Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47phox, a major cytosolic component of this oxidase. Protein kinase C ζ (PKC ζ), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC ζ in p47phox phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47phox with recombinant PKC ζ induced a time- and concentration-dependent phosphorylation of p47phox with an apparent Km value of 2 μM. Phosphopeptide mapping analysis of p47phox showed that PKC ζ phosphorylated fewer selective sites in comparison to “conventional” PKCs. Serine 303/304 and serine 315 were identified as targets of PKC ζ by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC ζ that correlated to that of p47phox. A cell-permeant-specific peptide antagonist of PKC ζ inhibited both fMLP-induced phosphorylation of p47phox and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC50 of 10 μM), but not that induced by PMA. The inhibition of PKC ζ expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 μM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47phox is a substrate for PKC ζ and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.

Triethylene glycol dimethacrylate (TEGDMA) is a dentin-bonding agent and a major component of various dental restorative biomaterials. TEGDMA monomers are released from dental resins and induce dental pulp inflammation and necrosis. In this study, we have investigated the mechanism of TEGDMA-induced cytotoxicity of fibroblasts. Treatment of cultured human gingival and pulpal fibroblasts with 0.1-3 mM of TEGDMA for 24 h induced a concentration-dependent and variable cytotoxic effect. Fifty percent of toxicity (TC(50)) was obtained with 1.2 +/- 0.9 and 2.6 +/- 1.1 mM of TEGDMA for gingival and pulpal fibroblasts, respectively. Moreover, TEGDMA-induced cytotoxicity was associated with an early and drastic depletion of cellular glutathione (GSH), which started at 15-30 min and was almost complete at 4-6 h. Antioxidants, such as Trolox (0.01 mM), ascorbate (0.2 mM), and N-acetylcysteine (NAC) (5 mM) prevented the TEGDMA-induced cytotoxicity while GSH depletion was partially inhibited. Finally, a late production of reactive oxygen species (ROS) occurred in fibroblasts treated with TEGDMA for 3-4 h, as determined by 2',7'-dichlorofluorescein fluorescence, and was completely inhibited by Trolox (5 microM). The data show that TEGDMA induced a drastic GSH depletion followed by production of ROS, which may contribute to the toxicity of gingival and pulpal fibroblasts. Antioxidants, such as NAC, ascorbate, and particularly Trolox, appear useful in preventing cell damage mediated by resin-containing dental restorative materials.

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of ␤-catenin from mice exhibiting constitutive activated ␤-catenin in the liver. Adenovirus-mediated expression of oncogenic ␤-catenin was used to isolate early targets of ␤-catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell-derived chemotaxin 2 (LECT2) gene as a direct target of ␤-catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated ␤-catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a ␤-catenin-independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by ␤-catenin. We further characterized the chemotactic property of LECT2 for human neutrophils. Finally, we have shown an up-regulation of LECT2 in human liver tumors that expressed aberrant activation of ␤-catenin signaling; these tumors constituted a subset of hepatocellular carcinomas (HCC) and most of the hepatoblastomas that were studied. In conclusion, our results show that LECT2, which encodes a protein with chemotactic properties for human neutro- H epatocellular carcinoma (HCC), the major primary liver cancer, is becoming increasingly common worldwide. 1 The prognosis for patients with HCC is rather poor. The molecular changes underlying HCC remain largely unknown despite the fact that major risk factors, such as chronic hepatitis B or C infection and exposure to hepatocarcinogens like aflatoxin B1, are well recognized. Several genetic changes have been implicated in at least 3 pathways of carcinogenesis, specifically, the p53, RB and Wnt/␤-catenin signaling pathways. 2 Deregulation of the Wnt pathway appears to be most frequent of these changes in human HCC; it occurs in about 30% to 40% of patients. 2,3 It also occurs in more than 90% of hepatoblastomas, which are rare embryonal liver tumors. 4 Mutations affecting 2 partners of the Wnt pathway have been found in liver cancers. One is a mutation that activates the ␤-catenin gene. Such mutations occur mainly in hepatitis B-negative HCC 5 and in more than 50% of hepatoblastomas. 6,7 The other is a mutation that inactivates the axin 1, and, less commonly, the axin 2 gene. 5,8,9 Mutations that activate the Wnt pathway result in ␤-catenin accumulation in the nucleus. This process, in association with LEF/TCF transcription factors, modulates the transcription of target genes. 10,11 It is now clear that the genetic program triggered by activation of ␤-catenin signaling depends on the cellular context. The ␤-catenin target genes c-myc and cyclin D1 are well

Cirrhosis is a multisystemic disease wherein inflammatory responses originating from advanced liver disease and its sequelae affect distant compartments. Patients with cirrhosis are susceptible to bacterial infections, which may precipitate acute decompensation and acute-on-chronic liver failure, both of which are associated with high short-term mortality. Innate immune cells are an essential first line of defence against pathogens. Activation of liver macrophages (Kupffer cells) and resident mastocytes generate proinflammatory and vaso-permeating mediators that induce accumulation of neutrophils, lymphocytes, eosinophils and monocytes in the liver, and promote tissue damage. During cirrhosis progression, damage-and pathogen-associated molecular patterns activate immune cells and promote development of systemic inflammatory responses which may involve different tissues and compartments. The antibacterial function of circulating neutrophils and monocytes is gradually and severely impaired as cirrhosis worsens, contributing to disease progression. The mechanisms underlying impaired antimicrobial responses are complex and incompletely understood. This review focuses on the continuous and distinct perturbations arising in innate immune cells during cirrhosis, including their impact on disease progression, as well as reviewing potential therapeutic targets.

␤-arrestins (␤arrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two ␤arrs (␤arr1 and ␤arr2) is that ␤arr1 is evenly distributed throughout the cell, whereas ␤arr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. ␤arr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in ␤arr1. In addition, using a nuclear import assay in yeast we showed that ␤arr2 is actively imported into the nucleus, suggesting that ␤arr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing ␤arr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in ␤arr2, which was sufficient to redistribute ␤arr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of ␤arr2 controls the subcellular distribution of JNK3.

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Background and AimsPatients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients.Material and MethodsBlood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions.ResultsSeveral features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion.ConclusionsGenomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies.

Cirrhosis is commonly accompanied by impaired defense functions of polymorphonuclear leucocytes (PMNs), increased patient susceptibility to infections, and hepatocellular carcinoma (HCC). PMN antimicrobial activity is dependent on a massive production of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) 2 (NADPH oxidase 2; NOX2), termed respiratory burst (RB). Rapamycin, an antagonist of mammalian target of rapamycin (mTOR), may be used in the treatment of HCC and in transplanted patients. However, the effect of mTOR inhibition on the PMN RB of patients with cirrhosis remains unexplored and was studied here using the bacterial peptide, formyl‐Met‐Leu‐Phe (fMLP), as an RB inducer. fMLP‐induced RB of PMN from patients with decompensated alcoholic cirrhosis was strongly impaired (30%‐35% of control) as a result of intracellular signaling alterations. Blocking mTOR activation (phospho‐S2448‐mTOR) with rapamycin further aggravated the RB defect. Rapamycin also inhibited the RB of healthy PMNs, which was associated with impaired phosphorylation of the NOX2 component, p47phox (phox: phagocyte oxidase), on its mitogen‐activated protein kinase (MAPK) site (S345) as well as a preferential inhibition of p38‐MAPK relative to p44/42‐MAPK. However, rapamycin did not alter the fMLP‐induced membrane association of p47phox and p38‐MAPK in patients' PMNs, but did prevent their phosphorylation at the membranes. The mTOR contribution to fMLP‐induced RB, phosphorylation of p47phox and p38‐MAPK was further confirmed by mTOR knockdown in HL‐60 cells. Finally, rapamycin impaired PMN bactericidal activity, but not bacterial uptake. Conclusion: mTOR significantly up‐regulates the PMN RB of patients with cirrhosis by p38‐MAPK activation. Consequently, mTOR inhibition by rapamycin dramatically aggravates their PMN RB defect, which may increase patients' susceptibility to infection. Thus, concerns should be raised about the use of rapamycin in immuno‐depressed patients. (HEPATOLOGY 2013)