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Loss of liver function in chronic liver disease: An identity crisis

  • Carmen Berasain
    Correspondence
    Corresponding author. Address: Program of Hepatology, CIMA, University of Navarra, Avda, Pio XII, n55, 31008, Pamplona, Spain.
    Affiliations
    Program of Hepatology, CIMA, University of Navarra, Pamplona, Spain

    Centro de Investigación Biomédica en Red, CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

    Instituto de Investigaciones Sanitarias de Navarra, IdiSNA, Pamplona, Spain
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  • Maria Arechederra
    Affiliations
    Program of Hepatology, CIMA, University of Navarra, Pamplona, Spain

    Centro de Investigación Biomédica en Red, CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

    Instituto de Investigaciones Sanitarias de Navarra, IdiSNA, Pamplona, Spain
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  • Josepmaria Argemí
    Affiliations
    Centro de Investigación Biomédica en Red, CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

    Instituto de Investigaciones Sanitarias de Navarra, IdiSNA, Pamplona, Spain

    Liver Unit, Clinica Universidad de Navarra, Pamplona, Spain
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  • Maite G. Fernández-Barrena
    Affiliations
    Program of Hepatology, CIMA, University of Navarra, Pamplona, Spain

    Centro de Investigación Biomédica en Red, CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

    Instituto de Investigaciones Sanitarias de Navarra, IdiSNA, Pamplona, Spain
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  • Matías A. Avila
    Correspondence
    Corresponding author. Address: Program of Hepatology, CIMA, University of Navarra, Avda, Pio XII, n55, 31008, Pamplona, Spain.
    Affiliations
    Program of Hepatology, CIMA, University of Navarra, Pamplona, Spain

    Centro de Investigación Biomédica en Red, CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

    Instituto de Investigaciones Sanitarias de Navarra, IdiSNA, Pamplona, Spain
    Search for articles by this author
Open AccessPublished:September 14, 2022DOI:https://doi.org/10.1016/j.jhep.2022.09.001

      Summary

      Adult hepatocyte identity is constructed throughout embryonic development and fine-tuned after birth. A multinodular network of transcription factors, along with pre-mRNA splicing regulators, define the transcriptome, which encodes the proteins needed to perform the complex metabolic and secretory functions of the mature liver. Transient hepatocellular dedifferentiation can occur as part of the regenerative mechanisms triggered in response to acute liver injury. However, persistent downregulation of key identity genes is now accepted as a strong determinant of organ dysfunction in chronic liver disease, a major global health burden. Therefore, the identification of core transcription factors and splicing regulators that preserve hepatocellular phenotype, and a thorough understanding of how these networks become disrupted in diseased hepatocytes, is of high clinical relevance. In this context, we review the key players in liver differentiation and discuss in detail critical factors, such as HNF4α, whose impairment mediates the breakdown of liver function. Moreover, we present compelling experimental evidence demonstrating that restoration of core transcription factor expression in a chronically injured liver can reset hepatocellular identity, improve function and ameliorate structural abnormalities. The possibility of correcting the phenotype of severely damaged and malfunctional livers may reveal new therapeutic opportunities for individuals with cirrhosis and advanced liver disease.

      Keywords

      Introduction

      The liver performs vital roles in systemic homeostasis, including bile acid and cholesterol metabolism, detoxification of endo- and xenobiotics, glucose synthesis and storage, lipid turnover, hormone metabolism and plasma protein secretion. Most of these functions are carried out by hepatocytes, parenchymal cells constituting 80% of the liver mass and 60% of its cellular composition.
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      Besides hepatocytes, there are at least six other types of liver cells: biliary epithelial cells, sinusoidal endothelial cells, stellate cells, dendritic cells, macrophages and additional immune cell types. These cells support hepatocellular function, and together with hepatocytes are arranged in three-dimensional anatomical units in the form of hexagonal columns called liver lobules.
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      Near the central vein, pericentral hepatocytes are responsible for bile acid synthesis and contribute to ammonia metabolism, whereas hepatocytes in the remaining mid zone are involved in a variety of cytochrome P450-mediated metabolic reactions.
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      • The liver performs a series of metabolic and secretory functions that are essential for the preservation of systemic homeostasis. Hepatic identity is typified by the expression of a unique complement of genes.
      • Hepatocellular identity is developmentally established and post-natally preserved by a specific network of transcription factors and mRNA splicing regulators.
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      • Re-expression of key transcription factors such as HNF4α in the decompensated liver can restore liver function and attenuate liver damage in preclinical models.

      Establishment and maintenance of hepatocellular identity

      Cellular signals and pioneer and non-pioneer TFs

      During embryonic development liver organogenesis arises from the foregut endoderm, a region from which pancreatic progenitors are also derived. Signals from adjacent cardiac mesoderm and the septum transversum mesenchyme, particularly fibroblast growth factor (FGF) and bone morphogenic protein (BMP), respectively, suppress the pancreatic programme and, together with Nodal signalling, specify the hepatic fate, leading to the emergence of hepatoblasts, the liver parenchymal progenitor cells.
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      The expansion of the hepatoblast pool is to a great extent regulated by BMP, FGF, WNT and transforming growth factor β (TGFβ) signalling from the nearby endothelium and the septum transversum mesenchyme.
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      Subsequently, hepatoblasts proliferate and can differentiate into hepatocytes or biliary epithelial cells (cholangiocytes). This process is controlled by signalling gradients that depend on hepatoblasts’ location within the emerging liver parenchyma. Only cells in contact with the portal vein, a region known as the ductal plate, will give rise to cholangiocytes in response to TGFβ and Notch signals emanating from the periportal mesenchyme, signals that concomitantly inhibit hepatocyte differentiation.
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      The remaining hepatoblasts will undergo differentiation into hepatocytes, in a process involving the WNT/β-catenin signalling pathway.
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      The Hippo/Yes-associated protein (YAP) pathway is relevant for both hepatocyte and cholangiocyte differentiation,
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      Another set of regulatory proteins almost concomitantly involved in hepatoblast specification is the GATA family of TFs, particularly GATA4 and GATA6. FOXA and GATA factors are needed for lineage specification, and their mutual interaction is required for their binding to regulatory DNA regions (i.e. enhancers and promoters).
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      ,
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      Moreover, OC-1, OC-2, HNF1α, and to a lesser extent HNF1β, regulate the expression of the P2 promoter-derived HNF4α7 isoform during development, while at later stages of liver maturation this P2-driven foetal isoform will be repressed by HNF4α1 (a P1 promoter-driven form).
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      (Fig. 1A). The complexity and redundancy of this network becomes more intricate as post-natal hepatocyte differentiation progresses.
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      • Iwafuchi-Doi M.
      Molecular regulation of mammalian hepatic architecture.
      This complexity, and a certain functional redundancy contributes to the stability of the expression of the individual factors, and therefore to the ultimate maintenance of the adult liver phenotype.
      • Tachmatzidi E.C.
      • Galanopoulou O.
      • Talianidis I.
      Transcription control of liver development.
      Figure thumbnail gr1
      Fig. 1Establishment of the adult liver phenotype.
      (A) Pioneer factors FOXA and GATA6 unmask chromatin domains (“bookmarking” activity) enabling the access of core liver TFs. A cross-regulated network of core TFs established during development binds to CRMs in bookmarked genes. Expression of these genes, together with stage-specific pre-mRNA splicing, will define the embryonic liver. (B) The complexity of this TF network markedly increases towards post-natal stages. Additional transcriptional regulators complete the maturation process, and maintain the gene expression pattern of differentiated hepatocytes. Bookmarking permits the re-establishment of hepatic gene expression after cell division. Developmentally regulated SFs contribute to the post-transcriptional definition of the mature liver phenotype, generating the adult isoforms of alternatively spliced pre-mRNAs. C/EBP, CAAT/enhancer binding protein; CRM, cis-regulatory module; ESRP2, epithelial splicing regulatory protein 2; FOXA, forkhead box A; FXRα, farnesoid X receptor α; HNF, hepatocyte nuclear factor; LRH1, liver receptor homolog-1; PXR, pregnane X receptor; SFs, splicing factors; SRSF, serine and arginine-rich splicing factor; TFs, transcription factors.

      Epigenetic regulation and bookmarking

      Changes in the chromatin landscape are a central process in organogenesis. The establishment of the gene expression pattern of fully differentiated hepatocytes also involves significant modifications in chromatin structure.
      • Xu C.-R.
      • Zaret K.S.
      Chromatin “pre-pattern” and epigenetic modulation in the cell fate choice of liver over pancreas in the endoderm.
      As mentioned, in early development, pioneer factors bind closed chromatin and introduce nucleosomal changes to create a permissive state for gene regulation in a stepwise manner. This process entails epigenetic modifications including loss of DNA methylation, or the increase in histone H3K4 methylation as observed upon FOXA binding. Pioneer factors interact with chromatin remodelling proteins, such as the SWI/SNF complex and the histone N-methyltransferase MLL3, and induce the displacement of the linker histone H1.
      • Mayran A.
      • Drouin J.
      Pioneer transcription factors shape the epigenetic landscape.
      ,
      • Iwafuchi-Doi M.
      The mechanistic basis for chromatin regulation by pioneer transcription factors.
      These modifications prepare enhancer regions for the interaction with other non-pioneer TFs when the cells receive the environmental signals that promote the acquisition of hepatic identity. This process involves the deposition of activating chromatin marks, such as H3K9ac/H3K14ac (introduced by the histone acetyltransferase p300), in genes that will be expressed in hepatoblasts,
      • Macchi F.
      • Sadler K.C.
      Unraveling the epigenetic basis of liver development, regeneration and disease.
      and repressing marks like H3K27me3 (deposited by the histone methyltransferase EZH2) in those that will only be expressed in cells that undergo pancreatic differentiation.
      • Huppert S.S.
      • Iwafuchi-Doi M.
      Molecular regulation of mammalian hepatic architecture.
      The sequential interaction of pioneer and non-pioneer TFs progressively shapes chromatin throughout the maturation process, generating a conformation that is competent for gene expression. These binding and chromatin modification activities may not be immediately followed by gene transcription, but label specific genomic sequences for future activation either during development or in the adult. This process is called “bookmarking” or epigenetic memory, and contributes to the preservation of cell identity during cell division, allowing for the re-establishment of regulatory networks.
      • Elsherbiny A.
      • Dobreva G.
      Epigenetic memory of cell fate commitment.
      In liver development, chromatin bookmarking activity has been demonstrated for non-pioneer factors such as HNF4α and C/EBPα which remain bound to mitotic chromatin in cycling cells.
      • Karagianni P.
      • Moulos P.
      • Schmidt D.
      • Odom D.T.
      • Talianidis I.
      Bookmarking by non-pioneer transcription factors during liver development establishes competence for future gene activation.
      In the embryonic liver, early binding of HNF4α and C/EBPα progressively increases the levels of H3K27ac and the formation of open chromatin domains well before transcription activation, thereby preventing heterochromatin formation and bookmarking genes for future expression.
      • Karagianni P.
      • Moulos P.
      • Schmidt D.
      • Odom D.T.
      • Talianidis I.
      Bookmarking by non-pioneer transcription factors during liver development establishes competence for future gene activation.
      Interestingly, while many of these HNF4α and C/EBPα marked genes belong to pathways related to metabolic processes, and thus will continue to be highly expressed in adult life,
      • Thakur A.
      • Wong J.C.H.
      • Wang E.Y.
      • Lotto J.
      • Kim D.
      • Cheng J.C.
      • et al.
      Hepatocyte nuclear factor 4-alpha is essential for the active epigenetic state at enhancers in mouse liver.
      others will be silenced after birth. As postulated by Tallianidis and collaborators, the HNF4α-C/EBPα bookmark on these post-natally silenced genes can make them competent for future activation in specific conditions, as observed for oncofoetal genes in liver cancer.
      • Karagianni P.
      • Moulos P.
      • Schmidt D.
      • Odom D.T.
      • Talianidis I.
      Bookmarking by non-pioneer transcription factors during liver development establishes competence for future gene activation.

      Post-natal hepatic identity: cis-regulatory modules (CRMs), TFs and splicing regulators

      While our molecular understanding of embryonic liver development has markedly increased, less is known about the mechanisms behind the full acquisition of hepatic functions and their maintenance in post-natal life. Interestingly, it has become apparent that even molecular signals coming from outside the liver could be involved. Indeed, a fundamental role for the gut microbiome as a critical contributor to post-natal hepatic programming and the preservation of adult liver function is increasingly being recognised.
      • Almeida J.I.
      • Tenreiro M.F.
      • Martinez-Santamaria L.
      • Guerrero-Aspizua S.
      • Gisbert J.P.
      • Alves P.M.
      • et al.
      Hallmarks of the human intestinal microbiome on liver maturation and function.
      Nevertheless, evidence indicates that a core set of TFs interact in the adult liver, regulating each other’s expression, restricting cell proliferation and stabilising the commitment to hepatocyte identity.
      • Tachmatzidi E.C.
      • Galanopoulou O.
      • Talianidis I.
      Transcription control of liver development.
      ,
      • Berasain C.
      • Avila M.A.
      Regulation of hepatocyte identity and quiescence.
      The individual contribution of some hepatic TFs to the preservation of adult liver function has been established in the corresponding knockout mouse models. Early studies showed that hepatocyte-specific deletion of Foxa2 resulted in a relatively mild phenotype, with intrahepatic cholestasis and liver injury.
      • Bochkis I.M.
      • Rubins N.E.
      • White P.
      • Furth E.E.
      • Friedman J.R.
      • Kaestner K.H.
      Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress.
      More recently it was shown that the combined depletion of Foxa1, Foxa2 and Foxa3 in the adult mouse liver had a dramatic effect on the expression of the hepatocellular transcriptome.
      • Reizel Y.
      • Morgan A.
      • Gao L.
      • Lan Y.
      • Manduchi E.
      • Waite E.L.
      • et al.
      Collapse of the hepatic gene regulatory network in the absence of FoxA factors.
      These findings indicate that pioneer factors involved in early liver specification such as the Foxa genes also play a critical role in adult liver function. Interestingly, FOXA proteins were shown to be necessary for the binding of HNF4α to enhancers co-occupied by both factors in mouse hepatocytes.
      • Reizel Y.
      • Morgan A.
      • Gao L.
      • Lan Y.
      • Manduchi E.
      • Waite E.L.
      • et al.
      Collapse of the hepatic gene regulatory network in the absence of FoxA factors.
      Consistently, it was recently observed that FOXA2 was required for HNF4α and C/EBPα expression in adult human hepatocytes. Moreover, FOXA2 also acted as a pioneer factor in these cells, facilitating the binding of HNF4α and C/EBPα to ALB gene enhancers and driving its expression.
      • Feng R.
      • Kan K.
      • Sticht C.
      • Li Y.
      • Wang S.
      • Liu H.
      • et al.
      A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.
      Assemblies containing multiple TFs collaboratively bind tissue-specific CRMs, i.e. enhancers and promoters, to regulate cell identity genes in different tissues, including the liver.
      • Ballester B.
      • Medina-Rivera A.
      • Schmidt D.
      • Gonzàlez-Porta M.
      • Carlucci M.
      • Chen X.
      • et al.
      Multi-species, multi-transcription factor binding highlights conserved control of tissue-specific biological pathways.
      ,
      • Dubois-Chevalier J.
      • Dubois V.
      • Dehondt H.
      • Mazrooei P.
      • Mazuy C.
      • Sérandour A.A.
      • et al.
      The logic of transcriptional regulator recruitment architecture at cis-regulatory modules controlling liver functions.
      Enhancers may be found in clusters known as super-enhancers – regions of open chromatin conformation and active epigenetic marks that synergistically drive gene transcription. The presence of super-enhancer signatures has been linked to genes that define cellular identity.
      • Dubois-Chevalier J.
      • Dubois V.
      • Staels B.
      • Lefebvre P.
      • Eeckhoute J.
      Perspectives on the use of super-enhancers as a defining feature of cell/tissue-identity genes.
      In this context, the composition of the central network of hepatic TFs that cooperatively bind the CRMs of genes that maintain liver-specific functions is currently being elucidated.
      • Gérard C.
      • Tys J.
      • Lemaigre F.P.
      Gene regulatory networks in differentiation and direct reprogramming of hepatic cells.
      ,
      • Joo M.S.
      • Koo J.H.
      • Kim T.H.
      • Kim Y.S.
      • Kim S.G.
      LRH1-driven transcription factor circuitry for hepatocyte identity: super-enhancer cistromic analysis.
      So far, and besides FOXA2, this network includes other hepatic TFs such as HNF1α, HNF1β, HNF6, LRH-1, C/EBPα and HNF4α (Fig. 1B).
      • Tachmatzidi E.C.
      • Galanopoulou O.
      • Talianidis I.
      Transcription control of liver development.
      ,
      • Dubois V.
      • Staels B.
      • Lefebvre P.
      • Verzi M.P.
      • Eeckhoute J.
      Control of cell identity by the nuclear receptor HNF4 in organ pathophysiology.
      ,
      • Ballester B.
      • Medina-Rivera A.
      • Schmidt D.
      • Gonzàlez-Porta M.
      • Carlucci M.
      • Chen X.
      • et al.
      Multi-species, multi-transcription factor binding highlights conserved control of tissue-specific biological pathways.
      ,
      • Dubois-Chevalier J.
      • Dubois V.
      • Dehondt H.
      • Mazrooei P.
      • Mazuy C.
      • Sérandour A.A.
      • et al.
      The logic of transcriptional regulator recruitment architecture at cis-regulatory modules controlling liver functions.
      ,
      • Joo M.S.
      • Koo J.H.
      • Kim T.H.
      • Kim Y.S.
      • Kim S.G.
      LRH1-driven transcription factor circuitry for hepatocyte identity: super-enhancer cistromic analysis.
      Among these TFs, HNF4α has emerged as a fundamental player.
      • Kyrmizi I.
      • Hatzis P.
      • Katrakili N.
      • Tronche F.
      • Gonzalez F.J.
      • Talianidis I.
      Plasticity and expanding complexity of the hepatic transcription factor network during liver development.
      Deletion of Hnf4α in adult mouse hepatocytes resulted in loss of the epithelial polarised phenotype and an epithelial-to-mesenchymal transition, accompanied by a profound dysregulation in the expression of multiple metabolic genes and increased proliferation.
      • Hayhurst G.P.
      • Lee Y.-H.
      • Lambert G.
      • Ward J.M.
      • Gonzalez F.J.
      Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis.
      • Martinez-Jimenez C.P.
      • Kyrmizi I.
      • Cardot P.
      • Gonzalez F.J.
      • Talianidis I.
      Hepatocyte nuclear factor 4alpha coordinates a transcription factor network regulating hepatic fatty acid metabolism.
      • Santangelo L.
      • Marchetti A.
      • Cicchini C.
      • Conigliaro A.
      • Conti B.
      • Mancone C.
      • et al.
      The stable repression of mesenchymal program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4α.
      • Bonzo J.A.
      • Ferry C.H.
      • Matsubara T.
      • Kim J.H.
      • Gonzalez F.J.
      Suppression of hepatocyte proliferation by hepatocyte nuclear factor 4α in adult mice.
      • Wu H.
      • Reizel T.
      • Wang Y.J.
      • Lapiro J.L.
      • Kren B.T.
      • Schug J.
      • et al.
      A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver.
      Indeed, HNF4α binds to the CRMs of almost half of the genes expressed in the adult liver,
      • Odom D.T.
      • Zizlsperger H.
      • Gordon D.B.
      • Bell G.W.
      • Rinaldi N.J.
      • Murray H.L.
      • et al.
      Control of pancreas and liver gene expression by HNF transcription factors.
      contributing to the establishment of an active chromatin conformation at liver-characteristic genes.
      • Thakur A.
      • Wong J.C.H.
      • Wang E.Y.
      • Lotto J.
      • Kim D.
      • Cheng J.C.
      • et al.
      Hepatocyte nuclear factor 4-alpha is essential for the active epigenetic state at enhancers in mouse liver.
      ,
      • Dubois-Chevalier J.
      • Dubois V.
      • Staels B.
      • Lefebvre P.
      • Eeckhoute J.
      Perspectives on the use of super-enhancers as a defining feature of cell/tissue-identity genes.
      ,
      • Palierne G.
      • Fabre A.
      • Solinhac R.
      • Le Péron C.
      • Avner S.
      • Lenfant F.
      • et al.
      Changes in gene expression and estrogen receptor cistrome in mouse liver upon acute E2 treatment.
      Notably, HNF4α expression, and its transcriptional activity on hepatocyte-specific genes, i.e. those defining hepatocellular identity, are transiently downregulated early after partial hepatectomy (PH) in mice, permitting hepatocytes to leave quiescence and enter the cell cycle.
      • Wu H.
      • Reizel T.
      • Wang Y.J.
      • Lapiro J.L.
      • Kren B.T.
      • Schug J.
      • et al.
      A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver.
      ,
      • Alvarez-Sola G.
      • Uriarte I.
      • Latasa M.U.
      • Urtasun R.
      • Bárcena-Varela M.
      • Elizalde M.
      • et al.
      Fibroblast growth factor 15/19 in hepatocarcinogenesis.
      ,
      • Dubois V.
      • Gheeraert C.
      • Vankrunkelsven W.
      • Dubois-Chevalier J.
      • Dehondt H.
      • Bobowski-Gerard M.
      • et al.
      Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver.
      On the other hand, reintroduction of HNF4α into liver-specific Hnf4α knockout mice restored hepatocellular gene expression and quiescence after PH, which was shown to be essential for survival.
      • Huck I.
      • Gunewardena S.
      • Espanol-Suner R.
      • Willenbring H.
      • Apte U.
      Hepatocyte nuclear factor 4 alpha activation is essential for termination of liver regeneration in mice.
      Moreover, HNF4α was also found to repress the epithelial-to-mesenchymal transition regulators Snail, Slug and Hmga2, and to indirectly suppress the expression of oncogenic and inflammatory genes.
      • Santangelo L.
      • Marchetti A.
      • Cicchini C.
      • Conigliaro A.
      • Conti B.
      • Mancone C.
      • et al.
      The stable repression of mesenchymal program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4α.
      ,
      • Hatziapostolou M.
      • Polytarchou C.
      • Aggelidou E.
      • Drakaki A.
      • Poultsides G.A.
      • Jaeger S.A.
      • et al.
      An HNF4α-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis.
      • Ning B.F.
      • Ding J.
      • Liu J.
      • Yin C.
      • Xu W.P.
      • Cong W.M.
      • et al.
      Hepatocyte nuclear factor 4α-nuclear factor-κB feedback circuit modulates liver cancer progression.
      • Morimoto A.
      • Kannari M.
      • Tsuchida Y.
      • Sasaki S.
      • Saito C.
      • Matsuta T.
      • et al.
      An HNF4α-microRNA-194/192 signaling axis maintains hepatic cell function.
      Besides its involvement in liver development, Hippo signalling also plays a fundamental role in the preservation of adult hepatocellular quiescence, differentiation and metabolic zonation.
      • Russell J.O.
      • Camargo F.D.
      Hippo signalling in the liver: role in development, regeneration and disease.
      Interestingly, while Hippo-YAP signalling is activated in hepatocytes after PH, the precise role of this pathway in liver regeneration upon partial resection or acute injury needs to be better defined.
      • Russell J.O.
      • Camargo F.D.
      Hippo signalling in the liver: role in development, regeneration and disease.
      However, constitutive YAP activation triggers hepatocyte proliferation and dedifferentiation to cells expressing cholangiocyte biomarkers. NOTCH signalling was identified early as a key downstream effector of Hippo-YAP-mediated hepatocellular dedifferentiation.
      • Yimlamai D.
      • Christodoulou C.
      • Galli G.G.
      • Yanger K.
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      • Gurung B.
      • et al.
      Hippo pathway activity influences liver cell fate.
      This NOTCH-Hippo-YAP crosstalk may be implicated in the progression of chronic liver disease and carcinogenesis.
      • Zhu C.
      • Tabas I.
      • Schwabe R.F.
      • Pajvani U.B.
      Maladaptive regeneration - the reawakening of developmental pathways in NASH and fibrosis.
      ,
      • Hu S.
      • Molina L.
      • Tao J.
      • Liu S.
      • Hassan M.
      • Singh S.
      • et al.
      NOTCH-YAP1/TEAD-DNMT1 Axis drives hepatocyte reprogramming into intrahepatic cholangiocarcinoma.
      Although much less studied than the role of TFs, post-transcriptional mechanisms such as pre-mRNA alternative splicing also play a fundamental role in liver development, particularly in the post-natal transition and the preservation of adult liver identity.
      • Navi L.R. Ben
      • Tsukerman A.
      • Feldman A.
      • Melamed P.
      • Tomic M.
      • Stojilkovic S.S.
      • et al.
      Alternative RNA splicing in the pathogenesis of liver disease.
      The sequential replacement of foetal-to-adult mRNA isoforms is essential for the functional maturation of hepatocytes, as the protein variants resulting from these isoforms can have very different characteristics, including subcellular localisation, kinetic properties and biological functions.
      • Berasain C.
      • Goñi S.
      • Castillo J.
      • Latasa M.U.
      • Prieto J.
      • Ávila M.A.
      Impairment of pre-mRNA splicing in liver disease: mechanisms and consequences.
      Substantial changes in alternatively spliced variants, and the expression levels of splicing factors, have been described during the post-natal transition in mouse and human livers (Fig. 1B).
      • Navi L.R. Ben
      • Tsukerman A.
      • Feldman A.
      • Melamed P.
      • Tomic M.
      • Stojilkovic S.S.
      • et al.
      Alternative RNA splicing in the pathogenesis of liver disease.
      ,
      • Bhate A.
      • Parker D.J.
      • Bebee T.W.
      • Ahn J.
      • Arif W.
      • Rashan E.H.
      • et al.
      ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation.
      Among the best studied is epithelial splicing regulatory protein 2 (ESRP2). ESRP2 expression is markedly upregulated during foetal to post-natal maturation and is thought to control up to 20% of the splice isoform transitions in this period.
      • Bhate A.
      • Parker D.J.
      • Bebee T.W.
      • Ahn J.
      • Arif W.
      • Rashan E.H.
      • et al.
      ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation.
      Its deletion results in hepatocellular proliferation, loss of parenchymal zonation, persistent expression of foetal markers and impaired liver-specific gene expression.
      • Bhate A.
      • Parker D.J.
      • Bebee T.W.
      • Ahn J.
      • Arif W.
      • Rashan E.H.
      • et al.
      ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation.
      Hepatocyte-specific knockout of other splicing regulators, such as serine and arginine-rich splicing factor (SRSF)2 and SRSF3. also results in dedifferentiation, loss of hepatic metabolic functions and reduced expression of key liver-enriched TFs.
      • Cheng Y.
      • Luo C.
      • Wu W.
      • Xie Z.
      • Fu X.
      • Feng Y.
      Liver-specific deletion of SRSF2 caused acute liver failure and early death in mice.
      ,
      • Sen S.
      • Jumaa H.
      • Webster N.J.G.
      Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.
      Another splicing factor involved in the preservation of adult hepatocellular differentiation and quiescence is SLU7. As we reported, its downregulation in adult mouse liver causes a loss in liver metabolic functions accompanied by hepatocellular proliferation and the reactivation of a foetal gene expression pattern.
      • Elizalde M.
      • Urtasun R.
      • Azkona M.
      • Latasa M.U.
      • Goñi S.
      • García-Irigoyen O.
      • et al.
      Splicing regulator SLU7 is essential for maintaining liver homeostasis.
      Interestingly, we found that SLU7 regulates the splicing of SRSF3, preventing the generation of truncated dominant-negative isoforms of this splicing factor.
      • Elizalde M.
      • Urtasun R.
      • Azkona M.
      • Latasa M.U.
      • Goñi S.
      • García-Irigoyen O.
      • et al.
      Splicing regulator SLU7 is essential for maintaining liver homeostasis.
      ,
      • Jiménez M.
      • Urtasun R.
      • Elizalde M.
      • Azkona M.
      • Latasa M.U.
      • Uriarte I.
      • et al.
      Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins.
      On the other hand, loss of SRSF3 expression is known to result in a marked downregulation of ESRP2 expression.
      • Sen S.
      • Jumaa H.
      • Webster N.J.G.
      Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.
      Together, these observations highlight the existence of a hierarchical and intricate network of splicing regulators involved in the preservation of the adult liver phenotype.

      Loss of hepatic function in liver disease

      Deterioration and loss of hepatic functions is a hallmark of severe acute injury and chronic damage to the organ.
      • Haj M.
      • Rockey D.C.
      Predictors of clinical outcomes in cirrhosis patients.
      ,
      • Lemmer P.
      • Pospiech J.C.
      • Canbay A.
      Liver failure-future challenges and remaining questions.
      In the clinic, the scores most commonly used to predict patient outcomes, the Child-Turcotte-Pugh and model of end-stage liver disease (MELD) scores, include the serum levels and the activity of proteins produced by hepatocytes such as albumin and coagulation factors, as well as bilirubin concentration, which are indicative of liver biosynthetic and secretory functions.
      • Haj M.
      • Rockey D.C.
      Predictors of clinical outcomes in cirrhosis patients.
      Severity of liver disease, independently from the causative agent, can be estimated with these biochemical tools which, by definition, closely reflect overall liver functional capacity. Hepatocyte death, which can be massive in the context of acute liver injury, undoubtedly contributes to the failure of liver functions.
      • Engelmann C.
      • Clària J.
      • Szabo G.
      • Bosch J.
      • Bernardi M.
      Pathophysiology of decompensated cirrhosis: portal hypertension, circulatory dysfunction, inflammation, metabolism and mitochondrial dysfunction.
      ,
      • Lin T.
      • Feng R.
      • Liebe R.
      • Weng H.L.
      Liver progenitor cells in massive hepatic necrosis-how can a patient survive acute liver failure?.
      However, as mentioned, it is now believed that liver dysfunction cannot be solely ascribed to hepatocytes’ demise and the substitution of parenchyma by fibrous tissue. In a context of impaired perfusion, hypoxia, inflammation, oxidative and endoplasmic reticulum (ER) stress, and under abundant mitogenic signals, the surviving hepatocytes undergo profound coping adaptations, changing their metabolic, bioenergetic and quiescent balance.
      • Engelmann C.
      • Clària J.
      • Szabo G.
      • Bosch J.
      • Bernardi M.
      Pathophysiology of decompensated cirrhosis: portal hypertension, circulatory dysfunction, inflammation, metabolism and mitochondrial dysfunction.
      ,
      • Berasain C.
      • Avila M.A.
      Regulation of hepatocyte identity and quiescence.
      ,
      • Rutkowski D.T.
      Liver function and dysfunction - a unique window into the physiological reach of ER stress and the unfolded protein response.
      ,
      • Han H.
      • Desert R.
      • Das S.
      • Song Z.
      • Athavale D.
      • Ge X.
      • et al.
      Danger signals in liver injury and restoration of homeostasis.
      Indeed, more than two decades ago, we described a marked reduction in the expression of genes involved in methionine, homocysteine and one carbon metabolism, which correlated with disease severity, in the livers of individuals with cirrhosis.
      • Avila M.A.
      • Berasain C.
      • Torres L.
      • Martín-Duce A.
      • Corrales F.J.
      • Yang H.
      • et al.
      Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma.
      The liver plays a unique role in systemic one carbon metabolism, and these findings helped to explain the hypermethioninemia and hyperhomocysteinemia commonly observed in individuals with cirrhosis.
      • Lu S.C.
      • Mato J.M.
      S-adenosylmethionine in liver health, injury, and cancer.
      Moreover, downregulation of methionine-adenosyltransferase 1A, a liver-enriched gene responsible for the synthesis of S-adenosylmethionine, in human and experimental cirrhosis was accompanied by the hypermethylation of its gene promoter.
      • Avila M.A.
      • Berasain C.
      • Torres L.
      • Martín-Duce A.
      • Corrales F.J.
      • Yang H.
      • et al.
      Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma.
      ,
      • Torres L.
      • Ávila MatA.
      • Carretero M.V.
      • Latasa M.U.
      • Caballería J.
      • López-Rodas G.
      • et al.
      Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation.
      Since these original observations, the gradual decrease in the expression of hepato-specific genes in association with liver disease progression has been repeatedly confirmed in experimental and clinical studies.
      • Dubois V.
      • Gheeraert C.
      • Vankrunkelsven W.
      • Dubois-Chevalier J.
      • Dehondt H.
      • Bobowski-Gerard M.
      • et al.
      Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver.
      ,
      • Mirpuri E.
      • García-Trevijano E.R.
      • Castilla-Cortazar I.
      • Berasain C.
      • Quiroga J.
      • Rodriguez-Ortigosa C.
      • et al.
      Altered liver gene expression in CCl4-cirrhotic rats is partially normalized by insulin-like growth factor-I.
      • Liu L.
      • Yannam G.R.
      • Nishikawa T.
      • Yamamoto T.
      • Basma H.
      • Ito R.
      • et al.
      The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis.
      • Nishikawa T.
      • Bell A.
      • Brooks J.M.
      • Setoyama K.
      • Melis M.
      • Han B.
      • et al.
      Resetting the transcription factor network reverses terminal chronic hepatic failure.
      • Guzman-Lepe J.
      • Cervantes-Alvarez E.
      • Collin de l’Hortet A.
      • Wang Y.
      • Mars W.M.
      • Oda Y.
      • et al.
      Liver-enriched transcription factor expression relates to chronic hepatic failure in humans.
      • Argemi J.
      • Latasa M.U.
      • Atkinson S.R.
      • Blokhin I.O.
      • Massey V.
      • Gue J.P.
      • et al.
      Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.
      • Loft A.
      • Alfaro A.J.
      • Schmidt S.F.
      • Pedersen F.B.
      • Terkelsen M.K.
      • Puglia M.
      • et al.
      Liver-fibrosis-activated transcriptional networks govern hepatocyte reprogramming and intra-hepatic communication.
      • Guldiken N.
      • Argemi J.
      • Gurbuz B.
      • Atkinson S.R.
      • Oliverius M.
      • Fila P.
      • et al.
      Serum transferrin as a biomarker of hepatocyte nuclear factor 4 alpha activity and hepatocyte function in liver diseases.
      • Bou Saleh M.
      • Louvet A.
      • Ntandja-Wandji L.C.
      • Boleslawski E.
      • Gnemmi V.
      • Lassailly G.
      • et al.
      Loss of hepatocyte identity following aberrant YAP activation: a key mechanism in alcoholic hepatitis.
      These findings suggest that active reprogramming of the hepatocellular transcriptome during chronic liver injury leads to the loss of hepatocyte identity. Moreover, as mentioned, reduced expression of hepato-specific genes is accompanied by the reactivation of foetal isoforms, further contributing to the abandonment of fundamental metabolic functions by the injured liver.
      • Nishikawa T.
      • Bell A.
      • Brooks J.M.
      • Setoyama K.
      • Melis M.
      • Han B.
      • et al.
      Resetting the transcription factor network reverses terminal chronic hepatic failure.
      ,
      • Argemi J.
      • Latasa M.U.
      • Atkinson S.R.
      • Blokhin I.O.
      • Massey V.
      • Gue J.P.
      • et al.
      Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.
      ,
      • Berasain C.
      • Herrero J.I.
      • García-Trevijano E.R.
      • Avila M.A.
      • Esteban J.I.
      • Mato J.M.
      • et al.
      Expression of Wilms’ tumor suppressor in the liver with cirrhosis: relation to hepatocyte nuclear factor 4 and hepatocellular function.
      ,
      • Hyun J.
      • Oh S.H.
      • Premont R.T.
      • Guy C.D.
      • Berg C.L.
      • Diehl A.M.
      Dysregulated activation of fetal liver programme in acute liver failure.
      The loss of liver-enriched and zonated gene expression, and the upregulation of foetal-specific genes, have recently been confirmed by single-cell RNA-sequencing analyses of hepatocytes isolated from experimental models of liver injury and regeneration, as well as in clinical samples from individuals with cirrhosis or alcoholic hepatitis.
      • Loft A.
      • Alfaro A.J.
      • Schmidt S.F.
      • Pedersen F.B.
      • Terkelsen M.K.
      • Puglia M.
      • et al.
      Liver-fibrosis-activated transcriptional networks govern hepatocyte reprogramming and intra-hepatic communication.
      ,
      • Pepe-Mooney B.J.
      • Dill M.T.
      • Alemany A.
      • Ordovas-Montanes J.
      • Matsushita Y.
      • Rao A.
      • et al.
      Single-cell analysis of the liver epithelium reveals dynamic heterogeneity and an essential role for YAP in homeostasis and regeneration.
      • Ben-Moshe S.
      • Veg T.
      • Manco R.
      • Dan S.
      • Papinutti D.
      • Lifshitz A.
      • et al.
      The spatiotemporal program of zonal liver regeneration following acute injury.
      • Kim A.
      • Wu X.
      • Allende D.S.
      • Nagy L.E.
      Gene deconvolution reveals aberrant liver regeneration and immune cell infiltration in alcohol-associated hepatitis.
      • He Z.
      • Peng C.
      • Li T.
      • Li J.
      Cell differentiation trajectory in liver cirrhosis predicts hepatocellular carcinoma prognosis and reveals potential biomarkers for progression of liver cirrhosis to hepatocellular carcinoma.
      Importantly, the reactivation of foetal hepatic genes and foetal isoforms is not only implicated in liver dysfunction but may also herald the development of cancer. For instance, a recent study in individuals with chronic liver disease, including more than 8,200 people, reported that elevated serum levels of alpha-fetoprotein, encoded by a liver oncofoetal gene,
      • Galle P.R.
      • Foerster F.
      • Kudo M.
      • Chan S.L.
      • Llovet J.M.
      • Qin S.
      • et al.
      Biology and significance of alpha-fetoprotein in hepatocellular carcinoma.
      can be observed more than 10 years before HCC detection.
      • Hughes D.M.
      • Berhane S.
      • Emily de Groot C.A.
      • Toyoda H.
      • Tada T.
      • Kumada T.
      • et al.
      Serum levels of α-fetoprotein increased more than 10 Years before detection of hepatocellular carcinoma.
      As summarised in the previous section, the preservation of hepatocellular phenotype relies on a complex network of interconnected TFs interacting with the CRMs of genes that define the liver identity. Although this complexity provides stability, pathophysiological changes in expression and/or activity of key TFs are likely to impact on the hepatic transcriptional equilibrium. Early reports showed marked downregulation of FOXA2 and C/EBPβ expression in experimental cirrhosis.
      • Mirpuri E.
      • García-Trevijano E.R.
      • Castilla-Cortazar I.
      • Berasain C.
      • Quiroga J.
      • Rodriguez-Ortigosa C.
      • et al.
      Altered liver gene expression in CCl4-cirrhotic rats is partially normalized by insulin-like growth factor-I.
      Reduced FOXA2 expression was subsequently found in the livers of individuals with fibrosis and cirrhosis,
      • Wang W.
      • Yao L.J.
      • Shen W.
      • Ding K.
      • Shi P.M.
      • Chen F.
      • et al.
      FOXA2 alleviates CCl 4-induced liver fibrosis by protecting hepatocytes in mice.
      as well as cholestatic syndromes, with FOXA2 downregulation contributing to disease progression.
      • Bochkis I.M.
      • Rubins N.E.
      • White P.
      • Furth E.E.
      • Friedman J.R.
      • Kaestner K.H.
      Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress.
      ,
      • McDaniel K.
      • Meng F.
      • Wu N.
      • Sato K.
      • Venter J.
      • Bernuzzi F.
      • et al.
      Forkhead box A2 regulates biliary heterogeneity and senescence during cholestatic liver injury in mice.
      Conversely, more recent studies did not find lower FOXA2 gene expression in cirrhotic human livers of various non-cholestatic aetiologies.
      • Feng R.
      • Kan K.
      • Sticht C.
      • Li Y.
      • Wang S.
      • Liu H.
      • et al.
      A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.
      ,
      • Guzman-Lepe J.
      • Cervantes-Alvarez E.
      • Collin de l’Hortet A.
      • Wang Y.
      • Mars W.M.
      • Oda Y.
      • et al.
      Liver-enriched transcription factor expression relates to chronic hepatic failure in humans.
      However, FOXA2 was shown to be essential for the preservation of ALB gene expression in decompensated cirrhosis,
      • Feng R.
      • Kan K.
      • Sticht C.
      • Li Y.
      • Wang S.
      • Liu H.
      • et al.
      A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.
      as well as for bilirubin excretion and albumin production in conditions of acute liver failure.
      • Feng R.
      • Kan K.
      • Sticht C.
      • Li Y.
      • Wang S.
      • Liu H.
      • et al.
      A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.
      ,
      • Wang S.
      • Feng R.
      • Wang S.S.
      • Liu H.
      • Shao C.
      • Li Y.
      • et al.
      FOXA2 prevents hyperbilirubinaemia in acute liver failure by maintaining apical MRP2 expression.
      The hepatic expression of FOXA1 and FOXA3 is also lower in experimental and human chronic liver injury,
      • Moya M.
      • Benet M.
      • Guzmán C.
      • Tolosa L.
      • García-Monzón C.
      • Pareja E.
      • et al.
      Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver.
      ,
      • Dong R.
      • Yang Y.
      • Shen Z.
      • Zheng C.
      • Jin Z.
      • Huang Y.
      • et al.
      Forkhead box A3 attenuated the progression of fibrosis in a rat model of biliary atresia.
      as is C/EBPα in individuals with acute and chronic liver damage, including severe alcoholic hepatitis.
      • Joo M.S.
      • Koo J.H.
      • Kim T.H.
      • Kim Y.S.
      • Kim S.G.
      LRH1-driven transcription factor circuitry for hepatocyte identity: super-enhancer cistromic analysis.
      ,
      • Hyun J.
      • Sun Z.
      • Ahmadi A.R.
      • Bangru S.
      • Chembazhi U.V.
      • Du K.
      • et al.
      Epithelial splicing regulatory protein 2-mediated alternative splicing reprograms hepatocytes in severe alcoholic hepatitis.
      Among the core TFs investigated for their involvement in liver failure, the hepatic expression of HNF1α is reduced in experimental cirrhosis and hepatocarcinogenesis, as well as in human cirrhotic and tumoral liver tissues.
      • Joo M.S.
      • Koo J.H.
      • Kim T.H.
      • Kim Y.S.
      • Kim S.G.
      LRH1-driven transcription factor circuitry for hepatocyte identity: super-enhancer cistromic analysis.
      ,
      • Lazarevich N.L.
      • Cheremnova O.A.
      • Varga E.V.
      • Ovchinnikov D.A.
      • Kudrjavtseva E.I.
      • Morozova O.V.
      • et al.
      Progression of HCC in mice is associated with a downregulation in the expression of hepatocyte nuclear factors.
      • Hellerbrand C.
      • Amann T.
      • Schlegel J.
      • Wild P.
      • Bataille F.
      • Spruss T.
      • et al.
      The novel gene MIA2 acts as a tumour suppressor in hepatocellular carcinoma.
      • Zeng X.
      • Lin Y.
      • Yin C.
      • Zhang X.
      • Ning B.F.
      • Zhang Q.
      • et al.
      Recombinant adenovirus carrying the hepatocyte nuclear factor-1alpha gene inhibits hepatocellular carcinoma xenograft growth in mice.
      • Teeli A.S.
      • Łuczyńska K.
      • Haque E.
      • Gayas M.A.
      • Winiarczyk D.
      • Taniguchi H.
      Disruption of tumor suppressors HNF4α/HNF1α causes tumorigenesis in liver.
      The impairment of HNF4α has attracted special attention. In 2003, we reported that HNF4α expression was markedly downregulated in human livers with advanced cirrhosis, inversely correlating with the MELD score.
      • Berasain C.
      • Herrero J.I.
      • García-Trevijano E.R.
      • Avila M.A.
      • Esteban J.I.
      • Mato J.M.
      • et al.
      Expression of Wilms’ tumor suppressor in the liver with cirrhosis: relation to hepatocyte nuclear factor 4 and hepatocellular function.
      Interestingly, in that early study we also described how TGFβ downregulated HNF4α levels in hepatocytes through the induction of the transcription factor WT1 (Wilm’s tumour 1), a foetal gene reactivated during hepatocellular dedifferentiation in human cirrhosis.
      • Berasain C.
      • Herrero J.I.
      • García-Trevijano E.R.
      • Avila M.A.
      • Esteban J.I.
      • Mato J.M.
      • et al.
      Expression of Wilms’ tumor suppressor in the liver with cirrhosis: relation to hepatocyte nuclear factor 4 and hepatocellular function.
      ,
      • Perugorria M.J.
      • Castillo J.
      • Latasa M.U.
      • Goñi S.
      • Segura V.
      • Sangro B.
      • et al.
      Wilms’ tumor 1 gene expression in hepatocellular carcinoma promotes cell dedifferentiation and resistance to chemotherapy.
      Since then, a plethora of studies have confirmed and extended these original findings, describing the impairment of HNF4α expression and regulatory activity in human chronic liver disease of viral, alcoholic or metabolic origin,
      • Feng R.
      • Kan K.
      • Sticht C.
      • Li Y.
      • Wang S.
      • Liu H.
      • et al.
      A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.
      ,
      • Joo M.S.
      • Koo J.H.
      • Kim T.H.
      • Kim Y.S.
      • Kim S.G.
      LRH1-driven transcription factor circuitry for hepatocyte identity: super-enhancer cistromic analysis.
      ,
      • Guzman-Lepe J.
      • Cervantes-Alvarez E.
      • Collin de l’Hortet A.
      • Wang Y.
      • Mars W.M.
      • Oda Y.
      • et al.
      Liver-enriched transcription factor expression relates to chronic hepatic failure in humans.
      ,
      • Argemi J.
      • Latasa M.U.
      • Atkinson S.R.
      • Blokhin I.O.
      • Massey V.
      • Gue J.P.
      • et al.
      Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.
      ,
      • Xu Y.
      • Zalzala M.
      • Xu J.
      • Li Y.
      • Yin L.
      • Zhang Y.
      A metabolic stress-inducible miR-34a-HNF4α pathway regulates lipid and lipoprotein metabolism.
      • Xu J.
      • Xu Y.
      • Li Y.
      • Jadhav K.
      • You M.
      • Yin L.
      • et al.
      Carboxylesterase 1 is regulated by hepatocyte nuclear factor 4α and protects against alcohol- and MCD diet-induced liver injury.
      • Sahoo S.
      • Singh D.
      • Chakraborty P.
      • Jolly M.K.
      Emergent properties of the HNF4α-PPARγ network may drive consequent phenotypic plasticity in NAFLD.
      as well as in acute liver failure.
      • Joo M.S.
      • Koo J.H.
      • Kim T.H.
      • Kim Y.S.
      • Kim S.G.
      LRH1-driven transcription factor circuitry for hepatocyte identity: super-enhancer cistromic analysis.
      ,
      • Dubois V.
      • Gheeraert C.
      • Vankrunkelsven W.
      • Dubois-Chevalier J.
      • Dehondt H.
      • Bobowski-Gerard M.
      • et al.
      Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver.
      ,
      • Lin T.
      • Wang S.
      • Munker S.
      • Jung K.
      • Macías-Rodríguez R.U.
      • Ruiz-Margáin A.
      • et al.
      Follistatin-controlled activin-HNF4α-coagulation factor axis in liver progenitor cells determines outcome of acute liver failure.
      ,
      • Gárate-Rascón M.
      • Recalde M.
      • Jimenez M.
      • Elizalde M.
      • Azkona M.
      • Uriarte I.
      • et al.
      Splicing factor SLU7 prevents oxidative stress-mediated hepatocyte nuclear factor 4α degradation, preserving hepatic differentiation and protecting from liver damage.
      Complementary studies in human tissues and experimental models also identified changes in the ratio of HNF4α P1- and P2-derived isoforms and in the subcellular localisation of HNF4α.
      • Yeh M.M.
      • Bosch D.E.
      • Daoud S.S.
      Role of hepatocyte nuclear factor 4-alpha in gastrointestinal and liver diseases.
      Upregulation of HNF4α P2 variants may have important functional consequences. These isoforms – normally expressed in the foetal liver – are induced in HCC,
      • Tanaka T.
      • Jiang S.
      • Hotta H.
      • Takano K.
      • Iwanari H.
      • Sumi K.
      • et al.
      Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.
      and display significant differences with P1-derived variants in their specificity and the potency with which they drive gene expression.
      • Nakhei H.
      • Lingott A.
      • Lemm I.
      • Ryffel G.U.
      An alternative splice variant of the tissue specific transcription factor HNF4alpha predominates in undifferentiated murine cell types.
      • Torres-Padilla M.E.
      • Fougère-Deschatrette C.
      • Weiss M.C.
      Expression of HNF4alpha isoforms in mouse liver development is regulated by sequential promoter usage and constitutive 3’ end splicing.
      • Briançon N.
      • Weiss M.C.
      In vivo role of the HNF4alpha AF-1 activation domain revealed by exon swapping.
      Cellular and in vivo models have helped to unravel the mechanisms involved in HNF4α dysregulation in liver injury, and to understand its central role in hepatoprotection. Early works reported that mitogen-activated protein kinase activation downregulates HNF4α expression in cultured human liver cells (HepG2).
      • Hatzis P.
      • Kyrmizi I.
      • Talianidis I.
      Mitogen-activated protein kinase-mediated disruption of enhancer-promoter communication inhibits hepatocyte nuclear factor 4alpha expression.
      Later on, we reported that epidermal growth factor receptor activation decreases HNF4α-P1 protein levels, potentiating the effect of TGFβ, and that TGFβ stimulated the expression of P2-derived HNF4α isoforms in human liver cell lines via c-Src-dependent signalling.
      • Argemi J.
      • Latasa M.U.
      • Atkinson S.R.
      • Blokhin I.O.
      • Massey V.
      • Gue J.P.
      • et al.
      Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.
      Furthermore, we demonstrated that these effects on HNF4α isoforms significantly influenced hepatocellular gene expression and function, and that P2–HNF4α variants were markedly upregulated in individuals with alcoholic hepatitis.
      • Argemi J.
      • Latasa M.U.
      • Atkinson S.R.
      • Blokhin I.O.
      • Massey V.
      • Gue J.P.
      • et al.
      Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.
      These findings, together with the recently identified key role for HNF4α in the transcriptional control of hepatic sulfur amino acid metabolism,
      • Xu Q.
      • Li Y.
      • Gao X.
      • Kang K.
      • Williams J.G.
      • Tong L.
      • et al.
      HNF4α regulates sulfur amino acid metabolism and confers sensitivity to methionine restriction in liver cancer.
      contribute to our understanding of the aforementioned impairment of methionine and homocysteine metabolism in chronic liver injury, including alcohol-related liver disease.
      • Avila M.A.
      • Berasain C.
      • Torres L.
      • Martín-Duce A.
      • Corrales F.J.
      • Yang H.
      • et al.
      Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma.
      ,
      • Lu S.C.
      • Mato J.M.
      S-adenosylmethionine in liver health, injury, and cancer.
      Interestingly, mislocalisation of HNF4α to the cytoplasm was recently observed in hepatocytes from individuals with advanced cirrhosis.
      • Florentino R.M.
      • Fraunhoffer N.A.
      • Morita K.
      • Takeishi K.
      • Ostrowska A.
      • Achreja A.
      • et al.
      Cellular location of HNF4α is linked with terminal liver failure in humans.
      Alterations in HNF4α acetylation and phosphorylation accompanied this increased cytosolic retention of HNF4α, which correlated with hepatocyte dysfunction.
      • Florentino R.M.
      • Fraunhoffer N.A.
      • Morita K.
      • Takeishi K.
      • Ostrowska A.
      • Achreja A.
      • et al.
      Cellular location of HNF4α is linked with terminal liver failure in humans.
      In this context, oxidative stress associated with fatty liver disease was recently reported to induce cytoplasmic retention of HNF4α upon protein kinase C-mediated phosphorylation.
      • Yu D.
      • Chen G.
      • Pan M.
      • Zhang J.
      • He W.
      • Liu Y.
      • et al.
      High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.
      Moreover, increased extracellular matrix stiffness can, via Rho/ROCK pathway signalling or YAP pathway activation,
      • Noce V.
      • Battistelli C.
      • Cozzolino A.M.
      • Consalvi V.
      • Cicchini C.
      • Strippoli R.
      • et al.
      YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation.
      lead to impaired HNF4α expression.
      • Desai S.S.
      • Tung J.C.
      • Zhou V.X.
      • Grenert J.P.
      • Malato Y.
      • Rezvani M.
      • et al.
      Physiological ranges of matrix rigidity modulate primary mouse hepatocyte function in part through hepatocyte nuclear factor 4 alpha.
      Therefore, environmental cues triggered during liver injury can indeed drive hepatocellular dedifferentiation. These signals include cytokines, chemokines, growth factors and extracellular matrix proteins, and are mostly produced by inflammatory cells and other non-parenchymal cells, such as activated stellate cells and sinusoidal endothelial cells.
      • Zhu C.
      • Tabas I.
      • Schwabe R.F.
      • Pajvani U.B.
      Maladaptive regeneration - the reawakening of developmental pathways in NASH and fibrosis.
      ,
      • Filliol A.
      • Schwabe R.F.
      Contributions of fibroblasts, extracellular matrix, stiffness, and mechanosensing to hepatocarcinogenesis.
      • Gibert-ramos A.
      • Sanfeliu-redondo D.
      • Aristu-zabalza P.
      • Martínez-alcocer A.
      • Gracia-sancho J.
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      • et al.
      The hepatic sinusoid in chronic liver disease: the optimal milieu for cancer.
      • Peiseler M.
      • Schwabe R.
      • Hampe J.
      • Kubes P.
      • Heikenwälder M.
      • Tacke F.
      Immune mechanisms linking metabolic injury to inflammation and fibrosis in fatty liver disease - novel insights into cellular communication circuits.
      Biliary epithelial cells, which are quiescent under physiological conditions, also undergo profound modifications during liver injury, adopting a proliferative, secretory and ultimately senescent phenotype. Activated cholangiocytes are a rich source of chemokines and cytokines, like tumour necrosis factor-α, interleukin 6 and TGFβ, which can have autocrine effects but may also act in a paracrine manner on other liver cells, including hepatocytes.
      • Pinto C.
      • Giordano D.M.
      • Maroni L.
      • Marzioni M.
      Role of inflammation and proinflammatory cytokines in cholangiocyte pathophysiology.
      ,
      • Molina L.
      • Nejak-Bowen K.
      • Monga S.P.
      Role of YAP1 signaling in biliary development, repair, and disease.
      Nevertheless, endogenous mechanisms are also involved. This is exemplified by the fundamental role of FOXA2 in the preservation of HNF4α expression, as recently described in human hepatocytes.
      • Feng R.
      • Kan K.
      • Sticht C.
      • Li Y.
      • Wang S.
      • Liu H.
      • et al.
      A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.
      We also demonstrated how HNF4α protein stability can be critically regulated by a novel antioxidant role of SLU7 in hepatocytes.
      • Gárate-Rascón M.
      • Recalde M.
      • Jimenez M.
      • Elizalde M.
      • Azkona M.
      • Uriarte I.
      • et al.
      Splicing factor SLU7 prevents oxidative stress-mediated hepatocyte nuclear factor 4α degradation, preserving hepatic differentiation and protecting from liver damage.
      Consequently, the downregulation of SLU7 in mouse livers results in reduced expression of P1 promoter HNF4α isoforms, and enhanced P2 promoter activity, a response exacerbated by chronic liver injury.
      • Elizalde M.
      • Urtasun R.
      • Azkona M.
      • Latasa M.U.
      • Goñi S.
      • García-Irigoyen O.
      • et al.
      Splicing regulator SLU7 is essential for maintaining liver homeostasis.
      ,
      • Gárate-Rascón M.
      • Recalde M.
      • Jimenez M.
      • Elizalde M.
      • Azkona M.
      • Uriarte I.
      • et al.
      Splicing factor SLU7 prevents oxidative stress-mediated hepatocyte nuclear factor 4α degradation, preserving hepatic differentiation and protecting from liver damage.
      In spite of being mostly gathered in pathophysiological contexts, these data indicate that hepatic HNF4α expression and activity are subjected to very precise regulation. This multifarious control may underpin the natural response of the liver to acute injury and inflammation, during which a transient loss of hepatocellular differentiation appears to be necessary to enable the activation of genes involved in stress control and cell cycle progression.
      • Dubois V.
      • Staels B.
      • Lefebvre P.
      • Verzi M.P.
      • Eeckhoute J.
      Control of cell identity by the nuclear receptor HNF4 in organ pathophysiology.
      ,
      • Wang A.W.
      • Wang Y.J.
      • Zahm A.M.
      • Morgan A.R.
      • Wangensteen K.J.
      • Kaestner K.H.
      The dynamic chromatin architecture of the regenerating liver.
      • Arechederra M.
      • Berasain C.
      • Avila M.A.
      • Fernández-Barrena M.G.
      Chromatin dynamics during liver regeneration.
      • Porukala M.
      • Vinod P.K.
      Systems-level analysis of transcriptome reorganization during liver regeneration.
      Interestingly, hepatocellular plasticity during regeneration also includes the activation of genes and splice variants characteristic of foetal, immature and transformed hepatocytes;
      • Hyun J.
      • Oh S.H.
      • Premont R.T.
      • Guy C.D.
      • Berg C.L.
      • Diehl A.M.
      Dysregulated activation of fetal liver programme in acute liver failure.
      ,
      • Bangru S.
      • Arif W.
      • Seimetz J.
      • Bhate A.
      • Chen J.
      • Rashan E.H.
      • et al.
      Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration.
      ; interference with the activation of this oncofoetal gene expression programme hampers liver regeneration.
      • Bangru S.
      • Arif W.
      • Seimetz J.
      • Bhate A.
      • Chen J.
      • Rashan E.H.
      • et al.
      Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration.
      As previously alluded to, reduced HNF4α expression occurs upon acute liver injury and ER stress,
      • Dubois V.
      • Gheeraert C.
      • Vankrunkelsven W.
      • Dubois-Chevalier J.
      • Dehondt H.
      • Bobowski-Gerard M.
      • et al.
      Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver.
      and a transient repression of HNF4α levels is also observed after PH in mice,
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      Dysregulation of the transcriptional coactivator YAP, and its paralogue transcriptional coactivator with PDZ-binding motif (TAZ), the major effectors of the Hippo signalling pathway, has emerged as a key event in multiple aspects of chronic liver disease.
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      YAP activation in the liver parenchyma has been observed in individuals with acute liver failure
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      • et al.
      Dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes hepatobiliary carcinogenesis in non-alcoholic fatty liver disease.
      Moreover, YAP activation is frequently observed in experimental hepatocarcinogenesis models and in early dysplastic liver nodules in humans.
      • Russell J.O.
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      Hippo signalling in the liver: role in development, regeneration and disease.
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      As previously discussed, YAP activity has profound effects on the hepatocyte transcriptome, promoting hepatocellular dedifferentiation and gain of progenitor cell features through the onset of an oncofoetal gene expression programme.
      • Hyun J.
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      Yap-Sox9 signaling determines hepatocyte plasticity and lineage-specific hepatocarcinogenesis.
      Mechanistically, Hippo-YAP signalling can affect HNF4α, FOXA2 and FOXA1 expression and transcriptional activity, thus influencing hepatocellular differentiation.
      • Russell J.O.
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      Hippo signalling in the liver: role in development, regeneration and disease.
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      Overexpression of YAP in the adult mouse liver significantly reduces HNF4α and FOXA2 occupancy in enhancers of adult liver-specific genes, decreasing their expression while promoting the induction of oncofoetal genes.
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      Notably, HNF4α in turn is able to negatively control YAP expression and activity, and thereby actively repress hepatocellular dedifferentiation.
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      Yes-associated protein/TEA domain family member and hepatocyte nuclear factor 4-alpha (HNF4α) repress reciprocally to regulate hepatocarcinogenesis in rats and mice.
      Nevertheless, YAP has been shown to drive liver growth, dedifferentiation and neoplastic conversion through multiple mechanisms,
      • Russell J.O.
      • Camargo F.D.
      Hippo signalling in the liver: role in development, regeneration and disease.
      including epigenetic remodelling, as recently described.
      • Wu B.-K.
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      • Chen E.H.
      • Zheng Y.
      • Pan D.
      YAP induces an oncogenic transcriptional program through TET1-mediated epigenetic remodeling in liver growth and tumorigenesis.
      Hippo-YAP signalling is modulated by a wide array of extracellular and intracellular inputs, many of which are still to be fully characterised.
      • Russell J.O.
      • Camargo F.D.
      Hippo signalling in the liver: role in development, regeneration and disease.
      Notably, the previously mentioned splicing regulator ESRP2, a guardian of the mature liver transcriptome, exerts tight control over developmentally regulated exons in major components of the Hippo pathway, including YAP.
      • Bhate A.
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      • et al.
      ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation.
      ESRP2 introduces protein segments characteristic of adult Hippo signalling components, while immature isoforms enabling higher YAP transcriptional activity are favoured in the absence of ESRP2.
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      Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration.
      Interestingly, the expression of ESRP2 is transiently reduced during experimental liver injury, which would contribute to Hippo-YAP activation and the onset of liver regeneration.
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      • Rashan E.H.
      • et al.
      Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration.
      However, persistent ESRP2 downregulation, as observed in chronic human liver disease in association with a strong pro-inflammatory microenvironment,
      • Hyun J.
      • Sun Z.
      • Ahmadi A.R.
      • Bangru S.
      • Chembazhi U.V.
      • Du K.
      • et al.
      Epithelial splicing regulatory protein 2-mediated alternative splicing reprograms hepatocytes in severe alcoholic hepatitis.
      ,
      • Hyun J.
      • Al Abo M.
      • Dutta R.K.
      • Oh S.H.
      • Xiang K.
      • Zhou X.
      • et al.
      Dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes hepatobiliary carcinogenesis in non-alcoholic fatty liver disease.
      results in an overall loss of adult isoforms and the reinduction of foetal splice variants, including those driving YAP signalling. Therefore, impairment of ESRP2 gene expression may also be a weighty determinant in the abandonment of liver functions (Fig. 2). This could be particularly relevant in individuals with alcohol-related liver disease, in whom hepatocellular ESRP2 levels are markedly reduced
      • Hyun J.
      • Sun Z.
      • Ahmadi A.R.
      • Bangru S.
      • Chembazhi U.V.
      • Du K.
      • et al.
      Epithelial splicing regulatory protein 2-mediated alternative splicing reprograms hepatocytes in severe alcoholic hepatitis.
      and YAP overactivation is strongly involved in the loss of hepatocellular identity.
      • Bou Saleh M.
      • Louvet A.
      • Ntandja-Wandji L.C.
      • Boleslawski E.
      • Gnemmi V.
      • Lassailly G.
      • et al.
      Loss of hepatocyte identity following aberrant YAP activation: a key mechanism in alcoholic hepatitis.
      Figure thumbnail gr2
      Fig. 2Loss of liver function during chronic injury: A case of forged identity.
      Besides a decrease in parenchymal mass, deterioration of liver function as disease progresses can now be attributed to profound changes in the hepatocellular transcriptome. Reduced levels of core liver TFs and SFs, upregulation of foetal TFs and altered expression of regulatory ncRNAs, result in impaired liver gene expression, loss of metabolic zonation and the emergence of a foetal phenotype (identity). ESRP2, epithelial splicing regulatory protein 2; FOXA, forkhead box A; GCK, glucokinase; GLS, glutaminase; HK2, hexokinase 2; HNF, hepatocyte nuclear factor; INSR, insulin receptor; LPK, liver pyruvate kinase; MAT1A/2A, methionine adenosyltransferase 1A and 2A; ncRNA, non-coding RNA; PKM2, pyruvate kinase M2; SFs, splicing factors; SRSF, serine and arginine-rich splicing factor; TAZ, transcriptional coactivator with PDZ-binding motif; TEAD, TEA domain transcription factor 1; YAP, Yes-associated protein.

      Loss of hepatocellular identity in organ-wide disease progression

      While dysregulation of the hepatocellular trancriptome can certainly impair liver function, growing evidence suggests that hepatocellular dedifferentiation also impinges on the development of liver inflammation and fibrogenesis. A recent experimental study described how shutdown of the hepatocellular transcriptome in advanced NASH was linked to the activation of a TF network that triggered the expression of pro-inflammatory and pro-fibrogenic cytokines, fostering liver disease progression.
      • Loft A.
      • Alfaro A.J.
      • Schmidt S.F.
      • Pedersen F.B.
      • Terkelsen M.K.
      • Puglia M.
      • et al.
      Liver-fibrosis-activated transcriptional networks govern hepatocyte reprogramming and intra-hepatic communication.
      Besides contributing to hepatocellular dedifferentiation and proliferation, YAP-TAZ activation during chronic liver injury also appears to be involved in the development of inflammation and fibrosis. Increased hepatocellular expression of TAZ in human and murine NASH livers was shown to promote oxidative stress, inflammation and fibrosis, eventually leading to HCC development.
      • Wang X.
      • Zheng Z.
      • Caviglia J.M.
      • Corey K.E.
      • Herfel T.M.
      • Cai B.
      • et al.
      Hepatocyte TAZ/WWTR1 promotes inflammation and fibrosis in nonalcoholic steatohepatitis.
      ,
      • Wang X.
      • Zeldin S.
      • Shi H.
      • Zhu C.
      • Saito Y.
      • Corey K.E.
      • et al.
      TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis.
      On the other hand, the upregulation of the YAP target gene CYR61, a potent macrophage chemoattractant, was critically involved in these responses, and a strong correlation between YAP activity, CYR61 expression and disease severity was observed in a cohort of individuals with NASH.
      • Mooring M.
      • Fowl B.H.
      • Lum S.Z.C.
      • Liu Y.
      • Yao K.
      • Softic S.
      • et al.
      Hepatocyte stress increases expression of yes-associated protein and transcriptional coactivator with PDZ-binding motif in hepatocytes to promote parenchymal inflammation and fibrosis.
      Nevertheless, perhaps one of the best examples of the connection between the loss of hepatocellular identity and histological liver deterioration is related to the downregulation of HNF4α levels. This TF was shown to be essential for maintaining the expression of paraoxonase 1 (PON1), a secreted protein produced by hepatocytes that has potent anti-inflammatory effects on macrophages and that acts as a negative modulator of fibrogenic cell activation.
      • Yang T.
      • Poenisch M.
      • Khanal R.
      • Hu Q.
      • Dai Z.
      • Li R.
      • et al.
      Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.
      Consistently, restoration of hepatic HNF4α levels, and thus of PON1 expression, reduced liver inflammation and injury in models of advanced fibrosis, as discussed in the subsequent section.
      • Yang T.
      • Poenisch M.
      • Khanal R.
      • Hu Q.
      • Dai Z.
      • Li R.
      • et al.
      Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.
      Together, these observations indicate that retaining hepatocellular identity holds importance beyond the preservation of metabolic homeostasis.

      Is it possible to recover hepatocellular identity in a chronically injured liver?

      Collectively, the summarised evidence strongly suggests that derangement of the TF network supporting hepatocellular identity contributes to the loss of liver function during degenerative disease and could foster inflammation, fibrosis and neoplasia. This notion also elicited the question of whether restoring the expression of key TFs in decompensated hepatocytes could reset hepatocellular gene expression and improve liver function. The remarkable plasticity of hepatocytes during acute injury and regeneration, reversibly changing their differentiated transcriptome to allow for proliferation, supported this possibility.
      • Chembazhi U.V.
      • Bangru S.
      • Hernaez M.
      • Kalsotra A.
      Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver.
      Evidence in this direction was already available a decade ago. Adenoviral gene transfer of HNF1α was shown to partially recover the expression of mature hepato-specific genes and inhibit the growth of HCC cells (Fig. 3).
      • Zeng X.
      • Lin Y.
      • Yin C.
      • Zhang X.
      • Ning B.F.
      • Zhang Q.
      • et al.
      Recombinant adenovirus carrying the hepatocyte nuclear factor-1alpha gene inhibits hepatocellular carcinoma xenograft growth in mice.
      Similarly, restoration of FOXA2 expression in hepatocytes using different viral vectors had significant hepatoprotective effects in mice subjected to different acute and chronic injuries, also attenuating liver fibrosis.
      • Wang W.
      • Yao L.J.
      • Shen W.
      • Ding K.
      • Shi P.M.
      • Chen F.
      • et al.
      FOXA2 alleviates CCl 4-induced liver fibrosis by protecting hepatocytes in mice.
      ,
      • Wang S.
      • Feng R.
      • Wang S.S.
      • Liu H.
      • Shao C.
      • Li Y.
      • et al.
      FOXA2 prevents hyperbilirubinaemia in acute liver failure by maintaining apical MRP2 expression.
      In vitro, FOXA2 expression also partially reversed HCC cell dedifferentiation and inhibited H-RAS12V-driven HCC progression in mice.
      • Chand V.
      • Pandey A.
      • Kopanja D.
      • Guzman G.
      • Raychaudhuri P.
      Opposing roles of the forkhead box factors FoxM1 and FoxA2 in liver cancer.
      Meanwhile, adenoviral vector-mediated hepatic expression of HNF4α in rodent models of chronic liver injury and carcinogenesis partially restored hepatocyte-specific gene expression and liver function, while also reducing fibrosis and HCC development (
      • Yue H.Y.
      • Yin C.
      • Hou J.L.
      • Zeng X.
      • Chen Y.X.
      • Zhong W.
      • et al.
      Hepatocyte nuclear factor 4alpha attenuates hepatic fibrosis in rats.
      and reviewed in
      • Berasain C.
      • Avila M.A.
      Regulation of hepatocyte identity and quiescence.
      ). However, more convincing evidence of the real therapeutic potential of this approach came from a rat model of irreversible and fatal chronic liver failure, resembling clinical ESLD.
      • Nishikawa T.
      • Bell A.
      • Brooks J.M.
      • Setoyama K.
      • Melis M.
      • Han B.
      • et al.
      Resetting the transcription factor network reverses terminal chronic hepatic failure.
      In this interesting study, HNF4α gene transfer using a recombinant adeno-associated viral vector (rAAV) improved liver function and survival, activating the expression of key TFs like HNF1α, C/EBPα and FOXA2, which in turn reactivated the expression of endogenous HNF4α. This reciprocal network interaction led to a sustained correction of the liver phenotype, and remarkably this was achieved by transducing a modest fraction of hepatocytes, suggesting the existence of beneficial “bystander” mechanisms.
      • Nishikawa T.
      • Bell A.
      • Brooks J.M.
      • Setoyama K.
      • Melis M.
      • Han B.
      • et al.
      Resetting the transcription factor network reverses terminal chronic hepatic failure.
      While this research provided proof-of-concept, the potential use of AAV vectors in individuals with advanced liver disease may raise safety concerns. rAAV administration elicits an immune response and transient hepatocellular injury, as observed in clinical trials,
      • Gardin A.
      • Remih K.
      • Gonzales E.
      • Andersson E.R.
      • Strnad P.
      Modern therapeutic approaches to liver-related disorders.
      and was shown to cause HCC in mice with chronic liver injury.
      • Dalwadi D.A.
      • Torrens L.
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      • Pinyol R.
      • Willoughby C.
      • Posey J.
      • et al.
      Liver injury increases the incidence of HCC following AAV gene therapy in mice.
      Another limitation of rAAV vectors is the previous existence, or development, of neutralising antibodies against a given rAAV serotype, which may limit first or sequential use of a specific serotype.
      • Gardin A.
      • Remih K.
      • Gonzales E.
      • Andersson E.R.
      • Strnad P.
      Modern therapeutic approaches to liver-related disorders.
      Figure thumbnail gr3
      Fig. 3Emerging therapeutic strategies to recover liver function: Differentiation therapies
      Recent preclinical studies demonstrate that the restoration of key transcription factors, and the inhibition of the YAP pathway, can reset the hepatocellular transcriptome, recovering hepatic function and improving histological degeneration in chronically damaged organs. C/EBP, CAAT/enhancer binding protein; FOXA2, forkhead box A2; saRNA, short-activating RNA; siRNA, small-interfering RNA; YAP, Yes-associated protein.
      Alternative approaches to restore expression of key liver TFs include the administration of short duplex RNA oligonucleotides that target specific promoter regions and mediate transcriptional activation, known as short-activating RNAs (saRNAs). Administration of saRNAs targeting C/EBPα in a rat model of cirrhosis-associated carcinogenesis reactivated C/EBPα transcription, induced HNF1α and HNF4α expression, and ameliorated liver function. Interestingly, these responses were accompanied by reduced tumour burden.
      • Reebye V.
      • Sætrom P.
      • Mintz P.J.
      • Huang K.W.
      • Swiderski P.
      • Peng L.
      • et al.
      Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo.
      More recently, administration of saRNAs targeting HNF4α P1 were shown to significantly improve lipid metabolism and glucose homeostasis in a rat model of NASH.
      • Huang K.W.
      • Reebye V.
      • Czysz K.
      • Ciriello S.
      • Dorman S.
      • Reccia I.
      • et al.
      Liver activation of hepatocellular nuclear factor-4α by small activating RNA rescues dyslipidemia and improves metabolic profile.
      mRNA-based therapy is an emerging approach with great potential for the treatment of liver diseases.
      • Berraondo P.
      • Martini P.G.V.
      • Avila M.A.
      • Fontanellas A.
      Messenger RNA therapy for rare genetic metabolic diseases.
      Indeed, systemic administration of engineered mRNAs formulated in lipid nanoparticles (mRNA-LNPs) shielded from humoral and cellular immunity showed high efficacy for the treatment of experimental hepatic porphyria in mouse and rabbit models.
      • Jiang L.
      • Berraondo P.
      • Jericó D.
      • Guey L.T.
      • Sampedro A.
      • Frassetto A.
      • et al.
      Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.
      ,
      • Jericó D.
      • Córdoba K.M.
      • Jiang L.
      • Schmitt C.
      • Morán M.
      • Sampedro A.
      • et al.
      mRNA-based therapy in a rabbit model of variegate porphyria offers new insights into the pathogenesis of acute attacks.
      When these mRNA-LNPs were tested in non-human primates the hepatic parenchyma was robustly transduced, and no signs of toxicity nor immune response were observed.
      • Jiang L.
      • Berraondo P.
      • Jericó D.
      • Guey L.T.
      • Sampedro A.
      • Frassetto A.
      • et al.
      Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.
      Most interestingly, a recent study showed the efficient delivery of HNF4α2 mRNA, the predominant P1-driven isoform expressed in adult human liver, to hepatocytes isolated from individuals with decompensated cirrhosis. HNF4α2 mRNA treatment upregulated the expression of HNF1α and C/EBPα, as well that of key serum proteins and metabolic genes.
      • Tafaleng E.N.
      • Mukherjee A.
      • Bell A.
      • Morita K.
      • Guzman-Lepe J.
      • Haep N.
      • et al.
      Hepatocyte nuclear factor 4 alpha 2 messenger RNA reprograms liver-enriched transcription factors and functional proteins in end-stage cirrhotic human hepatocytes.
      A concomitant study validated the restorative effect of HNF4α mRNA-LNP delivery on hepatocytes isolated from individuals with chronic liver injury, and the improvement of liver function, injury and cirrhosis in different mouse models of chronic liver disease.
      • Yang T.
      • Poenisch M.
      • Khanal R.
      • Hu Q.
      • Dai Z.
      • Li R.
      • et al.
      Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.
      This study also identified potential HNF4α gene targets that could mediate the beneficial effects of this TF on liver inflammation and fibrosis in a paracrine manner, such as the secreted enzyme PON1 mentioned earlier.
      • Yang T.
      • Poenisch M.
      • Khanal R.
      • Hu Q.
      • Dai Z.
      • Li R.
      • et al.
      Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.
      One potential limitation of mRNA-LNP-based therapy for chronic diseases is the transient expression of the mRNA; thus, repeated administrations would be required. That said, the need for repeat administration may enable a better control of the treatment, which is not always possible with rAAV-based strategies. This aspect may be relevant, as the restoration of the mature and quiescent transcriptome is usually accompanied by the inhibition of hepatocellular proliferation, and thus it might hamper the recovery of the hepatocellular mass. Therefore, individuals with advanced liver disease, in whom the proliferative capacity of hepatocytes is already exhausted, could benefit most from the use of transient delivery strategies. Interestingly, murine HNF1α and HNF4α expression was restored in cirrhotic mice treated with HNF4α mRNA-LNPs, suggesting the reactivation of the endogenous liver-specific TF network, and the likelihood of an amplified and longer lasting therapeutic effect. Whether this approach could improve hepatocyte differentiation and survival in the setting of acute or subacute liver failure syndromes, although compelling, still needs to be investigated. All these studies are summarised in Fig. 3.
      In view of the role of YAP in the progression of liver disease, the loss of hepatocellular identity, and ultimately in HCC development, numerous efforts are being made to develop drugs targeting the Hippo-YAP pathway.
      • Russell J.O.
      • Camargo F.D.
      Hippo signalling in the liver: role in development, regeneration and disease.
      Some approaches are aimed at inhibiting the interaction of YAP with the TEAD family of TFs;
      • Russell J.O.
      • Camargo F.D.
      Hippo signalling in the liver: role in development, regeneration and disease.
      ; however, interfering with protein-protein interactions in a specific manner is always challenging. Specific cell targeting is also important, given the central homeostatic role of this pathway in many cell types. In this regard, small-interfering RNA against YAP formulated in LNPs (siYAP-LNPs) showed preferential targeting of HCC cells, reactivating their differentiation programme and reducing tumour burden in a mouse model
      • Fitamant J.
      • Kottakis F.
      • Benhamouche S.
      • Tian H.S.
      • Chuvin N.
      • Parachoniak C.A.
      • et al.
      YAP inhibition restores hepatocyte differentiation in advanced HCC, leading to tumor regression.
      (Fig. 3). Interestingly, peritumoral regions of liver tissue also responded to siYAP-LNPs, recovering zonal gene expression and eliminating atypical ductal cells.
      • Fitamant J.
      • Kottakis F.
      • Benhamouche S.
      • Tian H.S.
      • Chuvin N.
      • Parachoniak C.A.
      • et al.
      YAP inhibition restores hepatocyte differentiation in advanced HCC, leading to tumor regression.
      The effect of drugs that indirectly inhibit YAP signalling, such as dobutamine, has been recently tested in primary hepatocytes isolated from individuals with alcoholic hepatitis with promising results.
      • Bou Saleh M.
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      • Ntandja-Wandji L.C.
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      • Gnemmi V.
      • Lassailly G.
      • et al.
      Loss of hepatocyte identity following aberrant YAP activation: a key mechanism in alcoholic hepatitis.
      Together, these observations suggest that YAP-interfering agents have potential as a strategy to restore hepatocellular differentiation and liver function.

      Conclusions

      Liver identity is established during embryonic development and maintained after birth by an interdependent network of TFs, splicing regulators and signalling pathways. Evidence accumulated over the past two decades suggests that the collapse of this TF network underlies the progressive loss of liver function during chronic liver disease. Concomitantly with the realisation of this phenomenon, it became apparent that a programmed and transient disabling of this network is required for liver parenchymal cells to trigger their outstanding proliferative capacity, and thus enable liver regeneration upon moderate injury.
      • Gadd V.L.
      • Aleksieva N.
      • Forbes S.J.
      Epithelial plasticity during liver injury and regeneration.
      ,
      • Michalopoulos G.K.
      • Bhushan B.
      Liver regeneration: biological and pathological mechanisms and implications.
      However, exacerbated and/or persistent disruption of the expression, protein turnover, post-translational modifications and subcellular localisation of liver-characteristic TFs and splicing regulators results in the perpetuation of a dedifferentiated phenotype. Among these TFs, cogent studies have identified HNF4α as a critical regulator of adult hepatocellular identity and demonstrated the relevance of its impairment in liver decompensation. These observations have prompted studies assessing the potential of re-expressing HNF4α as a strategy to reset the transcriptional network in damaged hepatocytes, correct chronic liver failure and improve fibrosis. Restoring hepatic function via ectopic expression of key TFs may also be useful in the context of acute liver injury, as recently demonstrated.
      • Wang S.
      • Feng R.
      • Wang S.S.
      • Liu H.
      • Shao C.
      • Li Y.
      • et al.
      FOXA2 prevents hyperbilirubinaemia in acute liver failure by maintaining apical MRP2 expression.
      Therefore, improving liver functional reserve may be beneficial to avoid postoperative liver failure, and also to increase the safety of systemic anti-HCC therapies in individuals with underlying cirrhosis.
      • D’Avola D.
      • Granito A.
      • de la Torre-Aláez M.
      • Piscaglia F.
      The importance of liver functional reserve in the non-surgical treatment of hepatocellular carcinoma.
      Gene delivery using rAAV vectors, as well as emerging mRNA-based therapies, have provided encouraging experimental results and warrant clinical evaluation. Although at a more preliminary stage from the pharmacological point of view, there is strong biological evidence that interfering with the YAP pathway can restore hepatocellular differentiation, justifying further translational research in this area.

      Abbreviations

      AF, activation function domain; BMP, bone morphogenic protein; C/EBP, CAAT/enhancer binding protein; CRM, cis-regulatory module; ER, endoplasmic reticulum; ESLD, end-stage liver disease; ESRP2, epithelial splicing regulatory protein 2; FGF, fibroblast growth factor; FOXA, forkhead box A; HCC, hepatocellular carcinoma; Hex, haematopoietically expressed homeobox; HNF, hepatocyte nuclear factor; LNP, lipid nanoparticles; MELD, model for end-stage liver disease; NASH, non-alcoholic steatohepatitis; OC, onecut; PH, partial hepatectomy; PON1, paraoxonase 1; Prox, prospero-related homeobox; rAAV, recombinant adeno-associated virus; saRNA, short-activating RNA; siYAP-LNP, small-interfering RNA against YAP formulated in LNPs; SRSF, serine and arginine-rich splicing factor; TAZ, transcriptional coactivator with PDZ-binding motif; TF, transcription factor; TGFβ, transforming growth factor-β.

      Financial support

      Work in the authors’ laboratories is supported by: CIBERehd Intramural funding 2021 call; grants PI19/00163 and PI20/01663 from Instituto de Salud Carlos III (ISCIII) co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER) “Una manera de hacer Europa”; grants PID2019-104878RB-100/AEI/10.13039/501100011033, PID2019-104265RB-I00/AEI/10.13039/501100011033 and PID2020-117116RB-I00, from Ministerio de Ciencia, Innovación y Universidades MICINN-Agencia Estatal de Investigación integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER, MCIU/AEI/FEDER; grants 55/2018, 42/2021 and PC082-083-084 EHGNA from Gobierno de Navarra; AECC post-doctoral fellowship POSTD18