Mechanisms Of Liver Disease Research Articles

Human hepatocytes response to pathological shifts in liver fibrosis

Protracted liver assault results in hepatic fibrosis, frequently advancing into cirrhosis, hepatic failure, portal hypertension and hepatocellular carcinoma or degeneration. Hepatic fibrosis emanates from the wound-healing response of the liver to persistent assault. Following acute liver insult, such as viral hepatitis, parenchymal cells regenerate and substitute the necrotic or apoptotic cells. A salient proportion of the affected population of NAFLD advances to nonalcoholic steatohepatitis, NASH, and is characterised by inflammation, hepatocellular swelling, with ensuing eruption of deteriorating fibrosis. An untreated or persistent NASH can progressively culminate in liver cirrhosis and hepatocellular carcinoma. Chronic liver derangement results in pathologic ECM protein or liver fibrosis aggregation. In essence, the encompassing characterisation of human ECM molecular composition provides for excursions into mechanisms of liver disease or hepatocellular degeneration. Hepatocytes are characterized by elevated mitochondrial concentrations. The difference between NASH and fibrosis is evident that the progression of NASH to cirrhosis commences as inflamed liver tissue evolves into scar tissue or fibrosis that is capable of obliterating the liver from optimum functionality. A third or more of persons presenting with NASH develop cirrhosis , and also elevates and accelerates the risk in the development of liver cancer. The hepatic stellate cells are responsible for liver fibrosis as they are substantially involved in the initiation, progression, and regression of liver fibrosis via the secretion of fibrogenic factors which promote portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts in the formation of collagen, and consequential propagation of fibrosis.

Alcohol and the mechanisms of liver disease.

Alcoholic liver disease (ALD), which is a leading cause of morbidity and mortality worldwide, covers a large spectrum of liver injuries ranging from simple steatosis to steatohepatitis, advanced fibrosis, cirrhosis, and hepatocellular carcinoma. The pathogenesis of ALD includes genetic and epigenetic alterations, oxidative stress, acetaldehyde-mediated toxicity and cytokine and chemokine-induced inflammation, metabolic reprogramming, immune damage, and dysbiosis of the gut microbiota. This review discusses the progress in the pathogenesis and molecular mechanism of ALD, which could provide evidence for further research on the potential therapeutic strategies targeting these pathways.

STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome

The activation of stimulator of interferon genes (STING) and NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnections between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown. STING and NLRP3 inflammasome signaling pathways are activated in fibrotic livers but are suppressed by Sting knockout. Sting knockout ameliorated hepatic pyroptosis, inflammation, and fibrosis. In vitro, STING induces pyroptosis in primary murine hepatocytes by activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) are identified to regulate NLRP3 expression in STING-overexpressing AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhances interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promotes STING-induced Nlrp3 transcription in hepatocytes. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gasdermin D (Gsdmd) knockout attenuate hepatic pyroptosis, inflammation, and fibrosis. RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes show that oxidative stress and metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. The STING-NLRP3-GSDMD axis inhibition suppresses hepatic ROS generation. In conclusion, this study describes a novel epigenetic mechanism by which the STING-WDR5/DOT1L/IRF3-NLRP3 signaling pathway enhances hepatocyte pyroptosis and hepatic inflammation in liver fibrosis.

Immune mechanisms in liver diseases

Immune mechanisms in liver diseases

MiR-516a-3P, a potential circulating biomarker in hepatocellular carcinoma, correlated with rs738409 polymorphism in PNPLA3.

Aim: The aim was to investigate the expression profile of miR-516a-3P and its correlation with thePNPLA3 rs738409 polymorphism in Egyptian hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) patients. Materials & methods: miR-516a-3P was quantified and rs738409 was genotyped by quantitative reverse transcription PCR. Results: miR-516a-3P was significantly upregulated in HCC patients compared with HCV patients (p=0.001). Receiver operating characteristic curve analysis confirmed that miR-516a-3P discriminates HCC from HCV (p=0.001). A significant (p=0.015) correlation between miR-516a-3p level and PNPLA3 rs738409 genotypes was recorded in HCV patients, yet it was not recorded in either healthy individualsor HCC patients. miR-516a-3p level was significantly (p=0.001) higher in HCV patients carrying the rs738409 GG genotype than in those carrying the CC genotype. Conclusion: miR-516a-3P is a potential biomarker in HCC. PNPLA3 rs738409 GG carriers affect miR-516a-3P expression in HCV, and this may highlight a new mechanism in liver disease.

The Regulatory Roles of Polysaccharides and Ferroptosis-Related Phytochemicals in Liver Diseases.

Liver disease is a global health burden with high morbidity and mortality worldwide. Liver injuries can develop into severe end-stage diseases, such as cirrhosis or hepatocellular carcinoma, without valid treatment. Therefore, identifying novel drugs may promote liver disease treatment. Phytochemicals, including polysaccharides, flavonoids, alkaloids, and terpenes, are abundant in foods and medicinal plants and have various bioactivities, such as antioxidation, immunoregulation, and tumor killing. Recent studies have shown that many natural polysaccharides play protective roles in liver disease models in vitro and in vivo, such as fatty liver disease, alcoholic liver disease, drug-induced liver injury, and liver cancer. The mechanisms of liver disease are complex. Notably, ferroptosis, a new type of cell death driven by iron and lipid peroxidation, is considered to be the key mechanism in many hepatic pathologies. Therefore, polysaccharides and other types of phytochemicals with activities in ferroptosis regulation provide novel therapeutic strategies for ferroptosis-related liver diseases. This review summarizes our current understanding of the mechanisms of ferroptosis and liver injury and compelling preclinical evidence of natural bioactive polysaccharides and phytochemicals in treating liver disease.

The Use of Rifaximin in Patients With Cirrhosis.

Rifaximin is an oral nonsystemic antibiotic with minimal gastrointestinal absorption and broad‐spectrum antibacterial activity covering both gram‐positive and gram‐negative organisms. Rifaximin is currently used worldwide in patients with cirrhosis for preventing recurrent HE because its efficacy and safety have been proven by large randomized clinical trials. In the last decade, experimental and clinical evidence suggest that rifaximin could have other beneficial effects on the course of cirrhosis by modulating the gut microbiome and affecting the gut‐liver axis, which in turn can interfere with major events of the pathophysiological cascade underlying decompensated cirrhosis, such as systemic inflammatory syndrome, portal hypertension, and bacterial infections. However, the use of rifaximin for prevention or treatment of other complications, including spontaneous bacterial peritonitis or other bacterial infections, is not accepted because evidence by clinical trials is still very weak. The present review deals in the first part with the potential impact of rifaximin on pathogenic mechanisms in liver diseases, whereas in the second part, its clinical effects are critically discussed. It clearly emerges that, because of its potential activity on multiple pathogenic events, the efficacy of rifaximin in the prevention or management of complications other than HE deserves to be investigated extensively. The results of double‐blinded, adequately powered randomized clinical trials assessing the effect of rifaximin, alone or in combination with other drugs, on hard clinical endpoints, such as decompensation of cirrhosis, acute‐on‐chronic liver failure, and mortality, are therefore eagerly awaited.

Cyclin-dependent kinase inhibitor roscovitine attenuates liver inflammation and fibrosis by influencing initiating steps of liver injury.

Liver diseases present a significant public health burden worldwide. Although the mechanisms of liver diseases are complex, it is generally accepted that inflammation is commonly involved in the pathogenesis. Ongoing inflammatory responses exacerbate liver injury, or even result in fibrosis and cirrhosis. Here we report that roscovitine, a cyclin-dependent kinase (CDK) inhibitor, exerts beneficial effects on acute and chronic liver inflammation as well as fibrosis. Animal models of lipopolysaccharide (LPS)/d-galactosamine- and acute or chronic CCl4-induced liver injury showed that roscovitine administration markedly attenuated liver injury, inflammation and histological damage in LPS/d-galactosamine- and CCl4-induced acute liver injury models, which is consistent with the results in vitro. RNA sequencing (RNA-seq) analysis showed that roscovitine treatment repressed the transcription of a broad set of pro-inflammatory genes involved in many aspects of inflammation, including cytokine production and immune cell proliferation and migration, and inhibited the TGF-β signaling pathway and the biological process of tissue remodeling. For further validation, the beneficial effect of roscovitine against inflammation was evaluated in chronic CCl4-challenged mice. The anti-inflammation effect of roscovitine was observed in this model, accompanied with reduced liver fibrosis. The anti-fibrotic mechanism involved inhibition of profibrotic genes and blocking of hepatic stellate cell (HSC) activation. Our data show that roscovitine administration protects against liver diseases through inhibition of macrophage inflammatory actions and HSC activation at the onset of liver injury.

Liver involvement in Gaucher disease – Review and clinical approach

Gaucher disease (GD), one of the most prevalent lysosomal storage diseases, is associated with glucocerebroside accumulation in cells of the monocyte-macrophage system in various organs, including the liver.Evaluating and managing liver disease in patients with Gaucher disease may be challenging. While hepatic involvement is common in Gaucher disease, its severity, and clinical significance span a wide spectrum, ranging from sub-clinical involvement to liver cirrhosis with its associated complications including portal hypertension. Apart from liver involvement in Gaucher disease, patients with may also suffer from other comorbidities involving the liver. That Gaucher disease itself can mimic hepatic lesions, affect laboratory tests used to characterize liver disease, and may be associated with non-cirrhotic portal hypertension, complicates the diagnostic approach even more.Better understanding of liver involvement in Gaucher disease can spare patients unnecessary invasive testing, and assist physicians in decision making when evaluating patients with Gaucher disease suspected for significant liver disease.This review describes the various clinical manifestations, laboratory and imaging abnormalities that may be encountered when following patients with Gaucher disease for liver involvement. The mechanism for liver disease are discussed, as well as the possible hepato-protective effect of glucocerebroside, and the a diagnostic and treatment approaches.

Reprogramming factor Oct4 is crucial for Trans differentiation of Hepatocytes to Biliary epithelial cells

Trans differentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such trans differentiation of terminally differentiated epithelial cells are not fully elucidated. Recently we reported that adult rat liver expresses reprogramming factors Oct4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factor Oct4 plays a role in trans differentiation of hepatocytes to BECs, we used the hepatocyte organoid culture: an in vitro hepatocyte to biliary trans differentiation model. In this model, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, trans differentiate to form BECs between 6–15 days in culture. We found that Oct4 is upregulated at day 6 in this model as assessed by mRNA and protein levels suggesting its possible involvement in trans differentiation. When expression of Oct4 was blocked in hepatocytes using adenovirus containing Oct4 shRNA, there was a significant decrease in the number of biliary cells as compared to controls as assessed by biliary marker HNF1‐beta, strongly supporting the role of Oct4 in trans differentiation of hepatocyte to biliary epithelial cells. Moreover, Oct4 inhibited group shows significantly less SOX9, YAP, and Notch (also shown to be involved in trans differentiation) expression as compared to controls. Experiments to investigate mechanisms through which Oct4 might regulate trans differentiation are under way.Support or Funding Information Center for Chronic Disorders of Aging. American Association of Colleges of Pharmacy New Investigator Award.

Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution.

Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.

Organ Culture Model of Liver for the Study of Cancer Treatment for Hepatocellular Carcinoma

The liver, the largest organ of the human body, is a multifunctional organ with various metabolic activities that plays a fundamental role in maintaining the body and in sustaining life. Although the liver has great regenerative capacity and recovery, the damage caused by chronic diseases such as cancer or viral infections can lead to permanent loss of liver function. Studies on the mechanism of liver disease, have focused on the selection of cell and tissue culture techniques, including strategies based on in vitro models. The organ culture is a promising tool for the study of liver diseases, because it can mimic the complex of the microenvironment in vivo using a three-dimensional model of human liver tissue. These models allow a better study of the specific functions of the liver. In this context, we have analyzed the development of a hepatocarcinoma, obtained by inoculating a murine hepatocarcinoma cell line, Hepa 1/A1s, in the liver of 10 mice of the strain C57BL / 6. After 20 days from the inoculation, the portion of liver invaded by the tumor was removed from the animals and cultured. A group of 5 liver explants were used as a control and other 5 explants were cultured for 4 weeks in a complete medium containing 10% Citozym , a food supplement with reported antioxidant properties. The cancer-invaded hepatic lobes, treated with Citozym , showed a clear reduction of the weight and the volume of the hepatic tumors, when compared with the control explants.

Reprogramming Factor OCT4 is Crucial for Transdifferentiation of Hepatocytes to Biliary Epithelial Cells.

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial cells are not fully elucidated. Recently we reported that adult rat liver expresses reprogramming factors OCT4 and its expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factor OCT4 plays a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid culture. In this model, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6‐8 days in culture. We found that OCT4 is upregulated at day 6 in this model as assessed by mRNA and protein levels suggesting its possible involvement in transdifferentiation. When expression of OCT4 was blocked in hepatocytes using adenovirus containing OCT4 shRNA, there was a significant decrease in the number of biliary cells as compared to controls as assessed by biliary marker HNF1‐beta, strongly supporting the role of OCT4 in transdifferentiation of hepatocyte to biliary epithelial cells. Moreover, OCT4 inhibited group showed significantly less SOX9 expression as compared to controls. Experiments to investigate mechanisms through which OCT4 might regulate transdifferentiation and mechanisms upstream of OCT4 are under way.

Asymmetric dimethylarginine as a mediator of vascular dysfunction in cirrhosis.

Cirrhosis is associated with marked abnormalities in the circulatory function that involve a reduction in systemic vascular resistance. An important cause of this vasodilatation is the increased production or activity of nitric oxide (NO) in the splanchnic circulation. During portal hypertension and cirrhosis an increased endothelial NO synthase (eNOS) activity is demonstrated in splanchnic vessels. In contrast, the activity of eNOS in the cirrhotic liver is decreased, which suggests a different regulation of eNOS in the liver and in the splanchnic vessels. Asymmetric dimethylarginine (ADMA) is an endogenous NO inhibitor and higher plasma levels of ADMA are related to increased cardiovascular risk in both the general population and among patients with cirrhosis. It has been demonstrated that the liver is a key player in the metabolism of ADMA. This observation was further supported by investigations in human patients, showing a close correlation between ADMA plasma levels and the degree of hepatic dysfunction. ADMA is degraded to citrulline and dimethylamine by dimethylarginine dimethylaminohydrolases (DDAHs). DDAHs are expressed as type 1 and 2 isoforms and are widely distributed in various organs and tissues, including the liver. In this review, we discuss experimental and clinical data that document the effects of dimethylarginines on vascular function in cirrhosis. Our increasing understanding of the routes of synthesis and metabolism of methylarginines is beginning to provide insights into novel mechanisms of liver disease and allowing us to identify potential therapeutic opportunities.

Mo1996 Inflammatory Cytokines Are Associated With Hepatic Steatosis and Fibrosis Measured by Computer-Assisted Morphometry (CAM) in Patients With Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH)

BACKGROUND: Systemic inflammation has been shown to play an important role in the development and progression of NASH and related fibrosis. AIM: To compare inflammationassociated cytokines with the degree of liver fibrosis and steatosis as measured by computerassisted morphometry (CAM). METHODS: Liver biopsies, clinical data and fasting serum samples had been collected after informed consent. Biopsies were semiquantitatively graded for histopathologic features, and digital images of Sirius red stained sections was used to determine the fraction of collagen and steatosis on a continuous scale by CAM. Serum from the time of liver biopsy was examined with a Bio-Plex Pro Human Cytokine 17-plex assay (Bio-Rad) run on a Bio-Plex 200 (Bio-Rad). RESULTS: 66 NAFLD subjects are included [22 (33.3%) male, 51 (76.6%) Caucasian, 23 (34.8%) diabetes mellitus, 28 (42.4%) with histologic NASH, and 5 (7.6%) with histologic cirrhosis]. The amount of collagen as measured by CAM correlated moderately with the semiquantitative stage of portal fibrosis (r = 0.49, p < 0.0001). The percentage of fat as measured by CAM highly correlated with semiquantitative grade of steatosis (r=0.74, p < 0.0001). Percentage of collagen as measured bymorphometry correlated with IL-1β and IL-7 (r = 0.37, p < 0.01; r = 0.39, p=0.001, respectively). On the other hand, TNF-α was positively associated with both percentage of fat (CAM) and grade of steatosis (r = 0.32, p < 0.01; r = 0.35, p < 0.01, respectively). CONCLUSIONS: These results show that CAM can provide a new dimension to standard histology with the capability of uncovering unsuspected associations with circulating biochemical markers, and may lead to a better understanding of pathogenetic mechanisms in liver disease

Transdifferentiation of hepatocytes to biliary epithelial cells requires expression of reprogramming factor Oct3/4 (398.8)

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial are not known. Recently we reported that adult rat liver expresses reprogramming factors Oct3/4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factor Oct3/4 plays a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid culture: an in vitro hepatocyte to biliary transdifferentiation model. In this model, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6‐8 days in culture. We found that Oct3/4 is upregulated at day 6 in this model as assessed by mRNA and protein levels suggesting its possible involvement in transdifferentiation. When expression of Oct3/4 was blocked in hepatocytes using adenovirus containing Oct shRNA, there was a significant decrease in appearance of biliary cells (compared to controls) as assessed by biliary marker HNF1‐beta, strongly supporting the role of Oct3/4 in transdifferentiation of hepatocyte to biliary epithelial cells. Experiments to investigate mechanisms through which Oct3/4 might regulate transdifferentiation are under way.Grant Funding Source: 1. Pathology Post‐doctoral Research Training grant, 2. Center for chronic diseases of aging grant

Isolation and culture of hepatic stellate cells from mouse liver

Hepatic stellate cells (HSCs) are the primary extracellular matrix-producing cells within the liver and have numerous vital functions. A robust protocol for the isolation and culture of HSCs is important for further investigations of cell functions and related mechanisms in liver disease. The volume of the mouse liver is much smaller than that of the rat liver, which makes it much more difficult to isolate mouse HSCs (mHSCs) than rat HSCs. At present, isolating mHSCs is still a challenge because there is no efficient, robust method to isolate and culture these cells. In the present study, C57BL/6J mice were intravenously injected with liposome-encapsulated dichloromethylene diphosphate (CL2MDP) to selectively eliminate Kupffer cells from the liver. The mouse livers were then perfused in situ, and the mHSCs were isolated with an optimized density gradient centrifugation technique. In the phosphate buffer solution (PBS)-liposome group, the yield of mHSCs was (1.37 ± 0.23) × 10(6)/g liver, the cell purity was (90.18 ± 1.61)%, and the cell survival rate was (94.51 ± 1.61)%. While in the CL2MDP-liposome group, the yield of mHSCs was (1.62 ± 0.34) × 10(6)/g liver, the cell purity was (94.44 ± 1.89)%, and the cell survival rate was (94.41 ± 1.50)%. Based on the yield and purity of mHSCs, the CL2MDP-liposome treatment was superior to the PBS-liposome treatment (P < 0.05, P < 0.01). This study established successfully a robust and efficient protocol for the separation and purification of mHSCs, and both a high purity and an adequate yield of mHSCs were obtained.

Cooperation of Liver Cells in the Metabolism Regulation of Bioactive Substances

Hepatocyte and nonparenchymal cells are the key foundation of liver structure and function. Hepatocyte is one of the main liver cell types, involved in large physiological processes, such as material metabolism, detoxication, and homeostasis. On the other hand, the nonparenchymal cells, including hepatic stellate cell, liver sinusoidal endothelial cell, kupffer cell, natural killer cell and pit cell, perform the material transportation, phagocytose, antigen presentation and immune tolerance. However, at present there is few research on cell cooperation between hepatocyte and the nonparenchymal cells in liver physiological and pathological process. This paper aims to comprehensively summarize the interaction between different kinds of liver cells both in health and disease conditions to provide references for further studies of the specific molecular mechanism of liver diseases.

Abstract 100A: Prognostic significance of the expression levels of target genes of retinoic acid in hepatocellular carcinoma.

Abstract Hepatocellular carcinoma (HCC), a common and fatal malignancy, is increasing in incidence in developed countries. HCC generally arises in cirrhotic livers and has a poor prognosis because of the high incidence of disease recurrence after curative treatment. To improve patient prognosis, identification of new targets for early detection, chemoprevention, and treatment is urgently required. In the pathogenesis of chronic liver disease, activation of hepatic stellate cells accompanied by decreasing vitamin A storage has been observed, leading to development of the hypothesis that the loss of retinoid content is closely associated with the progression of chronic liver diseases including HCC. In a previous study, we observed steatohepatitis and HCC development in transgenic mice that express a dominant-negative form of RAR in a liver-specific manner. These findings indicate that identifying genes regulated by retinoid signaling is important for understanding the molecular mechanisms of liver disease leading to hepatocarcinogenesis. To identify novel retinoic acid (RA)-responsive genes facilitating the antitumor activity of RA in hepatocellular carcinoma (HCC), we performed a gene screening based on in silico analysis of retinoic acid response elements (RAREs), chromatin immunoprecipitation analysis of the retinoic acid receptor (RAR), and gene expression analysis. We identified 201 candidate genes with promoter regions containing consensus RAREs using in silico analysis. We identified 126 candidate genes with functional RAREs which are occupied by RAR and finally selected 26 RA-responsive genes in 2 human HCC and 1 human breast cancer cell lines. Among these genes, we identified OTU domain-containing 7B (OTUD7B) and frizzled family receptor 4 (FZD4) as potential inducers of RA-mediated antitumor activity. OTUD7B and FZD4 were involved in the regulation of nuclear factor κB and Wnt/β-catenin signaling, respectively, and affected the viability of HCC cell lines. To evaluate the clinical significance of RA-responsive genes in HCC, gene expression analysis was performed using tumor and nontumor tissues from 171 patients. In univariate and multivariate analyses, low OTUD7B mRNA expression in tumor tissue was associated with a poor prognosis forHCC patients. Conclusion: We identified RA-responsive genes that exhibit antitumor activity against HCC cells. These findings provide useful information to understand the molecular mechanisms underlying the chemopreventive effects of RA against liver diseases, including HCC. Citation Format: Goshi Shiota, Keita Kanki. Prognostic significance of the expression levels of target genes of retinoic acid in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 100A. doi:10.1158/1538-7445.AM2013-100A

Role of reprogramming factors in transdifferentiation of hepatocytes to biliary epithelial cells

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial are not known. Recently we reported that adult rat liver expresses reprogramming factors Oct3/4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factors play a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid cultures. In this system, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6–8 days in culture. When we looked at the expression of reprogramming factors in this system, we found that Oct3/4, Nanog, and Klf4 are upregulated at day 6 in this system as assessed by mRNA levels suggesting the possible involvement of reprogramming factors in transdifferentiation. Further analysis of protein levels by Western blot, immunohistochemical staining, and intervention studies by blocking reprogramming factors are in progress to elucidate the role of reprogramming factors in transdifferentiation.