ZHX2 Inhibits Liver Regeneration By Repressing Mitochondrial Oxidative Phosphorylation

The liver is the only organ in the mammalian body that has the potent ability to restore its mass after injury.In the setting of liver injuries such as infection with acute liver injury or exposure to liver toxins,hepatocytes divide to restore lost parenchyma.The capacity of liver regeneration(LR)is not only essential for liver to fulfill its function in metabolic homeostasis,detoxification,and storage of nutrients,but also important in surgery when partial hepatectomy(PHx)and liver transplantation are performed.However,the capacity of LR is usually impaired in liver diseases,such as fatty liver,hepatitis,fibrosis and post-hepatectomy liver failure.Therefore,defining the molecular mechanisms and pathways that govern LR is urgently required to prevent complications in liver diseases therapy.Liver regeneration is exquisitely controlled by well-orchestrated signaling pathways involving cytokines,growth factors and metabolic networks.Transcription factor activation is a signature response to liver regeneration after hepatocyte injury.Therefore,significant progress has been made in the identification of transcription factors that control liver regeneration.To fulfi11 the demands of rapid cell proliferation,LR requires a large amount of energy production and metabolites.Different signal pathways will lead to the activation of transcription factors that either stimulate hepatocyte proliferation or promote cell survival to promote liver regeneration,whether they also regulate transcription factors in this novel pathway by metabolic reprogramming needs to be further investigated.A transcriptional repressor of postnatal AFP was identified in mice,which is named as zinc fingers and homeoboxes 2(ZHX2).ZHX2 has been found to be involved in regulation of liver lipid homeostasis.Loss of ZHX2 was partially responsible for high-fat diet-induced lipid accumulation and liver damage.Further studies demonstrated that ZHX2 inhibited exogenous lipid uptake and de novo lipid synthesis.However,whether ZHX2 plays a certain role in mitochondrial energy production and LR remains completely unknown.Here,we identified ZHX2,a transcription factor critical for regulating liver regeneration.Therefore,aiming to ZHX2 manipulation may be a helpful strategy to enhance hepatic regeneration for treatment of liver diseases.1.Hepatocyte-specific deletion of Zhx2 accelerates liver regenerationTo assess the potential role of ZHX2 in LR,2/3 PHx and CC14 was performed on mice with hepatocyte-specific Zhx2 knockout(Zhx2-KOhep)and their littermate controls(Zhx2-WT).The liver/body weight ratios following 2/3 PHx were recovered faster in Zhx2-KOhep mice than Zhx2-WT mice,and the liver mass of Zhx2-KOhep mice was larger than Zhx2-WT mice after 2/3 PHx.In line with the rapid cell proliferation during LR,we observed obviously higher levels of cell-cycle-related genes(Cyclin D1)during 3 days after PHx,and the expression of these genes were significantly increased in Zhx2-KOhep mice compared to those of Zhx2-WT mice.Similarly,the numbers of Ki67-positive cells and BrdU-positive cells in the livers of Zhx2-KOhep mice were higher than those of Zhx2-WT mice.Moreover,the activities of AST and ALT,indicators of liver damage,were markedly decreased in Zhx2-KOhep mice compared to Zhx2-WT mice.CC14 models gave the same result.Taken together,these results demonstrate that hepatic Zhx2 deficiency promotes LR in different mouse models.2.Zhx2 deficiency enhances mitochondrial OXPHOS activity in hepatocytesTo investigate the mechanisms by which Zhx2 deficiency accelerates liver regeneration,RNA sequencing(RNA-seq)and quantitative proteomics were performed with liver tissues from Zhx2-KOhep and Zhx2-WT mice 48 h after 2/3 PHx GSEA analysis many metabolism-related gene sets,such as OXPHOS and mitochondrial metabolism,were up-regulated in Zhx2-KOhep mice during LR.To verify the ZHX2-mediated regulation of mitochondrial OXPHOS,as indicated by transcriptomics and proteomics in LR,mitochondrial functional assays were performed in mouse and human hepatocytes with manipulation of ZHX2.ATP level was higher in Zhx2-KOhep hepatocytes than that of Zhx2-WT hepatocytes.Consistently,knockout of Zhx2 increased mitochondrial membrane potential and oxygen consumption in mouse hepatocytes.Zhx2-KOhep mice displayed increased hepatic expression of hepatocytes mitochondrial complex genes.Numbers of mitochondria and mtDNA copy number was increased in the livers of Zhx2-KOhep mice compared to that of Zhx2-WT mice.To validate above results in human hepatocytes,both human liver cancer cell line Huh7 cells and hepatic organoids derived from human liver tissues were included.ZHX2 overexpression greatly inhibited mitochondrial OXPHOS,presented as lower JC-1 aggregates,decreased ATP generation and reduced oxygen consumption rate(OCR).Ectopic expression of ZHX2 in Huh7 cells greatly decreased the expression of ETC genes and mitochondrial mass,as evidenced by lower copy number of mtDNA,weaker intensities of Mito Tracker Deep Red,less total mitochondrial area as well as fewer mitochondrial filamentous network in ZHX2-overexpressing Huh7 cells than those of control cells.To determine whether Zhx2 knockout facilitates LR by regulating mitochondrial OXPHOS,we use metformin,a widely used mitochondrial ETC complex I inhibitor.Metformin administration abolished the enhanced liver regeneration,seen as increased liver/body weight ratio in Zhx2-KOhep mice.Consistently,metformin treatment eliminated the elevated levels of PCNA protein and increased numbers of Ki67-and BrdU-positive cells in Zgx2-KOhep mice,metformin dampens Zhx2 deficiency-accelerated cell proliferation during LR.More importantly,metformin not only decreased the ATP levels in Zhx2-KOhep and Zhx2-WT mice,but also eliminated the difference of ATP levels between Zhx2-KOhep and Zhx2-WT mice.Taken together,these data demonstrate that abolishes Zhx2 deficiency-enhanced LR,suggesting that ZHX2 inhibits LR through reducing OXPHOS.3.The molecular mechanisms of ZHX2 regulates mitochondrial oxidative phosphorylation.Since ZHX2 has been identified as a transcriptional factor,we performed ChIP sequencing(ChIP-seq)of Huh7 cells with inducible ZHX2-HA overexpression,ZHX2-bound genes were enriched in OXPHOS pathway,which in agreement with RNA-seq data from Zhx2-KOhep and Zhx2-WT mice livers.Six ETC genes(including NDUFB9,SHDA,COX7C)were overlapped in both sequencing data,indicating that these genes are the potential targets of ZHX2.Interestingly,ChIP-seq data showed that 4 of these 6 ETC genes contain a common ZHX2-binding motif.ChIP and dual luciferase assays performed with anti-HA confirmed the occupancy of ZHX2 in the promoter regions of these ETC genes in Huh7 cells.EMSA and pull-down assay confirmed the interaction of ZHX2 with the biotin-labeled motif.These observations support that ZHX2 binds to the promoter region of ETC genes,and represses their expression.Mitochondrial OXPHOS is controlled by some mitochondrial transcription factors including PPAR-γ coactivators(PGC-1α),nuclear respiratory factor 1(NRF1),and mitochondrial transcription factor A(TFAM).Since only a small subset of ETC genes contain ZHX2-binding motif and are identified as the direct targets of ZHX2,we therefore asked whether ZHX2 regulates mitochondrial OXPHOS activity through other intermediators.ZHX2 markedly decreased PGC-1α protein level in Huh7 cells,tissue and hepatic organoids.More importantly,in vitro ubiquitination assay demonstrated that FBXW7 knockdown abolished the increased ubiquitination of PGC-la initiated by ZHX2 overexpression in Huh7 cells.Zhx2-WT and Zhx2-KOhep mice were pretreated with PGC-1α inhibitor(SR18292),and were then subjected to 2/3 PHx.The difference of liver/body weight ratio between Zhx2-WT and Zhx2-KOhep mice was abolished by SR18292 treatment.Consistently,the increases of Ki67 positive cells caused by Zhx2 deficiency were eliminated after SR18292 treatment.In line with the evidence showing that ZHX2 suppressed OXPHS and restrained energy supply for rapid cell proliferation during LR,the augmented ATP levels in Zhx2-KOhep mice were largely eliminated by SR18292 treatment.Above data suggest that ZHX2 represses LR in PGC-1α dependent manner.Altogether,our results imply that ZHX2 restricts mitochondrial OXPHS and LR via promoting FBXW7-mediated PGC-la degradation.Conclusion and Significance:Our results showed that hepatocyte-specific Zhx2 knockout(Zhx2-KOhep)enhanced LR after 2/3 partial hepatectomy(PHx)and carbon tetrachloride(CC14)exposure in mice.Loss of Zhx2 resulted in increased mitochondrial oxidative phosphorylation(OXPHOS)and higher production of ATP in hepatocytes,suppling energy for hepatocyte proliferation.ZHX2 represses mitochondrial OXPHOS through two mechanisms.First,ZHX2 binds to the promoters of a subset of electron transport chain(ETC)genes for inhibition.Second,ZHX2 reduces stability of peroxisome proliferator-activated receptor-γ coactlvator-1α(PGC-1α)to decrease mitochondrial mass and OXPHOS.Inhibition of PGC-lα or ETC abolishes the enhanced LR caused by ZHX2 loss.This study highlights a novel and important mechanism underlying mitochondrial regulation during LR.