TIPE1 Promotes Hepatocyte Autophagy And Liver Regeneration By Activating ROS-mediated FoxO1 Acetylation

BackgroundAs the largest organ in the human body,the liver has a strong regeneration and repair ability.When the adult liver is subjected to 70%hepatotomy,it can be completely repaired within a few months and at the same time,its normal function can be restored.Based on this significant characteristic,partial liver resection and liver transplantation have become the effective strategy for the treatment of end-stage liver diseases.Thus,to elucidate the molecular basis and signal transduction mechanism involved in liver regeneration will provide a new intervention strategy for liver functional recovery after surgical resection of malignant tumors and cirrhotic liver tissues.Under normal conditions,hepatocytes are mainly in a static state(GO stage),with only 0.0012%-0.1%of hepatocytes undergoing mitosis.After partial hepatectomy or drug-induced and virus-induced liver injury,the residual hepatocytes would immediately enter the mitotic phase and begin to proliferate.Cytokines,such as TNF-α,IL-6,and HGF,play an important role in this initiation process.When the liver recovers to sufficient size and function,liver regeneration slows down,and cytokines,such as TGF-β family,would be expressed,inhibit cell proliferation and promote cell apoptosis.In addition to the regulation of hepatocytes themselves,liver resection or injury can induce the expression of angiogenic factors,improve the liver microenvironment,and promote liver regeneration.Moreover,the inflammatory process and innate immune response after liver surgery are also key factors participating in liver regeneration.In conclusion,a variety of cells and signal transduction pathways are involved in regulating the process of liver regeneration,and the explanation of detailed mechanisms might provide potential targets for clinical intervention.TIPE1 is a member of the tumor necrosis factor-α-inductive protein 8(TNFAIP8)family,and its encoded gene is located on human chromosome 19.TIPE1 is widely expressed almost in all tissues and cells throughout the body and highly expressed in adult liver,suggesting that TIPE1 may be involved in regulating the functional state of liver.The first published study showed that TIPE1 interference inhibited both apoptosis and necroptosis,so it is defined as a candidate molecule to regulate cell death.Our previous study also showed that TIPE1 inhibited the growth of HCC cells by inhibiting RAC1 activation.Thus,TIPE1 is thought to be involved in the regulation of cell death and proliferation balance.Combined with the research progress and our previous study,we hypothesize that TIPE1 may affect the liver regeneration process by regulating cell death and proliferation balance.The present study aims to clarify the role of TIPE1 in liver regeneration and to explore its molecular mechanism by using liver regeneration mouse model and in vitro experiment.Objectives:In this study,we focus to unravel the roles of a novel cell death regulator TIPE1 in the process of liver regeneration and to explore the underlying molecular mechanism.Our study might provide a potential target for clinical intervention of liver regeneration.Specific objectives are as follows:1.To clarify the expression changes of TIPE1 during liver regeneration by establishing different kinds of mouse model;2.To determine the role of hepatocyte TIPE1 in liver regeneration by using conditional knockout mice and in vitro cultured primary hepatocytes;3.To explore the molecular mechanism of TIPE1 regulation in liver regeneration through RNA sequencing techniques and and in vivo validation experiments.Methods and results1.TIPE1 expression was upregulated during liver regeneration in miceFirstly,we used wild-type(WT)mice to perform 70%hepatotomy,which is a widely used model to simulate the liver regeneration process.The mice were sacrificed at 24h,48h,72h,120h and 168h after 70%PHx respectively.Liver tissues were collected and weighed.HE staining,immunohistochemistry staining for PCNA,immunofluorescence staining for BrdU and serum ALT and AST analysis were performed to evaluate the degree of liver regeneration.Results showed that the ratio of liver weight to body weight gradually recovered with time after 70%PHx;the hepatocytes were in a state of rapid proliferation at 24h,48h and 72h after hepatectomy,and the levels of serum ALT and AST increased at 24h and then recovered.Simutaneouly,RT-qPCR,Western blot and immunohistochemistry staining showed that the expression of TIPE1 in the liver tissue was upregulated at 24h,48h,72h and 120h after 70%PHX.Then,we established liver injury regeneration mouse model by intraperitoneal injection of 10%CCL4.Mice were sacrificed at 12h,24h,72h and 120h respectively.We collected serum and liver tissue.Serum ALT and AST level and HE staining were used to evaluate the degree of liver injury.Results showed that liver injury was most serious after 24h,and then gradually recovered.Western blot analysis displayed the increased expression of TIPE1 in liver tissue of mice with 10%CCL4 induced liver injury.2.Hepatocyte Tipel knockout retards liver regenerationWe bred hepatocyte specific Tipel knockout C56BL/6 mice(ALB-Cre;Tipe1flox/flox,referred to as HKO).We established a 70%PHx model in 6-to 8-week-old male WT and HKO mice.The mice were sacrificed at 24h,48h and 72h later.The proliferation of hepatocytes and liver regeneration were observed.It was found that,compared to control mice,hepatocyte specific knockout of Tipel significantly down-regulated the ratio of liver weight to body weight,the positive proportion of BrdU and PCNA expression in liver tissue after hepatectomy.In addition,primary hepatocytes were isolated from WT and HKO mice and stimulated by HGF.Western blot analysis showed that PCNA expression in Tipel knockout primary hepatocytes was significantly lower than that in wild type hepatocytes.These results suggest that Tipel knockout can inhibit the liver regeneration process after 70%PHx and in vitro hepatocyte proliferation.The liver injury model induced by CCL4 was established in WT and HKO mice.The indicators related to liver injury and regeneration were detected after 72h.The results showed that compared to those of WT mice,the liver weight/body weight ratio of HKO mice was decreased,the liver injury and apoptosis in HKO mice were decreased,Ki67 and PCNA expression were significantly downregulated in the liver tissues of HKO mice.These results suggest that hepatocyte specific knockout inhibits the regeneration process after CCL4-induced liver injury.3.TIPE1 promoteshepatocyte autophagy and liver regeneration through up-regulation of ROS/ac-FoxO1 axis3.1 TIPE1 upregulates ROS level in hepatocytesWe performed RNA sequencing for the liver tissues from HKO mice and WT mice after 70%PHx.GSEA analysis revealed that the reactive oxygen species pathway was significantly enriched in liver tissues from HKO mice.Next,we detected ROS levels in WT and HKO hepatocytes after 70%PHx.Results showed that ROS level in hepatocytes of HKO mice was significantly lower than that of WT mice.Concordantly,ROS levels in primary HKO hepatocytes and TIPE1 knockdown Huh7 cells were significantly lower than those in control cells,while TIPE1 overexpression in Huh-cells up-regulated ROS levels.The intracellular ROS level is determined by the balance between the production and scavenging pathways of ROS.Mitochondrial respiratory chain and nicotinamide adenine dinucleotide phosphate(NADPH)oxidase complex are the main sources of ROS.We first found that TIPE1 did not affect mitochondrial ROS level,mitochondrial number and membrane potential.Importantly,blocking the NADPH oxidase pathway by DPI reversed the TIPE1-upregulated ROS levels in Huh7 cells.Moreover,TIPE1 increased the expression of NOX2 and NOX4 in hepatocytes.In addition,Tipel knockout or knockdown significantly upregulated d the expression of NRF2 in liver tissues,primary progenytes and Huh7 cells.These results suggest that TIPE1 may up-regulate ROS in liver cells by activating the NADPH oxidase pathway and inhibiting the expression of NRF2.3.2 Tipel knockout retarded liver regeneration in a ROS-dependent mannerIn order to further explore the role of ROS in TIPE1 regulation on liver regeneration.We pretreated WT and HKO mice with NAC and established 70%PHx model in control or NAC treated mice.The ROS levels and liver regeneration degree was evaluated after 70%PHx for 24h.Results showed that NAC treatment almost neutralized the difference of ROS levels,liver weight/body weight ratio,the proportion of dividing and BrdU positive hepatocytes,and PCNA expression level between WT and HKO mice after 70%PHx.These results suggest that ROS is involved in the delay of liver regeneration caused by Tipe1 gene deletion in hepatocytes.3.3 ROS-activated FoxO1 acetylation and hepatocyte autophagy are responsible for the regulation of TIPE1 on liver regenerationAs the second messenger molecule,ROS regulates a variety of biological processes by affecting intracellular signal transduction.We further analyzed the differentially expressed genes of liver tissues in WT and HKO mice after 70%PHx by RNAseq,and GSEA enrichment analysis revealed significant enrichment of FoxO signaling pathway in HKO mice.It has been reported that the FoxO1 signaling pathway regulates a variety of cellular biological processes,and FoxO1 acetylation is partially responsible for the regulation of ROS on biological processes.Western blot analysis showed that compared to WT mice,there was no significant difference in the expression of total FoxO1 protein in the liver tissues of HKO mice after 70%PHx,while the acetylated FoxO1(ac-FoxO1)level was significantly reduced.Consistent with this,Huh7 cells overexpressing TIPE1 showed higher levels of ac-FoxO1 either in normal culture or with H2O2 stimulation.More importantly,there was no significant difference in ac-FoxO1 levels in liver tissue of NAC treated WT and HKO mice after 70%PHx.These results suggest that TIPE1 up-regulates FoxO1 acetylation by increasing ROS level in hepatocytes.Then,we pretreated WT and HKO mice with FoxO1 inhibitor AS 1842856,and then observed its effect on TIPE1 regulation of liver regeneration process.These results showed that AS 1842856 significantly down-regulated the ratio of liver weight to body weight,the proportion of dividing and BrdU positive hepatocytes and PCNA expression in the liver tissues of WT mice at 24h after 70%PHx.However,there was no significant difference in these indicators for liver regeneration between AS 1842856 treated WT and HKO mice.These results suggest that FoxO1 pathway may be involved in the inhibition of liver regeneration induced by Tipe1 gene knockout.Previous studies have shown that FoxO1 acetylation plays an important role in autophagy activation,and hepatocyte autophagy is an important way in maintaining the ability of liver regeneration.Therefore,we further found that p62 levels were increased and LC3II/I levels were decreased in liver tissue of HKO mice before and after PHx compared to those in WT mice.Consistent with this,increased p62 and decreased LC3II/I level were found in cultured primary Tipel knockout hepatocytes when compared to that in wild-type hepatocytes.More importantly,NAC treatment and AS 1842856 treatment leveled p62 and LC3II/I changes in liver tissue of WT and HKO mice.These results suggest that TIPE1 may regulate hepatocyte autophagy through ROS activation on FoxO1 acetylation,and then participate in the regulation of liver regeneration process.4.Hepatocyte specific Tipel overexpression significantly promotes the liver regenerationFinally,we performed 70%PHx in WT mice by hydrodynamic injection of hepatocyte specific empty adeno-associated virus(AAV-EV)or TIPE1 overexpressing adeno-associated virus(AAV-TIPE1)by the tail vein.Results showed that the ratio of liver weight to body weight,the proportion of dividing and proliferating hepatocytes were significantly higher in AAV-TIPE1 treated mice than in AAV-EV injected mice.Further analysis showed that AAV-TIPE1 significantly upregulated the ROS level,which is accompanied by the increased levels of ac-Fox01 and LC3II/I and the decreased p62 level in liver tissues.These results confirm that hepatocellular specific overexpression of Tipel could up-regulate ROS levels,FoxO1 acetylation and autophagy of hepatocytes,which in turn promote liver regeneration.Conclusions and significances:Based on the above experimental results,we found that TIPE1 was involved in liver regeneration,and preliminarily explored the molecular mechanism,and made the following conclusions:TIPE1 expression is up-regulated during liver regeneration,and TIPE1 promotes liver regeneration through up-regulation of ROS level,activation of FoxO1 acetylation and autophagy in hepatocytes.Innovations and significances:In this study,we detected the expression changes of TIPE1 in liver regeneration for the first time,and confirmed that the expression of TIPE1 in liver cells contributes to liver regeneration process.It was preliminarily proved that TIPE1 promoted liver regeneration by increasing ROS level,up-regulating FoxO1 acetylation and autophagy in hepatocytes.This study revealed a new biological role and molecular mechanism of TIPE1 involving in the regulation of liver functional homeostasis,which is expected to provide novel targets for clinical intervention of liver regeneration.