c-Met Signaling Is Essential for Mouse Adult Liver Progenitor Cells Expansion After Transforming Growth Factor-ß-Induced Epithelial-Mesenchymal Transition and Regulates Cell Phenotypic Switch.

Adult hepatic progenitor cells (HPCs)/oval cells are bipotential progenitors that participate in liver repair responses upon chronic injury. Recent findings highlight HPCs plasticity and importance of the HPCs niche signals to determine their fate during the regenerative process, favoring either fibrogenesis or damage resolution. Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) are among the key signals involved in liver regeneration and as component of HPCs niche regulates HPCs biology. Here, we characterize the TGF-ß-triggered epithelial-mesenchymal transition (EMT) response in oval cells, its effects on cell fate in vivo, and the regulatory effect of the HGF/c-Met signaling. Our data show that chronic treatment with TGF-ß triggers a partial EMT in oval cells based on coexpression of epithelial and mesenchymal markers. The phenotypic and functional profiling indicates that TGF-ß-induced EMT is not associated with stemness but rather represents a step forward along hepatic lineage. This phenotypic transition confers advantageous traits to HPCs including survival, migratory/invasive and metabolic benefit, overall enhancing the regenerative potential of oval cells upon transplantation into a carbon tetrachloride-damaged liver. We further uncover a key contribution of the HGF/c-Met pathway to modulate the TGF-ß-mediated EMT response. It allows oval cells expansion after EMT by controlling oxidative stress and apoptosis, likely via Twist regulation, and it counterbalances EMT by maintaining epithelial properties. Our work provides evidence that a coordinated and balanced action of TGF-ß and HGF are critical for achievement of the optimal regenerative potential of HPCs, opening new therapeutic perspectives. Stem Cells 2019;37:1108-1118.

Matrilin-2 prevents the TNFα-induced apoptosis of WB-F344 cells via suppressing JNK pathway.

Oval cells, a kind of hepatic progenitor cell quiescent at normal condition, activates to proliferate and differentiate into hepatocytes under severe and long-term liver injury, which usually raises severe inflammation. However, how oval cell survives in the inflammatory milieu interne is still unclear. Tumor necrosis factor α (TNFα), mimicking inflammatory hepatic milieu interne, was used to treat oval cell line, WB-F344, to test the protective function of matrilin-2. In this study, our data suggested that matrilin-2 prevented TNFα-induced apoptosis in WB-F344 cells via inhibiting ASK1/MKK7/JNK pathway. In conclusion, we determined that matrilin-2 plays the key role in maintaining the survival of oval cell and guarantees its proliferation under various injury factors.

Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

BACKGROUND & AIMS: Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. METHODS: WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. RESULTS: Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. CONCLUSIONS: We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases.

Potential ultrastructure predicting factors for hepatocellular carcinoma in HCV infected patients.

Hepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Although the early diagnosis of HCC is vital for successful curative treatment, the majority of lesions are diagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study of experimentally gradually induced HCC, surgically resected HCC, and potential premalignant lesions from HCV-infected patients, with the prospect to detect cellular criteria denoting premalignant transformation. Among the main detected pathological changes which are postulated to precede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmentation, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of different electron density loaded with small sized rounded monotonous mitochondria, increase junctional complexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellular proteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER), sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation of lipolysosomes, large autophagosomes, and micro-vesicular fat deposition. In conclusion, the present work has visualized new hepatocytic division or regenerative process that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, new observations that may be of value or assist in predicting HCC and identifying the appropriate patient for surveillance have been reported. Moreover, it has pointed to the possible malignant potentiality of liver stem/progenitor cells. For reliability, the results can be subjected to cohort longitudinal study.

Activin A-Smad Signaling Mediates Connective Tissue Growth Factor Synthesis in Liver Progenitor Cells.

Liver progenitor cells (LPCs) are activated in chronic liver damage and may contribute to liver fibrosis. Our previous investigation reported that LPCs produced connective tissue growth factor (CTGF/CCN2), an inducer of liver fibrosis, yet the regulatory mechanism of the production of CTGF/CCN2 in LPCs remains elusive. In this study, we report that Activin A is an inducer of CTGF/CCN2 in LPCs. Here we show that expression of both Activin A and CTGF/CCN2 were upregulated in the cirrhotic liver, and the expression of Activin A positively correlates with that of CTGF/CCN2 in liver tissues. We go on to show that Activin A induced de novo synthesis of CTGF/CCN2 in LPC cell lines LE/6 and WB-F344. Furthermore, Activin A contributed to autonomous production of CTGF/CCN2 in liver progenitor cells (LPCs) via activation of the Smad signaling pathway. Smad2, 3 and 4 were all required for this induction. Collectively, these results provide evidence for the fibrotic role of LPCs in the liver and suggest that the Activin A-Smad-CTGF/CCN2 signaling in LPCs may be a therapeutic target of liver fibrosis.

Chronic liver inflammation and hepatocellular carcinogenesis are independent of S100A9.

The S100A8/A9 heterodimer (calprotectin) acts as a danger signal when secreted into the extracellular space during inflammation and tissue damage. It promotes proinflammatory responses and drives tumor development in different models of inflammation-driven carcinogenesis. S100A8/A9 is strongly expressed in several human tumors, including hepatocellular carcinoma (HCC). Apart from this evidence, the role of calprotectin in hepatocyte transformation and tumor microenvironment is still unknown. The aim of this study was to define the function of S100A8/A9 in inflammation-driven HCC. Mice lacking S100a9 were crossed with the Mdr2(-/-) model, a prototype of inflammation-induced HCC formation. S100a9(-/-) Mdr2(-/-) (dKO) mice displayed no significant differences in tumor incidence or multiplicity compared to Mdr2(-/-) animals. Chronic liver inflammation, fibrosis and oval cell activation were not affected upon S100a9 deletion. Our data demonstrate that, although highly upregulated, calprotectin is dispensable in the onset and development of HCC, and in the maintenance of liver inflammation.

Expression and activation of EGFR and STAT3 during the multistage carcinogenesis of intrahepatic cholangiocarcinoma induced by 3'-methyl-4 dimethylaminoazobenzene in rats.

The purpose of this study was to investigate whether the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription-3 (STAT3) signal pathway contributes to the carcinogenesis of intrahepatic cholangiocarcinoma (ICC) induced by 3'-methyl-4 dimethylaminoazobenzene (3'Me-DAB) in rats. EGFR, TGFα, STAT3 and p-STAT3 in different stages of carcinogenesis were detected by immunohistochemistry (IHC). In situ hybridization (ISH) was applied to investigate the expression of STAT3 mRNA. Oval cells were verified by the immunohistochemical staining of alpha-fetoprotein (AFP), CD133 and epithelial cell adhesion molecules (EpCAM). Sequential development of necrosis, oval cell proliferation, cholangiofibrosis (CF) and ICC was observed in the liver of rats administered 3'Me-DAB. Oval cells showed positive expression of AFP, CD133 and EpCAM. The expression of EGFR was significantly higher in the ICC than in oval cells, CF or normal bile ducts (p<0.05), but there was no difference in EGFR expression between the other groups. The highest expression of p-STAT3 and TGFα was observed in CF. The expression of these two molecules in the ICC and oval cells was significantly higher than in normal bile ducts (p<0.05). Elevation of STAT3 mRNA was detected during carcinogenesis as shown by ISH, strong intensity was observed in the ICC and moderate intensity was observed in oval cells and CF. These observations suggest that the EGFR and STAT3 signal pathway contributes to the carcinogenesis of ICC. High activity of STAT3 during the carcinogenesis of ICC may be the result of high activity of EGFR triggered by TGFα.

Biliary proliferative lesions in the Sprague-Dawley rat: adverse/non-adverse.

Whether biliary proliferative lesions in nonclinical species are predictive of potential hepatotoxicity in humans depends, at least in part, on the nature and severity of such changes in the nonclinical species. We reviewed published literature (clinical and nonclinical) and experimental data from rat toxicology studies conducted by GlaxoSmithKline and the National Institute of Environmental Health Sciences' National Toxicology Program in an effort to better characterize the relative risk of hepatobiliary effects in humans. Available evidence supports the interpretation that minimal "typical" appearing bile duct hyperplasia limited to the portal triads may be considered non-adverse in the rat and is of little to no concern to humans. The toxicological relevance of mild to moderate "typical" hyperplasia is less certain, and may be considered adverse in the rat and potentially pose a risk for humans, particularly if accompanied by evidence of hepatobiliary injury or functional compromise. In addition, any proliferative lesion that includes atypical or dysplastic epithelial changes, oval cell proliferation, and/or significant extension beyond the portal tracts is considered more ominous and may be considered adverse in the rat.

Propranolol, a ß-adrenoceptor antagonist, worsens liver injury in a model of non-alcoholic steatohepatitis.

Prazosin an α1-adrenoceptor (AR) antagonist has been shown to reduce liver injury in a mouse model of non-alcoholic steatohepatitis (NASH) and is suggested as a potential treatment of NASH especially given its concomitant anti-fibrotic properties. The effect however, of ß-AR blockade in non-cirrhotic NASH is unknown and is as such investigated here. In the presence of the ß-blocker propranolol (PRL), mice fed normal chow or a half methionine and choline deficient diet, supplemented with ethionine (HMCDE), to induce NASH, showed significantly enhanced liver injury, as evidenced by higher hepatic necrosis scores and elevated serum aminotransferases (ALT). Mechanistically, we showed that murine hepatocytes express α and ß adrenoceptors; that PRL directly induces hepatocyte injury and death as evidenced by increased release of lactate dehydrogenase, FASL and TNF-α from hepatocytes in the presence of PRL; and that PRL activated the apoptotic pathway in primary hepatocyte cultures, as indicated by upregulation of Fas receptor and caspase-8 proteins. The ß-AR antagonist PRL therefore appears to enhance liver injury through induction of hepatocyte death via the death pathway. Further studies are now required to extrapolate these findings to humans but meanwhile, ß-AR antagonists should be avoided or used with caution in patients with non-cirrhotic NASH as they may worsen liver injury.

Expression and significance of histone methyltransferase SET domain containing 2 with histone H3 lysine 36 trimethylation in mouse hepatic oval cells differentiated into bile duct epithelial cells in vitro.

The present study aimed to identify the function and expression of trimethylated protein histone H3 lysine 36 (H3K36)me3 and the upstream specific enzyme histone methyltransferase SET domain containing 2 (SETD2), during the differentiation of hepatic oval cells (HOCs) into cholangiocytes in mice following partial liver resection and fed with 2­acetamidofluorene. HOCs were isolated from Kunming male mice fed with 2­acetamidofluorene for 10 days. Their liver tissues were then isolated following partial liver resection and another week of 2­acetamidofluorene treatment. HOCs were collected following a two­step enzyme digestion procedure involving protease E and collagenase 4. The target cells were cultured in DMEM/F12 supplemented with 10 µg/ml EGF, 5 µg/ml stem cell growth factor and 5 µg/ml leukemia inhibitory factor. Target cells using the markers OV­6, CK­19, SETD2, H3K36me3, were detected with flow cytometry and immunofluorescence microscopy; reverse transcription­quantitative PCR and western blotting were used to quantify the protein levels of SETD2 and H3K36me3. The retrieved primary hepatocytes developed into cholangiocytes with increasing CK­19 and decreasing OV­6 expression in each subsequent passage, whereas the SETD2 and H3K36me3 levels gradually increased, suggesting the possible involvement of both of these factors in differentiation.

Troglitazone inhibits hepatic oval cell proliferation by inducing cell cycle arrest through Hippo/YAP pathway regulation.

Hepatic oval cells have strong proliferation and differentiation capabilities and are activated when chronic liver injury occurs or when liver function is severely impaired. Peroxisome proliferation-activated receptors (PPARs) are ligand-dependent, sequence-specific nuclear transcription factors. PPARγ is closely related to liver diseases (such as liver cancer, liver fibrosis and non-alcoholic fatty liver disease). As the main effector downstream of the Hippo signaling pathway, YAP can activate the hepatic progenitor cell program, and different expression or activity levels of YAP can determine different liver cell fates. We found that troglitazone (TRO), a classic PPARγ activator, can inhibit the growth of hepatic oval cells, and flow cytometry results showed that TRO inhibited the growth of WB-F344 cells by arresting the cells in the G0/1 phase. Western blot results also confirmed changes in G0/1 phase-related protein expression. Further experiments showed that PPARγ agonists induced hepatic oval cell proliferation inhibition and cell cycle G0/1 phase arrest through the Hippo/YAP pathway. Our experiment demonstrated, for the first time, the relationship between PPARγ and the Hippo/YAP pathway in liver oval cells and revealed that PPARγ acts as a negative regulator of liver regeneration by inhibiting the proliferation of oval cells.

Dkk1 inhibits malignant transformation induced by Bmi1 via the ß-catenin signaling axis in WB-F344 oval cells.

Dickkopf-1 (Dkk1) is an inhibitor of Wnt signaling involved in cancer cell proliferation, apoptosis, and migration and angiogenesis. It was previously reported that B cell-specific Moloney mouse leukemia virus integration site 1 (Bmi1) activates the Wnt pathway by inhibiting the expression of DKK1 in breast cancer cell lines and 293T cells. Bmi1 and DKK1 are highly expressed in liver samples taken by biopsy from patients with hepatitis B virus-related hepatocellular carcinoma (HCC), but the effect of both Bmi1 and DKK1 on the carcinogenesis of adult hepatic stem cells (oval cells) has not previously been reported. In this study, we used WB-F344 cells to explore the function and regulation of Dkk1 upon Bmi1 treatment. Overexpression of Dkk1 repressed differentiation, proliferation, and migration induced by Bmi1 but promoted the apoptosis of hepatic WB-F344 oval cells. In addition, Dkk1 reduced the enhancement of ß-catenin levels induced by Bmi1. Finally, we used transcriptome sequencing to perform a comprehensive evaluation of the transcriptome-related changes in WB-F344 oval cells induced by Dkk1 and Bmi1. These results may provide evidence for future studies of the pathogenesis of HCC and the design of possible therapies.

Liver Progenitors and Adult Cell Plasticity in Hepatic Injury and Repair: Knowns and Unknowns.

The liver is a complex organ performing numerous vital physiological functions. For that reason, it possesses immense regenerative potential. The capacity for repair is largely attributable to the ability of its differentiated epithelial cells, hepatocytes and biliary epithelial cells, to proliferate after injury. However, in cases of extreme acute injury or prolonged chronic insult, the liver may fail to regenerate or do so suboptimally. This often results in life-threatening end-stage liver disease for which liver transplantation is the only effective treatment. In many forms of liver injury, bipotent liver progenitor cells are theorized to be activated as an additional tier of liver repair. However, the existence, origin, fate, activation, and contribution to regeneration of liver progenitor cells is hotly debated, especially since hepatocytes and biliary epithelial cells themselves may serve as facultative stem cells for one another during severe liver injury. Here, we discuss the evidence both supporting and refuting the existence of liver progenitor cells in a variety of experimental models. We also debate the validity of developing therapies harnessing the capabilities of these cells as potential treatments for patients with severe and chronic liver diseases.

A Signaling Crosstalk between BMP9 and HGF/c-Met Regulates Mouse Adult Liver Progenitor Cell Survival.

During chronic liver disease, hepatic progenitor cells (HPC, oval cells in rodents) become activated, proliferate, and differentiate into cholangiocytes and/or hepatocytes contributing to the final outcome of the regenerative process in a context-dependent fashion. Here, we analyze the crosstalk between the hepatocyte growth factor (HGF)/c-Met signaling axis, key for liver regeneration, and bone morphogenetic protein (BMP)9, a BMP family ligand that has emerged as a critical regulator of liver pathology. Our results show that HGF/c-Met signaling blocks BMP9-mediated apoptotic cell death, while it potentiates small mothers against decapentaplegic (SMAD)1 signaling triggered by BMP9 in oval cells. Interestingly, HGF-induced overactivation of SMAD1, -5, -8 requires the upregulation of TGF-ß type receptor activin receptor-like kinase (ALK)1, and both ALK1 and SMAD1 are required for the counteracting effect of HGF on BMP9 apoptotic activity. On the other hand, we also prove that BMP9 triggers the activation of p38MAPK in oval cells, which drives BMP9-apoptotic cell death. Therefore, our data support a model in which BMP9 and HGF/c-Met signaling axes establish a signaling crosstalk via ALK1 that modulates the balance between the two pathways with opposing activities, SMAD1 (pro-survival) and p38 mitogen-activated protein kinases (p38MAPK; pro-apoptotic), which determines oval cell fate. These data help delineate the complex signaling network established during chronic liver injury and its impact on the oval cell regenerative response.

Nrf2 Ameliorates DDC-Induced Sclerosing Cholangitis and Biliary Fibrosis and Improves the Regenerative Capacity of the Liver.

The Nrf2 pathway protects against oxidative stress and induces regeneration of various tissues. Here, we investigated whether Nrf2 protects from sclerosing cholangitis and biliary fibrosis and simultaneously induces liver regeneration. Diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was fed to Nrf2-KO mice (Nrf2-/-), mice with liver-specific hyperactivated Nrf2 (HKeap1-/-) and wild-type (WT) littermates to induce cholangitis, liver fibrosis, and oval cell expansion. HKeap1-/--mice were protected from almost all DDC-induced injury compared with WT and Nrf2-/-. Liver injury in Nrf2-/- and WT mice was mostly similar, albeit Nrf2-/- suffered more from DDC diet as seen for several parameters. Nrf2 activity was especially important for the expression of the hepatic efflux transporters Abcg2 and Abcc2-4, which are involved in hepatic toxin elimination. Surprisingly, cell proliferation was more enhanced in Nrf2-/-- and HKeap1-/--mice compared with WT. Interestingly, Nrf2-/--mice failed to sufficiently activate oval cell expansion after DDC treatment and showed almost no resident oval cell population under control conditions. The resident oval cell population of untreated HKeap1-/--mice was increased and DDC treatment resulted in a stronger oval cell expansion compared with WT. We provide evidence that Nrf2 activation protects from DDC-induced sclerosing cholangitis and biliary fibrosis. Moreover, our data establish a possible role of Nrf2 in oval cell expansion.

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis.

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival.

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation.

Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.

Distinct role of interleukin-6 and tumor necrosis factor receptor-1 in oval cell- mediated liver regeneration and inflammation-associated hepatocarcinogenesis.

Interleukin 6 (IL6), tumor necrosis factor α (TNFα) and TNF receptor-1(TNFR1) have been shown to involve in oval cell proliferation and hepatocellular carcinoma (HCC) development. However, their role in these processes is still unclear. In the present study, by using hepatocytes-specific DDB1 deletion mouse models, we explored the role and mechanism of IL6, TNFα and TNFR1 in oval cell proliferation and HCC development in the context of inflammation, which is the common features of HCC pathogenesis in humans. Our results showed that IL6 promotes oval cell proliferation and liver regeneration, while TNFα/TNFR1 does not affect this process. Deletion of IL6 accelerates HCC development and increases tumor burden. The number of natural killer(NK) cells is significantly decreased in tumors without IL6, implying that IL6 suppresses HCC by NK cells. In contrast to IL6, TNFR1-mediated signaling pathway promotes HCC development, and deletion of TNFR1 reduced tumor incidence. Increased apoptosis, compensatory proliferation and activation of MAPK/MEK/ERK cascade contribute to the oncogenic function of TNFR1-mediated signaling pathway. Intriguingly, deletion of TNFα accelerates tumor development, which shows divergent roles of TNFα and TNFR1 in hepatocarcinogenesis.

Calligonum comosum extract inhibits diethylnitrosamine-induced hepatocarcinogenesis in rats.

Calligonum comosum (C. comosum) is an Egyptian desert plant that contains polyphenol antioxidants. The present study examined the chemopreventive effect of an extract of C. comosum in a rat model of hepatocarcinogenesis. Male Wistar rats (n=40) were administered 100 mg/kg diethylnitrosamine (DEN) by intraperitoneal (i.p.) injection once a week for 3 weeks. Subsequently, depending on whether the rats received further administration of 0.8 mg/kg carbon tetrachloride (CCl4) i.p. once a week for 7 weeks and 100 mg/kg C. comosum extract in their diet for 7 weeks, the rats were divided into four groups as follows: Group 1, treatment with DEN alone; group 2, treatment with DEN and C. comosum extract; group 3, treatment with DEN and CCl4; and group 4, treatment with DEN, CCl4 and C. comosum extract. The supplementation of C. comosum extract significantly suppressed the elevation in serum liver enzyme levels, including aspartate aminotransferase, alanine transaminase and γ-glutamyl transferase, and reduced the degree of oval cell proliferation induced by DEN and CCl4. In addition, C. comosum extract significantly decreased the number and area of glutathione S-transferase placental form-positive preneoplastic hepatic foci induced by DEN, with or without CCl4 treatment. To the best of our knowledge, the present study is the first to provide definitive evidence of the hepatoprotective and chemopreventive effects of C. comosum.