The hippo pathway: A master regulator of liver metabolism, regeneration, and disease

The FASEB JournalVolume 35, Issue 5 e21570 REVIEWOpen Access The hippo pathway: A master regulator of liver metabolism, regeneration, and disease Anh Thu Nguyen-Lefebvre, orcid.org/0000-0001-6247-7101 Department of Medicine, Multi-Organ Transplant Program, Toronto General Hospital, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute, Toronto, ON, CanadaSearch for more papers by this authorNazia Selzner, Department of Medicine, Multi-Organ Transplant Program, Toronto General Hospital, Toronto, ON, CanadaSearch for more papers by this authorJeffrey L. Wrana, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, CanadaSearch for more papers by this authorMamatha Bhat, Corresponding Author mamatha.bhat@uhn.ca Department of Medicine, Multi-Organ Transplant Program, Toronto General Hospital, Toronto, ON, Canada Correspondence Mamatha Bhat, Multiorgan Transplant Program, University Health Network, University of Toronto, 585 University Avenue, 11PMB-183, Toronto, Ontario, Canada. Email: mamatha.bhat@uhn.caSearch for more papers by this author Anh Thu Nguyen-Lefebvre, orcid.org/0000-0001-6247-7101 Department of Medicine, Multi-Organ Transplant Program, Toronto General Hospital, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute, Toronto, ON, CanadaSearch for more papers by this authorNazia Selzner, Department of Medicine, Multi-Organ Transplant Program, Toronto General Hospital, Toronto, ON, CanadaSearch for more papers by this authorJeffrey L. Wrana, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, CanadaSearch for more papers by this authorMamatha Bhat, Corresponding Author mamatha.bhat@uhn.ca Department of Medicine, Multi-Organ Transplant Program, Toronto General Hospital, Toronto, ON, Canada Correspondence Mamatha Bhat, Multiorgan Transplant Program, University Health Network, University of Toronto, 585 University Avenue, 11PMB-183, Toronto, Ontario, Canada. Email: mamatha.bhat@uhn.caSearch for more papers by this author First published: 08 April 2021 https://doi.org/10.1096/fj.202002284RRCitations: 5AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The liver is the only visceral organ in the body with a tremendous capacity to regenerate in response to insults that induce inflammation, cell death, and injury. Liver regeneration is a complicated process involving a well-orchestrated activation of non-parenchymal cells in the injured area and proliferation of undamaged hepatocytes. Furthermore, the liver has a Hepatostat, defined as adjustment of its volume to that required for homeostasis. Understanding the mechanisms that control different steps of liver regeneration is critical to informing therapies for liver repair, to help patients with liver disease. The Hippo signaling pathway is well known for playing an essential role in the control and regulation of liver size, regeneration, stem cell self-renewal, and liver cancer. Thus, the Hippo pathway regulates dynamic cell fates in liver, and in absence of its downstream effectors YAP and TAZ, liver regeneration is severely impaired, and the proliferative expansion of liver cells blocked. We will mainly review upstream mechanisms activating the Hippo signaling pathway following partial hepatectomy in mouse model and patients, its roles during different steps of liver regeneration, metabolism, and cancer. We will also discuss how targeting the Hippo signaling cascade might improve liver regeneration and suppress liver tumorigenesis. Abbreviations AKT Protein kinase B, PKB AMOT Angiomotin AMOTL Angiomotin ligand AMPK AMP-activated protein kinase ANGPT-2 Angiopoietin-2 BEC Biliary Epithelial Cell BMP-4 Bone Morphogenetic Protein 4 CCA Cholangiocarcinoma CP Capping Protein ECM Extracellular Matrix EGFR Epidermal Growth Factor Receptor EMT Epithelial Mesenchymal Transition ERK Extracellular signal-Regulated Kinase FGF Fibroblast Growth Factor GPCR G Protein-Coupled Receptor HCC Hepatocellular Carcinoma HGF Hepatocyte Growth Factor HIF Hypoxia Inducible Factor HNF-4α Hepatocyte Nuclear Factor-4 alpha HSC Hepatic Stellate Cell HSP90 Heat shock protein 90 IL-6 Interleukin-6 JAG-1 Jagged-1 JAK Janus Kinase JNK c-Jun N-Terminal Kinase KC Kupffer Cell LATS1/2 Large Tumor Suppressor Homolog 1/2 LKB1 Liver Kinase B1 LPS Lipopolysaccharide MAP4K Mitogen-activated protein kinase kinase kinase kinase MOB1A/B MPS one binder kinase activator-like 1A/B MRTF-A Myocardin-Related Transcription Factor A MSC Mesenchymal Stem Cell MST1/2 Mammalian Sterile Twenty-like 1/2 mTOR Mammalian target of rapamycin NAFLD Nonalcoholic fatty liver disease NASH Nonalcoholic steatohepatitis NEKB Nuclear Factor Kappa-light-chain-enhancer of activated B cells NF2 Neurofibromin 2 PKA/B Protein Kinase A/B PKC Protein Kinase C PP1/2 Protein phosphatases 1 and 2 PTx Partial hepatectomy SAV1 Salvador Homolog 1 SFSS Small-For-Size Syndrome SOH Silence of Hippo Signaling SRC Sarcoma SREBP Sterol Regulatory Element-Binding Protein STAT Signal Transducer and Activator of Transcription protein TAZ Transcriptional co-Activator with PDZ-binding motif TEAD Transcriptional Enhancer Associate Domain TGF-β Transforming Growth Factor beta TNF-α Tumor Necrosis Factor-alpha WNT Wingless and Int-1 WW45 Th mammalian Salvador homolog YAP Yes-Associated Protein 1 INTRODUCTION 1.1 Liver turnover The liver is a central organ for maintaining systemic health and homeostasis: it performs hundreds of essential metabolic reactions, eliminates toxic metabolites, xenobiotics, and infectious agents.1, 2 Additional critical functions include production of clotting factors, hormones, growth factors, and bile. The liver is unique among solid organs in its tremendous capacity to rapidly regenerate. Under homeostasis, cell turnover in the liver is minimal: adult liver cells are mainly quiescent, and their turnover is estimated to be between 180 and 400 days, meaning these cells divide once or twice a year.3 However, after partial hepatectomy (PHx), regeneration is completed by 3 months in humans and 7-10 days in rodents.4, 5 Slower liver regeneration occurs in response to chronic insults such as viral hepatitis, alcohol, and fat that induce damage.6, 7 Liver regeneration is a complicated process involving a well-orchestrated activation of non-parenchymal cells in the injured area and proliferation of undamaged hepatocytes. 1.2 The Hippo signaling pathway The Hippo signaling pathway and its components were first identified in Drosophila melanogaster as a critical regulator of cell proliferation, apoptosis, and organ/tissue size.8-14 Mammal homologs of the fly Hippo signaling pathway were identified and their functions were characterized in the following years.15-21 The core pathway in mammals is composed of the MST1/2 (Mammalian Ste20-like kinases) and LATS1/2 (Large tumor suppressor) kinase cassette16, 18 that acts to phosphorylate and inhibit the transcriptional regulators YAP (Yes-Associated Protein)15 and TAZ (Transcriptional co-activator with PDZ-binding motif)17, 19 in cooperation with WW45 (mammalian ortholog of Salvator (Sav)) and MOB1 (MOB kinase activator 1). YAP is an essential transcription coactivator that regulates organ size and is considered a potential oncogene. Its activation is tightly regulated by the Hippo pathway kinase cascade, especially by LATS1/2, which recognizes the five consensus HXRXXS motifs present in YAP. The phosphorylation on Ser 127 was identified as the crucial signal leading to YAP retention in the cytoplasm by the 14-3-3 protein.15, 22 Phosphorylation on Ser 397 creates a phosphodegron motif for SCF-β-TRCP E3 ubiquitin ligase binding, leading to YAP ubiquitination and degradation by proteasome.23 Furthermore, phosphorylation of the Ser 381 was found to be crucial for inducing phosphorylation of a phosphodegron in YAP by CK1δ/ε (Casein Kinase 1), following by recruitment of SCF-β-TRCP E3 ubiquitin ligase.22 Parallel to LATS1/2, AKT (Protein Kinase B, Ser 127), and AMPK (AMO-activated protein kinase, Ser 61, Ser 94, and Thr 119) were reported to have the capacity to phosphorylate YAP to regulate apoptosis upon cellular damage and to modulate glucose homeostasis, respectively.24-26 Studies on phosphorylation-independent regulation of YAP/TAZ have shown that AMOT (Angiomotin), AMOTL (Angiomotin ligand), and α-catenin negatively regulate YAP/TAZ activity by sequestering them in the cytoplasm at the tight and adherens junctions, respectively.27-31 The interaction of AMOT and AMOTL with YAP and TAZ is mediated by the PPXY motifs of AMOT and AMOTL, and the WW domain of TAZ and YAP.27, 28 Alpha-catenin was identified as one of the principal component of the adherens junctions32 and its role as YAP regulator was discovered this last decade. Data from murine model demonstrated that α-catenin interacts with YAP1 and 14-3-3 to keep YAP1 in the cytoplasmic compartment. This interaction requires the WW domains of YAP and the participation of 14-3-3.30 Conversely, Guan's group demonstrated that PP1 (protein phosphatase 1) dephosphorylates TAZ at Ser 89 and Ser 311, promoting also TAZ nuclear translocation and stabilizing TAZ by disrupting its binding to the SCF-β-TRCP.33 Furthermore, PP2 (protein phosphatase 2) dephosphorylates YAP at Ser 127, triggering its activation and nuclear recruitment.30 MST1/2 and LATS1/2 kinases are the core kinases of the Hippo pathway and their activity is regulated at different levels by several kinases and regulators, including MAP4K family (Mitogen-activated protein kinase kinase kinase kinase),34-36 RASSF1A (Ras association domain family member 1A),37, 38 TAOK1-3 (Tao kinases 1-3),39, 40 AMPK (PRKKA1/PRKKA2),25 PKA (protein kinase A: PRKACA/PRKCB),25, 26, 41 PKC (protein kinase C),39, 42 PP2,43-45 RhoA (Ras homology family member A),46-49 NF2 (Neurofibromin 2),15, 50-52 and Ajuba LIM proteins.53-55 More recently, a new regulator of LATS1/2 was identified as HSP90 (heat shock protein 90).56-58 Inhibition of HSP90 by its chemical inhibitor 17-AAG led to depletion LATS1/2 kinases by disrupting LATS chaperoning and targeting LATS for proteasomal degradation.56 In vitro study revealed that the heat shock rapidly induces through SRC, the formation of aggregates including LATS kinases and protein phosphatases, such as PP1 (protein phosphatase 1), where LATS are dephosphorylated and inactivated. Four hours upon heat shock, HSP90 plays a crucial role for the dissociation of LATS aggregates and their reactivation in the cytoplasm.58 Another study on physiological and pathological stress induced by heat shock demonstrated another function of HSP90 as a negative regulator of LATS. Upon heat shock, LATS kinases were rapidly bound and dephosphorylated by PP5, and HSP90 acts as a scaffold to facilitate the interaction between LATS and PP5 (protein phosphatase 5).57 The Hippo pathway and its regulators are summarized in Figure 1. FIGURE 1Open in figure viewer Schematic figure of the Hippo pathway in mammals and its regulators. The core pathway is composed of the NF2 (Neurofibromin 2), MST1/2 (Mammalian Dte20-like kinases), and LATS1/2 (Large tumor suppressor) kinase cassette that acts to phosphorylate and inhibit the transcriptional regulators YAP (Yes-associated protein) and TAZ (Transcriptional co-activator with PDZ-binding motif). MST1/2 cooperate with WW45 (mammalian ortholog of Salvator) to phosphorylate and activate LATS1/2, which become active with MOB1 to and in turn phosphorylate YAP (on Serine 127) and TAZ, to induce their cytoplasmic sequestration by 14-3-3 protein. This mechanism of regulation is reversible. Other important phosphorylation sites on YAP are Serines 381 and 397, which prime YAP for subsequent phosphorylation by Casein Kinase 1 (CK1) (Serine 381), leading both to its poly-ubiquitination by SCF-b-TRCP E3 ubiquitin ligase, and their degradation by the proteasome. Different regulators of the Hippo core kinases and YAP/TAZ were identified. Inactivation of the Hippo signaling pathway results on the importation of the unphosphorylated YAP/TAZ complex into the nucleus to bind to TEADs (TEA domain transcriptional enhancer factors) factors for activating transcription of YAP/TAZ target genes, which display roles in antiapoptotic process, cell proliferation, anti-differentiation of stem cells and progenitors, and cell migration The Hippo signaling pathway plays a fundamental role in regulation of various processes, including organ size, regeneration, cell proliferation, apoptosis, and carcinogenesis.44, 52, 59-65 As a result, dynamic changes in the Hippo signaling pathway and YAP activation are crucial in the maintenance of liver homeostasis and the liver-to-body ratio. In this review, we will first cover the breadth of literature on the critical role of the Hippo signaling pathway on liver metabolism, proliferation, size, cell fate, and homeostasis. Then, we will discuss on multiple signals regulating modulation of the Hippo pathway in liver regeneration and how to target this pathway to improve liver regeneration. Finally, we will discuss its role in liver cancer and its central function as target of liver cancer treatment. 2 HIPPO PATHWAY TIGHTLY REGULATES LIVER METABOLISM AND CELL PROLIFERATION The Hippo pathway responds not only to biochemical, but also to physical changes.26, 46, 59, 66-70 Its activity is regulated by a network of upstream components that have roles in other processes, such as establishment of cell adhesion, morphology, and polarity.15, 66, 67, 70-72 In this section, we will describe the multiple modulations of liver metabolism and cell proliferation by the Hippo pathway by interacting with other signals. Hepatocytes exhibit different metabolic functions based on their location on the pericentral-periportal axis: pericentral hepatocytes are mainly involved in glycolysis, bile synthesis, and glutaminogenesis, whereas periportal hepatocytes participate actively in gluconeogenesis and ammonia clearance (Figure 2).73 These metabolic activities of hepatocytes are intricately linked to the Hippo signaling pathway. For example, in vitro experiments on HepG2 and Huh7 cell lines demonstrated that hypoxia and HIFs (Hypoxia inducible factors) modulate the Hippo pathway components at different levels. Hypoxia increases YAP binding to HIF-1α in the nucleus, which promoted accelerated glycolysis.74 With liver-specific deletion of Mst1 or Mst2, glutamine synthase zone was reduced, suggesting a role for this gene in metabolic zonation.75 FIGURE 2Open in figure viewer Metabolic zonation of the Liver. The liver is composed of remarkable uniform hexagonal lobules named acinus. At the periphery of each acinus, there is a portal triad containing one portal vein, one hepatic artery, and one bile duct. From these portal triads, a network of smallest capillaries of the liver runs to the central vein, where the blood drains into the hepatic venules. Three different zones were identified from the portal triad to the central vein: zone 1 (periportal), zone 2 (transition), and zone 3 (pericentral). Periportal zone is highly oxygenated compared to the pericentral zone. Consequently, hepatocytes from these zones exhibit different metabolic functions: periportal hepatocytes are mainly involved in glycolysis, bile synthesis whereas, pericentral hepatocytes participate actively in gluconeogenesis, glycolysis, and lipogenesis. Under physiological conditions, Hippo pathway follows the gradient of oxygen, it is mainly activated in the periportal zone. In the contrast, YAP activity is highly concentrated in the pericentral zone. Following liver injury, Hippo-dependent liver repair will be treated differently by hepatocytes from periportal, transitional, and pericentral zones Moreover, Hippo signaling pathway and its downstream targets, including MST1/2, LATS1/2, and YAP/TAZ are regulated by metabolic networks, such as by glycolysis,25, 48 hexosamine biosynthesis,76, 77 and mevalonate synthesis,78, 79 as well as by nutrient-sensing pathways including AMPK,25, 26, 41 and mTOR (mammalian target of rapamycin).80-83 Inversely, Hippo pathway affects gluconeogenesis, as well as cholesterol and lipid biosynthesis.84-87 Dysregulation of the Hippo pathway has been associated with metabolic diseases, such as obesity, type 2 diabetes, NAFLD, cardiovascular disorders, and cancer.2, 88, 89 This association may be beneficial from a therapeutic standpoint, given the more than 422 million people affected by diabetes worldwide.90 Previous studies have demonstrated that glucose metabolism tightly regulates the activation of YAP/TAZ at different levels.76, 77, 91, 92 Glucose metabolism is essential for growing or functionally active cells. Increased glucose uptake enhances the binding between YAP/TAZ and TEADs transcription factors to activating YAP-dependent genes expression, and cell proliferation.25, 26, 48, 93 Elevated levels of extracellular glucose and other metabolic nutrient induce O-GlcNAcetylation of YAP, at Ser109 and Thr241, by O-GlcNAc transferase (OGT), a key enzyme of the hexosamine biosynthesis pathway (HBP). This translational modification prevents LATS-induced YAP phosphorylation and increases YAP recruitment in the nucleus to activate transcription of its target genes including OGT and genes implicating in glycolysis pathway, such as glucose transporter 3 (GLUT3) to enhance glucose uptake, lactate production, and cell growth.25, 76, 77 Recently, another target gene of YAP was discovered as insulin receptor substrate 2 (IRS2), an integral component of insulin signaling pathway. YAP/TAZ complex positively regulates the expression of IRS2 in human HCC in humans and similarly leads to NAFLD and subsequently HCC in mice.94 NAFLD is characterized by an accumulation of fat in liver which results in inflammation and destruction of hepatocytes, also promoting the development of cirrhosis and HCC.95-97 Increased YAP/TAZ levels have been shown in mouse models of NAFLD and in NAFLD patient biopsies. In a mouse model, overexpression of Taz-induced liver inflammation, NAFLD, and tumor formation.98, 99 Activation of the Hippo pathway negatively modulates lipid metabolism: LATS2 cooperates with the tumor-suppressor transcription factor p53 to impair SREBPs, a key transcription factor of lipogenesis.84 The LATS-p53 complex disrupts endoplasmic reticulum-located SREBP1 and SREBP2, which inhibits the transcriptional activity of mature cleaved nuclear SREBPs and the expression of lipogenic enzymes.84 Mice having liver-specific disruption of Lts2 showed increased expression of SREBP (Sterol Regulatory Element-Binding Protein) target genes. SREBP family contains three members: SREBP-1a, SREBP-1c, and SREBP-2. The main isoforms found in liver were identified as SREBP-1c and SREBP-2.100-102 The SREBP family transcription factors tightly control the transcription of genes encoding enzymes, cellular-binding proteins, and import/export factors involved in the production and trafficking of cholesterol, fatty acids, and other lipids.103 In the physiological conditions, SREBP-1c favors fatty acid biosynthesis via ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase, the enzyme of the fatty acid elongase complex,23 stearoyl-CoA desaturase, and glycerol-3-phosphate acyltransferase. SREBP-2 promotes cholesterologenesis, including the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase.104 Increased expression of SREBP target genes in liver is associated with several metabolic disorders, including obesity, type 2 diabetes (T2B), dyslipidemia, nonalcoholic fatty liver disease (NAFLD, hepatic steatosis), and nonalcoholic steatohepatitis (NASH, NAFLD associated with hepatic inflammation and fibrosis).105-107 The loss of Lats2 in mice hepatocytes induces excessive cholesterol synthesis, de novo lipogenesis, the development of steatosis and fatty liver disease, due to chronic metabolic stress.84 This process is YAP-independent, suggesting the contribution of the Hippo pathway to regulation of metabolic homeostasis through multiple effectors.2, 84 Furthermore, deletion of Mst1 in mice liver also results in enhanced lipid droplet accumulation, ballooning, and liver degeneration when these mice were fasted for 48 hours or fed with high-fat diet.108 Conversely, MST1 action is not direct on SREBPs: MST1 stabilizes sirtuin 1 (SIRT1), an important regulator of hepatic glucose and lipid metabolism,91, 109 and a negative regulator of SREBPs.110, 111 When mice were fasted, hepatic Sirt1 expression is induced, but this process was abolished in Mst1 knockout mice. Clinical studies demonstrated that reduced LATS2 and abnormal increase of SREBPs were observed in patients diagnosed with advanced fatty liver disease,84 liver cirrhosis, and cancer.112 SREBP-1c and SREBP-2 regulate lipogenic process and cholesterol homeostasis in liver by activating genes involved in these processes as described above.113 However, here is a YAP-dependent activation of SREBPs target genes: The SREBPs transcription factors are recruited into the nucleus, form a complex with YAP, and together they bind to the sterol regulatory elements (SREs) present in the promotor of their own and target genes, thereby promoting the lipogenic process.85 YAP was showed to interact directly with SREBP-1c and SREBP-2 on the promotors of the fatty acid synthase (Fas) and 30-hydroxylmethyl glutaryl coenzyme A reductase (Hmgcr), the key enzymes implicated in the synthesis of triglyceride and cholesterol, to promote their transcription, leading to hepatocyte lipogenesis and cholesterol synthesis.114 Studies on C57BL/6 mice fed with the diabetogenic diet containing high fat and high sucrose (HFHS) demonstrated that these mice develop obesity and T2D.114 These mice showed accumulation of YAP in the hepatocyte nucleus. When overexpression of Lats was induced in these mice, mRNA levels of Fas and Hmgcr were significantly reduced. Furthermore, administration of sh-Yap to insulin-resistant mice after 16 weeks of HFHS diet, decreases the total plasma cholesterol and triglyceride levels.114 Together, these studies suggest that the Hippo pathway negatively regulates hepatic lipogenesis by suppressing YAP-SREBPs complexes, thereby playing an important role in metabolism diseases. The mammalian liver exhibits a tremendous regenerative capacity. After partial hepatectomy of up to 70% in humans, and 75% in rodents, reconstitution of the original liver mass proceeds through compensatory hypertrophy and then, proliferation of residual hepatocytes. YAP plays a crucial role during the early stage of liver regeneration since its expression level and activity were shown to increase significantly within 24 hours, before the onset of hepatocyte proliferation, and remained elevated for 72 hours.115 This process goes along with a decreased activation of MST and LATS kinases, until the hepatostat is restored.116 The dynamic of the Hippo pathway is tightly regulated to maintain hepatocytes in differentiated state and is essential for the cell fate determination in liver62, 117, 118: increased expression of Yap led to hepatocyte hyperproliferation and liver enlargement,119, 120 while inactivation of Yap-induced loss of hepatocytes and biliary epithelial cells.121, 122 Moreover, Hippo signaling may play an essential role in liver development, while Lats1/2 deleted mouse embryos exhibit accumulation of immature cholangiocytes and a lack of functional mature hepatocytes.123 Oval cells are reported to be one of the sources of liver progenitor cells and their expansion after liver injury is associated with liver regeneration. These cells are considered to have a hepatic bipotent capacity allowing them to differentiate into hepatocytes and bile duct cells.124 In liver-specific deleted Mst1/2, Ww45, or Nf2 mice, as well as in Yap transgenic animals, over-proliferation of oval cells-induced liver enlargement and liver cancer formation.125 Inactive Hippo signaling leads to translocation of YAP and TAZ into the nuclear compartment. YAP/TAZ complex interacts with the transcription factors, such as the TEA/ATTS domain transcription factor family (TEAD1-4).126 The transcriptional complex YAP/TAZ-TEAD binds to the core GTIIC or MCAT motifs in gene promoters and enhancers to activate the expression downstream target genes involved in cell proliferation and survival.126 Recent studies identified molecular mechanisms by which YAP activates key downstream genes and pathways contributing to liver cell proliferation. Wang and colleagues recently found in mouse model and in HepG2 cell line, that YAP associated to TEAD4 binds directly to ERBB2 gene promoter and induces its expression. Inhibition of YAP expression, in contrast, decreases the mRNA and protein levels of ERBB2, EGF-induced ERBB2-mediated HepG2 cell proliferation, and PI3K/AKT activation.127 Additionally, YAP positively modulates the binding of the transcription factors HNF4A and FOX2 to mouse embryonic enhancers during hepatocyte differentiation to increase transcription of specific genes.128 In parallel, YAP is very important for cholangiocyte proliferation: conditional knockout of Yap or of both Yap and Taz in mouse model results in defects in bile duct morphogenesis with irregularly shaped and deformed intrahepatic bile ducts.129, 130 In the mouse model of cholestatic liver damage by bile duct ligation, the reactive proliferation and expansion of bile duct cells are mainly dependent of YAP activation, while liver-specific deleted Yap mice demonstrated significant reduction of these processes.131 Furthermore, YAP is essential for the maintenance of cholangiocyte phenotype, while decreased activation of YAP led in reduced expression of cholangiocyte markers.129 Tightly controlled regulation of liver metabolism is essential for controlling liver growth and homeostasis, and in this context the Hippo pathway regulators play a key role in modulating liver organ size, cell fate, and homeostasis.2, 62, 64, 117, 118, 128, 132, 133 These complex modulations are delineated in detail in the following section, with the emphasis on the role of YAP/TAZ. 3 THE HIPPO PATHWAY IN REGULATION OF LIVER ORGAN SIZE, CELL FATE, AND HOMEOSTASIS The function of the Hippo signaling pathway was initially discovered in D melanogaster. Afterward, studies in a mouse model showed that inactivation of Hippo kinases or hyperactivation of YAP/TAZ proteins drive overgrowth of multiple organs, including liver and heart.69, 134, 135 Here, we will examine tightly the role of the Hippo pathway on controlling liver organ size, liver cell fate, and liver homeostasis. During mouse embryogenesis, YAP and TAZ play an essential role in development: ubiquitous Yap-deleted mice cannot survive beyond embryonic day 8.5 and present defects in york sac vasculogenesis, chorioallantoic attachment, and body axis elongation136 ; and ubiquitous Taz-deleted mice develop renal cysts and emphysema.137, 138 Liver-specific Yap- and Taz-deleted mice exhibited hepatocyte with macrophage infiltration necrosis. Liver injury in this mouse model also occurred in the form of defective biliary structures due to the accumulation of toxic bile acids, resulting in hepatocyte death.130 Other studies in the liver using mouse genetic models have shown a key role for the Hippo pathway in controlling liver size and regeneration. The hepatocyte nuclear factor-4 alpha (HNF-4α) is a key regulator of hepatocyte differentiation process during embryonic development and of maintenance of differentiated phenotype in the adult liver.139-141 Its expression is negatively regulated by YAP-1 through a ubiquitin proteasome pathway. Seminal studies showed that ectopic YAP activity led to liver overgrowth,132 while genetic analyses showed active, nuclear YAP together with Notch signaling drive a ductal/progenitor fate, and Hippo activity enforces the hepatocyte fate.118 Thus, Hippo regulates dynamic cell fates in the liver, and in the absence of Yap and Taz (YAP/TAZ), liver regeneration is severely impaired130 and the proliferative expansion of hepatic stellate cells blocked.142 YAP/TAZ also play a key role in regulating liver regeneration and phenotypic plasticity in the liver. Obstinate Yap liver-specific overexpression resulted in hepatomegaly in mice. However, restoration of endogenous YAP levels, by activation of the Hippo pathway, led to rapid reversal of the hepatomegaly, and normalization of the parenchymal architecture. These findings strongly suggest the role of Hippo pathway as an important regulator of overall liver size.143 Unlike the skin or intestinal epithelial cells, where rapid cell renewal is driven by multipotent stem cells, on site differentiated adult hepatocytes represent the source of tissue renewal under homeostasis in the liver. During liver injury, a small population of cells emerges and is called oval cells based on their morphology. These cells participate actively in liver repair. These oval cells are normally not present in healthy liver, and give rise to both hepatocytes and cholangiocytes, demonstrated by cell lineage tracing studies after liver injury.144, 145 Two hypotheses have been reported concerning the origin of t