Yosuke Osawa

TLR4 enhances TGF-beta signaling and hepatic fibrosis.

Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-β pseudoreceptor Bambi to sensitize HSCs to TGF-β–induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-β is mediated by a MyD88–NF-κB–dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-β signaling by a TLR4-MyD88–NF-κB axis provides a novel link between proinflammatory and profibrogenic signals.

Roles for C16-ceramide and Sphingosine 1-Phosphate in Regulating Hepatocyte Apoptosis in Response to Tumor Necrosis Factor-α

Tumor necrosis factor (TNF)-α signals cell death and simultaneously induces the generation of ceramide, which is metabolized to sphingosine and sphingosine 1-phosphate (S1P) by ceramidase (CDase) and sphingosine kinase. Because the dynamic balance between the intracellular levels of ceramide and S1P (the “ceramide/S1P rheostat”) may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-α-induced apoptosis of primary hepatocytes. Endogenous C16-ceramide was elevated during TNF-α-induced apoptosis in both rat and mouse primary hepatocytes. The putative acid sphingomyelinase (ASMase) inhibitor imipramine inhibited TNF-α-induced apoptosis and C16-ceramide increase as did the knock out of ASMase. Overexpression of neutral CDase (NCDase) inhibited the TNF-α-induced increase of C16-ceramide and apoptosis in rat primary hepatocytes. Moreover, NCDase inhibited liver injury and hepatocyte apoptosis in mice treated with d-galactosamine plus TNF-α. This protective effect was abrogated by the sphingosine kinase inhibitor N,N-demethylsphingosine, suggesting that the survival effect of NCDase is due to not only C16-ceramide reduction but also S1P formation. Administration of S1P or overexpression of NCDase activated the pro-survival kinase AKT, and overexpression of dominant negative AKT blocked the survival effect of NCDase. In conclusion, activation of ASMase and generation of C16-ceramide contributed to TNF-α-induced hepatocyte apoptosis. NCDase prevented apoptosis both by reducing C16-ceramide and by activation of AKT through S1P formation. Therefore, the cross-talk between sphingolipids and AKT pathway may determine hepatocyte apoptosis by TNF-α.

Anandamide induces necrosis in primary hepatic stellate cells.

The endogenous cannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces apoptosis in many cell types. Although AEA levels are elevated in liver fibrosis, its role in fibrogenesis remains unclear. This study investigated effects of AEA in primary hepatic stellate cells (HSCs). Anandamide blocked HSC proliferation at concentrations of 1 to 10 μmol/L but did not affect HSC proliferation or activation at nanomolar concentrations. At higher concentrations (25–100 μmol/L), AEA rapidly and dose‐dependently induced cell death in primary culture‐activated and in vivo‐activated HSCs, with over 70% cell death after 4 hours at 25 μmol/L. In contrast to treatment with Fas ligand or gliotoxin, AEA‐mediated death was caspase independent and showed typical features of necrosis such as rapid adenosine triphosphate depletion and propidium iodide uptake. Anandamide‐induced reactive oxygen species (ROS) formation, and an increase in intracellular Ca2+. Pretreatment with the antioxidant glutathione or Ca2+‐chelation attenuated AEA‐induced cell death. Although the putative endocannabinoid receptors CB1, CB2, and VR1 were expressed in HSCs, specific receptor blockade failed to block cell death. Depletion of membrane cholesterol by methyl‐β‐cyclodextrin inhibited AEA binding, blocked ROS formation and intracellular Ca2+‐increase, and prevented cell death. In primary hepatocytes, AEA showed significantly lower binding and failed to induce cell death even after prolonged treatment. In conclusion, AEA efficiently induces necrosis in activated HSCs, an effect that depends on membrane cholesterol and a subsequent increase in intracellular Ca2+ and ROS. The anti‐proliferative effects and the selective killing of HSCs, but not hepatocytes, indicate that AEA may be used as a potential anti‐fibrogenic tool. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:1085–1095.)

TNF-α-Induced Sphingosine 1-Phosphate Inhibits Apoptosis Through a Phosphatidylinositol 3-Kinase/Akt Pathway in Human Hepatocytes

Human hepatocytes usually are resistant to TNF-α cytotoxicity. In mouse or rat hepatocytes, repression of NF-κB activation is sufficient to induce TNF-α-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IκBα (Ad5IκB), which almost completely blocks NF-κB activation, >80% of the cells survived 24 h after TNF-α stimulation. Here, we report that TNF-α activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-α in Ad5IκB-infected Huh-7 and Hc cells. TNF-α-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-α-induced apoptosis. Although Akt has been reported to activate NF-κB, DMS and LY 294002 failed to prevent TNF-α-induced NF-κB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-κB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-κB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-α and probably FasL.

Roles for C16-ceramide and sphingosine-1-phosphate in regulating hepatocyte apoptosis in response to TNF-α

Tumor necrosis factor (TNF)-α signals cell death and simultaneously induces the generation of ceramide, which is metabolized to sphingosine and sphingosine 1-phosphate (S1P) by ceramidase (CDase) and sphingosine kinase. Because the dynamic balance between the intracellular levels of ceramide and S1P (the “ceramide/S1P rheostat”) may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-α-induced apoptosis of primary hepatocytes. Endogenous C16-ceramide was elevated during TNF-α-induced apoptosis in both rat and mouse primary hepatocytes. The putative acid sphingomyelinase (ASMase) inhibitor imipramine inhibited TNF-α-induced apoptosis and C16-ceramide increase as did the knock out of ASMase. Overexpression of neutral CDase (NCDase) inhibited the TNF-α-induced increase of C16-ceramide and apoptosis in rat primary hepatocytes. Moreover, NCDase inhibited liver injury and hepatocyte apoptosis in mice treated with d-galactosamine plus TNF-α. This protective effect was abrogated by the sphingosine kinase inhibitor N,N-demethylsphingosine, suggesting that the survival effect of NCDase is due to not only C16-ceramide reduction but also S1P formation. Administration of S1P or overexpression of NCDase activated the pro-survival kinase AKT, and overexpression of dominant negative AKT blocked the survival effect of NCDase. In conclusion, activation of ASMase and generation of C16-ceramide contributed to TNF-α-induced hepatocyte apoptosis. NCDase prevented apoptosis both by reducing C16-ceramide and by activation of AKT through S1P formation. Therefore, the cross-talk between sphingolipids and AKT pathway may determine hepatocyte apoptosis by TNF-α.

Fatty Acid Amide Hydrolase Determines Anandamide-induced Cell Death in the Liver

The endocannabinoid anandamide (AEA) induces cell death in many cell types, but determinants of AEA-induced cell death remain unknown. In this study, we investigated the role of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in AEA-induced cell death in the liver. Primary hepatocytes expressed high levels of FAAH and were completely resistant to AEA-induced cell death, whereas primary hepatic stellate cells (HSCs) expressed low levels of FAAH and were highly sensitive to AEA-induced cell death. Hepatocytes that were pretreated or with the FAAH inhibitor URB597 isolated from FAAH-/- mice displayed increased AEA-induced reactive oxygen species (ROS) formation and were susceptible to AEA-mediated death. Conversely, overexpression of FAAH in HSCs prevented AEA-induced death. Since FAAH inhibition conferred only partial AEA sensitivity in hepatocytes, we analyzed additional factors that might regulate AEA-induced death. Hepatocytes contained significantly higher levels of glutathione (GSH) than HSCs. Glutathione depletion by dl-buthionine-(S,R)-sulfoximine rendered hepatocytes susceptible to AEA-mediated ROS production and cell death, whereas GSH ethyl ester prevented ROS production and cell death in HSCs. FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24 h at 50 μm AEA and lowering the threshold for cell death to 500 nm. Following bile duct ligation, FAAH-/- mice displayed increased hepatocellular injury, suggesting that FAAH protects hepatocytes from AEA-induced cell death in vivo. In conclusion, FAAH and GSH are determinants of AEA-mediated cell death in the liver.

Tumor Necrosis Factor Alpha-Induced Interleukin-8 Production via NF-κB and Phosphatidylinositol 3-Kinase/Akt Pathways Inhibits Cell Apoptosis in Human Hepatocytes

ABSTRACT Tumor necrosis factor alpha (TNF-α) not only induces apoptotic signals but also causes antiapoptotic and regenerative responses in the liver. However, the molecular mechanism(s) of the latter events remains unclear. In the present study, we examined TNF-α-induced genes in Hc human normal (unsensitized) hepatocytes by cDNA microarray analysis. Interleukin-8 (IL-8) induction was the most pronounced of the upregulated genes. The IL-8 protein level was also increased. IL-8 belongs to the ELR-CXC chemokine family and appears to exert mitogenic and antiapoptotic functions in other cell systems. IL-8 expression by TNF-α was inhibited when two survival signals, nuclear factor κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt, were inhibited by a mutant form of inhibitor of NF-κB (IκB); by dominant negative (kinase-dead) Akt; or by treatment with LY 294002, an inhibitor of PI3K. TNF-α induced apoptosis in Hc cells that were sensitized by inhibition of NF-κB and PI3K activation. IL-8 administration protected mice against concanavalin A-induced hepatitis in vivo. IL-8 also rescued the sensitized Hc cells, at least in part, from TNF-α-induced apoptosis in vitro. TNF-α inhibited DNA synthesis in unsensitized Hc cells in the absence of serum. Exogenous IL-8 reversed, though anti-IL-8 neutralization antibody enhanced, growth inhibition by TNF-α. These results indicate that IL-8, the production of which is stimulated by TNF-α, inhibits apoptosis of sensitized hepatocytes and releases normal (unsensitized) hepatocytes from growth inhibition induced by TNF-α.

Antiapoptotic Effect of c-Jun N-terminal Kinase-1 through Mcl-1 Stabilization in TNF-Induced Hepatocyte Apoptosis

BACKGROUND & AIMS c-Jun N-terminal Kinase (JNK) is a key regulator in tumor necrosis factor (TNF)-mediated liver injury. However, distinct roles for JNK1 and JNK2 in hepatocyte apoptosis are still unresolved. Although myeloid cell leukemia-1 (Mcl-1) has been reported as a substrate of JNK, the role of Mcl-1 and its functional regulation by JNK in TNF-induced hepatocyte apoptosis and liver injury remain to be elucidated. METHODS TNF-induced hepatocyte apoptosis was investigated in wild-type, jnk1-/- and jnk2-/- mice in vitro and in the galactosamine/TNF (GalN/TNF) liver injury model. For further analysis, we used adenoviruses expressing wild-type Mcl-1 or its substitution mutant, and the Cre/loxP system (mcl-1f/f) to delete mcl-1. RESULTS jnk2-/- Hepatocytes showed increased Mcl-1 expression and were more resistant to TNF-induced apoptosis compared with wild-type or jnk1-/- hepatocytes. Increased Mcl-1 expression in jnk2-/- hepatocytes correlated with their JNK activity, which is mediated by residual JNK1 and higher than in wild-type or jnk1-/- hepatocytes. JNK activation led to phosphorylation of Mcl-1 in hepatocytes, and this increased the half-life of the Mcl-1 protein. Overexpression of Mcl-1 confirmed its antiapoptotic effect in TNF-induced hepatocyte apoptosis in vitro and in vivo. Deletion of mcl-1 in jnk2-/- hepatocytes increased TNF-induced hepatocyte apoptosis both in vitro and in GalN/TNF-induced liver injury model. CONCLUSIONS jnk2-/- Hepatocytes are resistant to TNF-induced apoptosis. Activated JNK1 contributes to this antiapoptotic phenotype of jnk2-/- hepatocytes through phosphorylation-mediated stabilization of Mcl-1.

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

Indoleamine 2,3-dioxygenase, the l-tryptophan–degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO−/−) and 1-methyl-d-l-tryptophan–treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO−/− mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO−/− mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO−/− mice or 1-methyl-d-l-tryptophan–treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO−/− mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

Lethal hepatic apoptosis mediated by tumor necrosis factor receptor, unlike Fas-mediated apoptosis, requires hepatocyte sensitization in mice

BACKGROUND/AIMS Tumor necrosis factor a (TNF-alpha) and Fas ligand are apoptotic cell-death mediators that act by binding to their responsive receptors. The aims of this study were to assess the differences between liver cell deaths induced by TNF-alpha and anti-Fas antibody, and to investigate the mechanism by which GalN sensitizes the hepatocyte to injury by TNF-alpha. METHODS TNF-alpha or anti-Fas antibody was injected into BALB/c mice sensitized or unsensitized by D-galactosamine (GalN). Liver injury was assessed biochemically and histologically. The expressions of TNF receptor (TNFR)1 and TNFR2 mRNA in the liver were determined by Northern blot analysis. Nuclear factor-kappaB (NF-kappaB) DNA binding activity was determined by gel shift assay. RESULTS In GalN-sensitized mice, hepatocyte apoptosis and liver failure were observed after TNF-alpha injection, but neither occurred in unsensitized mice. Microscopically, GalN preceding TNF-alpha caused massive hemorrhagic liver damage with fragmented hepatocyte nuclei resembling effects of anti-Fas antibody, but GalN largely failed to sensitize to injury by this antibody. TNFR1 mRNA expression in the liver was upregulated within 3 h after GalN administration, and anti-TNFR1 antibody protected GalN-sensitized mice from hepatotoxic effects of TNF-alpha. GalN treatment failed to affect TNF-alpha-induced NF-kappaB activation. CONCLUSIONS Unlike Fas-related apoptosis, TNFR-mediated apoptosis requires hepatocyte sensitization involving TNFR1 upregulation.