The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity.

Ubiquitin and ubiquitin-like chains are finely balanced by conjugating and de-conjugating enzymes. Alterations in this balance trigger the response to stress conditions and are often observed in pathologies. How such changes are detected is not well understood. We identify the HSP70 chaperone as a sensor of changes in the balance between mono- and poly-NEDDylation. Upon DNA damage, the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerization and apoptosis induction, a step that requires the HSP70 ATPase activity. HSP70 binds to NEDD8, and, in vitro, the conversion of NEDD8 chains into mono-NEDD8 stimulates HSP70 ATPase activity. This effect is independent of NEDD8 conjugation onto substrates. The study indicates that the NEDD8 cycle is a regulatory module of HSP70 function. These findings may be important in tumorigenesis, as we find decreased NEDP1 levels in hepatocellular carcinoma with concomitant accumulation of NEDD8 conjugates.

Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression.

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual's level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values=0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention.

The role of stem cells/progenitor cells in liver carcinogenesis in glycine N-methyltransferase deficient mice.

Regeneration of the liver is inhibited as a result of a sustained increase in S-adenosylmethionine levels in glycine N-methyltransferase (GNMT)-/- mice. This sets the stage for normally dormant stem cells/progenitor cells to replicate and differentiate to replenish the liver parenchyma with liver cells. With time the stem cells/progenitor cells may aggregate and ultimately form liver tumors. This transformation of stem cells persists within the tumors that form in order to maintain the growth of the tumors that have formed. To test this hypothesis, GNMT-/- mice were maintained for 18 months and their livers were studied at intervals, in order to document the process of tumors formation and the identification of stem cells/progenitor cells involved in the process. Progenitor cell (OV-6 positive cells) hyperplasia was already established at 8 months in the livers of the GNMT-/- mice. This process was expanded at 18 months when liver tumors had formed. Stem cells which stained positive in the livers at 8 months and within tumors at 18 months (Oct 4 and CK 19 positive cells) were found. Fat 10, a marker for progenitor liver cells, was uniformly expressed by all tumors that developed at 8 and 18 months in GNMT-/- mice.

Salermide, a Sirtuin inhibitor with a strong cancer-specific proapoptotic effect.

Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2) belong to the family of NAD+ (nicotinamide adenine dinucleotide-positive)-dependent class III histone deacetylases and are involved in regulating lifespan. As cancer is a disease of ageing, targeting Sirtuins is emerging as a promising antitumour strategy. Here we present Salermide (N-{3-[(2-hydroxy-naphthalen-1-ylmethylene)-amino]-phenyl}-2-phenyl-propionamide), a reverse amide with a strong in vitro inhibitory effect on Sirt1 and Sirt2. Salermide was well tolerated by mice at concentrations up to 100 muM and prompted tumour-specific cell death in a wide range of human cancer cell lines. The antitumour activity of Salermide was primarily because of a massive induction of apoptosis. This was independent of global tubulin and K16H4 acetylation, which ruled out a putative Sirt2-mediated apoptotic pathway and suggested an in vivo mechanism of action through Sirt1. Consistently with this, RNA interference-mediated knockdown of Sirt1, but not Sirt2, induced apoptosis in cancer cells. Although p53 has been reported to be a target of Sirt1, genetic p53 knockdowns showed that the Sirt1-dependent proapoptotic effect of Salermide is p53-independent. We were finally able to ascribe the apoptotic effect of Salermide to the reactivation of proapoptotic genes epigenetically repressed exclusively in cancer cells by Sirt1. Taken together, our results underline Salermide's promise as an anticancer drug and provide evidence for the molecular mechanism through which Sirt1 is involved in human tumorigenesis.

Insulin-like growth factor I improves intestinal barrier function in cirrhotic rats.

BACKGROUND AND AIMS: In liver cirrhosis, disruption of the intestinal barrier facilitates bacterial translocation and spontaneous bacterial peritonitis. Insulin-like growth factor I (IGF-I) is an anabolic hormone synthesised by hepatocytes that displays hepatoprotective activities and trophic effects on the intestine. The aim of this study was to investigate the effect of IGF-I on intestinal barrier function in cirrhotic rats. METHODS: In rats with carbon tetrachloride induced cirrhosis, we investigated the effect of IGF-I therapy on: (a) portal pressure; (b) intestinal histology and permeability to endotoxin and bacteria; (c) intestinal expression of cyclooxygenase 2 (COX-2) and tumour necrosis factor alpha (TNF-alpha), two factors that influence in a positive and negative manner, respectively, the integrity of the intestinal barrier; (d) intestinal permeability to 3H-mannitol in rats with bile duct ligation (BDL); and (e) transepithelial electrical resistance (TER) of polarised monolayers of rat small intestine epithelial cells. RESULTS: IGF-I therapy reduced liver collagen expression and portal pressure in cirrhotic rats, induced improvement in intestinal histology, and caused a reduction in bacterial translocation and endotoxaemia. These changes were associated with diminished TNF-alpha expression and elevated COX-2 levels in the intestine. IGF-I reduced intestinal permeability in BDL rats and enhanced barrier function of the monolayers of epithelial intestinal cells where lipopolysaccharide (LPS) caused a decrease in TER that was reversed by IGF-I. This effect of IGF-I was associated with upregulation of COX-2 in LPS treated enterocytes. CONCLUSIONS: IGF-I enhances intestinal barrier function and reduces endotoxaemia and bacterial translocation in cirrhotic rats. IGF-I therapy might be useful in the prevention of spontaneous bacterial peritonitis in liver cirrhosis.

Expression of insulin-like growth factor I by activated hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury.

BACKGROUND/AIM: Hepatic stellate cells (HSCs) express alpha-smooth muscle actin (alphaSMA) and acquire a profibrogenic phenotype upon activation by noxious stimuli. Insulin-like growth I (IGF-I) has been shown to stimulate HSCs proliferation in vitro, but it has been reported to reduce liver damage and fibrogenesis when given to cirrhotic rats. METHODS: The authors used transgenic mice (SMP8-IGF-I) expressing IGF-I under control of alphaSMA promoter to study the influence of IGF-I synthesised by activated HSCs on the recovery from liver injury. RESULTS: The transgene was expressed by HSCs from SMP8-IGF-I mice upon activation in culture and in the livers of these animals after CCl4 challenge. Twenty four hours after administration of CCl4 both transgenic and wild type mice showed similar extensive necrosis and increased levels of serum transaminases. However at 72 hours SMP8-IGF-I mice exhibited lower serum transaminases, reduced hepatic expression of alphaSMA, and improved liver morphology compared with wild type littermates. Remarkably, at this time all eight CCl4 treated wild type mice manifested histological signs of liver necrosis that was severe in six of them, while six out of eight transgenic animals had virtually no necrosis. In SMP8-IGF-I mice robust DNA synthesis occurred earlier than in wild type animals and this was associated with enhanced production of HGF and lower TGFbeta1 mRNA expression in the SMP8-IGF-I group. Moreover, Colalpha1(I) mRNA abundance at 72 hours was reduced in SMP8-IGF-I mice compared with wild type controls. CONCLUSIONS: Targeted overexpression of IGF-I by activated HSCs restricts their activation, attenuates fibrogenesis, and accelerates liver regeneration. These effects appear to be mediated in part by upregulation of HGF and downregulation of TGFbeta1. The data indicate that IGF-I can modulate the cytokine response to liver injury facilitating regeneration and reducing fibrosis.

Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase.

Rat liver methionine adenosyltransferase incorporated 8 mol of N-ethylmaleimide per mol of subunit upon denaturation in the presence of 8 M urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme.

Regulation of rat liver S-adenosylmethionine synthetase during septic shock: role of nitric oxide.

We investigated the modulation of rat liver S-adenosylmethionine (SAM) synthetase in a model of acute sepsis. Our results show that animals treated with bacterial lipopolysaccharide experience a marked decrease in liver SAM synthetase activity. No changes were detected in the hepatic levels of SAM synthetase protein, suggesting that inactivation of the existing enzyme was the cause of the observed activity loss. Lipopolysaccharide treatment resulted in the expression of calcium-independent/cytokine-inducible nitric oxide (NO) synthase in liver and the accumulation in plasma of the NO-derived species nitrite and nitrate. NO implication in the in vivo regulation of SAM synthetase was evaluated in animals treated with the NO donor molecule 3-morpholinosydnonimine. The analysis of liver enzymatic activity, along with protein and messenger RNA levels yielded results similar to those obtained with lipopolysaccharide treatment. To assess directly the sensitivity of SAM synthetase to NO, the rat liver-purified high- and low-molecular weight forms of the enzyme were exposed to various doses of 3-morpholinosydnonimine and other NO donors such as S-nitroso-N-acetylpenicillamine, resulting in a dose-dependent inhibition of enzymatic activity. This effect was reversed by addition of the reducing agents beta-mercaptoethanol and glutathione. Finally, cysteine 121 was identified as the site of molecular interaction between NO and rat liver SAM synthetase that is responsible for the inhibition of the enzyme. To reach this conclusion, the 10 cysteine residues of the enzyme were changed to serine by site-directed mutagenesis, and the effect of NO on the various recombinant enzymes was measured.

Role of thioltransferases on the modulation of rat liver S-adenosylmethionine synthetase activity by glutathione.

Rat liver S-adenosylmethionine synthetase, high- and low-Mr forms, are regulated in vitro by the GSH/GSSG ratio at pH 8. The inhibition and oxidation constants for both forms have been calculated in the presence of thioltransferases. The mechanism of the reaction appeared to involve the formation of intramolecular disulfides. Increases of 3- to 4-fold in the oxidation constants for both S-adenosylmethionine synthetase isoenzymes in the presence of protein disulfide isomerase suggested the possibility of a thiol-disulfide exchange regulatory mechanism for this enzyme in vivo. The significance of these results is discussed on the light of the data available relating glutathione changes and modulation of enzyme activities, either in vivo and in vitro.