first CCl4 injection.（3）Non-specific enhancement of FOXA2 in CCl4-induced fibrotic livers in mice:The lentiviral vector pCDH-CMV-MCS-EF1-copGFP（System Biosciences）was used to construct the lentivirus LV-FOXA2 for non-specific FOXA2 upregulation in liver.The lenti-PCDH-FOXA2 or control virus was injected via tail vein 2 weeks after the first CCl4injection.3.FOXA2 regulates liver fibrogenesis involved in ER stress and oxidative stressThe liver-specific genes were detected by Real-time RT-PCR in isolated hepatocytes from FOXA2^loxP/loxP and FOXA2^H-KO with or without CCl₄ treated mice.ER stress:Western blot and immunohistochemistry were used to analyze the changes of ER stress marker（CHOP）in liver tissues of above three fibrotic mouse models,respectively;Oxidative stress:Malondialdehyde（MDA）kit was performed to detect the changes of MDA in liver lysis.The protein of N-nitrotyrosine was analyzed in above fibrotic mouse model using Western blot.4.To investigate the situation of cell apoptosis in liver fibrosisTUNEL（Td T-mediated dUTP Nick-End Labeling）kit was conducted to evaluate the cell apoptosis in FOXA2^H-KO CCl4-treated model mice;The changes of apoptosis related genes（Bax and Cleaved caspase 3）in liver tissues of different types of mice models were detected by Western blot.5.Statistical AnalysisStatistical calculation of values was performed using software SPSS 18.0 version.All data were analyzed by ANOVA and t test and the results were presented as mean±standard deviation.The P<0.05 was considered as the test standard of statistically significant.【Results】?1.FOXA2 modulates liver fibrogenesis related to ER stress（1）Hepatocyte-specific deletion of FOXA2 aggravates hepatic fibrosis by promoting endoplasmic reticulum stress.We investigated liver-specific genes involved in glucose,lipid,amino acid,xenobiotic,and drug metabolism as a consequence of liver function disorder in these mice.In relative to WT mice,hepatocytes derived from FOXA2^H-KO showed no obvious differences in the liver metabolism genes.But these genes were to some extend affected by CCl₄ administration.More importantly,when induced by CCl₄,hepatocytes from FOXA2^H-KO mice indicated a more severely reduction in hepatic function genes expression including Apoa2,ApoE,Gys2,Gck,F7,Trf,Cyp1a1 and Cyp2e1,compared with control fibrotic group.Thus,we conclude that in the fibrotic livers,FOXA2 is required for maintenance of hepatocytes.Next,we examined the expression of molecular markers of ER stress,CHOP/Gadd153.The trend of CHOP changes was consistent with the liver-specific genes alterations.The protein levels of CHOP showed no changes in the livers between FOXA2^H-KO and WT mice.And CCl₄-treatment could elevate the CHOP expression,which was further aggravated by FOXA2 knockout specifically in hepatocytes.Consistent with the results of Western blot.,immunohistochemistry showed distinctly higher CHOP staining was observed at the lobular areas of FOXA2^H-KO fibrotic livers.（2）Hepatocyte-specific upregulation of FOXA2 suppresses liver fibrosis by inhibiting ER stress.The results of Western blot showed that the expression of CHOP in liver tissues of the AAV8-TBG-FOXA2 group were significantly lower than those of the control group,which indicated that elevating FOXA2 specifically in hepatocytes in liver significantly protected the liver against ER stress effect.（3）FOXA2 overexpression in liver can alleviate liver fibrosis through repressing ER stress.Western blot showed that the expression of CHOP in the livers of mice could be induced by CCl₄,overexpression of FOXA2 in liver efficiently reduced the accumulation of the protein in fibrotic livers in mice.In conclusion,these results suggest that FOXA2 modulates the process of liver fibrosis involved in ER stress.2.FOXA2 inhibits hepatic chronic fibrosis involved in oxidative stress（1）Hepatocyte-specific deletion of FOXA2 promotes hepatic fibrosis by increasing oxidative stress.The level of oxidative damage marker,MDA,was slightly elevated in the livers treated with CCl₄ injection.FOXA2^H-KO mice exhibited dramatically enhanced oxidative stress following CCl₄ administration.Additionally,the results showed that the protein of nitro-tyrosine,another indicator of oxidative stress,was also increased significantly in FOXA2^H-KO fibrotic livers compared with controls.（2）Hepatocyte-specific upregulation of FOXA2 suppresses liver fibrosis by reducing oxidative stress.Western blot was performed to show that the Nitrotyrosine expression in liver tissues of the AAV8-TBG-FOXA2 group were obviously lower than those of the control group,which suggested that elevating FOXA2 specifically in hepatocytes in liver significantly protected the liver against oxidative stress effect.（3）FOXA2 overexpression in liver can alleviate liver fibrosis via inhibition of oxidative stress.Western blot showed that the expression of Nitrotyrosine in the livers of mice could be induced by CCl₄,overexpression of FOXA2 in liver efficiently reduced the accumulation of the proteins in mice fibrotic livers.Together,these results suggest that FOXA2 alleviates hepatic fibrosis via inhibition of oxidative stress.3.FOXA2 mitigates hepatic fibrosis through repressing hepatocyte apoptosis（1）Hepatocyte-specific loss of FOXA2 promotes liver fibrosis by increasing hepatocyte apoptosis.TUNEL kit was performed to measure the hepatic apoptosis.WT and FOXA2^H-KO mice without CCl₄ inducement showed no spontaneous apoptosis of hepatocytes,but in the CCl₄-induced model,more pronouncedly TUNEL-positive hepatocytes were scattered in the liver lobules of FOXA2^H-KO fibrotic mice compared to control fibrotic littermates.To further investigate the signal components of potential apoptotic pathway,Bax and activated caspase 3 were measured.Compared with the matched controls,all of them were significantly elevated in FOXA2^H-KO fibrotic livers in relative to matched groups.In addition,the occurrence of apoptosis in FOXA2^H-KO fibrotic livers was confirmed by activated caspase 3 staining as well as Tunel staining.Cytoplasm cleaved caspase 3mostly located in hepatocyte was enforced remarkably at the periphery of the centrilobular areas of FOXA2^H-KO CCl₄-treated livers.The data suggests FOXA2 deletion renders hepatocytes more vulnerable towards apoptosis in vivo.（2）Hepatocyte-specific overexpression of FOXA2 inhibits hepatic fibrosis by decreasing hepatocyte apoptosis.As predicted,all induction of Bax and active caspase 3 were attenuated in fibrotic livers after injection of AAV-TBG-FOXA2.Consistent with the results of Western blot,immunohistochemistry showed obviously lower Cleaved caspase 3 staining was observed at the lobular areas of fibrotic livers with the AAV8-TBG-FOXA2 administration.（3）Upregulation of FOXA2 in liver alleviates hepatic fibrosis via suppressing hepatocytes apoptosis.All accumulation of Bax and active caspase 3 were declined in fibrotic livers after LV-FOXA2 delivery.Collectively,these results confirm that during liver fibrogenesis,FOXA2 plays an indispensable role in hepatocytes apoptosis.【Conclusion】1.During liver fibrogenesis,CCl4-induced chronic hepatic injury is sufficient to trigger ER stress and oxidative stress,which ultimately induced hepatocyte apoptosis.2.FOXA2 ameliorates ER stress in the fibrotic liver.3.FOXA2 relieves oxidative stress during the development of hepatic fibrosis.4.FOXA2 alleviates hepatic fibrosis via repressing hepatocyte apoptosis.5.FOXA2 protects liver from fibrogenesis via inhibiting CHOP-and ROS-induced hepatocyte apoptosis.These data suggest that FOXA2 and hepatocyte protection are potential therapeutic targets for liver fibrosis.Our data also ignites the light of hepatocyte nuclear factor therapy in liver diseases.