Joy X. Jiang

Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo.

Hepatic stellate cell activation is a main feature of liver fibrogenesis. We have previously shown that phagocytosis of apoptotic bodies by stellate cells induces procollagen α1 (I) and transforming growth factor beta (TGF‐β) expression in vitro. Here we have further investigated the downstream effects of phagocytosis by studying NADPH oxidase activation and its link to procollagen α1 (I) and TGF‐β1 expression in an immortalized human stellate cell line and in several models of liver fibrosis. Phagocytosis of apoptotic bodies in LX‐1 cells significantly increased superoxide production both in the extracellular and intracellular milieus. By confocal microscopy of LX‐1 cells, increased intracellular reactive oxygen species (ROS) were detected in the cells with intracellular apoptotic bodies, and immunohistochemistry documented translocation of the NADPH oxidase p47phox subunit to the membrane. NADPH oxidase activation resulted in upregulation of procollagen α1 (I); in contrast, TGF‐β1 expression was independent of NADPH oxidase activation. This was also confirmed by using siRNA to inhibit TGF‐β1 production. In addition, with EM studies we showed that phagocytosis of apoptotic bodies by stellate cells occurs in vivo. In conclusion, these data provide a mechanistic link between phagocytosis of apoptotic bodies, production of oxidative radicals, and the activation of hepatic stellate cells. (HEPATOLOGY 2006;43:435–443.)

Liver fibrosis causes downregulation of miRNA-150 and miRNA-194 in hepatic stellate cells, and their overexpression causes decreased stellate cell activation

Activation of hepatic stellate cells (HSC) results in their proliferation and in the secretion of extracellular matrix (ECM) proteins, which leads to hepatic fibrosis. microRNAs (miRNAs) have been shown to regulate various cell functions, such as proliferation, differentiation, and apoptosis. Hence, we have analyzed the miRNAs that were differentially expressed in HSC isolated from sham-operated and bile duct-ligated rats. Expression of two miRNAs, miRNA-150 and miRNA-194, was reduced in HSC isolated from fibrotic rats compared with sham-operated animals. These two miRNAs were overexpressed in LX-2 cells, and their ability to inhibit cell proliferation, the expression of smooth muscle alpha-actin (SMA), a marker for activation, and collagen type I, a marker for ECM secretion, was determined. Overexpression of these two miRNAs resulted in a significant inhibition of proliferation (P < 0.05) and reduced SMA and collagen I levels compared with either untreated cells or nonspecific miRNA-expressing cells. Next, the protein targets of these two miRNAs were found using bioinformatics approaches. C-myb was found to be a target for miRNA-150, and rac 1 was found to be one of the targets for miRNA-194. Therefore, we studied the expression of these two proteins by overexpressing these two miRNAs in LX-2 cells and found that overexpression of miRNA-150 and miRNA-194 resulted in a significant inhibition of c-myb and rac 1 expression, respectively. We conclude that both miRNA-150 and miRNA-194 inhibit HSC activation and ECM production, at least in part, via inhibition of c-myb and rac 1 expression.

Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831 a novel NOX4/NOX1 inhibitor in vivo

Reactive oxygen species (ROS) play a key role in chronic liver injury and fibrosis. Homologs of NADPH oxidases (NOXs) are major sources of ROS, but the exact role of the individual homologs in liver disease is unknown. Our goal was to determine the role of NOX4 in liver fibrosis induced by bile duct ligation (BDL) with the aid of the pharmacological inhibitor GKT137831, and genetic deletion of NOX4 in mice. GKT137831 was either applied for the full term of BDL (preventive arm) or started at 10 day postoperatively (therapeutic arm). Primary hepatic stellate cells (HSC) from control mice with and without BDL were analyzed and the effect of NOX4 inhibition on HSC activation was also studied. FasL or TNFα/actinomycin D-induced apoptosis was studied in wild-type and NOX4(-/-) hepatocytes. NOX4 was upregulated by a TGF-β/Smad3-dependent mechanism in HSC. Downregulation of NOX4 decreased ROS production and the activation of NOX4(-/-) HSC was attenuated. NOX4(-/-) hepatocytes were more resistant to FasL or TNFα/actinomycin D-induced apoptosis. Similarly, after pharmacological NOX4 inhibition, ROS production, the expression of fibrogenic markers, and hepatocyte apoptosis were reduced. NOX4 was expressed in human livers with stage 2-3 autoimmune hepatitis. Fibrosis was attenuated by the genetic deletion of NOX4. BDL mice gavaged with GKT137831 in the preventive or the therapeutic arm displayed less ROS production, significantly attenuated fibrosis, and decreased hepatocyte apoptosis. In conclusion, NOX4 plays a key role in liver fibrosis. GKT137831 is a potent inhibitor of fibrosis and hepatocyte apoptosis; therefore, it is a promising therapeutic agent for future translational studies.

Apoptotic body engulfment by hepatic stellate cells promotes their survival by the JAK/STAT and Akt/NF-κB-dependent pathways ☆

BACKGROUND/AIMS We have previously shown that phagocytosis of apoptotic bodies (AB) by hepatic stellate cells (HSC) is profibrogenic. As HSC survival is central to the progression of liver fibrosis, our goal was to investigate if phagocytosis induces HSC survival. METHODS Apoptosis of phagocytosing HSC was studied in the presence of known apoptotic agents. The JAK/STAT- and PI3K/Akt-dependent pathways, NF-kappaB activation and expression of the anti-apoptotic proteins Mcl-1 and A1 were evaluated. Apoptosis was assessed after blocking A1 by an siRNA approach. RESULTS Phagocytosing HSC were resistant to FasL/cycloheximide or TRAIL-induced apoptosis. Inhibition of the JAK/STAT or PI3K-mediated pathways induced apoptosis of HSC. Phagocytosis induced JAK1/STAT3 phosphorylation, and this was prevented by inhibiting JAK. Translocation of STAT3 to the nucleus was also blocked by JAK inhibition. Mcl-1 expression was upregulated in a JAK-dependent manner. PI3K-dependent phosphorylation of Akt depended on NADPH oxidase activity and superoxide production. NF-kappaB activation and subsequent upregulation of A1 was observed, and A1 inhibition induced apoptosis of HSC. CONCLUSION Phagocytosis of AB promotes HSC survival by two pathways, of which the A1 dependent is more significant. This represents a new mechanism by which engulfment of AB contributes to the propagation of liver fibrosis.

Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 Plays a Key Role in Stellate Cell Activation and Liver Fibrogenesis In Vivo

BACKGROUND & AIMS Hepatocyte apoptosis and activation of hepatic stellate cells (HSC) are critical events in fibrogenesis. We previously demonstrated that phagocytosis of apoptotic hepatocytes by HSC is profibrogenic. Based on this, as well as the observation that reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase induction is central to fibrogenesis, our aim was to study the phagocytic NADPH oxidase NOX2. METHODS An in vivo phagocytosis model was developed by injecting wild type (wt) or NOX2(-/-) mice with lentiviral-green fluorescence protein (GFP) containing a hepatocyte-specific promoter, and adeno-tumor necrosis factor-related apoptosis-inducing ligand (ad-TRAIL). Fibrosis was evaluated in bile duct ligated (BDL) wt and NOX2(-/-) mice with or without gadolinium treatment. NOX2 expression was studied in human liver samples and in HSC isolated from fibrotic livers. The fibrogenic activity of NOX2 was assessed by collagen reporter assays. RESULTS In the phagocytosis model, engulfment of GFP-labeled apoptotic bodies was seen, and the expression of α-smooth muscle actin (α-SMA) and collagen I increased significantly in the wt but not in the NOX2(-/-) mice. Inhibiting apoptosis decreased the profibrogenic response. NOX2(-/-) animals exhibited significantly less fibrosis following BDL. Inactivating macrophages in wt BDL mice did not lower collagen production to the level observed in NOX2(-/-) mice, suggesting that NOX2-expressing HSC are important in fibrogenesis. NOX2 was up-regulated in HSC from fibrotic livers, and phagocytosis-induced NOX2 expression and activity were demonstrated. Based on reporter assays, production of NOX2-mediated reactive oxygen species directly induced collagen promoter activity in HSC. CONCLUSIONS Apoptosis and phagocytosis of hepatocytes directly induce HSC activation and initiation of fibrosis. NOX2, the phagocytic NADPH oxidase, plays a key role in this process and in liver fibrogenesis in vivo.

Epigenetic Modulation of miR-122 Facilitates Human Embryonic Stem Cell Self-Renewal and Hepatocellular Carcinoma Proliferation

The self-renewal capacity ascribed to hESCs is paralleled in cancer cell proliferation, suggesting that a common network of genes may facilitate the promotion of these traits. However, the molecular mechanisms that are involved in regulating the silencing of these genes as stem cells differentiate into quiescent cellular lineages remain poorly understood. Here, we show that a differentiated cell specific miR-122 exemplifies this regulatory attribute by suppressing the translation of a gene, Pkm2, which is commonly enriched in hESCs and liver cancer cells (HCCs), and facilitates self-renewal and proliferation. Through a series of gene expression analysis, we show that miR-122 expression is highly elevated in quiescent human primary hepatocytes (hPHs) but lost or attenuated in hESCs and HCCs, while an opposing expression pattern is observed for Pkm2. Depleting hESCs and HCCs of Pkm2, or overexpressing miR-122, leads to a common deficiency in self-renewal and proliferation. Likewise, during the differentiation process of hESCs into hepatocytes, a reciprocal expression pattern is observed between miR-122 and Pkm2. An examination of the genomic region upstream of miR-122 uncovered hyper-methylation in hESCs and HCCs, while the same region is de-methylated and occupied by a transcription initiating protein, RNA polymerase II (RNAPII), in hPHs. These findings indicate that one possible mechanism by which hESC self-renewal is modulated in quiescent hepatic derivatives of hESCs is through the regulatory activity of a differentiated cell-specific miR-122, and that a failure to properly turn “on” this miRNA is observed in uncontrollably proliferating HCCs.

Leptin induces phagocytosis of apoptotic bodies by hepatic stellate cells via a Rho guanosine triphosphatase–dependent mechanism

Leptin, a profibrogenic cytokine, plays an important role in the development of non‐alcoholic steatohepatitis. Leptin also regulates immune responses, including macrophage phagocytic activity. Stellate cells are key elements in liver fibrogenesis, and previously we have demonstrated that phagocytosis of apoptotic bodies by stellate cells is profibrogenic. To study the effects of leptin on the phagocytic activity of hepatic stellate cells, we exposed both LX‐2 cells and primary stellate cells to leptin, and we have observed increased phagocytic activity. In stellate cells isolated from Zucker (fa/fa) rats, the rate of phagocytosis was significantly decreased. To investigate the mechanism by which leptin induces phagocytosis, we focused on the role of Rho‐guanosine triphosphate (GTP)‐ases. We found that leptin induced the PI3K‐dependent activation of Rac1, and that nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase activation was also implicated in this process. Leptin also induced RhoA activation and translocation to the phagosomes. Expression of the constitutive active Rac1 and RhoA both increased the phagocytic rate, whereas inhibition of the Rho‐dependent kinase decreased the phagocytic activity. Conclusion: We describe a novel role of leptin in the fibrogenic process, the induction of phagocytosis of apoptotic bodies by hepatic stellate cells. The data provide strong evidence of a Rho‐GTPase–mediated regulation of the cytoskeleton during stellate cell phagocytosis. Leptin‐mediated phagocytic activity of stellate cells therefore could be an important mechanism responsible for progression of fibrosis in non‐alcoholic steatohepatitis. (HEPATOLOGY 2008.)

Hepatocyte Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice

BACKGROUND & AIMS Reactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. METHODS Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. RESULTS Levels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity. CONCLUSIONS NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

The Aryl Hydrocarbon Receptor: A Key Bridging Molecule of External and Internal Chemical Signals

The aryl hydrocarbon receptor (AhR) is a highly evolutionary conserved, ligand-activated transcription factor that is best known to mediate the toxicities of dioxins and dioxin-like compounds. Phenotype of AhR-null mice, together with the recent discovery of a variety of endogenous and plant-derived ligands, point to the integral roles of AhR in normal cell physiology, in addition to its roles in sensing the environmental chemicals. Here, we summarize the current knowledge about AhR signaling pathways, its ligands and AhR-mediated effects on cell specialization, host defense and detoxification. AhR-mediated health effects particularly in liver, immune, and nervous systems, as well as in tumorgenesis are discussed. Dioxin-initiated embryotoxicity and immunosuppressive effects in fish and birds are reviewed. Recent data demonstrate that AhR is a convergence point of multiple signaling pathways that inform the cell of its external and internal environments. As such, AhR pathway is a promising potential target for therapeutics targeting nervous, liver, and autoimmune diseases through AhR ligand-mediated interventions and other perturbations of AhR signaling. Additionally, using available laboratory data obtained on animal models, AhR-centered adverse outcome pathway analysis is useful in reexamining known and potential adverse outcomes of specific or mixed compounds on wildlife.

Wilson's disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease.

Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilsons disease (WD) through the inhibition of S‐adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S‐adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx‐j mouse (tx‐j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down‐regulated at baseline in tx‐j mice, further down‐regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx‐j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf‐α) levels. Dnmt3b was down‐regulated in tx‐j mice together with global DNA hypomethylation. PCA treatment of tx‐j mice reduced Tnf‐α and ALT levels, betaine treatment increased S‐adenosylmethionine and up‐regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. Conclusion: Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx‐j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD. (HEPATOLOGY 2013)