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Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD.

The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the alpha-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the truncated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif [Ceschini et al. (2001) EMBO J. 20, 6601-6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Deltaydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full-length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA 'phenotype'. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe.

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

Background Sympathetic nervous system (SNS) signalling regulates murine hepatic fibrogenesis through effects on hepatic stellate cells (HSC), and obesity-related hypertension with SNS activation accelerates progression of non-alcoholic fatty liver disease (NAFLD), the commonest cause of chronic liver disease. NAFLD may lead to cirrhosis. The effects of the SNS neurotransmitters norepinephrine (NE), epinephrine (EPI) and neuropeptide Y (NPY) on human primary HSC (hHSC) function and in NAFLD pathogenesis are poorly understood. Aims to determine the mechanistic effects of NE/EPI/NPY on phenotypic changes in cultured hHSC, and to study SNS signalling in human NAFLD livers. Methods Freshly isolated hHSC were assessed for expression of cathecholamine/neuropeptide Y receptors and for the synthesis of NE/EPI. The effects of NE/EPI/NPY and adrenoceptor antagonists prazosin (PRZ)/propranolol (PRL) on hHSC fibrogenic functions and the involved kinases and interleukin pathways were examined. Human livers with proven NAFLD were then assessed for upregulation of SNS signalling components. Results Activated hHSC express functional α/β-adrenoceptors and NPY receptors, which are upregulated in the livers of patients with cirrhotic NAFLD. hHSC in culture synthesize and release NE/EPI, required for their optimal basal growth and survival. Exogenous NE/EPI and NPY dose-dependently induced hHSC proliferation, mediated via p38 MAP, PI3K and MEK signalling. NE and EPI but not NPY increased expression of collagen-1α2 via TGF-β without involvement of the pro-fibrogenic cytokines leptin, IL-4 and IL-13 or the anti-fibrotic cytokine IL-10. Conclusions hHSC synthesize and require cathecholamines for optimal survival and fibrogenic functionality. Activated hHSC express directly fibrogenic α/β-adrenoceptors and NPY receptors, upregulated in human cirrhotic NAFLD. Adrenoceptor and NPY antagonists may be novel anti-fibrotic agents in human NAFLD.

Mutations in the mitochondrial genome leading to mitochondrial dysfunction have been reported in a variety of cancers. However, the potential implication of these findings in the cellular response to cancer therapeutic agents is unclear. To examine the importance of mitochondrial DNA (mitDNA) encoded functions in cancer therapeutic response, we determined the clonogenic survival of HSL2 (Rho+, HeLa subline), and its derivative cell line lacking mitDNA (Rho0) after exposure to different anticancer agents. We found that isogenic Rho0 cells lacking mitDNA were extremely resistant to adriamycin and photodynamic therapy (PDT) induced cell death, whereas the Rho+ cell line was sensitive. However, there was no measurable difference in the responses of these cell lines to either alkylating agent or gamma-radiation. We show that the development of resistance to adriamycin was not due to changes in apoptotic cell death, cell cycle response or to the uptake of adriamycin in isogenic Rho0 cells. We also demonstrate that exposure of HeLa cells to adriamycin leads to mutations in mitDNA. These studies provide direct evidence that mitDNA plays an important role in cellular sensitivity to cancer therapeutic agents.