Satdarshan P.S. Monga

WNT/β‐catenin signaling in liver health and disease

Wnt/β‐catenin signaling is emerging as a forerunner for its critical roles in many facets of human biology. Its roles in embryogenesis, organogenesis, and maintaining tissue and organ homeostasis demonstrate its munificent character. Its roles in pathological conditions such as cancer and other human disorders such as inflammatory disorders and fibrosis reveal its villainous disposition. In liver, it also maintains its dual personality and is clearly of essence in several physiological events such as development, regeneration, and growth. Its aberrant activation is also evident in many different tumors of the liver, and recent studies are beginning to identify its role in additional hepatic pathological conditions. It is contributing to liver physiology and pathology by regulating various basic cellular events, including differentiation, proliferation, survival, oxidative stress, morphogenesis, and others. This review discusses the contribution of the Wnt/β‐catenin signaling pathway in these events and simultaneously provides an essential overview of the major developments in the field of Wnt/β‐catenin and liver pathobiology. In addition, areas that are currently deficient or understudied are identified and discussed along with the avenues of translational and clinical relevance. (HEPATOLOGY 2007;45:1298–1305.)

Wnt/β-Catenin Signaling Promotes Renal Interstitial Fibrosis

Wnts compose a family of signaling proteins that play an essential role in kidney development, but their expression in adult kidney is thought to be silenced. Here, we analyzed the expression and regulation of Wnts and their receptors and antagonists in normal and fibrotic kidneys after obstructive injury. In the normal mouse kidney, the vast majority of 19 different Wnts and 10 frizzled receptor genes was expressed at various levels. After unilateral ureteral obstruction, all members of the Wnt family except Wnt5b, Wnt8b, and Wnt9b were upregulated in the fibrotic kidney with distinct dynamics. In addition, the expression of most Fzd receptors and Wnt antagonists was also induced. Obstructive injury led to a dramatic accumulation of beta-catenin in the cytoplasm and nuclei of renal tubular epithelial cells, indicating activation of the canonical pathway of Wnt signaling. Numerous Wnt/beta-catenin target genes (c-Myc, Twist, lymphoid enhancer-binding factor 1, and fibronectin) were induced, and their expression was closely correlated with renal beta-catenin abundance. Delivery of the Wnt antagonist Dickkopf-1 gene significantly reduced renal beta-catenin accumulation and inhibited the expression of Wnt/beta-catenin target genes. Furthermore, gene therapy with Dickkopf-1 inhibited myofibroblast activation; suppressed expression of fibroblast-specific protein 1, type I collagen, and fibronectin; and reduced total collagen content in the model of obstructive nephropathy. In summary, these results establish a role for Wnt/beta-catenin signaling in the pathogenesis of renal fibrosis and identify this pathway as a potential therapeutic target.

Wnt/β-Catenin Signaling Promotes Podocyte Dysfunction and Albuminuria

Podocyte dysfunction, one of the major causes of proteinuria, leads to glomerulosclerosis and end stage renal disease, but its underlying mechanism remains poorly understood. Here we show that Wnt/beta-catenin signaling plays a critical role in podocyte injury and proteinuria. Treatment with adriamycin induced Wnt and activated beta-catenin in mouse podocytes. Overexpression of Wnt1 in vivo activated glomerular beta-catenin and aggravated albuminuria and adriamycin-induced suppression of nephrin expression, whereas blockade of Wnt signaling with Dickkopf-1 ameliorated podocyte lesions. Podocyte-specific knockout of beta-catenin protected against development of albuminuria after injury. Moreover, pharmacologic activation of beta-catenin induced albuminuria in wild-type mice but not in beta-catenin-knockout littermates. In human proteinuric kidney diseases such as diabetic nephropathy and focal segmental glomerulosclerosis, we observed upregulation of Wnt1 and active beta-catenin in podocytes. Ectopic expression of either Wnt1 or stabilized beta-catenin in vitro induced the transcription factor Snail and suppressed nephrin expression, leading to podocyte dysfunction. These results suggest that targeting hyperactive Wnt/beta-catenin signaling may represent a novel therapeutic strategy for proteinuric kidney diseases.

Beta-catenin signaling, liver regeneration and hepatocellular cancer: sorting the good from the bad.

Among the adult organs, liver is unique for its ability to regenerate. A concerted signaling cascade enables optimum initiation of the regeneration process following insults brought about by surgery or a toxicant. Additionally, there exists a cellular redundancy, whereby a transiently amplifying progenitor population appears and expands to ensure regeneration, when differentiated cells of the liver are unable to proliferate in both experimental and clinical scenarios. One such pathway of relevance in these phenomena is Wnt/β-catenin signaling, which is activated relatively early during regeneration mostly through post-translational modifications. Once activated, β-catenin signaling drives the expression of target genes that are critical for cell cycle progression and contribute to initiation of the regeneration process. The role and regulation of Wnt/β-catenin signaling is now documented in rats, mice, zebrafish and patients. More recently, a regenerative advantage of the livers in β-catenin overexpressing mice was reported, as was also the case after exogenous Wnt-1 delivery to the liver paving the way for assessing means to stimulate the pathway for therapeutics in liver failure. β-Catenin is also pertinent in hepatic oval cell activation and differentiation. However, aberrant activation of the Wnt/β-catenin signaling is reported in a significant subset of hepatocellular cancers (HCC). While many mechanisms of such activation have been reported, the most functional means of aberrant and sustained activation is through mutations in the β-catenin gene or in AXIN1/2, which encodes for a scaffolding protein critical for β-catenin degradation. Intriguingly, in experimental models hepatic overexpression of normal or mutant β-catenin is insufficient for tumorigenesis. In fact β-catenin loss promoted chemical carcinogenesis in the liver due to alternate mechanisms. Since most HCC occur in the backdrop of chronic hepatic injury, where hepatic regeneration is necessary for maintenance of liver function, but at the same time serves as the basis of dysplastic changes, this Promethean attribute exhibits a Jekyll and Hyde behavior that makes distinguishing good regeneration from bad regeneration essential for targeting selective molecular pathways as personalized medicine becomes a norm in clinical practice. Could β-catenin signaling be one such pathway that may be redundant in regeneration and indispensible in HCC in a subset of cases?

Wnt/β-Catenin Signaling Mediates Oval Cell Response in Rodents

Adult hepatic stem cells or oval cells are facultative stem cells in the liver that are activated during regeneration only during inhibition of innate hepatocyte proliferation. On the basis of its involvement in liver cancer, regeneration, and development, we investigated the role of the Wnt/β‐catenin pathway in oval cell response, which was initiated in male Fisher rats with 2‐acetylaminofluorine and two‐third partial hepatectomy (PHX). Extensive oval cell activation and proliferation were observed at 5 and 10 days post‐PHX, as indicated by hematoxylin‐eosin and proliferating cell nuclear antigen analysis. A noteworthy increase in total and active β‐catenin was observed at this time, which was localized to the oval cell cytoplasm and nuclei by immunohistochemistry and confirmed by double immunofluorescence. A concomitant increase in Wnt‐1 in hepatocytes along with increased expression of Frizzled‐2 in oval cells was observed. This paracrine mechanism coincided with a decrease in Wnt inhibitory factor‐1 and glycogen synthase kinase‐3β down‐regulation leading to β‐catenin stabilization. To strengthen its role, β‐catenin conditional knockout mice were treated with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine to induce oval cell activation. A dramatic decrease in the A6‐positive oval cell numbers in the absence of β‐catenin demonstrated a critical role of β‐catenin in oval cell biology. Conclusion: The Wnt/β‐catenin pathway plays a key role in the normal activation and proliferation of adult hepatic stem cells. (HEPATOLOGY 2007.)

Expression of Notch-1 and its Ligand Jagged-1 in Rat Liver During Liver Regeneration

The Notch/Jagged signaling pathway is important for cellular differentiation and proliferation. Its dysfunction is associated with human pathologies in several tissues including liver. Point mutations in Jagged‐1 gene are the cause for Alagille syndrome, associated with paucity of intrahepatic bile ducts. To determine the putative role of the trans‐membrane receptor Notch and its ligand Jagged‐1 in liver regeneration, we investigated the expression of Notch and Jagged‐1 in rat liver following 2/3 partial hepatectomy. Immunohistochemical staining of normal rat liver showed that Notch was expressed in hepatocytes, bile duct cells and endothelial cells, whereas Jagged‐1 was expressed in bile duct cells and hepatocytes. Both Notch‐1 and Jagged‐1 proteins were upregulated in hepatocytes after partial hepatectomy up to day 4. After partial hepatectomy, nuclear translocation of the intracellular cytoplasmic domain of Notch (NICD) increased and peaked within 15 minutes, indicating the activation of Notch. Expression of the Notch‐dependent target gene (HES‐1) expression increased within 30–60 minutes. Addition of recombinant Jagged‐1 protein to primary cultures of hepatocytes stimulated hepatocyte DNA synthesis. Furthermore, injection of silencing RNA for Notch and Jagged‐1 to livers 2 days before partial hepatectomy significantly suppressed proliferation of hepatocytes at days 2 to 4 of the regenerative response. In conclusion, Notch/Jagged signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting cell growth and differentiation. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;39:1056–1065.)

High‐mobility group box 1 activates caspase‐1 and promotes hepatocellular carcinoma invasiveness and metastases

Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. The exact mechanisms related to hypoxia‐induced invasion and metastasis remain unclear. We elucidated the mechanism by which the nuclear‐damage–associated molecular pattern molecule, high‐mobility group box 1 (HMGB1), released under hypoxic stress, can induce an inflammatory response to promote invasion and metastasis in hepatocellular carcinoma (HCC) cells. Caspase‐1 activation was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of HMGB1 and subsequent activation of both Toll‐like receptor 4 (TLR4)‐ and receptor for advanced glycation endproducts (RAGE)‐signaling pathways. Downstream from hypoxia‐induced caspase‐1 activation, cleavage and release of proinflammatory cytokines interleukin (IL)‐1β and ‐18 occurred. We further demonstrate that overexpression of HMGB1 or treatment with recombinant HMGB1 enhanced the invasiveness of HCC cells, whereas stable knockdown of HMGB1 remarkably reduced HCC invasion. Moreover, in a murine model of HCC pulmonary metastasis, stable knockdown of HMGB1 suppressed HCC invasion and metastasis. Conclusion: These results suggest that in hypoxic HCC cells, HMGB1 activates TLR4‐ and RAGE‐signaling pathways to induce caspase‐1 activation with the subsequent production of multiple inflammatory mediators, which, in turn, promote cancer invasion and metastasis. (HEPATOLOGY 2012;55:1866–1875)

β-Catenin Signaling and Roles in Liver Homeostasis, Injury, and Tumorigenesis

β-catenin (encoded by CTNNB1) is a subunit of the cell surface cadherin protein complex that acts as an intracellular signal transducer in the WNT signaling pathway; alterations in its activity have been associated with the development of hepatocellular carcinoma and other liver diseases. Other than WNT, additional signaling pathways also can converge at β-catenin. β-catenin also interacts with transcription factors such as T-cell factor, forkhead box protein O, and hypoxia inducible factor 1α to regulate the expression of target genes. We discuss the role of β-catenin in metabolic zonation of the adult liver. β-catenin also regulates the expression of genes that control metabolism of glucose, nutrients, and xenobiotics; alterations in its activity may contribute to the pathogenesis of nonalcoholic steatohepatitis. Alterations in β-catenin signaling may lead to activation of hepatic stellate cells, which is required for fibrosis. Many hepatic tumors such as hepatocellular adenomas, hepatocellular cancers, and hepatoblastomas have mutations in CTNNB1 that result in constitutive activation of β-catenin, so this molecule could be a therapeutic target. We discuss how alterations in β-catenin activity contribute to liver disease and how these might be used in diagnosis and prognosis, as well as in the development of therapeutics.

Enhanced liver regeneration following changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase

Following liver regeneration after partial hepatectomy, liver grows back precisely to its original mass and does not exceed it. The mechanism regulating this “hepatostat” is not clear and no exceptions have been found to date. Although pathways initiating liver regeneration have been well studied, mechanisms involved in the termination of liver regeneration are unclear. Here, we report that integrin‐linked kinase (ILK) (involved in transmission of the extracellular matrix [ECM] signaling by way of integrin receptors) and/or hepatic adaptations that ensue following ILK hepatocyte‐targeted removal are critical for proper termination of liver regeneration. Following partial hepatectomy (PHx), mice with a liver‐specific ILK ablation (ILK‐KO‐Liver) demonstrate a termination defect resulting in 58% larger liver than their original pre‐PHx mass. This increase in post‐PHx liver mass is due to sustained cell proliferation driven in part by increased signaling through hepatocyte growth factor (HGF), and the β‐catenin pathway and Hippo kinase pathways. Conclusion: The data indicate that ECM‐mediated signaling by way of ILK is essential in proper termination of liver regeneration. This is the first evidence of a defect leading to impaired termination of regeneration and excessive accumulation of liver weight following partial hepatectomy. (HEPATOLOGY 2009.)

Role of Wnt/β-catenin signaling in liver metabolism and cancer.

Wnt/β-catenin signaling is known for its role in embryogenesis as well as carcinogenesis. In the liver, it plays many critical roles during hepatic development and regeneration, and its dysregulation is evident in aberrant hepatic growth during various liver tumors. Its chief cellular roles in the liver include regulation of processes of cell proliferation, apoptosis, oxidative stress and differentiation, which in turn contributes to hepatic growth, zonation, xenobiotic metabolism and other metabolic processes inherent to the liver. Most of these functions of the Wnt/β-catenin signaling are dictated through the highly temporal and tissue-specific or non-specific transcriptional targets of the pathway. In addition, some of the critical functions such as cell-cell adhesion and perhaps maintenance of various junctions that are critical from an epithelial cell biology perspective are also a function of β-catenin, which is the central component of the canonical Wnt pathway. Various animal models and clinical studies have demonstrated the spectra of Wnt/β-catenin signaling in liver health and disease. Thus therapeutic modulation of this pathway for improved hepatic health is inevitable in the future. The current review discusses the advances in our understanding of the Wnt/β-catenin signaling in liver physiology and pathology especially in hepatic metabolism and various tumors in adult liver and goes on to extrapolate the pre-clinical significance and possible translational implications of such findings.