Expression of proteins upregulated in hepatocellular carcinoma in patients with alcoholic hepatitis (AH) compared to non-alcoholic steatohepatitis (NASH): An immunohistochemical analysis of candidate proteins.

Both non-alcoholic steatohepatitis (NASH) and alcoholic hepatitis (AH) can lead to cirrhosis and hepatocellular carcinoma. However, the rate of progression to cirrhosis and tumorigenesis in AH is greater than that in NASH. We asked whether there are differences between the two conditions in the expression levels of proteins involved in the pathogenesis of hepatocellular carcinoma. The proteins tested were presented at the 2017 American Association for the Study of Liver Diseases (AASLD) Liver Meeting as overexpressed in hepatocellular carcinoma: KLF4, SCL19A1, FANCG, HRH-1, DNMT1, DNMT3B, TNFR2, DUSP4, EGFR, Integrin α6, HDACII, PDE3A, BCL-XL, and MTCO2. The expression of these proteins was measured in liver biopsy sections from NASH and AH patients using immunohistochemical staining with fluorescent antibodies and then quantifying the fluorescence intensity morphometrically. In AH patients, levels of all tested proteins except HRH-1 were elevated compared to normal patients. In NASH patients, KLF4, SCL19A1, FANCG, HDACII, BCL-XL levels were increased compared to normal controls while HRH-1, DNMT1 and PDE3A levels were decreased. The relative expression of all proteins studied except BCL-XL was significantly higher in AH compared to NASH. In conclusion, proteins involved in hepatocellular cancer development are more highly expressed in AH compared to NASH and normal liver, which corresponds with the higher rate of tumorigenesis in AH patients compared to NASH patients.

Altered regulation of miR-34a and miR-483-3p in alcoholic hepatitis and DDC fed mice.

MicroRNAs are small noncoding RNAs that negatively regulate gene expression by binding to the untranslated regions of their target mRNAs. Deregulation of miRNAs is shown to play pivotal roles in tumorigenesis and progression. Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. By comparing AH livers where MDBs had formed with normal livers, there were significant changes of miR-34a and miR-483-3p by RNA sequencing (RNA-Seq) analyses. Real-time PCR further shows a 3- and 6-fold upregulation (respectively) of miR-34a in the AH livers and in the livers of DDC re-fed mice, while miR-483-3p was significantly downregulated in AH and DDC re-fed mice livers. This indicates that miR-34a and miR-483-3p may be crucial for liver MDB formation. P53 mRNA was found to be significantly downregulated both in the AH livers and in the livers of DDC re-fed mice, indicating that the upregulation of miR-34a is permitted by the decrease of p53 in AH since miR-34a is a main target of p53. Overexpression of miR-34a leads to an increase of p53 targets such as p27, which inhibits the cell cycle leading to cell cycle arrest. Importantly, BRCA1 is a target gene of miR-483-3p by RNA-Seq analyses and the downregulation of miR-483-3p may be the mechanism for liver MDB formation since the BRCA1 signal was markedly upregulated in AH livers. These results constitute a demonstration of the altered regulation of miR-34a and miR-483-3p in the livers of AH and mice fed DDC where MDBs formed, providing further insight into the mechanism of MDB formation mediated by miR-34a and miR-483-3p in AH.

Increased DNA methylation in the livers of patients with alcoholic hepatitis.

Epigenetic regulation of gene expression has been suggested to play a critical role in the development of alcoholic hepatitis (AH). Although it has been shown that ethanol-induced damage in hepatocytes resulted from a change in methionine metabolism causes global gene expression changes in hepatocytes, the role of the epigenetic machinery in such processes has, however, been barely investigated. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are major molecules of epigenetic DNA modification that play an important role in the control of gene expression. Using antibodies against 5mC and 5hmC, the DNA methylation in patients with AH was examined by immunohistochemistry and quantified by morphometric image analysis. The immunoreactivity intensity of 5mC in patients with AH was significantly higher than that seen in normal controls. While there was a trend of decreased 5-hmC in patients with AH, the difference between patients with AH and normal control was not significant. Our study suggests that aberrant DNA-methylation is associated with pathogenesis of AH.

Levels of metacaspase1 and chaperones related to protein quality control in alcoholic and nonalcoholic steatohepatitis.

Efficient management of misfolded or aggregated proteins in ASH and NASH is crucial for continued hepatic viability. Cellular protein quality control systems play an important role in the pathogenesis and progression of ASH and NASH. In a recent study, elevated Mca1 expression counteracted aggregation and accumulation of misfolded proteins and extended the life span of the yeast Saccharomyces cerevisiae (Hill et al, 2014). Mca1 may also associate with Ssa1 and Hsp104 in disaggregation and fragmentation of aggregated proteins and their subsequent degradation through the ER-associated degradation (ERAD) pathway. If degradation is not available, protection of the cellular environment from a misfolded protein is accomplished by its sequestration into two distinct inclusion bodies (Kaganovich et al., 2008) called the JUNQ (JUxta Nuclear Quality control compartment) and the IPOD (Insoluble Protein Deposit). Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 all play important roles in protein quality control systems. This study aims to measure the expression of Mca1 and related chaperones involved in protein quality control in alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) compared with normal control liver biopsies. Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 expressions were measured in three to six formalin-fixed paraffin embedded ASH and NASH liver biopsies and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. Mca1, Hsp104, Ydj1 and p62 were significantly upregulated compared to control (p<0.05) in ASH specimens. Hsp40 and VCP/p97 were also uptrending in ASH. In NASH, the only significant difference was the increased expression of Hsp104 compared to control (p<0.05). Ssa1 levels were uptrending in both ASH and NASH specimens. The upregulation of Mca1, Hsp104, Ydj1 and p62 in ASH may be elicited as a response to the chronic exposure of the hepatocytes to the toxicity of alcohol. Recruitment of Mca1, Hsp104, Ydj1 and p62 may indicate that autophagy, the ERAD, JUNQ, and IPOD systems are active in ASH. Whereas in NASH, elevated Hsp104 and uptrending Ssa1 levels may indicate that autophagy and IPOD may be the only active protein quality control systems involved.

IL-8 signaling is up-regulated in alcoholic hepatitis and DDC fed mice with Mallory Denk Bodies (MDBs) present.

Chemokines and their receptors are involved in oncogenesis and in tumor progression, invasion, and metastasis. Various chemokines also promote cell proliferation and resistance to apoptosis of stressed cells. The chemokine CXCL8, also known as interleukin-8 (IL-8), is a proinflammatory molecule that has functions within the tumor microenvironment. Deregulation of IL-8 signaling is shown to play pivotal roles in tumorigenesis and progression. Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. By comparing AH livers where MDBs had formed with normal livers, there were significant changes of IL-8 signaling by RNA sequencing (RNA-Seq) analyses. Real-time PCR analysis of CXCR2 further shows a 6-fold up-regulation in AH livers and a 26-fold up-regulation in the livers of DDC re-fed mice. IL-8 mRNA was also significantly up-regulated in AH livers and DDC re-fed mice livers. This indicates that CXCR2 and IL-8 may be crucial for liver MDB formation. MDB containing balloon hepatocytes in AH livers had increased intensity of staining of the cytoplasm for both CXCR2 and IL-8. Overexpression of IL-8 leads to an increase of the mitogen activated protein kinase (MAPK) cascade and exacerbates the inflammatory cycle. These observations constitute a demonstration of the altered regulation of IL-8 signaling in the livers of AH and mice fed DDC where MDBs formed, providing further insight into the mechanism of MDB formation mediated by IL-8 signaling in AH.

FAT10 suppression stabilizes oxidized proteins in liver cells: Effects of HCV and ethanol.

FAT10 belongs to the ubiquitin-like modifier (ULM) family that targets proteins for degradation and is recognized by 26S proteasome. FAT10 is presented on immune cells and under the inflammatory conditions, is synergistically induced by IFNγ and TNFα in the non-immune (liver parenchymal) cells. It is not clear how viral proteins and alcohol regulate FAT10 expression on liver cells. In this study, we aimed to investigate whether FAT10 expression on liver cells is activated by the innate immunity factor, IFNα and how HCV protein expression in hepatocytes and ethanol-induced oxidative stress affect the level of FAT10 in liver cells. For this study, we used HCV(+) transgenic mice that express structural HCV proteins and their HCV(-) littermates. Mice were fed Lieber De Carli diet (control and ethanol) as specified in the NIH protocol for chronic-acute ethanol feeding. Alcohol exposure enhanced steatosis, induced oxidative stress and decreased proteasome activity in the liversof these mice, with more robust response to ethanol in HCV(+) mice. IFNα induced transcriptional activation of FAT10 in liver cells, which was dysregulated by ethanol feeding. Accordingly, IFNα-activated expression of FAT10 in hepatocytes (measured by indirect immunofluorescent of liver tissue) was also suppressed by ethanol exposure in both HCV(+) and HCV(-) mice. This suppression was accompanied with ethanol-mediated induction of lipid peroxidation marker, 4-HNE. All aforementioned effects of ethanol were attenuated by in vivo feeding of mice with the pro-methylating agent, betaine, which exhibits strong anti-oxidant properties. Based on this study, we hypothesize that FAT10 targets oxidatively modified proteins for proteasomal degradation, and that the reduction in FAT10 levels along with decreased proteasome activity may contribute to stabilization of these altered proteins in hepatocytes. In conclusion, IFNα induced FAT10 expression, which is suppressed by ethanol feeding in both HCV(+) and HCV(-) mice. Betaine treatment reverses HCV-ethanol induced dysregulation of protein methylation and oxidative stress, thereby restoring the FAT10 expression on liver cells.

Increased activity of the complement system in the liver of patients with alcoholic hepatitis.

Inflammation has been suggested as a mechanism underlying the development of alcoholic hepatitis (AH). The activation of the complement system plays an important role in inflammation. Although it has been shown that ethanol-induced activation of the complement system contributes to the pathophysiology of ethanol-induced liver injury in mice, whether ethanol consumption activates the complement system in the human liver has not been investigated. Using antibodies against C1q, C3, and C5, the immunoreactivity of the complement system in patients with AH was examined by immunohistochemistry and quantified by morphometric image analysis. The immunoreactivity intensity of C1q, C3, and C5 in patients with AH was significantly higher than that seen in normal controls. Further, the gene expression of C1q, C3, and C5 was examined using real-time PCR. There were increases in the levels of C1q and C5, but not C3 mRNA in AH. Moreover, the immunoreactivity of C5a receptor (C5aR) also increased in AH. To explore the functional implication of the activation of the complement system in AH, we examined the colocalization of C5aR in Mallory-Denk bodies (MDBs) forming balloon hepatocytes. C5aR was focally overexpressed in the MDB forming cells. Collectively, our study suggests that alcohol consumption increases the activity of the complement system in the liver cells, which contributes to the inflammation-associated pathogenesis of AH.

Mallory-Denk Body (MDB) formation modulates Ufmylation expression epigenetically in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH).

Promoter CpG island hypermethylation is an important mechanism for inactivating key cellular enzymes that mediate epigenetic processes in hepatitis-related hepatocellular carcinoma (HCC). The ubiquitin-fold modifier 1 (Ufm1) conjugation pathway (Ufmylation) plays an essential role in protein degradation, protein quality control and signal transduction. Previous studies showed that the Ufmylation pathway was downregulated in alcoholic hepatitis (AH), non-alcoholic steatohepatitis (NASH) and in mice fed DDC, resulting in the formation of Mallory-Denk Bodies (MDBs). In this study, we further discovered that betaine, a methyl donor, fed together with DDC significantly prevents the increased expression of Ufmylation in drug-primed mice fed DDC. Betaine significantly prevented transcript silencing of Ufm1, Uba5 and UfSP1 where MDBs developed and also prevented the increased expression of FAT10 and LMP7 caused by DDC re-fed mice. Similar downregulation of Ufmylation was observed in multiple AH and NASH biopsies which had formed MDBs. The DNA methylation levels of Ufm1, Ufc1 and UfSP1 in the promoter CpG region were significantly increased both in AH and NASH patients compared to normal subjects. DNA (cytosine-5-)-methyltransferase 1 (DNMT1) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) mRNA levels were markedly upregulated in AH and NASH patients, implying that the maintenance of Ufmylation methylation might be mediated by DNMT1 and DNMT3B together. These data show that MDB formation results from Ufmylation expression epigenetically in AH and NASH patients. Promoter CpG methylation may be a major mechanism silencing Ufmylation expression.

The inflammasome in alcoholic hepatitis: Its relationship with Mallory-Denk body formation.

Recent studies indicate that the inflammasome activation plays important roles in the pathogenesis of alcoholic hepatitis (AH). Nod-like receptor protein 3 (NLRP3) is a key component of the macromolecular complex that is so called the inflammasome that triggers caspase 1-dependent maturation of the precursors of IL-1ß and IL-18 cytokines. It is also known that the adaptor proteins including apoptosis-associated speck-like protein containing CARD (ASC) and the mitochondrial antiviral signaling protein (MAVS) are necessary for NLRP3-dependent inflammasome function. Steatohepatitis frequently includes Mallory-Denk body (MDB) formation. In the case of alcoholic steatohepatitis, MDB formation occurs in 80% of biopsies (French 1981; French 1981). While previous studies have focused on in vitro cell lines and mouse models, we are the first group to investigate inflammasome activation in AH liver biopsy specimen and correlate it with MDB formation. Expression of NOD1, NLRP3, ASC, NAIP, MAVS, caspase 1, IL-1ß, IL-18, and other inflammatory components including IL-6, IL-10, TNF-α, IFN-γ, STAT3, and p65 was measured in three to eight formalin-fixed paraffin-embedded AH specimens and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. The specimens were double stained with ubiquitin to demonstrate the relationship between inflammasome activation and MDB formation. MAVS, caspase1, IL-18, and TNF-α showed increases in expression in AH compared to the controls (p<0.05), and NAIP expression markedly increased in AH compared to the controls (p<0.01). There was a trend that levels of NLRP3, ASC, caspase1, IL-18, IL-10, and p65 expression correlated with the number of MDBs found in the same field of measurement (correlation coefficients were between 0.62 and 0.93, p<0.05). Our results demonstrate the activation of the inflammasome in AH and suggest that MDB could be an indicator of the extent of inflammasome activation.

TLR3/4 signaling is mediated via the NFκB-CXCR4/7 pathway in human alcoholic hepatitis and non-alcoholic steatohepatitis which formed Mallory-Denk bodies.

Activation of Toll-like receptor (TLR) signaling which stimulates inflammatory and proliferative pathways is the key element in the pathogenesis of Mallory-Denk bodies (MDBs) in mice fed DDC. However, little is known as to how TLR signaling is regulated in MDB formation during chronic liver disease development. The first systematic study of TLR signaling pathway transcript regulation in human archived formalin-fixed, paraffin-embedded (FFPE) liver biopsies with MDB formation is presented here. When compared to the activation of Toll-like signaling in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH) patients, striking similarities and obvious differences were observed. Similar TLRs (TLR3 and TLR4, etc.), TLR downstream adaptors (MyD88 and TRIF, etc.) and transcript factors (NFκB and IRF7, etc.) were all upregulated in the patients' livers. MyD88, TLR3 and TLR4 were significantly induced in the livers of AH and NASH compared to normal subjects, while TRIF and IRF7 mRNA were only slightly upregulated in AH patients. This is a different pathway from the induction of the TLR4-MyD88-independent pathway in the AH and NASH patients with MDBs present. Importantly, chemokine receptor 4 and 7 (CXCR4/7) mRNAs were found to be induced in the patients livers in FAT10 positive hepatocytes. The CXCR7 pathway was significantly upregulated in patients with AH and the CXCR4 was markedly upregulated in patients with NASH, indicating that CXCR4/7 is crucial in liver MDB formation. This data constitutes the first demonstration of the upregulation of the MyD88-dependent TLR4/NFκB pathway in AH and NASH where MDBs formed, via the NFκB-CXCR4/7 pathway, and provides further insight into the mechanism of MDB formation in human liver diseases.

Alcoholic and non-alcoholic steatohepatitis.

This paper is based upon the "Charles Lieber Satellite Symposia" organized by Manuela G. Neuman at the Research Society on Alcoholism (RSA) Annual Meetings, 2013 and 2014. The present review includes pre-clinical, translational and clinical research that characterize alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH). In addition, a literature search in the discussed area was performed. Strong clinical and experimental evidence lead to recognition of the key toxic role of alcohol in the pathogenesis of ALD. The liver biopsy can confirm the etiology of NASH or alcoholic steatohepatitis (ASH) and assess structural alterations of cells, their organelles, as well as inflammatory activity. Three histological stages of ALD are simple steatosis, ASH, and chronic hepatitis with hepatic fibrosis or cirrhosis. These latter stages may also be associated with a number of cellular and histological changes, including the presence of Mallory's hyaline, megamitochondria, or perivenular and perisinusoidal fibrosis. Genetic polymorphisms of ethanol metabolizing enzymes such as cytochrome p450 (CYP) 2E1 activation may change the severity of ASH and NASH. Alcohol mediated hepatocarcinogenesis, immune response to alcohol in ASH, as well as the role of other risk factors such as its co-morbidities with chronic viral hepatitis in the presence or absence of human immunodeficiency virus are discussed. Dysregulation of hepatic methylation, as result of ethanol exposure, in hepatocytes transfected with hepatitis C virus (HCV), illustrates an impaired interferon signaling. The hepatotoxic effects of ethanol undermine the contribution of malnutrition to the liver injury. Dietary interventions such as micro and macronutrients, as well as changes to the microbiota are suggested. The clinical aspects of NASH, as part of metabolic syndrome in the aging population, are offered. The integrative symposia investigate different aspects of alcohol-induced liver damage and possible repair. We aim to (1) determine the immuno-pathology of alcohol-induced liver damage, (2) examine the role of genetics in the development of ASH, (3) propose diagnostic markers of ASH and NASH, (4) examine age differences, (5) develop common research tools to study alcohol-induced effects in clinical and pre-clinical studies, and (6) focus on factors that aggravate severity of organ-damage. The intention of these symposia is to advance the international profile of the biological research on alcoholism. We also wish to further our mission of leading the forum to progress the science and practice of translational research in alcoholism.

SAMe treatment prevents the ethanol-induced epigenetic alterations of genes in the Toll-like receptor pathway.

Prior studies showed that Toll-like receptor (TLR) signaling pathway genes were upregulated in the liver of rats fed ethanol, but not in rats fed ethanol plus S-adenosylmethionine (SAMe). These results were obtained using a PCR microplate array analysis for TLRs and associated proteins such as proinflammatory cytokines and chemokine mRNA levels. A large number of genes were upregulated by the ethanol diet, but not the ethanol plus SAMe diet. In the present study, using the same experimental rat livers, DNA methylation analysis was done by using an Epitect Methyl DNA Restriction Kit (Qiagen, 335451) (24 genes). The results of all the genes combined show a highly significant increase in methylation in the ethanol-fed group of rats, but not in the dextrose-fed, SAMe-fed or ethanol plus SAMe-fed groups of rats. There was also an increase in DNA methylation in rats with high blood alcohol levels compared to a rat with a low blood alcohol level. The individual genes that were upregulated as indicated by the increased mRNA measured by qPCR correlated positively with the increased methylation of the DNA of the corresponding genes as follows: Cd14, Hspa1a, Irf1, Irak1, Irak2, Map3k7, Myd88, Pparα, Ripk2, Tollip and Traf6.

Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.

BACKGROUND & AIMS: The tumor suppressor PTEN inhibits AKT2 signaling; both are aberrantly expressed in liver tumors. We investigated how PTEN and AKT2 regulate liver carcinogenesis. Loss of PTEN leads to spontaneous development of liver tumors from progenitor cells. We investigated how the loss of PTEN activates liver progenitor cells and induces tumorigenesis. METHODS: We studied mice with liver-specific disruptions in Pten and the combination of Pten and Akt2 to investigate mechanisms of liver carcinogenesis. RESULTS: PTEN loss leads to hepatic injury and establishes selective pressure for tumor-initiating cells (TICs), which proliferate to form mixed-lineage tumors. The Pten-null mice had increasing levels of hepatic injury before proliferation of hepatic progenitors. Attenuation of hepatic injury by deletion of Akt2 reduced progenitor cell proliferation and delayed tumor development. In Pten/Akt2-null mice given 3,5-diethoxycarbonyl-1,4 dihydrocollidine (DDC), we found that the primary effect of AKT2 loss was attenuation of hepatic injury and not inhibition of progenitor-cell proliferation in response to injury. CONCLUSIONS: Liver carcinogenesis in Pten-null mice requires not only the transformation of TICs but selection pressure from hepatic injury and cell death, which activates TICs. Further research is required to elucidate the mechanism for hepatic injury and its relationship with TIC activation.

Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice.

UNLABELLED: Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. CONCLUSION: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes.

Proteasome inhibitor up regulates liver antioxidative enzymes in rat model of alcoholic liver disease.

Oxidative stress occurs in the liver of rats fed with alcohol chronically due to ethanol metabolism by CYP2E1, causing liver injury. The proteasome is considered as an antioxidant defense in the cell because of its activity in removing damaged and oxidized proteins, but a growing body of evidence shows that proteasome inhibitor treatment, at a non toxic low dose, provides protection against oxidative stress. In the present study, rats were fed with ethanol for 4 weeks and were treated with the proteasome inhibitor PS-341 (Bortezomib, Velcade®). Exposure to proteasome inhibitor elicited the elevation of antioxidative defense by enhancing the levels of mRNA and protein expression transcripts of glutathione reductase (GSR), glutathione synthetase (GSS), glutathione peroxidase 2 (GPX2), and superoxide dismutase 2 (SOD2) in the liver of rats fed with ethanol chronically, while ethanol alone did not increase these genes' mRNA. Our results also showed that glutamate cysteine ligase catalytic subunit (GCLC), a rate-limiting enzyme in glutathione biosynthesis, was also up regulated in the liver of rats fed with ethanol and injected with PS-431. Nrf2 mRNA level was significantly decreased in the liver of ethanol fed rats, as well as in the livers of animal fed with ethanol and treated with proteasome inhibitor, indicating that the mechanism by which proteasome inhibitor up regulates the antioxidant response element is not due to regulation of Nrf2. However, ATF4, a major regulator of antioxidant response elements, was significantly up regulated by proteasome inhibitor treatment. The beneficial effects of proteasome inhibitor treatment also reside in the reversibility of the drug because the proteasome activity was significantly increased 72 h post treatment. In conclusion, proteasome inhibitor treatment used at a non toxic low dose has potential protective effects against oxidative stress due to chronic ethanol feeding.

S-adenosylmethionine prevents the up regulation of Toll-like receptor (TLR) signaling caused by chronic ethanol feeding in rats.

Toll-like receptors (TLR) play a role in mediating the proinflammatory response, fibrogenesis and carcinogenesis in chronic liver diseases such as alcoholic liver disease, non-alcoholic liver disease, hepatitis C and hepatocellular carcinoma. This is true in experimental models of these diseases. For this reason, we investigated the TLR proinflammatory response in the chronic intragastric tube feeding rat model of alcohol liver disease. The methyl donor S-adenosylmethionine was also fed to prevent the gene expression changes induced by ethanol. Ethanol feeding tended to increase the up regulation of the gene expression of TLR2 and TLR4. SAMe feeding prevented this. TLR4 and MyD88 protein levels were significantly increased by ethanol and this was prevented by SAMe. This is the first report where ethanol feeding induced TLR2 and SAMe prevented the induction by ethanol. CD34, FOS, interferon responsive factor 1 (IRF-1), Jun, TLR 1,2,3,4,6 and 7 and Traf-6 were found to be up regulated as seen by microarray analysis where rats were sacrificed at high blood alcohol levels compared to pair fed controls. Il-6, IL-10 and IFNγ were also up regulated by high blood levels of ethanol. The gene expression of CD14, MyD88 and TNFR1SF1 were not up regulated by ethanol but were down regulated by SAMe. The gene expression of IL-1R1 and IRF1 tended to be up regulated by ethanol and this was prevented by feeding SAMe. The results suggest that SAMe, fed chronically prevents the activation of TLR pathways caused by ethanol. In this way the proinflammatory response, fibrogenesis, cirrhosis and hepatocellular carcinoma formation due to alcohol liver disease could be prevented by SAMe.

The identification of stem cells in human liver diseases and hepatocellular carcinoma.

UNLABELLED: Liver stem cells are thought to preside in bile ducts and the canals of Hering. They extend into the liver parenchyma at a time when normal liver cell proliferation is suppressed and liver regeneration is stimulated. In the present study 69 liver biopsies and surgically excised liver tumors were studied for the presence of liver stem cells. It was found that human cirrhotic livers and hepatocellular carcinomas (HCC) frequently exhibited isolated single scattered hepatocyte stem cells within the liver parenchyma rather than in the portal tract, bile duct or the canal of Hering. These cells expressed liver stem cell markers. HCCs also contained isolated tumor cell which expressed the same stem cell markers. The markers used were GST-P, OV-6, CK-19, Oct-3/4 and FAT10. They were identified by immunofluorescent antibody staining. HGF, EGF, CK19, AIR, H19, Nanog, Oct-3/4 and FAT10 were identified by RNA-FISH. H19 is a non-coding RNA, which is expressed in most HCCs. RESULTS: Immunohistochemistry and RNA-FISH performed on human livers identified isolated stem cells in liver parenchyma as follows: Stem cells identified by immunohistochemical markers (OV-6 and GST-P) and RNA-FISH markers (HGF, EGF, CK19 and H19) were found scattered in the liver parenchyma of cirrhotic livers and within hepatocellular carcinomas (HCCs). Precirrhotic ASH or NASH all stained negative for these stem cells. In HCCs, 13 out of 15 had stem cells located within the tumor (78%). In cirrhotic livers, 12 out of 28 (37%) had liver parenchymal stem cells present. In one case of stage 3 precirrhosis, stem cells were also found. Double staining for the markers showed colocalization of the markers in stem cells. Stem cells were found in 33% of HBV, 47% of HCV, 25% of alcoholic steatohepatitis (ASH) and 17% of non-alcoholic steatohepatitis (NASH). The frequency of stem cells found in the different disease categories correlates with the frequency of HCC occurring in these different diseases.

SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

Mallory-Denk bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway's (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFalpha and IFNgamma receptors. SAMe prevented the increase in the TNFalpha and IFNgamma receptors, supporting the idea that TNFalpha and IFNgamma were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome.

Independent phenotype of binuclear hepatocytes and cellular localization of UbD.

Mice fed DDC (0.1%) for 10 weeks, and then withdrawn from the drug for 1 month, retain the ability to form Mallory-Denk bodies (MDBs) when the drug is refed for 7 days. The number of liver cells that form MDBs increased and partially replaced normal liver cells, at the end of 7 days of refeeding DDC. The MDBs that formed were associated with increased expression of UbD (also called FAT10) in the Mallory-Denk body forming cells. UbD is over expressed in 70% of human HCCs, but its cellular localization is not well established. UbD belongs to the UbL family (ubiquitin-like), and can be linked to others proteins with their 2 C-terminal glycine to lysine. By Western Blot, UbD was found to be covalently linked with proteins. We performed immunohistochemistry on tissue from mouse liver and found that UbD was located in the cytoplasm and in one or two nuclei of the same hepatocyte. However, in primary cell culture, UbD formed speckles within the cytoplasm of the liver cell. A similar pattern of cytoplasmic localization was observed in the Hepa 1-6 cell lines, which over expressed UbD fused with GFP at the C-Terminal. The localization and the control of UbD localization remain unclear. The identification of proteins that interact with UbD and the post translational modification of UbD would help to determine the regulation of this localization and function.

The role of cytokines in UbD promoter regulation and Mallory-Denk body-like aggresomes.

Mallory-Denk bodies (MDBs) are found in chronic liver diseases. Previous studies showed that diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) induced formation of MDBs and the up regulation of UbD expression in mouse liver. UbD is a protein over expressed in hepatocellular carcinomas. It is a potential preneoplastic marker in the mouse. It is hypothesized that inflammatory cytokines play a critical role in UbD up regulation and MDB formation. TNFa and IFNg treatment of HCC cell line Hepa 1-6, induced the expression of UbD and the expression of genes coding for the immunoproteasome (LMP2, LMP7, and MECL-1 subunits). TNFa and IFNg induced the activity of the UbD promoter, using a luciferase assay. The cotreatment with TNFa and IFNg induced the activity of the UbD promoter through an Interferon Sequence Responsive Element (ISRE). In addition, long term treatment with TNFa and IFNg induced the formation of MDB-like aggresomes in Hepa 1-6 cells, which emphasizes the role of inflammation in the formation of MDBs leading to the formation of liver tumors, in the mouse. Identifying the mechanism that regulates gene expression of UbD supports the hypothesis that down regulation of UbD and the proinflammatory gene expression would prevent MDB and HCC formations. Previous studies indicate that S-adenosylmethionine or betaine prevented IFNg induced UbD and MDB formations.