Valerio Pazienza

The hepatitis C virus core protein of genotypes 3a and 1b downregulates insulin receptor substrate 1 through genotype-specific mechanisms†

Both molecular and clinical evidence support a link between HCV infection and insulin resistance. We examined the in vitro interaction between the HCV core protein of genotypes 3a and 1b with the insulin‐signaling pathway. We measured the expression levels of insulin receptor substrate 1 (IRS‐1), IRS‐2, and other factors involved in the insulin signal pathway in a human hepatoma cell line (Huh‐7) transiently expressing the HCV core protein of genotypes 3a or 1b by molecular biology and biochemical techniques. The IRS‐1 (but not IRS‐2) protein level was significantly reduced in Huh‐7 expressing the core protein of both genotypes 3a and 1b, as compared to cells transfected with the empty vector. However, while the core protein of genotype 3a promoted IRS‐1 degradation through the downregulation of peroxisome proliferator‐activated receptor γ (PPARγ) and by upregulating the suppressor of cytokine signal 7 (SOCS‐7), the core protein of genotype 1b activated the mammalian target of rapamycin (mTOR). We confirmed these findings by using agonists for PPARγ (rosiglitazone) or short interfering RNAs for SOCS‐7. Conclusion: Despite the small sequence divergence of the HCV core proteins of genotypes 3a and 1b, the 2 proteins appear to interfere with the insulin signaling pathway using genotype‐specific mechanisms. (HEPATOLOGY 2007;45:1164–1171.)

Mirna Expression Profiles Identify Drivers in Colorectal and Pancreatic Cancers

Background and Aim Altered expression of microRNAs (miRNAs) hallmarks many cancer types. The study of the associations of miRNA expression profile and cancer phenotype could help identify the links between deregulation of miRNA expression and oncogenic pathways. Methods Expression profiling of 866 human miRNAs in 19 colorectal and 17 pancreatic cancers and in matched adjacent normal tissues was investigated. Classical paired t-test and random forest analyses were applied to identify miRNAs associated with tissue-specific tumors. Network analysis based on a computational approach to mine associations between cancer types and miRNAs was performed. Results The merge between the two statistical methods used to intersect the miRNAs differentially expressed in colon and pancreatic cancers allowed the identification of cancer-specific miRNA alterations. By miRNA-network analysis, tissue-specific patterns of miRNA deregulation were traced: the driving miRNAs were miR-195, miR-1280, miR-140-3p and miR-1246 in colorectal tumors, and miR-103, miR-23a and miR-15b in pancreatic cancers. Conclusion MiRNA expression profiles may identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of tissue specific cancers. miRNA-network analysis help identify altered miRNA regulatory networks that could play a role in tumor pathogenesis.

Peroxisome proliferator-activated receptor-α and -γ mRNA levels are reduced in chronic hepatitis C with steatosis and genotype 3 infection

Steatosis in chronic hepatitis C is associated with inflammation and accelerated fibrogenesis.

mIGF‐1/JNK1/SirT1 signaling confers protection against oxidative stress in the heart

Oxidative stress contributes to the pathogenesis of aging‐associated heart failure. Among various signaling pathways mediating oxidative stress, the NAD+‐dependent protein deacetylase SirT1 has been implicated in the protection of heart muscle. Expression of a locally acting insulin‐like growth factor‐1 (IGF‐1) propeptide (mIGF‐1) helps the heart to recover from infarct and enhances SirT1 expression in cardiomyocytes (CM) in vitro, exerting protection from hypertrophic and oxidative stresses. To study the role of mIGF‐1/SirT1 signaling in vivo, we generated cardiac‐specific mIGF‐1 transgenic mice in which SirT1 was depleted from adult CM in a tamoxifen‐inducible and conditional fashion. Analysis of these mice confirmed that mIGF‐1‐induced SirT1 activity is necessary to protect the heart from paraquat (PQ)‐induced oxidative stress and lethality. In cultured CM, mIGF‐1 increases SirT1 expression through a c‐Jun NH(2)‐terminal protein kinase 1 (JNK1)‐dependent signaling mechanism. Thus, mIGF‐1 protects the heart from oxidative stress via SirT1/JNK1 activity, suggesting new avenues for cardiac therapy during aging and heart failure.

Monocyte chemoattractant protein‐1 secreted by adipose tissue induces direct lipid accumulation in hepatocytes

For many years, adipose tissue has been mainly considered as an inert reservoir for storing triglycerides. Since the discovery that adipocytes may secrete a variety of bioactive molecules (hormones, chemokines, and cytokines), an endocrine and paracrine role for white adipose tissue (WAT) in the regulation of energy balance and other physiological processes has been established, particularly with regard to brain and muscle. In contrast, little is known about the interactions of WAT with liver. Hence, we examined the effect of the secretory products of WAT on hepatocytes. Conditioned medium of human WAT explants induced significant steatosis in hepatocyte cell lines. Factor(s) responsible for the conditioned medium‐induced steatosis were screened by a battery of blocking antibodies against different cytokines/chemokines shown to be secreted by WAT. In contrast to interleukin‐8 and interleukin‐6, the monocyte chemoattractant protein‐1 was capable of inducing steatosis in hepatocytes in a time‐dependent manner at concentrations similar to those found in conditioned medium. Incubation of conditioned medium with antimonocyte chemoattractant protein‐1 antibodies prevented triglyceride accumulation. Investigation of the mechanism leading to the triglyceride accumulation showed that both a diminution of apolipoprotein B secretion and an increase in phosphoenolpyruvate carboxykinase messenger RNA may be involved. Conclusion: The monocyte chemoattractant protein‐1 secreted by adipose tissue may induce steatosis not only recruiting macrophages but also acting directly on hepatocytes. (HEPATOLOGY 2008.)

Hepatitis delta virus inhibits alpha interferon signaling

Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected with hepatitis B virus. Interferon‐α (IFN‐α) is the only treatment reported to be effective in chronic hepatitis delta, albeit in a minority of patients. The molecular mechanisms underlying resistance to therapy are unclear. IFN‐α–induced activation of the Janus kinase‐signal transducer and activator of transcription (JAK‐STAT) signaling cascade is essential for the induction of an antiviral state. Interference of HDV with the JAK‐STAT pathway could be responsible for the IFN‐α resistance in chronic hepatitis delta patients. We analyzed IFN‐α–induced signal transduction through the JAK‐STAT pathway in human hepatoma cells transfected with the complete HDV genome. The expression of IFN‐α–stimulated genes was investigated with reverse transcription real‐time polymerase chain reaction (PCR). STATs and JAKs activations were examined by immunofluorescence and immunoblot. The IFN‐α–stimulated genes coding for the antiviral proteins myxovirus resistance A, double‐stranded RNA (dsRNA)‐activated protein kinase and 2′,5′‐oligoadenylate synthetase were down‐regulated in HDV‐transfected hepatoma cells in response to IFN‐α treatment. HDV severely impaired the phosphorylation of both STAT1 and STAT2, thus preventing their accumulation in the nucleus. Furthermore, HDV blocked the IFN‐α–stimulated tyrosine phosphorylation of IFN receptor‐associated JAK kinase Tyk2, without affecting either the tyrosine phosphorylation of Jak1 or the expression of type I IFN receptor subunits. Conclusions: IFN‐α–induced intracellular signaling is impaired in HDV‐transfected human hepatoma cells. HDV subverts the effect of IFN‐α by blocking Tyk2 activation, thereby resulting in selective impairment of activation and translocation to the nucleus of STAT1 and STAT2. Interference of HDV with IFN‐α signaling could represent an important mechanism of viral persistence and treatment resistance. (HEPATOLOGY 2008.)

Clock Gene Expression Levels and Relationship With Clinical and Pathological Features in Colorectal Cancer Patients

The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1Ε expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p = .002), PER1 (p = .002), PER2 (p = .011), PER3 (p = .003), and CRY2 (p = .012) were lower, whereas the expression level of TIM (p = .044) was higher. No significant difference was observed in the expression levels of CLOCK (p = .778), CRY1 (p = .600), CSNK1Ε (p = .903), and TIPIN (p = .136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p = .026), and female sex (p = .005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p = .015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p = .013) and associated with TNM stages III–IV (p = .005) and microsatellite instability (p = .015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p = .010), PER3 (p = .010), and CSNKIE (p = .024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions. (Author correspondence: g.mazzoccoli@tin.it)

Microarray analyses and molecular profiling of steatosis induction in immortalized human hepatocytes

Hepatic steatosis is an important risk factor for the development of inflammation, fibrosis and impaired liver regeneration. The factors regulating lipid accumulation and driving hepatic steatosis toward inflammation, fibrosis and impaired regeneration are largely unknown. The aim of this study was to identify major alterations in gene expression occurring in steatotic hepatocytes, and to analyze how these changes impact cellular processes associated with steatosis. Microarray gene chips and RT-PCR were performed to analyze changes in gene expression induced in fatty human immortalized hepatocytes after treatment with 50 μM oleic acid for 7 days. Lipid metabolism and triglyceride accumulation in these cells was examined by Oil-Red-O staining, thin-layer chromatography (TLC) and immunofluorescence. Caspase 3 activity, BrdU incorporation and trypan blue exclusion were used to study apoptosis, proliferation and cell viability. Finally, quantitative analysis of signalling induced by insulin was performed by Western blot. Characterization of steatosis in three hepatocyte-derived cell lines indicated that the immortalized human hepatocytes (IHH) line was the most appropriate cell line for this study. Gene expression analysis showed significant alterations in the transcription of two major classes of genes involved either in cholesterol and fatty acid biosynthesis, as well as lipid export, or in apoptosis and cell proliferation. Such changes were functionally relevant, since TLC indicated that synthesis and accumulation of triglycerides were increased in steatotic cells, while synthesis of cholesterol and fatty acids were decreased. Lipid accumulation in IHH was associated with an increased apoptosis and an inhibition of cell proliferation and viability. No detectable changes in genes associated with insulin resistance were observed in steatotic cells, but signalling induced by insulin was more efficient in steatotic IHH as compared to control cells. We conclude that IHH represent a new valuable model of steatosis, not associated with insulin resistance, to study at both the genetic and functional level factors involved in the process of lipid accumulation and steatosis-associated liver injury.

Redox homeostasis and epigenetics in non-alcoholic fatty liver disease (NAFLD)

Non-alcoholic fatty liver disease (NAFLD), an accumulation of intra-hepatic triglycerides that is often considered the hepatic manifestation of insulin resistance, is the most common cause of chronic liver disease in the Western countries with up to one third of the population affected. NAFLD is a spectrum of disturbances that encompasses various degrees of liver damage ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). NASH is characterized by hepatocellular injury/inflammation with or without fibrosis. The individuals with NAFLD develop NASH in 10% of the cases, and are also at risk of developing hepatocellular carcinoma (HCC). Epigenetic mechanisms of nuclear chromatin remodeling, such as DNA methylation, post-translational modifications of histones, and incorporation of histone variants into the chromatin are increasingly recognized as crucial factors in the pathophysiology of NAFLD. NAFLD is often accompanied by oxidative stress: reactive oxygen species (ROS) are implicated in altered reduction/oxidation (redox) reactions that attack cellular macromolecules and are detected in the liver of patients and animal models of NAFLD. In this review, we summarize recent knowledge advancements in the hepatic epigenetic and redox mechanisms, and their possible links, involved in the pathogenesis and treatment of NAFLD.

Immunopositivity for Histone MacroH2A1 Isoforms Marks Steatosis-Associated Hepatocellular Carcinoma.

Background Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. Methods We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN) and the phosphatase and tensin homolog (PTEN) liver specific knock-out (KO) mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. Results Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01) in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2), when compared to steatosis (<2% of hepatocytes positive for either isoform). The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. Conclusions These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.