Hepatocyte nuclear factor 4␣ (HNF-4␣), a liver-specific transcription factor, plays a significant role in many liver-specific functions, including lipid, glucose, drug, and ammonia metabolism, and also in embryonal liver development. However, its functions and regulation are not yet clearly understood. In this study, we constructed an adenovirus vector carrying rat HNF-4␣ cDNA and transfected the adenovirus to human hepatoma cells, HuH-7, to enforce expression of the exogenous HNF-4␣ gene. We analyzed HNF-4␣-induced genes using cDNA microarray technology, which included over 9000 genes. As a result, 62 genes showed a greater than 2.0-fold change in expression level after the viral transfection. Fifty-six genes were consistently induced by HNF-4␣ overexpression, and six genes were repressed. To assess HNF-4␣ function, we attempted to classify the genes, which had been classified by their encoding protein functions in a previous report. We could classify 45 genes. The rest of the HNF-4␣-sensitive genes were unclassified (4 genes) or not identified (13 genes). Among the classified genes, almost half of the induced genes (26 of 40) were related to metabolism genes and particularly to lipid metabolism-related genes. This cDNA microarray analysis showed that HNF-4␣ is one of the central liver metabolism regulators.Differentiation of mammalian cells is associated with changes in gene expression that are primarily controlled at the level of transcription. Tissue-specific gene transcription is regulated based on the recognition of cis-elements of the target genes, accomplished by transcription factors that have restricted tissue distributions. Transcription factors that control embryonic cell differentiation are often required to maintain and regulate gene expression in the adult cell. Liver-specific gene expression is governed by the combinatorial action of a small set of liver-enriched transcription factors as follows: hepatocyte nuclear factor-1 (HNF-1), 1 a member of the POU homeobox gene family (1); the leucine zipper dimerization family, including CCAAT/enhancer-binding protein (C/EBP) ␣ (2); Dsite-binding protein (3); and C/EBP␤/liver activator protein (4); HNF-4, a member of the steroid hormone receptor superfamily (5); and HNF-3, the DNA binding domain, which is very similar to that of the Drosophila homeotic forkhead gene (6). Although many of these factors have been shown to be important components of the differentiation process that culminates in the fully functional liver, the manner in which different members of these families participate in the determination of cell phenotypes is poorly understood. Dedifferentiated hepatoma variants (7) and intertypic rat hepatoma-human fibroblast hybrids that show extinction of liver-specific gene expression (8) are deficient for the expression only of HNF-4 and HNF-1, and re-expression of liverspecific genes in revertants correlates with the re-expression of both liver-enriched transcriptional factors. We have demonstrated that when hepatocytes are plated onto a model ba...

Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) initiates apoptosis and simultaneously activates the transcription factor, nuclear factor-kappaB (NF-kappaB), which suppresses apoptosis by an unknown mechanism. Pretreatment with TNF-alpha or interleukin-1beta (IL-1beta), which activated NF-kappaB in the liver, dramatically prevented TNF-alpha-induced liver-cell apoptosis in D-galactosamine (GalN)-sensitized mice, but not anti-Fas antibody-induced hepatotoxicity. This protective effect of TNF-alpha continued for 5 hours after TNF-alpha administration, a time course similar to that found in NF-kappaB activation after TNF-alpha administration. In mice treated with adenoviruses expressing a mutant form of IkappaB, the antiapoptotic effect of TNF-alpha was inhibited in part. Prior TNF-alpha administration was not found to block the activation of caspase-8, although caspase-3 was inhibited in mice treated with TNF-alpha plus GalN/TNF-alpha compared with mice treated with GalN/TNF-alpha. These results indicate that TNFR and Fas independently regulate murine apoptotic liver failure, and that a rapid defense mechanism induced by the activation of NF-kappaB blocks death-signaling at the initiation stage of hepatic apoptosis mediated by TNFR, probably downstream of caspase-8, but not by Fas.

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-␣) and interleukin 2 concentrations, and also reduced TNF-␣ 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-␣. Activation of nuclear factor B (NF-B), which regulates TNF-␣ production, was inhibited in the liver of mice treated with leflunomide, resulting in a reduction of TNF-␣ 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-␣ production in the liver through inhibition of NF-B activation caused by leflunomide.

Intravenous administration of tumor necrosis factor-alpha (TNF-alpha) (0.5 microg/mouse) caused hepatocyte apoptosis in BALB/c mice when they were sensitized with D-galactosamine (GalN, 20 mg/mouse). Activation of nuclear factor kappa B (NF-kappa B) and expression of apoptotic Bcl-2 family members were not significantly different between livers of mice treated with TNF-alpha alone and GalN + TNF-alpha, indicating that neither activation of NF-kappa B nor expression of Bcl-2 family is involved in the sensitization by GalN against TNF-alpha-induced hepatocyte apoptosis. To identify differentially expressed genes implicated in GalN-induced hepatocyte sensitization, we adopted mRNA fingerprinting using an arbitrarily primed polymerase chain reaction. The present analysis revealed that mRNA expression of extracellular antioxidant, selenoprotein P, was up-regulated in the livers after GalN administration. GalN-induced increase in its protein level was confirmed by Western blotting. Increased expression of this gene was also observed in the liver of mice treated with concanavalin A, but not anti-Fas antibody. mRNA of another antioxidant, glutathione peroxidase-1, was also up-regulated, and lipid peroxides were produced in the liver after GalN administration. Selenoprotein P mRNA level also increased in Huh-7 human hepatoma cells incubated with GalN (5 or 10 mM). Accordingly, formation of reactive oxygen species (ROS) was observed in GalN-treated Huh-7 cells. H(2)O(2) induced up-regulation of selenoprotein P mRNA and sensitized Huh-7 cells to TNF-alpha-induced apoptosis. These results suggest that ROS produced by GalN may play a pivotal role in hepatocyte sensitization toward TNF-alpha-induced apoptosis.

The inducible activation of NF-kappaB and constitutive activation of Akt regulate hepatocyte survival against TNF-alpha, which occurs independent of Bcl-2 families.