Hisataka Moriwaki

Expression of the NF-κB Target Gene X-Ray-Inducible Immediate Early Response Factor-1 Short Enhances TNF-α-Induced Hepatocyte Apoptosis by Inhibiting Akt Activation

Using a cDNA microarray analysis, we identified x-ray-inducible immediate early response factor-1 (IEX-1) as a proapoptotic gene which was induced by TNF-α and also depend on NF-κB activation in Hc human hepatocytes. In these cells only the original form of IEX-1, termed IEX-1S, but not its longer transcript IEX-1L, was expressed. Overexpression of IEX-1S resulted in promotion of TNF-α-induced apoptosis in Hc cells expressing a mutant form of IκB. This proapoptotic action can be explained by its inhibitory findings on survival signals; inhibition of TNF-α-induced activation and expression of phosphatidylinositol 3-kinase (PI3K)/Akt, and also blockage of expression of Mcl-1, an antiapoptotic Bcl-2 family member which is located downstream of Akt, was inhibited by IEX-1S. LY 294002, an inhibitor of PI3K, increased IEX-1S expression induced by TNF-α and accelerated TNF-α-induced apoptosis in IκB-treated Hc cells. Overexpression of the dominant-negative Akt enhanced, but the constitutively active Akt suppressed, TNF-α-induced IEX-1S expression, suggesting that PI3K/Akt negatively regulated IEX-1S expression. These results demonstrate that NF-κB-dependent recruitment of IEX-1S may play a proapoptotic role in TNF-α-stimulated hepatocytes through blockage of the PI3K/Akt pathway. Moreover, the reciprocal cross-talk between IEX-1S and PI3K/Akt may closely be involved in the regulation of TNF-α-induced hepatocyte apoptosis.

Leflunomide protects from T‐cell–mediated liver injury in mice through inhibition of nuclear factor κB

Leflunomide is a novel immunosuppressive and anti‐inflammatory agent for the treatment of autoimmune disease. The aim of this study was to investigate whether leflunomide protects from liver injury induced by concanavalin A (Con A), a T‐cell–dependent model of liver damage. BALB/c mice were injected with 25 mg/kg Con A in the presence or absence of 30 mg/kg leflunomide. Liver injury was assessed biochemically and histologically. Levels of circulating cytokines and expressions of cytokine messenger RNA (mRNA) in the liver and the spleen were determined. Treatment with leflunomide markedly reduced serum transaminase activities and the numbers of dead liver cells. Leflunomide significantly inhibited increases in plasma tumor necrosis factor alpha (TNF‐α) and interleukin 2 concentrations, and also reduced TNF‐α mRNA expression in the liver after administration of Con A. These findings were supported by the results in which leflunomide administration decreased the number of T lymphocytes infiltrating the liver as well as inhibiting their production of TNF‐α. Activation of nuclear factor κB (NF‐κB), which regulates TNF‐α production, was inhibited in the liver of mice treated with leflunomide, resulting in a reduction of TNF‐α production from lymphocytes infiltrating the liver. In conclusion, leflunomide is capable of regulating T‐cell–mediated liver injury in vivo and that this event may depend on the decrease of TNF‐α production in the liver through inhibition of NF‐κB activation caused by leflunomide. (HEPATOLOGY 2004.)

Normal liver regeneration and liver cell apoptosis after partial hepatectomy in tumor necrosis factor-α-deficient mice

Abstract: Aims/Background: Tumor necrosis factor‐α (TNF‐α) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF‐α also influences liver repair following hepatotoxic damage, and regeneration following partial hepatectomy (PH). The aim of this study was to assess the mechanism by which TNF‐α influences liver cell apoptosis and regeneration following PH in TNF‐α‐deficient (TNF‐α−/−) mice.

Inhibition of nuclear factor κB and phosphatidylinositol 3-kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt.

Differential caspase-9-dependent signaling pathway between tumor necrosis factor receptor- and Fas-mediated hepatocyte apoptosis in mice

Abstract: Background/Aims: Two apoptosis signaling pathways, which are used by different cell types, are identified. The activation of caspases is critical for the apoptosis process. The aim of this study was to investigate the effects of the caspase‐9 inhibitor Ac‐LEHD‐CHO on tumor necrosis factor receptor (TNFR)‐ and Fas‐mediated hepatocyte apoptosis in vivo, in order to evaluate the similarities and distinctions between TNFR‐ and Fas‐mediated signaling pathways.

Blockade of neutrophil elastase attenuates severe liver injury in hepatitis B transgenic mice.

ABSTRACT Serine proteinases produced by polymorphonuclear neutrophils play important roles in neutrophil-mediated tissue injury at inflammatory sites. Although neutrophil recruitment to the liver has been shown to be involved in the exacerbation of liver inflammation, the function of neutrophil elastase (NE) in liver injury remains unclear. Here, we found that administration of an NE inhibitor (NEI) reduced serum alanine aminotransferase (sALT) activity and inflammatory cell infiltration into the liver from 8 to 24 h after injection of antigen-specific cytotoxic T lymphocytes (CTLs) into hepatitis B virus transgenic mice. Furthermore, the NEI treatment reduced the expressions of inflammatory cytokines and chemokines in the liver and tumor necrosis factor alpha production by macrophages. In addition, the NEI treatment suppressed the mRNA expressions of CC chemokine ligand 3 (CCL-3), CCL-4, and macrophage inflammatory protein 2 (MIP-2) in neutrophils in the liver at 8 h after the CTL injection. In support of these results, we confirmed that administration of anti-CCL-3, anti-CCL-4, and anti-MIP-2 monoclonal antibodies suppressed sALT activity and leukocyte migration into the liver. In conclusion, the present results suggest that NE contributes to the early step of the inflammatory cascade in acute viral hepatitis and that NEIs may have potential as therapeutic drugs against acute severe viral hepatitis.

Functional Activity of Human Hepatoma Cells Transfected with Adenovirus-Mediated Hepatocyte Nuclear Factor (HNF)-4 Gene:

Fulminant hepatic failure (FHF) is still associated with high mortality despite recent advances in medical management. There is need of an effective and safe bioartificial liver (BAL) support to help keep patients with FHF alive until an organ becomes available for transplantation or the native liver recovers. The aim of this study was to establish highly functional liver cells by means of transfecting hepatocyte nuclear factor (HNF)-4 gene for the development of BAL. We constructed adenovirus vector carrying rat HNF-4 cDNA, and transfected to hepatoma-derived cell lines, HepG2 and HuH-7, to enforce expression of the exogenous HNF-4 gene. We analyzed expression of HNF-4, HNF-1, and liver-specific genes in cells infected by the adenovirus vector expressing HNF-4. Adenovirus-mediated HNF-4 gene transfer resulted in increases in expressions of HNF-4, HNF-1, and liver-specific genes such as apolipoproteins, α1-antitrypsin (α1-AT), phosphoenolpyruvate carboxy-kinase, cytochrome P450 families, and glutamine synthetase in transfected hepatoma cells. Cells overexpressing HNF-4 removed ammonia from medium supplemented with NH4Cl to a greater extent than control cells. These findings demonstrated that transfected cell lines restored differentiated gene expressions and liver-specific function by the overproduction of HNF-4. HNF-4-overexpressing hepatocyte cell lines are useful for bioreactor of BAL systems.

Pivotal role of nuclear factor κB signaling in anti‐CD40–induced liver injury in mice

Nuclear factor κB (NF‐κB) has a central role in coordinating the expression of a wide variety of genes that control immune responses and is also recognized as an antiapoptotic transcription factor. Here, we focused on the role of the NF‐κB signaling pathway in the interaction between inflammatory cells and hepatocytes in liver inflammation. We found that pretreatment of mice with adenoviruses expressing a mutant form of the inhibitor κB superrepressor (Ad5IκB), a NF‐κB inhibitor, reduced the migration of inflammatory cells and cytokine and chemokine expression in the liver 12 hours after a single intravenous injection of an anti‐CD40 antibody (αCD40) compared with mice infected with control adenoviruses (Ad5LacZ). We also confirmed reductions in cytokine production by macrophages, T cells, and natural killer (NK) cells in the liver of Ad5IκB‐treated mice by FACS analysis. However, αCD40 treatment in Ad5IκB‐infected mice induced elevation of serum alanine aminotransferase at 24 hours, and the liver injury was associated with massive hepatocyte apoptosis. Furthermore, interferon gamma (IFN‐γ) production by NK cells and T cells was increased and stimulated tumor necrosis factor alpha (TNF‐α) production by macrophages in the Ad5IκB‐infected liver. Moreover, the liver injury was completely suppressed by the administration of anti–IFN‐γ and anti–TNF‐α. These results suggest that inhibition of NF‐κB activity suppressed αCD40‐induced liver inflammation at an early phase, resulting in a reduction in cytokine and chemokine production, whereas it sensitized hepatocytes to TNF‐α–induced apoptosis and exacerbated liver injury at the late phase. In conclusion, NF‐κB exerts pivotal activities at inflammatory sites, and caution should be exercised in NF‐κB–targeted therapy of liver disease. (HEPATOLOGY 2004;40:1180–1189.)