Dysregulated Autophagy and Lysosome Function Are Linked to Exosome Production by Micro-RNA 155 in Alcoholic Liver Disease.

Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3-II levels. Alcohol reduced autophagy flux in vivo in chloroquine-treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol-related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy-related protein 7 (Atg7), proteins involved in phagophore-autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal-associated membrane protein 1 (LAMP1) and lysosomal-associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro-RNA 155 (miR-155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR-155-deficient mice were protected from alcohol-induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down-regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol-induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR-155 as a mediator of alcohol-related regulation of autophagy and exosome production in hepatocytes and macrophages.

Higher odds of irritable bowel syndrome among hospitalized patients using cannabis: a propensity-matched analysis.

BACKGROUND: The endogenous cannabinoid system modulates many brain-gut and gut-brain physiologic pathways, which are postulated to be dysfunctional in irritable bowel syndrome (IBS). Herein, we examine the relationship between cannabis use disorder (CUD) and having IBS. PATIENTS AND METHODS: After selecting patients aged 18 years and above from the 2014 Nationwide Inpatient Survey, we used the International Classification of Diseases, 9th ed. codes to identify individuals with CUD, IBS, and the established risk factors for IBS. We then estimated the crude and adjusted odds ratios of having a diagnosis of IBS with CUD and assessed for the interactions of CUD with other risk factors (SAS 9.4). We confirmed our findings in two ways: conducting a similar analysis on a previous Nationwide Inpatient Survey data (2012); and using a greedy algorithm to design a propensity-scored case-control (1 : 10) study, approximating a pseudorandomized clinical trial. RESULTS: Out of 4 709 043 patients evaluated, 0.03% had a primary admission for IBS and 1.32% had CUD. CUD was associated with increased odds of IBS [adjusted odds ratio: 2.03; 95% confidence interval (CI): 1.53-2.71]. CUD was related to higher odds for IBS among males compared with females (3.48; 1.98-6.12 vs. 1.48; 0.88-2.50), and Hispanics and Caucasians compared with Blacks (5.28; 1.77-15.76, 1.80; 1.02-3.18 vs. 1.80; 0.65-5.03). On propensity-matching, CUD was associated with 80% increased odds for IBS (1.82; 1.27-2.60). CONCLUSION: Our findings suggest that CUD is significantly associated with IBS among the general population. Males, Caucasians, and Hispanics might be more impacted by CUD associated IBS. Additional biomedical studies are required to elucidate this relationship.

Cannabis use is associated with reduced prevalence of progressive stages of alcoholic liver disease.

BACKGROUND: Abusive alcohol use has well-established health risks including causing liver disease (ALD) characterized by alcoholic steatosis (AS), steatohepatitis (AH), fibrosis, cirrhosis (AC) and hepatocellular carcinoma (HCC). Strikingly, a significant number of individuals who abuse alcohol also use Cannabis, which has seen increased legalization globally. While cannabis has demonstrated anti-inflammatory properties, its combined use with alcohol and the development of liver disease remain unclear. AIM: The aim of this study was to determine the effects of cannabis use on the incidence of liver disease in individuals who abuse alcohol. METHODS: We analysed the 2014 Healthcare Cost and Utilization Project-Nationwide Inpatient Sample (NIS) discharge records of patients 18 years and older, who had a past or current history of abusive alcohol use (n = 319 514). Using the International Classification of Disease, Ninth Edition codes, we studied the four distinct phases of progressive ALD with respect to three cannabis exposure groups: non-cannabis users (90.39%), non-dependent cannabis users (8.26%) and dependent cannabis users (1.36%). We accounted for the complex survey sampling methodology and estimated the adjusted odds ratio (AOR) for developing AS, AH, AC and HCC with respect to cannabis use (SAS 9.4). RESULTS: Our study revealed that among alcohol users, individuals who additionally use cannabis (dependent and non-dependent cannabis use) showed significantly lower odds of developing AS, AH, AC and HCC (AOR: 0.55 [0.48-0.64], 0.57 [0.53-0.61], 0.45 [0.43-0.48] and 0.62 [0.51-0.76]). Furthermore, dependent users had significantly lower odds than non-dependent users for developing liver disease. CONCLUSIONS: Our findings suggest that cannabis use is associated with a reduced incidence of liver disease in alcoholics.

Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease.

UNLABELLED: The spectrum of alcoholic liver disease (ALD) is a major cause of mortality with limited therapies available. Because alcohol targets numerous signaling pathways in hepatocytes and in immune cells, the identification of a master regulatory target that modulates multiple signaling processes is attractive. In this report, we assessed the role of spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, which has a central modulatory role in multiple proinflammatory signaling pathways involved in the pathomechanism of ALD. Using mouse disease models that represent various phases in the progression of human ALD, we found that alcohol, in all of these models, induced SYK activation in the liver, both in hepatocytes and liver mononuclear cells. Furthermore, significant SYK activation also occurred in liver samples and peripheral blood mononuclear cells of patients with ALD/alcoholic hepatitis compared to controls. Functional inhibition of SYK activation in vivo abrogated alcohol-induced hepatic neutrophil infiltration, resident immune cell activation, as well as inflammasome and extracellular signal-regulated kinase 1 and 2-mediated nuclear factor kappa B activation in mice. Strikingly, inhibition of SYK activation diminished alcohol-induced hepatic steatosis and interferon regulatory factor 3-mediated apoptosis. CONCLUSION: Our data demonstrate a novel, functional, and multicellular role for SYK phosphorylation in modulating immune cell-driven liver inflammation, hepatocyte cell death, and steatosis at different stages of ALD. These novel findings highlight SYK as a potential multifunctional target in the treatment of alcoholic steatohepatitis. (Hepatology 2016;64:1057-1071).

Therapeutic Benefits of Spleen Tyrosine Kinase Inhibitor Administration on Binge Drinking-Induced Alcoholic Liver Injury, Steatosis, and Inflammation in Mice.

BACKGROUND: Binge drinking is increasingly recognized as an important cause of liver disease with limited therapeutic options for patients. Binge alcohol use, similar to chronic alcohol consumption, induces numerous deregulated signaling events that drive liver damage, steatosis, and inflammation. In this article, we evaluated the role of spleen tyrosine kinase (SYK), which modulates numerous signaling events previously identified linked in the development alcohol-induced liver pathology. METHODS: A 3-day alcohol binge was administered to C57BL/6 female mice, and features of alcoholic liver disease were assessed. Some mice were treated daily with intraperitoneal injections of a SYK inhibitor (R406; 5 to 10 mg/kg body weight) or drug vehicle control. Liver and serum samples were collected and were assessed by Western blotting, biochemical, ELISA, electrophoretic mobility shift assays, real-time quantitative polymerase chain reaction, and histopathological analysis. RESULTS: We found that binge drinking induced significant SYK activation (SYK(Y525/526) ) with no change in total SYK expression in the liver. Functional inhibition of SYK activation using a potent SYK inhibitor, R406, was associated with a significant decrease in alcohol-induced hepatic inflammation as demonstrated by decreased phospho-nuclear factor kappa beta (NF-κB) p65, NF-κB nuclear binding, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 mRNA in the liver. Compared to vehicle controls, SYK inhibitor treatment decreased alcohol binge-induced hepatocyte injury indicated by histology and serum alanine aminotransferase. Strikingly, SYK inhibitor treatment also resulted in a significant reduction in alcohol-induced liver steatosis. CONCLUSIONS: Our novel observations demonstrate the role of SYK, activation in the pathomechanism of binge drinking-induced liver disease highlighting SYK a potential multifaceted therapeutic target.

Versican: a novel modulator of hepatic fibrosis.

Little is known about the deposition and turnover of proteoglycans in liver fibrosis, despite their abundance in the extracellular matrix. Versican plays diverse roles in modulating cell behavior in other fibroproliferative diseases, but remains poorly described in the liver. Hepatic fibrosis was induced by carbon tetrachloride treatment of C57BL/6 mice over 4 weeks followed by recovery over a 28-day period. Primary mouse hepatic stellate cells (HSCs) were activated in culture and versican was transiently knocked down in human (LX2) and mouse HSCs. Expression of versican, A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs (ADAMTS)-1, -4, -5, -8, -9, -15, and -20, and markers of fibrogenesis were studied using immunohistochemistry, real-time quantitative PCR, and western blotting. Immunohistochemistry showed increased expression of versican in cirrhotic human livers and the mouse model of fibrosis. Carbon tetrachloride treatment led to significant increases in versican expression and the proteoglycanases ADAMTS-5, -9, -15, and -20, alongside TNF-α, α-smooth muscle actin (α-SMA), collagen-1, and TGF-ß expression. During recovery, expression of many of these genes returned to control levels. However, expression of ADAMTS-5, -8, -9, and -15 showed delayed increases in expression at 28 days of recovery, which corresponded with decreases in versican V0 and V1 cleavage products (G1-DPEAAE(1401) and G1-DPEAAE(441)). Activation of primary HSCs in vitro significantly increased versican, α-SMA, and collagen-1 expression. Transient knockdown of versican in HSCs led to decreases in markers of fibrogenesis and reduced cell proliferation, without inducing apoptosis. Versican expression increases during HSC activation and liver fibrosis, and proteolytic processing occurs during the resolution of fibrosis. Knockdown studies in vitro suggest a possible role of versican in modulating hepatic fibrogenesis.

Progression of non-alcoholic steatosis to steatohepatitis and fibrosis parallels cumulative accumulation of danger signals that promote inflammation and liver tumors in a high fat-cholesterol-sugar diet model in mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is becoming a pandemic. While multiple 'hits' have been reported to contribute to NAFLD progression to non-alcoholic steatohepatitis (NASH), fibrosis and liver cancer, understanding the natural history of the specific molecular signals leading to hepatocyte damage, inflammation and fibrosis, is hampered by the lack of suitable animal models that reproduce disease progression in humans. The purpose of this study was first, to develop a mouse model that closely mimics progressive NAFLD covering the spectrum of immune, metabolic and histopathologic abnormalities present in human disease; and second, to characterize the temporal relationship between sterile/exogenous danger signals, inflammation, inflammasome activation and NAFLD progression. METHODS: Male C57Bl/6 mice were fed a high fat diet with high cholesterol and a high sugar supplement (HF-HC-HSD) for 8, 27, and 49 weeks and the extent of steatosis, liver inflammation, fibrosis and tumor development were evaluated at each time point. RESULTS: The HF-HC-HSD resulted in liver steatosis at 8 weeks, progressing to steatohepatitis and early fibrosis at 27 weeks, and steatohepatitis, fibrosis, and tumor development at 49 weeks compared to chow diet. Steatohepatitis was characterized by increased levels of MCP-1, TNFα, IL-1ß and increased liver NASH histological score. We found increased serum levels of sterile danger signals, uric acid and HMGB1, as early as 8 weeks, while endotoxin and ATP levels increased only after 49 weeks. Increased levels of these sterile and microbial danger signals paralleled upregulation and activation of the multiprotein complex inflammasome. At 27, 49 weeks of HF-HC-HSD, activation of M1 macrophages and loss of M2 macrophages as well as liver fibrosis were present. Finally, similar to human NASH, liver tumors occurred in 41% of mice in the absence of cirrhosis and livers expressed increased p53 and detectable AFP. CONCLUSIONS: HF-HC-HSD over 49 weeks induces the full spectrum of liver pathophysiologic changes that characterizes the progression of NAFLD in humans. NAFLD progression to NASH, fibrosis and liver tumor follows progressive accumulation of sterile and microbial danger signals, inflammasome activation, altered M1/M2 cell ratios that likely contribute to NASH progression and hepatic tumor formation.

Alcohol and HCV: implications for liver cancer.

Liver cancers are one of the deadliest known malignancies which are increasingly becoming a major public health problem in both developed and developing countries. Overwhelming evidence suggests a strong role of infection with hepatitis B and C virus (HBV and HCV), alcohol abuse, as well as metabolic diseases such as obesity and diabetes either individually or synergistically to cause or exacerbate the development of liver cancers. Although numerous etiologic mechanisms for liver cancer development have been advanced and well characterized, the lack of definite curative treatments means that gaps in knowledge still exist in identifying key molecular mechanisms and pathways in the pathophysiology of liver cancers. Given the limited success with current therapies and preventive strategies against liver cancer, there is an urgent need to identify new therapeutic options for patients. Targeting HCV and or alcohol-induced signal transduction, or virus-host protein interactions may offer novel therapies for liver cancer. This review summarizes current knowledge on the mechanistic development of liver cancer associated with HCV infection and alcohol abuse as well as highlights potential novel therapeutic strategies.

A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry.

Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin-moesin-radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry.

Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90.

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.

Human ezrin-moesin-radixin proteins modulate hepatitis C virus infection.

UNLABELLED: Host cytoskeletal proteins of the ezrin-moesin-radixin (EMR) family have been shown to modulate single-stranded RNA virus infection through regulating stable microtubule formation. Antibody engagement of CD81, a key receptor for hepatitis C virus (HCV) entry, induces ezrin phosphorylation. Here we tested the role of EMR proteins in regulating HCV infection and explored potential therapeutic targets. We show that HCV E2 protein induces rapid ezrin phosphorylation and its cellular redistribution with F-actin by way of spleen tyrosine kinase (SYK). Therapeutically blocking the functional roles of SYK or F-actin reorganization significantly reduced Huh7.5 cell susceptibility to HCV J6/JFH-1 infection. Using gene regulation, real-time quantitative polymerase chain reaction, western blot, and fluorescent microscopy analysis, we found that proteins of the EMR family differentially regulate HCV infection in the J6/JFH-1/Huh7.5 cell system. Moesin and radixin, but not ezrin, expression were significantly decreased in chronic HCV J6/JFH-1-infected Huh7.5 cells and HCV-infected patient liver biopsies compared to controls. The decreases in moesin and radixin in HCV J6/JFH-1-infected Huh7.5 cells were associated with a significant increase in stable microtubules. Ezrin knockdown inhibited immediate postentry events in HCV infection. Overexpression of moesin or radixin significantly reduced HCV protein expression. In contrast, transient knockdown of moesin or radixin augmented HCV infection. Making use of the Con1 HCV replicon system, we tested the effect of EMR proteins on HCV replication. We found that transient knockdown of moesin increased HCV RNA expression while overexpression of EMR showed no significant effect on HCV replication. CONCLUSION: Our findings demonstrate the important role of EMR proteins during HCV infection at the postentry level and highlight possible novel targets for HCV treatment.

Ethanol facilitates hepatitis C virus replication via up-regulation of GW182 and heat shock protein 90 in human hepatoma cells.

UNLABELLED: Alcohol use and hepatitis C virus (HCV) infection synergize to cause liver damage, and microRNA-122 (miR-122) appears to play a key role in this process. Argonaute 2 (Ago2), a key component of the RNA-induced silencing complex (RISC), has been shown to be important in modulating miR-122 function during HCV infection. However, GW182, a critical component of processing bodies (GW bodies) that is recruited by Ago2 to target messenger RNA (mRNA), has not been assessed in HCV infection. To characterize the role of GW182 in the pathogenesis of HCV infection, we determined its transcription and protein expression in an HCV J6/JFH1 culture system. Transcript and protein levels of GW182 as well as HCV RNA and protein expression increased with alcohol exposure. Specific silencing of mRNA expression by small interfering RNA against GW182 significantly decreased HCV RNA and protein expression. Overexpression of GW182 significantly increased HCV RNA and protein expression in HCV J6/JFH1 infected Huh7.5 cells. Furthermore, GW182 colocalized and coimmunoprecipitated with heat shock protein 90 (HSP90), which increased upon alcohol exposure with and without HCV infection and enhanced HCV gene expression. The use of an HSP90 inhibitor or knockdown of HSP90 decreased GW182 and miR-122 expression and significantly reduced HCV replication. CONCLUSION: Overall, our results suggest that GW182 protein that is linked to miR-122 biogenesis and HSP90, which has been shown to stabilize the RISC, are novel host proteins that regulate HCV infection during alcohol abuse.

Alcohol facilitates HCV RNA replication via up-regulation of miR-122 expression and inhibition of cyclin G1 in human hepatoma cells.

BACKGROUND: Clinical studies demonstrate synergistic liver damage by alcohol and hepatitis C virus (HCV); however, the mechanisms by which alcohol promotes HCV infection remain obscure. The liver-specific microRNA-122 (miR-122) regulates HCV replication and expression of host genes, including Cyclin G1. Here, we hypothesized that alcohol regulates miR-122 expression and thereby modulates HCV RNA replication. METHODS: The J6/JFH/Huh-7.5 model of HCV infection was used in this study. Real-time quantitative polymerase chain reaction, Western blotting, electrophoretic mobility shift assay, and confocal microscopy were used for experimental analysis. RESULTS: We found that acute alcohol exposure (25 mM) significantly increased intracellular HCV RNA as well as miR-122 levels in Huh-7.5 and Huh-7.5/CYP2E1 expressing cells in the presence and absence of J6/JFH-HCV infection. Expression of the miR-122 target, Cyclin G1, was inhibited by alcohol both in J6/JFH-infected and uninfected Huh-7.5 cells. The use of a miR-122 inhibitor increased Cyclin G1 expression and prevented the alcohol-induced increase in HCV RNA and protein levels, suggesting a mechanistic role for alcohol-induced miR122 in HCV replication. We discovered that siRNA-mediated silencing of Cyclin G1 significantly increased intracellular HCV RNA levels compared with controls, suggesting a mechanistic role for Cyclin G1 in HCV replication. Alcohol-induced increase in miR-122 was associated with increased nuclear translocation and DNA binding of the nuclear regulatory factor-κB and could be prevented by NF-κB inhibition. CONCLUSIONS: Our novel data indicate a miR-122-mediated mechanism for alcohol increasing HCV RNA replication. We show for the first time that Cyclin G1, a miR-122 target gene, has regulatory effects on HCV replication and that alcohol increases HCV replication by regulating miR-122 and Cyclin G1.

Interplay between the HTLV-2 Tax and APH-2 proteins in the regulation of the AP-1 pathway.

BACKGROUND: In contrast with human T-cell leukemia virus type 1 (HTLV-1) that causes ATL (adult T-cell leukemia), HTLV-2 has not been causally linked to malignant disease. The minus strand of the HTLV genomes encode the regulatory proteins HTLV-1 bZIP factor (HBZ) for HTLV-1 and antisense protein of HTLV-2 (APH-2) for HTLV-2. Unlike the viral proteins Tax1 and Tax2, both HBZ and APH-2 are constitutively expressed in infected cells suggesting that they may play important roles in the pathogenesis of these viruses. To date, very little is known about the function of APH-2 except that it inhibits Tax2-mediated transcription of HTLV-2 genes. In the present study, we investigated the role of APH-2 in basal and Tax2B-mediated activation of the AP-1 pathway. RESULTS: We demonstrate that, unlike HBZ, APH-2 stimulates basal AP-1 transcription by interacting with c-Jun and JunB through its non-conventional bZIP domain. In addition, when Tax2 and APH-2 are co-expressed, they physically interact in vivo and in vitro and APH-2 acts as an inhibitor of Tax2-mediated activation of AP-1 transcription. CONCLUSIONS: This report is the first to document that HTLV-2 can modulate the AP-1 pathway. Altogether our results reveal that, in contrast with HBZ, APH-2 regulates AP-1 activity in a Tax2-dependant manner. As the AP-1 pathway is involved in numerous cellular functions susceptible to affect the life cycle of the virus, these distinct biological properties between HBZ and APH-2 may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2.

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.

BACKGROUND/AIMS: Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites. METHODS: Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays. RESULTS: Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase. CONCLUSION: We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation.

Lentivirus-associated MAPK/ERK2 phosphorylates EMD and regulates infectivity.

Infection of a cell by lentiviruses, such as human immunodeficiency virus type 1 or feline immunodeficiency virus, results in the formation of a reverse transcription complex, the pre-integration complex (PIC), where viral DNA is synthesized. In non-dividing cells, efficient nuclear translocation of the PIC requires the presence of the inner nuclear lamina protein emerin (EMD). Here, we demonstrate that EMD phosphorylation is induced early after infection in primary non-dividing cells. Furthermore, we demonstrate that EMD phosphorylation is dependent on virion-associated mitogen-activated protein kinase (MAPK). Specific inhibition of MAPK activity with kinase inhibitors markedly reduced EMD phosphorylation and resulted in decreased integration of the proviral DNA into chromatin. Similarly, when a MEK1 kinase-inactive mutant was expressed in virus-producer cells, virus-induced phosphorylation of EMD was impaired and viral integration reduced during the subsequent infection. Expression of constitutively active MEK1 kinase in producer cells did not result in modulation of EMD phosphorylation or viral integration during subsequent infection. These studies demonstrate that, in addition to phosphorylating components of the PICs at an early step of infection, virion-associated MAPK plays a role in facilitating cDNA integration after nuclear translocation through phosphorylation of target-cell EMD.