Udayan Apte

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.)

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.)

Wnt'er in liver: Expression of Wnt and frizzled genes in mouse

The Wnt signaling pathway is essential for a wide array of developmental and physiological processes. Wnts are extracellular ligands that bind to frizzled (Fz) receptors at the membrane, canonically inducing β‐catenin nuclear translocation and activation. Although β‐catenin has been shown to be critical in liver biology, the expression of the 19 Wnt and 10 Fz genes in liver remains undetermined. We report comprehensive analysis of Wnt and Fz expression in whole liver as well as individual cell types: freshly isolated and plated hepatocytes, biliary epithelial cells, normal and activated stellate and Kupffer cells, and sinusoidal endothelial cells (SECs). Oligonucleotides for the 19 Wnt, 10 frizzled receptors genes, and secreted Frizzled‐related protein‐1 (sFRP or Fzb) were synthesized based on the available sequences. A total of 11 Wnts and 8 Fz genes and Fzb were expressed in normal liver. Although only 6 Wnt and 5 Fz genes were expressed in freshly isolated hepatocytes, 8 Wnt genes, 7 Fz genes, and Fzb were expressed in plated hepatocytes. Although 12 Wnt and 7 Fz genes were expressed in biliary tree, additional Fz9 and Fzb were only expressed in cultured biliary epithelial cells. The same 14 Wnt and 7 Fz genes were expressed in both activated and normal stellate and Kupffer cells; only Fzb was expressed in their activated state. Also, 11 Wnt, seven Fz, and Fzb genes were expressed in SECs. Conclusion: These data indicate that most Wnt and frizzled genes are expressed in the liver and might be playing important roles in liver pathobiology via canonical and noncanonical pathways. HEPATOLOGY 2007;45:195–204.)

PK11195 labels activated microglia in Alzheimer's disease and in vivo in a mouse model using PET.

Activated microglia may promote neurodegeneration in Alzheimers disease (AD) and may also help in amyloid clearance in immunization therapies. In vivo imaging of activated microglia using positron emission tomography (PET) could assist in defining the role of activated microglia during AD progression and therapeutics. We hypothesized that PK11195, a ligand that binds activated microglia, could label these cells in postmortem AD tissues and in vivo in an animal model of AD using PET. [(3)H](R)-PK11195 binding was significantly higher in AD frontal cortex compared to controls and correlated mainly with the abundance of immunohistochemically labeled activated microglia. With age, the brains of APP/PS1 transgenic mice showed progressive increase in [(3)H](R)-PK11195 binding and [(11)C](R)-PK11195 retention in vivo assessed using microPET, which correlated with the histopathological abundance of activated microglia. These results suggest that PK11195 binding in AD postmortem tissue and transgenic mice in vivo correlates with the extent of microglial activation and may help define the role of activated microglia in the pathogenesis and treatment of AD.

Activation of Wnt/β-catenin pathway during hepatocyte growth factor–induced hepatomegaly in mice†

Hepatocyte growth factor (HGF) and β‐catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that β‐catenin forms a complex with c‐Met (HGF receptor) that undergoes dissociation because of β‐catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized β‐catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF‐induced hepatomegaly is mediated, at least in part, by activation of the Wnt/β‐catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via β‐catenin activation in the liver in 1‐ and 4‐week studies. The mechanisms of β‐catenin activation in the 1‐week study included loss of c‐Met–β‐catenin association as well as canonical β‐catenin activation, leading to its nuclear translocation. In the 4‐week study, β‐catenin activation was observed via canonical mechanisms, whereas the c‐Met–β‐catenin complex remained unchanged. In both studies there was an associated increase in the E‐cadherin–β‐catenin association at the membrane. In addition, we generated liver‐specific β‐catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these β‐catenin conditionally null mice. In conclusion, β‐catenin‐ and HGF‐mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma. (HEPATOLOGY 2006;44:992–1002.)

Liver-Specific β-Catenin Knockout Mice Exhibit Defective Bile Acid and Cholesterol Homeostasis and Increased Susceptibility to Diet-Induced Steatohepatitis

Although the role of Wnt/beta-catenin signaling in liver growth and development is well established, its contribution in non-neoplastic hepatic pathologies has not been investigated. Here, we examine the role of beta-catenin in a murine model of diet-induced liver injury. Mice with hepatocyte-specific beta-catenin deletion (KO) and littermate controls were fed the steatogenic methionine and choline-deficient (MCD) diet or the corresponding control diet for 2 weeks and characterized for histological, biochemical, and molecular changes. KO mice developed significantly higher steatohepatitis and fibrosis on the MCD diet compared with wild-type mice. Both wild-type and KO livers accumulated triglyceride on the MCD diet but, unexpectedly, higher hepatic cholesterol levels were observed in KO livers on both control and MCD diets. Gene expression analysis showed that hepatic cholesterol accumulation in KO livers was not attributable to increased synthesis or uptake. KO mice had lower expression of bile acid synthetic enzymes but exhibited higher hepatic bile acid and serum bilirubin levels, suggesting defects in bile export. Therefore, loss of beta-catenin in the liver leads to defective cholesterol and bile acid metabolism in the liver and increased susceptibility to developing steatohepatitis in the face of metabolic stress.

Liver‐specific ablation of integrin‐linked kinase in mice results in abnormal histology, enhanced cell proliferation, and hepatomegaly

Hepatocyte differentiation and proliferation are greatly affected by extracellular matrix (ECM). Primary hepatocytes cultured without matrix dedifferentiate over time, but matrix overlay quickly restores differentiation. ECM also is critical in liver regeneration where ECM degradation and reconstitution are steps in the regenerative process. Integrin‐linked kinase (ILK) is a cell‐ECM‐adhesion component implicated in cell–ECM signaling by means of integrins. We investigated the role of ILK in whole liver by using the LoxP/Cre model system. ILK was eliminated from the liver by mating homozygous ILK‐floxed animals with mice expressing Cre‐recombinase under control of the α fetoprotein enhancer and albumin promoter. After ablation of ILK, animals are born normal. Soon after birth, however, they develop histologic abnormalities characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells, and increased deposition of extracellular matrix. Cell proliferation is accompanied by increased cytoplasmic and nuclear stabilization of β‐catenin. After this transient proliferation of all epithelial components, proliferation subsides and final liver to body weight ratio in livers with ILK deficient hepatocytes is two times that of wild type. Microarray analysis of gene expression during the stage of cell proliferation shows up‐regulation of integrin and matrix‐related genes and a concurrent down‐regulation of differentiation‐related genes. After the proliferative stage, however, the previous trends are reversed resulting in a super‐differentiated phenotype in the ILK‐deficient livers. Conclusion: Our results show for the first time in vivo the significance of ILK and hepatic ECM‐signaling for regulation of hepatocyte proliferation and differentiation. (HEPATOLOGY 2008;48:1932‐1941.)

β-Catenin regulation during matrigel-induced rat hepatocyte differentiation

Hepatocytes in primary cultures de-differentiate and re-differentiate following addition of Engelbreth-Holm-Swarm mouse sarcoma (matrigel) to the cultures. The Wnt/β-catenin pathway has been shown to be important in liver growth and development. Here, we investigate changes in β-catenin and its mechanism, during matrigel-induced hepatocyte differentiation. Primary rat hepatocytes were cultured for 8 days, and matrigel was added to half of the cultures. Total and nuclear protein and total RNA were extracted at different days of culture and examined for β-catenin and other Wnt pathway components. A significant increase in total β-catenin protein was observed upon matrigel addition, during hepatocyte differentiation, despite a decrease in β-catenin and frizzled-1 (Wnt receptor) expression. A concurrent decrease in the glycogen synthase kinase-3β (GSK3β), axin, and ser45/thr41-phosphorylated β-catenin proteins was observed in matrigel-treated cultures, implying decreased degradation of β-catenin. Interestingly, a decrease in nuclear β-catenin and total active β-catenin was observed in the presence of matrigel. Matrigel also induced an increased association of β-catenin with Met (hepatocyte growth factor receptor), whereas association with E-cadherin remained unchanged. This coexisted with decreased β-catenin tyrosine phosphorylation. Thus, β-catenin undergoes multifactorial regulation during matrigel-induced hepatocyte differentiation and maturation; this induces its stabilization and membrane translocation, possibly contributing to hepatocyte differentiation.

Expression of hepatocytic- and biliary-specific transcription factors in regenerating bile ducts during hepatocyte-to-biliary epithelial cell transdifferentiation.

BackgroundUnder compromised biliary regeneration, transdifferentiation of hepatocytes into biliary epithelial cells (BEC) has been previously observed in rats, upon exposure to BEC-specific toxicant methylene dianiline (DAPM) followed by bile duct ligation (BDL), and in patients with chronic biliary liver disease. However, mechanisms promoting such transdifferentiation are not fully understood. In the present study, acquisition of biliary specific transcription factors by hepatocytes leading to reprogramming of BEC-specific cellular profile was investigated as a potential mechanism of transdifferentiation in two different models of compromised biliary regeneration in rats.ResultsIn addition to previously examined DAPM + BDL model, an experimental model resembling chronic biliary damage was established by repeated administration of DAPM. Hepatocyte to BEC transdifferentiation was tracked using dipetidyl dipeptidase IV (DDPIV) chimeric rats that normally carry DPPIV only in hepatocytes. Following DAPM treatment, ~20% BEC population turned DPPIV-positive, indicating that they are derived from DPPIV-positive hepatocytes. New ductules emerging after DAPM + BDL and repeated DAPM exposure expressed hepatocyte-associated transcription factor hepatocyte nuclear factor (HNF) 4α and biliary specific transcription factor HNF1β. In addition, periportal hepatocytes expressed biliary marker CK19 suggesting periportal hepatocytes as a potential source of transdifferentiating cells. Although TGFβ1 was induced, there was no considerable reduction in periportal HNF6 expression, as observed during embryonic biliary development.ConclusionsTaken together, these findings indicate that gradual loss of HNF4α and acquisition of HNF1β by hepatocytes, as well as increase in TGFβ1 expression in periportal region, appear to be the underlying mechanisms of hepatocyte-to-BEC transdifferentiation.

Margin of Exposure (MOE)

This article is a revision of the previous edition article by Udayan M. Apte and Harihara M. Mehendale, volume 3, pp 13–14,