Induction of choline kinase alpha by carbon tetrachloride (CCl4) occurs via increased binding of c-jun to an AP-1 element.

The mechanism by which treatment of mice with CCl4 induces an increase in choline kinase alpha has been investigated. Nuclear run on assays demonstrated a major increase in the transcript for choline kinase alpha in livers from mice 3 h and 6 h after administration of CCl4 compared to vehicle (olive oil). 5'deletion analyses of choline kinase alpha promoter-luciferase constructs expressed in Hepa-1 cells identified a promoter element between -875 and -866 that was nearly identical to an AP-1 consensus site. Mutation of this AP-1 site caused a striking decrease in the expression of choline kinase alpha promoter-luciferase constructs. Electromobility shift assays with nuclear extracts from mouse liver demonstrated that c-Jun, but not c-fos, bound oligonucleotides with the AP-1 site. The amount of c-jun bound was greatly increased when hepatic nuclear extracts from mice treated with CCl4 were used. Chromatin immunoprecipitation assays confirmed that c-jun binds to the choline kinase alpha promoter. The results from these studies provide strong evidence that the choline kinase alpha promoter has a distal element (-875/-867) that binds c-jun and the binding of c-jun is enhanced by treatment with CCl4.

Cytoprotective function of tetrahydrobiopterin in rat liver ischemia/reperfusion injury.

BACKGROUND: Tetrahydrobiopterin (BH(4)) is a key coenzyme of nitric oxide synthase (NOS), which is associated with a cytoprotective function in various ischemia-reperfusion (I/R) injury models. There have been a few reports on the efficacy of BH(4) in the treatment of I/R injury in other organs; therefore, the aim of this study was to investigate the effect of BH(4) related with NOS reaction in hepatic I/R injury. METHODS: A model of 70% liver I/R injury with a 100-minute ischemic time was created in rats, and the non-ischemic lobes were then resected. The rats were given BH(4) (BH(4) group) or saline solution (saline group) before reperfusion. The specific inducible NOS blocker 1400W was used to evaluate the effect of endogenous inducible NOS in the I/R hepatic injury. Survival, nitric oxide products (nitrate and nitrite), NOS expression, and nitrotyrosine (ie, the peroxynitrite product) were measured after reperfusion. RESULTS: On day 7, the survival rate was 62.5% in the BH(4) group, as opposed to 14.3% in the saline group (P = .0004); 1400W administration to the BH(4) group decreased the survival rate to 0% (P = .003). BH(4) prevented the significant increase in total bilirubin levels (P < .01) after 12-hour reperfusion. The increases in serum alanine transaminase levels (after 3 hours and 12 hours of reperfusion) were significantly (P < .01) attenuated in the BH(4) group. BH(4) increased the nitrate/nitrite concentrations in liver tissue (P < .05) and reduced nitrotyrosine production, and the protein assay showed that BH(4) increased inducible NOS and endothelial NOS expression. Histologic examination of the liver revealed that BH(4) mitigated the damage that was caused by liver I/R. CONCLUSION: Exogenous BH(4) increased nitric oxide production, which attenuated the hepatic I/R injury.

Deletion and alanine mutation analyses for the formation of active homo- or hetero-dimer complexes of mouse choline kinase-alpha and -beta.

Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (alpha1, alpha2 and beta) that are encoded by two separate genes termed ck-alpha and ck-beta. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-alpha1 and CK-beta tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424-K430 for CK-alpha1 and Q379-K385 for CK-beta) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-alpha1 was found to be essential for alpha1/alpha1 homo-dimerization but not for alpha1/beta hetero-dimerization. A mutation of the corresponding D276 of CK-beta to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.

Structure and function of choline kinase isoforms in mammalian cells.

Choline kinase (CK) catalyzes the first phosphorylation reaction in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC), yielding phosphocholine (P-Cho) from choline and ATP in the presence of Mg(2+). This enzyme exists in mammalian cells as at least three isoforms that are encoded by two separate genes termed ck-alpha and ck-beta. Each isoform is not active in its monomeric form. The active enzyme consists of either their homo- or hetero-dimeric (or oligomeric) forms. In recent years, the roles of CK in cell growth and cell stress/defense mechanisms have been intensely investigated. These functions of CK do not seem to be directly related to the net PC biosynthesis but predict another important role of this enzyme in certain cell physiology. This review summarizes briefly the recent progress of mammalian CK study which will include the gene structure of each isoform and its possible transcriptional regulation, the active configuration of the enzyme, induction of the particular isoform in chemically induced cell stress, and the possible role of this enzyme as well as of its reaction product, P-Cho, in cell growth and other cellular physiology.

Sialadenitis in patients with chronic hepatitis C is not directly related to hepatitis C virus.

Sjogren's syndrome has been suspected to be an extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. To evaluate the association of sialadenitis with HCV infection, serum levels of salivary amylase (s-isoamylase) and antibodies to Ro (SS-A) and La (SS-B) were analyzed in 114 patients with chronic hepatitis C. Serum s-isoamylase levels were monitored before and after HCV was eradicated by interferon therapy. Immunohistochemistry and Western blotting using anti-HCV antibodies, and in situ hybridization of HCV-RNA were performed in the salivary gland. Serum s-isoamylase levels were elevated in patients with chronic hepatitis C (P<0.0001). The s-isoamylase remained high even after HCV was eradicated. The in situ hybridization did not show the presence of HCV-RNA in the salivary gland from patients with chronic hepatitis C. A protein reacting with anti-HCV-E2 antibodies was found in the cytosol fraction of normal salivary gland from HCV-negative cases. Latent sialadenitis is frequently observed in chronic hepatitis C, which is not directly related to HCV per se. The presence of a common epitope between antigenic protein in the salivary gland and the HCV-derived protein may be a possible pathogenetic mechanisms such as molecular mimicry.

Fas-ligand and perforin expression in infiltrating cytotoxic T lymphocytes in the liver of chronic hepatitis C.

Cytotoxic T cells (CTLs) are believed to play an important role in the pathogenesis of chronic hepatitis C based on histological findings of the liver and in vivo experiments. Fas-ligand-Fas and perforin dependent pathways are two major killing systems when CTLs induce their target-cell death. Thus, the present study attempts to determine whether these pathways are activated, and if they are, how they are related in chronic hepatitis C. To investigate the expression of Fas-ligand and perforin, we performed double immunofluorescent staining of liver biopsy specimens from patients with chronic hepatitis C. Fas-ligand and perforin expression was observed in mononuclear cells, and the partial coexistence of the two proteins was observed. Cells expressing both proteins were positive for CD45RO(+) T cells (active T cells), whereas cells expressing perforin were negative for CD68 (macrophages). In the cases which had sustained negative HCV-RNA over 6 months after interferon treatment, Fas-ligand was not expressed, although perforin was slightly detectable. To quantitatively assess the balance of these pathways, hepatic mRNAs of Fas-ligand and perforin were measured by quantitative RT-PCR. The ratio of Fas-ligand-mRNA/perforin-mRNA was significantly correlated with serum alanine aminotransferase (ALT) levels (r=0.913). These results suggest that both pathways are activated in chronic hepatitis C and that Fas-ligand-Fas pathway may be predominant in active inflammation.

Expression and characterization of the active molecular forms of choline/ethanolamine kinase-alpha and -beta in mouse tissues, including carbon tetrachloride-induced liver.

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.