Norio Horiguchi

Paracrine Activation of Hepatic CB1 Receptors by Stellate Cell-Derived Endocannabinoids Mediates Alcoholic Fatty Liver

Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB1 receptors. Global or hepatocyte-specific CB1 knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB1 receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase beta selectively in hepatic stellate cells. In control but not CB1 receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB1 receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB1 receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.

Cell Type–Dependent Pro- and Anti-Inflammatory Role of Signal Transducer and Activator of Transcription 3 in Alcoholic Liver Injury

BACKGROUND & AIMS Signal transducer and activator of transcription 3 (STAT3) is known to be activated in human alcoholic liver disease, but its role in the pathogenesis of alcoholic liver injury remains obscure. METHODS The role of STAT3 in alcoholic liver injury was investigated in hepatocyte-specific STAT3 knockout (H-STAT3KO) mice and macrophage/neutrophil-specific STAT3 KO (M/N-STAT3KO) mice. Alcoholic liver injury was achieved by feeding mice a liquid diet containing 5% ethanol for up to 8 weeks. RESULTS Compared with wild-type mice, feeding H-STAT3KO mice with an ethanol-containing diet induced greater hepatic steatosis, hypertriglyceridemia, and hepatic expression of lipogenic genes (sterol regulatory element-binding protein, fatty acid synthase, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase 1), but less inflammation and lower expression of hepatic proinflammatory cytokines. In contrast, ethanol-fed M/N-STAT3KO mice showed more hepatic inflammation, worse injury, and increased hepatic expression of proinflammatory cytokines compared with wild-type mice. Kupffer cells isolated from ethanol-fed H-STAT3KO mice produced similar amounts of reactive oxygen species and tumor necrosis factor alpha, whereas Kupffer cells from M/N-STAT3KO mice produced more reactive oxygen species and tumor necrosis factor alpha compared with wild-type controls. CONCLUSIONS These findings suggest that STAT3 regulates hepatic inflammation in a cell type-dependent manner during alcoholic liver injury: STAT3 in hepatocytes promotes whereas STAT3 in macrophages/Kupffer cells suppresses inflammation. In addition, activation of hepatocellular STAT3 ameliorates alcoholic fatty liver via inhibition of sterol regulatory element-binding protein 1c expression.

Hepatocyte growth factor promotes hepatocarcinogenesis through c-Met autocrine activation and enhanced angiogenesis in transgenic mice treated with diethylnitrosamine.

Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, but it is not clear whether HGF stimulates or inhibits hepatocarcinogenesis. We previously reported that HGF transgenic mice under the metallothionein gene promoter developed benign and malignant liver tumors spontaneously after 17 months of age. To elucidate the role of HGF in hepatocarcinogenesis, diethylnitrosamine (DEN) was administered to HGF transgenic mice. HGF overexpression accelerated DEN-induced hepatocarcinogenesis, often accompanied by abnormal blood vessel formation. In this study, 59% of transgenic males (versus 20% of wild-type males) and 39% of transgenic females (versus 2% of wild-type females) developed either benign or malignant liver tumors by 48 weeks (P<0.005, P<0.001, respectively). Moreover, 33% of males and 23% of female transgenic mice developed hepatocellular carcinoma (HCC), while none of the wild-type mice developed HCC (P<0.001, P<0.005, respectively). Enhanced kinase activity of the HGF receptor, Met, was detected in most of these tumors. Expression of vascular endothelial growth factor (VEGF) was up-regulated in parallel with HGF transgene expression. Taken together, our results suggest that HGF promotes hepatocarcinogenesis through the autocrine activation of the HGF-Met signaling pathway in association with stimulation of angiogenesis by HGF itself and/or indirectly through VEGF.

A ligand for peroxisome proliferator activated receptor γ inhibits cell growth and induces apoptosis in human liver cancer cells

Background and aims: Induction of apoptosis of cancer cells through ligands of nuclear hormone receptors (NHRs) is a new approach in cancer therapy. Recently, one of the NHRs, peroxisome proliferator activated receptor γ (PPARγ), has been shown to influence cell growth in certain cancer cells although its effect on hepatocellular carcinoma (HCC) has not been analysed. Methods: Experiments were conducted using three human liver cancer cell lines, PLC/PRF/5, Hep G2 and HuH-7, in vitro. These cells were exposed to troglitazone, a synthetic ligand for PPARγ, and the effects on cell growth were analysed. Results: Expression of PPARγ mRNA was detected in all three liver cancer cell lines. Activation of PPARγ by troglitazone caused a marked growth inhibition in a dose dependent manner in three hepatoma cell lines. The DNA fragmentation ELISA assay and Hoechst 33258 staining revealed that the growth inhibitory effect by adding troglitazone was due to apoptosis of PLC/PRF/5, which strongly expressed PPARγ. Troglitazone also induced activation of the cell death protease, caspase 3, but not caspase 8, in PLC/PRF/5 cells. However, expression levels of antiapoptotic factor bcl-2 and apoptosis inducing factor bax were not affected. Conclusion: Our study showed that PPARγ was expressed in human liver cancer cells and that the ligand for PPARγ, troglitazone, inhibited the growth of these cells by inducing apoptosis through caspase 3 activation, indicating that troglitazone could be potentially useful as an apoptosis inducer for the treatment of HCC.

Molecular mechanisms of alcoholic liver disease: Innate immunity and cytokines

Alcohol consumption is a predominant etiological factor in the pathogenesis of chronic liver diseases worldwide, causing fatty liver, alcoholic hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma. In the past few decades, significant progress has been made in our understanding of the molecular mechanisms underlying alcoholic liver injury. Activation of innate immunity components such as Kupffer cells, LPS/TLR4, and complements in response to alcohol exposure plays a key role in the development and progression of alcoholic liver disease (ALD). LPS activation of Kupffer cells also produces IL-6 and IL-10 that may play a protective role in ameliorating ALD. IL-6 activates signal transducer and activator of transcription 3 (STAT3) in hepatocytes and sinusoidal endothelial cells, while IL-10 activates STAT3 in Kupffer cells/macrophages, subsequently protecting against ALD. In addition, alcohol consumption also inhibits some components of innate immunity such as natural killer (NK) cells, a type of cells that play key roles in anti-viral, anti-tumor, and anti-fibrotic defenses in the liver. Ethanol inhibition of NK cells likely contributes significantly to the pathogenesis of ALD. Understanding the roles of innate immunity and cytokines in alcoholic liver injury may provide insight into novel therapeutic targets in the treatment of alcoholic liver disease.

Inflammation-associated interleukin-6/signal transducer and activator of transcription 3 activation ameliorates alcoholic and nonalcoholic fatty liver diseases in interleukin-10–deficient mice†‡

Alcoholic and nonalcoholic steatohepatitis are characterized by fatty liver plus inflammation. It is generally believed that steatosis promotes inflammation, whereas inflammation in turn aggregates steatosis. Thus, we hypothesized the deletion of interleukin (IL)‐10, a key anti‐inflammatory cytokine, exacerbates liver inflammation, steatosis, and hepatocellular damage in alcoholic and nonalcoholic fatty liver disease models that were achieved via feeding mice with a liquid diet containing 5% ethanol for 4 weeks or a high‐fat diet (HFD) for 12 weeks, respectively. IL‐10 knockout (IL‐10−/−) mice and several other strains of genetically modified mice were generated and used. Compared with wild‐type mice, IL‐10−/− mice had greater liver inflammatory response with higher levels of IL‐6 and hepatic signal transducer and activator of transcription 3 (STAT3) activation, but less steatosis and hepatocellular damage after alcohol or HFD feeding. An additional deletion of IL‐6 or hepatic STAT3 restored steatosis and hepatocellular damage but further enhanced liver inflammatory response in IL‐10−/− mice. In addition, the hepatic expression of sterol regulatory element‐binding protein 1 and key downstream lipogenic proteins and enzymes in fatty acid synthesis were down‐regulated in IL‐10−/− mice. Conversely, IL‐10−/− mice displayed enhanced levels of phosphorylated adenosine monophosphate‐activated protein kinase and its downstream targets including phosphorylated acetyl‐coenzyme A carboxylase and carnitine palmitoyltransferase 1 in the liver. Such dysregulations were corrected in IL‐10−/−IL‐6−/− or IL‐10−/−STAT3Hep−/− double knockout mice. Conclusion: IL‐10−/− mice are prone to liver inflammatory response but are resistant to steatosis and hepatocellular damage induced by ethanol or HFD feeding. Resistance to steatosis in these mice is attributable to elevation of inflammation‐associated hepatic IL‐6/STAT3 activation that subsequently down‐regulates lipogenic genes but up‐regulates fatty acid oxidation‐associated genes in the liver. (HEPATOLOGY 2011; 54:846–856)

Overexpression of hepatocyte growth factor/scatter factor promotes vascularization and granulation tissue formation in vivo

The effect of hepatocyte growth factor/scatter factor (HGF/SF) during wound healing in the skin was investigated, using HGF/SF‐overexpressing transgenic mouse model. Histological analysis of HGF/SF transgenic mouse excisional wound sites revealed increased granulation tissue with marked vascularization. Northern blot analysis demonstrated that, relative to control, vascular endothelial growth factor (VEGF) expression in transgenic skin was significantly higher at baseline and was robustly up‐regulated during wound healing. Elevated levels of VEGF protein were detected immunohistochemically, predominantly in endothelial cells and fibroblasts within the granulation tissue of HGF/SF transgenic skin. Serum levels of VEGF were also elevated in HGF/SF transgenic mice. Thus, results from our study suggest that HGF/SF has a significant effect on vascularization and granulation tissue formation during wound healing in vivo, involving with induction of VEGF.

The role of the nuclear receptor constitutive androstane receptor in the pathogenesis of non-alcoholic steatohepatitis

Background: Non-alcoholic fatty liver disease is a common liver injury, but the pathophysiological mechanisms leading to the development of non-alcoholic steatohepatitis (NASH) remain unclear. The pathological roles of the nuclear receptor constitutive androstane receptor (CAR), a key regulator of drug-metabolising enzymes, in the development of NASH were investigated. Methods and results: CAR+/+ and CAR−/− mice were given a methionine and choline-deficient (MCD) diet to establish a dietary model of NASH. Increases in serum alanine aminotransferase (ALT) and in infiltration of inflammatory cells were dominant in CAR+/+ mice at 8 weeks. There was no significant difference in the lipid concentration of the liver—namely, the first hit between CAR+/+ and CAR−/− mice. The index of lipid peroxidation increased in liver of the CAR+/+ mice, as demonstrated by 8-iso-prostaglandin F2α (F2-isoprostanes). Western blotting analysis showed that nuclear translocation of CAR occurred in CAR+/+ mice fed the MCD diet. As a result, the CAR activation caused the lipid peroxidation—namely, the second hit. The expressions of cytochrome P450 (CYP)2B10, 2C29, 3A11 all increased considerably in the CAR+/+ mice. Furthermore, α smooth muscle actin immunohistochemistry and Sirius red staining showed an increase in the degree of fibrosis in CAR+/+ mice fed the MCD diet at 16 weeks. The mRNA expressions of collagen α1(1) and the tissue inhibitor of metalloproteinase-1 were found to be elevated in CAR+/+ mice. Conclusion: CAR caused the worsening of the hepatic injury and fibrosis in the dietary model of NASH. Our results suggest that the CAR nuclear receptor may thus play a critical role in the pathogenesis of NASH.

Cytokines and STATs in Liver Fibrosis

Liver fibrosis, or cirrhosis, is a common end-stage condition of many chronic liver diseases after incomplete recovery from hepatocyte damage. During fibrosis progression, hepatocellular damage and inflammation trigger complex cellular events that result in collagen deposition and the disruption of the normal liver architecture. Hepatic stellate cell activation and transdifferentiation into myofibroblasts are key events in liver fibrogenesis. Research findings from cell culture and animal models have revealed that the Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling pathway, which can be activated by many cytokines, growth factors, and hormones, plays a critical role in hepatic fibrogenesis. This review summarizes the biological significance of diverse cytokines and their downstream signaling protein STATs in hepatic fibrogenesis.

STAT1 contributes to dsRNA inhibition of liver regeneration after partial hepatectomy in mice

Increasing evidence suggests that liver regeneration is suppressed in patients with chronic HCV infection; however, the underlying mechanisms remain unclear. Previously, we demonstrated that injection of the synthetic double‐stranded RNA (dsRNA) poly I:C to mimic viral infection suppresses liver regeneration in the partial hepatectomy (PHx) model, whereby IFN‐γ contributes to the inhibition. In this study, we examined the role of the IFN‐γ‐activated downstream signal (STAT1) and genes (IRF‐1, p21cip1, and SOCS1) in liver regeneration and hepatocyte proliferation. Results show that disruption of the STAT1 gene abolished poly I:C suppression of liver regeneration and the inhibitory effect of poly I:C on liver regeneration was diminished in IRF‐1−/− and p21cip1‐/‐mice. Treatment with IFN‐γ in vitro inhibited cell proliferation of wild‐type mouse hepatocytes, but not STAT1−/− hepatocytes. The inhibitory effect of IFN‐γ on cell proliferation was also diminished in IRF‐1−/− and p21cip1−/− hepatocytes, but enhanced in SOCS1−/− hepatocytes. Hepatocyte proliferation was unaffected by treatment with poly I:C alone, but when hepatocytes were co‐cultured with liver lymphocytes, proliferation was inhibited by IFN‐γ/STAT1‐dependent mechanisms. Moreover, in HCV‐infected livers with cirrhosis, activation of STAT1 was detected and correlated positively with liver injury (elevated serum levels of AST) but negatively with hepatocyte proliferation (hepatocyte PCNA and Ki‐67 positive immunostaining). In conclusion, STAT1 is involved in dsRNA suppression of liver regeneration; not only does STAT1 activation contribute to liver injury, it may also block liver repair through inhibition of hepatocyte proliferation in HCV‐infected patients, playing an important role in the pathogenesis of disease. (HEPATOLOGY 2006;44:955–966.)