Splenectomy enhances liver regeneration through tumor necrosis factor (TNF)-alpha following dimethylnitrosamine-induced cirrhotic rat model.

BACKGROUND/AIMS: We demonstrated that partial splenic embolization for hematological disorders in cirrhotic patients also improved liver function. Therefore, we investigated the mechanism of the beneficial effects of splenectomy on a rat cirrhotic model. METHODOLOGY: 1) Rats were administered DMN (dimethylnitrosamine) after splenectomy (splenectomized DMN rats) or a sham operation (DMN rats). 2) After completion of DMN administration, a tumor necrosis factor-alpha inhibitor (E3330) was administered on the same day as the splenectomy. Histological examination and cytokine expressions were analyzed. RESULTS: The splenectomy apparently reduced liver damage. This may be partially due to the enhancement of liver regeneration since the proliferating cell nuclear antigen labeling index in the DMN-treated liver was significantly increased by splenectomy. Tumor necrosis factor-alpha was down-regulated in the DMN rats, whereas its expression was preserved in the splenectomized DMN rats. There were no apparent differences in the number of Kupffer cells between the splenectomized DMN and the DMN rats, suggesting that the down-regulation of tumor necrosis factor-alpha may contribute to the reduction of Kupffer cells' function. In addition, a tumor necrosis factor-alpha production inhibitor (E3330) significantly reduced the proliferating cell nuclear antigen labeling index after splenectomy. CONCLUSIONS: Splenectomy, in this model, may promote liver regeneration by preserving Kupffer cell function, especially the secretion of tumor necrosis factor-alpha.

Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient.

Although hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis, its pathogenic role in fulminant hepatitis remains controversial. A 32-year-old man contracted hepatitis. Serum ALT concentration was reached to 6,970 IU/L, the lowest prothrombin time value was 16% and jaundice and stage II encephalopathy were developed. HCV RNA was detected in this patient by reverse transcription polymerase chain reaction in sera at the acute phase, and it was undetectable during the remission phase when anti-HCV was found. The entire genome of infected HCV was recovered, cloned, and sequenced from this patient, and compared with the clones of six other chronic hepatitis patients. Phylogenetic analysis revealed a clustering around genotype 2a and a deviation from the other 2a chronic hepatitis strains. Calculating the genetic distance in each subgenomic region revealed that the 5'untranslated region (5'UTR), core, nonstructural (NS) 3, and NS5A were severely deviated. Of 20 clones of the hypervariable region (HVR), 17 showed an identical sequence with the others showing a difference of only one amino acid. HCV was isolated from a fulminant hepatitis patient and its entire genome was recovered; a clustering around genotype 2a was observed, but the sequence deviated especially in 5'UTR, core, NS3, and NS5A; and monoclonality of the HVR sequence was found not only in the fulminant hepatitis patient but in a certain percentage of chronic hepatitis patients.

Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex.

Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.

Association of insulin receptor substrate proteins with Bcl-2 and their effects on its phosphorylation and antiapoptotic function.

Insulin receptor substrate (IRS) proteins are docking proteins that couple growth factor receptors to various effector molecules, including phosphoinositide-3 kinase, Grb-2, Syp, and Nck. Here we show that IRS-1 associates with the loop domain of Bcl-2 and synergistically up-regulates antiapoptotic function of Bcl-2. IRS-2 but not IRS-3 binds to Bcl-2, and IRS-1 associates with Bcl-XL but not with Bax or Bik. Overexpression of IRS-1 suppresses phosphorylation of Bcl-2 induced by stimulation with insulin, and the hypophosphorylation may lead to its enhanced antiapoptotic activity. The binding site for Bcl-2 is located on the carboxyl half-domain of IRS-1. IRS-3, which lacks the corresponding region, dominant-negatively abrogates the survival effects of IRS-1 and Bcl-2. For the antiapoptotic activity of IRS-1, binding to Bcl-2 is more critical than activating phosphoinositide-3 kinase. Our results indicate that IRS proteins transmit signals from the insulin receptor to Bcl-2, thus regulating cell survival probably through regulating phosphorylation of Bcl-2.

Hepatocyte growth factor stimulates DNA synthesis in rat preneoplastic hepatocytes but not in liver carcinoma cells.

BACKGROUND & AIMS: It is not well clarified whether hepatocyte growth factor (HGF) stimulates the growth of preneoplastic hepatocytes and liver carcinoma cells in vivo. The effect of HGF on in vivo DNA synthesis in these cells and also its effect on tyrosine phosphorylation of the HGF receptor protein (c-Met) in liver carcinoma were examined. METHODS: Lesions were induced in rats using 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). The rats were given intravenous recombinant human HGF or vehicle, and DNA synthesis was assessed by the 5-bromo-2'-deoxyuridine labeling index. Tyrosine phosphorylation of c-Met by HGF was analyzed by Western blot. RESULTS: The labeling indices were significantly higher in the HGF group than in the vehicle control group in altered foci and hyperplastic nodules (preneoplastic hepatic lesions). No significant differences in the labeling indices were observed between the two groups with carcinoma. Tyrosine phosphorylation of c-Met in carcinoma cells was unaffected by HGF administration. CONCLUSIONS: HGF promotes the growth of preneoplastic hepatocytes but does not affect the growth of liver carcinoma cells in 3'-Me-DAB-treated rats.

Transgenic expression of hepatitis C virus structural proteins in the mouse.

Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the role of viral cytopathic effects remains unclear. To study the biosynthesis of HCV structural proteins and their pathogenic role, we constructed transgenic mice, expressing type 1b HCV structural proteins (core, E1, and E2) in liver tissues. Two liver-specific promoters were used. The mouse major urinary protein (MUP) promoter has been shown to be developmentally regulated with little or no expression in utero but high-level expression after birth. The albumin (Alb) promoter provides constitutive, high levels of transgenes in live. Expression of both HCV transgenes was detected in several lines by Northern blots, HCV-specific reverse transcriptase-polymerase chain reactions (RT-PCR), and Western immunoblotting. Alb HCV lines showed higher levels of HCV expression than the MUP HCV lines. Immunohistochemical analysis revealed a predominantly cytoplasmic presence of core protein with occasional nuclear staining, and both cytoplasmic and membrane expression of the E2 protein in the transgenic livers. In both transgenes, the highest levels of both antigens were seen in perivenular hepatocytes, suggesting potential processing specificity in those cells. At six months of age, the livers of all transgenic lineages remained histologically normal. We concluded that HCV structural proteins are not directly cytopathic in this animal model.

Anti-IRS-1 monoclonal antibody, 6G5, cross-reacts to IRS-2.

We previously reported the production of anti-IRS-1 monoclonal antibodies against human IRS-1 C-terminal portion. One of anti IRS-1 monoclonal antibodies, 6G5, was found to immunologically cross-react with IRS-2, which has been reported recently. The data presented here provide information arising from the use of the anti-IRS-1 MAb, 6G5; this MAb was found to be a useful reagent in studying the functional role of IRSs in the insulin-signaling system.

Ornithine decarboxylase induction during liver regeneration in IRS-1-deficient mice.

We investigated the induction of ornithine decarboxylase during liver regeneration after partial hepatectomy in IRS-1-deficient mice. There were no significant differences in ODC activity or the time course of changes in ODC activity between IRS-1-deficient mice and wild-type mice. PI 3'-kinase activity showed similar increases in both groups of mice. Furthermore, ODC induction in IRS-1 transfected CHO cells was studied after stimulation by addition of FCS. The maximal ODC activity was 2.5-fold greater in IRS-1-transfected CHO cells than in control CHO cells. Our results suggest that the IRS-1 pathway may be involved in ODC induction. The absence of a difference in ODC and PI 3'-kinase activity in the regenerating liver between IRS-1-deficient mice and wild-type mice may have been related to the compensatory effects of IRS-2/pp190 [Araki et al. Nature (1994) 372, 186-190; Tobe et al. J.Biol.Chem. (1995) 270, 5698-5701].

Expression of insulin receptor substrate-1 in hepatocytes: an investigation using monoclonal antibodies.

To investigate the expression and subcellular distribution of insulin receptor substrate-1 in hepatocytes, which are major targets of insulin along with muscle and adipose tissue, we obtained monoclonal antibodies by immunizing mice with a fusion protein consisting of the C-terminal portion of the human insulin receptor substrate-1 and glutathione-S-transferase. Two of the monoclonal antibodies (designated as 7B3 and 6G5) were found to be useful for immunohistochemical studies. Using 6G5 we demonstrate a high level of expression of insulin receptor substrate-1 in liver cirrhosis hepatocytes and variable expression in hepatocellular carcinoma cells. These results suggest that insulin receptor substrate-1 may play a role in liver regeneration during cirrhosis and that an insulin signaling cascade may be involved in hepatocarcinogenesis.