The Role And Mechanism Of Forkhead Box Protein A3 In Liver Regeneration After Acute/Chronic Liver Injury

Liver is the“core”metabolic organ,which plays vital roles in glucose,protein and lipid metabolism,bile acid synthesis and secretion,endogenous and xenobiotic toxins detoxification,and immune functionality maintenance.Liver injury is frequent in clinic including acute injury and chronic injury.Notably,liver characterizes the unique capability of robust regeneration upon injury,the only solid organ that use regenerative mechanisms to ensure that the liver-to-bodyweight ratio always at 100%.Liver regeneration（LR）from acute injury is beneficial and the liver will return equivalent size and weight to prior to injury via LR after partial hepatectomy（PH）or chemical insults.On the other hand,chronic injury also leads to chronic loss of hepatocytes,often has adverse consequences like fibrosis.Previous studies have shown that proliferation of hepatocytes failed,cholangiocytes or hepatic progenitor cells would proliferate to restore normal liver structure.However,cell turnover is very slow in normal liver.Forkhead box protein A（FOXA）transcription factors（also known as hepatocytenuclear factor 3,HNF3）family comprise three members,FOXA1,FOXA2 and FOXA3.Among them,Foxa1 and Foxa2 are essential for liver development since loss of Foxa1 and Foxa2 in the foregut endoderm leads to abrogated hepatic specification in mice.While Foxa3 is dispensable for liver development as Foxa3 deficient mice show no defect in liver growth.However,Foxa3 is the most highly expressed family member in adult liver,suggesting it may play different roles than Foxa1 and Foxa2 in liver.We have shown that FOXA3 play crucial roles in lipid and glucose homeostasis in liver.In 2015,using a hydrodynamic screen system in hereditary tyrosinemia mice model,Wangensteen et al screened 43 potential candidates and indicated that Foxa3 is among top candidates for liver regeneration,rather than Foxa1 and Foxa2.To clarify the role and mechanism of FOXA3 during liver regeneration,Gene Expression Omnibus（GEO）data for liver regeneration after PH were download and then RNA-seq analysis was performed.Foxa3 was identified as one of transcription factors that involved in cell proliferation regulation after PH.We found Foxa3features high and specific expression in hepatocytes and cholangiocytes via single cell sequencing analysis,which are major origins of cell proliferation during LR.Further analysis of single-cell RNA-seq suggested that Foxa3 expressed in zone 2 hepatocytes,which also been proved major source of cell proliferation during LR.Then we demonstrated that expression of Foxa3 was increased in acute liver injury model induced by PH or carbon tetrachloride（CCl₄）.We found FOXA3 deficient delayed proliferation including hepatocytes,cholangiocytes and stem cells during LR,exacerbates acute liver damage and delayed liver recovery after chronic injury.Ablation of Foxa3 in vitro inhibited proliferation and increased apoptosis of hepatocytes.Specific overexpression of FOXA3 in hepatocyte could attenuate liver damage and speeded hepatocytes proliferation and recovery.Mechanistically,via analysis of RNA-seq and Chip-seq,we found Cebpb（CCAAT/enhancer binding protein,beta）was direct downstream target of FOXA3.FOXA3 govern liver regeneration through regulating Cebpb transcription.Cebpb restore advanced liver injury and impaired cell proliferation in FOXA3 KO mice to the similar degree observed in WT mice.To further examine the role of FOXA3 in LR,we identified Cardamonin（CDN）,as a top Foxa3 transcription inducer,from 780 nature compounds.CDN could ameliorated liver injury and accelerate liver regeneration both in vivo and in vitro.As uncontrolled cell proliferation is a prelude to carcinogenesis.We further indicated that CDN speeded liver regeneration via activating Foxa3 without interfering with the normal termination of LR,which may due to CDN promote FOXA3-induced P53activation via chromatin remodeling at the late stage of LR.In summary,we demonstrate the crucial role of FOXA3 in liver regeneration after liver injury.FOXA3 governing speed of cell proliferation and degree of liver damage through regulating Cebpb transcription.Besides,we identified CDN as Foxa3pharmacological inducer which could accelerate liver regeneration and ameliorated liver damage without affecting determination of LR.This study provides important insight into the process and mechanism of liver regeneration,and also provides potential targets and theoretical basis for preclinical studies targeting transcription factors to treat liver injury and promote liver regeneration.