Beatrice Conti

The stable repression of mesenchymal program is required for hepatocyte identity: A novel role for hepatocyte nuclear factor 4α

The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial‐to‐mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal‐to‐epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real‐time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α‐mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. Conclusion: The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes. (HEPATOLOGY 2011;)

C242T Polymorphism of NADPH Oxidase p22phox and Recurrence of Cardiovascular Events in Coronary Artery Disease

Objectives—The common C242T polymorphism in the gene for the p22phox subunit of NADPH oxidase has been reported to be negatively associated with oxidative stress, but whether it confers prognostic information is not yet clear. Methods and Results—The incidence of major adverse cardiovascular events (MACE) were determined in 237 patients with coronary stenosis during a median follow-up of 7.8 years. The p22phox genotypes were evaluated in 213 patients (89.9%) by polymerase chain reaction and RsaI. digestion. Plasma levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative stress, were also measured. In the univariate analysis, patients with CT/TT genotypes showed reduced recurrence of cardiovascular deaths, nonfatal MI, and revascularization procedures compared with homozygous carriers of the C allele. After controlling for confounders, a significantly lower risk of new revascularization procedures (HR=0.31, 95% CI 0.12 to 0.70; P=0.014) remained associated with the T allele. The Kaplan–Meier analysis showed a longer survival free from fatal and nonfatal MI in carriers of T allele (P<0.001). The presence of the 242T allele was associated with significantly reduced plasma concentrations of 8-OHdG. Conclusions—The 242T allele was a predictor of lower risk of recurrence of cardiovascular events in high-risk patients and was associated with reduced systemic oxidative stress.

TGFβ overrides HNF4α tumor suppressing activity through GSK3β inactivation: implication for hepatocellular carcinoma gene therapy

BACKGROUND & AIMS The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity. METHODS Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression. RESULTS We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity. CONCLUSIONS Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.

Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.

Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT?

To the Editor: We read with great interest the article by Akkari et al. [1], who evaluated the EMT induction and correlated oncogenic transformation induced by HCV protein NS5A in primary hepatocyte precursors. Epithelial–mesenchymal transition (EMT) is a biological process occurring during tissue development and regeneration, moreover, it is known to be involved in tumor progression and metastasis. The EMT is a phenomenon, which well represents the cellular plasticity, i.e., the ability of polarized epithelial cells, which through multiple biochemical changes acquire a mesenchymal cell phenotype. These cells exhibit loss of cell adhesion, enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a great ability to produce extracellular matrix (ECM) components. Several molecular processes are involved in EMT onset and progression. Among them we have to mention: activation of transcription factors (e.g., Snail, Twist, etc.), expression of specific cell-surface proteins, remodelling of cytoskeletal components, production of ECM-degrading enzymes and changes in the expression of specific microRNAs (e.g., microRNA 200 family). Most of them are commonly used as biomarkers to highlight a cellular EMT [2,3]. Akkari et al. have evaluated how a viral protein can alter the typical epithelial architecture of hepatic cells. In particular, they demonstrated that the ectopic expression of hepatitis C virus (HCV) non-structural protein 5a (NS5A) is able to induce EMT in bipotential mouse embryonic liver (BMEL) cells which acquire mesenchymal phenotype, increased motility and invasiveness, and are featured by the downregulation of epithelial markers (e.g., E-Cadherin) and the upregulation of mesenchymal markers (like Vimentin and Twist2). They also confirmed their results in an in vivo xenograft mouse model. To unravel the molecular mechanisms involved in EMT occurrence, they focused their experimental investigation on transforming growth factor (TGF)-b, a main cytokine involved in EMT induction. They have shown that NS5A is not responsible for TGF-b pathway activation but it acts in a synergistic manner with the cytokine in inducing EMT. Furthermore, they also screened, in their in vitro model, the modulation of expression of EMT biomarkers, such as Snail, Slug, Zeb and Twist 1/2. They demonstrated that only Twist2 expression was specifically increased by NS5A protein, and that, accordingly, through short hairpin RNA against Twist2, they were able to counteract EMT occurrence. Interestingly, the TGF-b treatment of cells lacking of Twist2 is able to promote EMT in any case, suggesting that NS5A and TGF-b trigger EMT by distinct pathways and thus identifying Twist2 as an NS5A EMT specific effector.

Proteomic analysis reveals a major role for contact inhibition in the terminal differentiation of hepatocytes

BACKGROUND & AIMS Hepatocytes are considered an exception of the paradigmatic inverse correlation between cell proliferation and terminal differentiation. In fact, hepatic vital functions are guaranteed by proliferating parenchymal cells during liver regeneration. However, a fine molecular characterization of the relationship between proliferation and differentiation in hepatocytes has been hampered by the lack of reliable in vivo or in vitro models. METHODS The hepatocyte terminal differentiation program was characterized in the immortalized, untransformed and differentiated hepatocytic cell line MMH, using several techniques. Particularly, two-dimensional difference gel electrophoresis combined to tandem mass spectrometry proteomic approach was used. Cell cycle and cell adhesion properties of MMH have been altered using either myc-overexpression and MEK1/2 inhibition or a constitutive active beta-catenin mutant, respectively. RESULTS The hepatocyte terminal differentiation program is stimulated by the exit from the cell cycle induced by cell-cell contact. Comparative proteomic analysis of proliferating versus quiescent hepatocytes validated the importance of contact inhibition, identifying 68 differently expressed gene products, representing 49 unique proteins. Notably, enzymes involved in important liver functions such as detoxification processes, lipid metabolism, iron and vitamin A storage and secretion, anti-inflammatory response and exocytosis were found significantly up-regulated in quiescent hepatocytes. Finally, we found that: (i) cell cycle arrest induced by MEK1/2 inhibition is not sufficient to induce hepatic product expression; (ii) constitutive activation of beta-catenin counteracts the contact inhibition-induced terminal differentiation. CONCLUSION The hepatocyte terminal differentiation program requires a quiescent state maintained by cell-cell contact through the E-cadherin/beta-catenin pathway, rather than the inhibition of proliferation.

Lymphocytes as liver damage mirror of HCV related adipogenesis deregulation

Hepatitis C virus infection leads to a wide spectrum of liver diseases ranging from mild chronic hepatitis to end-stage cirrhosis and hepatocellular carcinoma. An intriguing aspect of the HCV infection is its close connection with lipid metabolism playing an important role in the HCV life cycle and in its pathogenesis. HCV is known to be a hepatotropic virus; however, it can also infect peripheral blood mononuclear cells (PBMCs). The goal of the current investigation is to compare the adipogenesis profile of liver tissues to lymphocytes of HCV infected patients, in order to understand if PBMCs may reflect the alterations of intracellular pathways occurring during HCV-related liver steatosis. Using the Human Adipogenesis PCR Array, gene expression was analyzed in liver samples and PBMCs of chronic HCV+, HBV+ and Healthy Donors (HDs) patients. We observed a similar modulation of lipid metabolism in HCV+ and HBV+liver tissues and lymphoid, cells suggesting that PBMCs reflect the liver adipogenesis deregulation related to infection, even if the two viruses have a different impact in the regulation of the adipogenesis mechanisms. In particular, some genes involved in lipid metabolism and inflammation, as well as in cell transformation, were up-regulated, in a similar way, in both HCV models analyzed. Interestingly, these genes were positively correlated to virological and hepatic functional parameters of HCV+ patients. On the contrary, HBV+ patients displayed a completely different profile. PBMCs of HCV+ patients seem to be useful model to study how HCV-related lipid metabolism deregulation occurs in liver. The obtained data suggest some molecules as new possible biomarkers of HCV-related liver damage progression.

1031 HCV INFECTION PROMOTES CYTOSKELETAL REORGANIZATION IN HEPATOCYTES AND A PRO-FIBROGENIC PHENOTYPE IN HEPATIC STELLATE CELLS BY THE ACTIVATION OF FOCAL ADHESION KINASE

1031 HCV INFECTION PROMOTES CYTOSKELETAL REORGANIZATION IN HEPATOCYTES AND A PRO-FIBROGENIC PHENOTYPE IN HEPATIC STELLATE CELLS BY THE ACTIVATION OF FOCAL ADHESION KINASE A. Alisi, M. Arciello, S. Petrini, B. Conti, C. Balsano. Liver Unit, Bambino Gesu Children’s Hospital, Laboratory of Molecular Virology and Oncology, Fondazione Andrea Cesalpino, Microscopy Unit, Bambino Gesu Children’s Hospital, Roma, Italy E-mail: anna.alisi@opbg.net

Regarding: Epithelial-Mesenchymal Transition Induced by Hepatitis C Virus Core Protein in Cholangiocarcinoma

We appreciate the article by Li et al. in a recent issue of the Annals of Surgical Oncology. In this study, the authors demonstrated that HCV, in particular HCV core protein (HCVc), was associated with epithelial and mesenchymal transition (EMT) in cholangiocarcinoma (CC). They demonstrated that HCVc may participate in CC invasion and metastasis with the downregulation of epithelial markers, as E-cadherin (E-cad), and the upregulation of mesenchymal markers, as vimentin and fibronectin. In addition, they investigated the role of lysil oxidase such as protein 2 (LOXL2), a new promoter of EMT and an important factor for HCV infection. They demonstrated that LOXL2 was highly expressed in CC tissues and was associated with invasion and metastasis through EMT. Thus, they hypothesized that HCVc can promote EMT in CC through LOXL2 activity. To test this hypothesis, LOXL2 expression was measured after HCVc gene transfection. They observed that LOXL2 level was increased in CC/HCVc, and after the inhibition of LOXL2 expression they observed a downregulation of LOXL2 and of mesenchymal markers and a simultaneous upregulation of epithelial markers. These results support the relationship between HCVc, LOXL2, and EMT. All these findings are very intriguing but they could be more interesting with the comprehension of the role of Snail, the EMT master gene. The transcription factor Snail controls epithelial-mesenchymal transitions (EMT) by repressing E-cad expression and other epithelial genes. Peinado et al. show that LOXL2 interacts and cooperates with Snail to downregulate E-cadherin expression. The functional collaboration of these two proteins to repress E-cadherin expression is strictly dependent on the presence in Snail protein of two specific lysine residues: K98 and K137. Snail’s lysine residues 98 and 137 are essential for Snail stability, functional cooperation with LOXL2, and induction of EMT. All these observations highlight that LOXL2 might be a key molecule during progression of tumors related or not to HCV infection. Furthermore, LOXL2 activity might be a crucial modulator of Snail, providing an additional control mechanism of EMT and tumor progression. It would be interesting to analyze the role of LOXL2 and Snail in hepatocarcinoma induced by HCV. In particular, it could examine how HCVc is really involved in the induction of LOXL2/Snail-mediated EMT. This evidence also raises the possibility of using LOXL2 expression as an additional predictive/prognostic marker for carcinoma progression.

Small heterodimer partner 1 directly interacts with NS5A viral protein and has a key role in HCV related liver cell transformation

HCV life cycle is strictly correlated with the hepatocyte lipid metabolism; moreover, the progression of HCV chronic hepatitis is accelerated by the presence of liver steatosis. Among the steatogenic genes deregulated during the HCV infection one of the most attractive is the Small Heterodimer Protein 1 (SHP1; NR0B2), that is involved in a remarkable number of metabolic functions. HCV NS5A is an essential and integral component of the HCV membranous-web replicon complex (RC) and plays an essential role to transfer the viral genome from the RCs to the surface of the lipid droplets (LDs) that, in turn, play a key function during HCV life cycle. With the help of a HCV infection model, we demonstrate a functional interaction between SHP1 and HCV NS5A protein. SHP1 silencing (siSHP1) reversed the pro-oncogenic effects of HCV infection, inducing a significant decrease in liver lipid accumulation and in NS5A protein expression. Moreover, siSHP1 causes a strong modulation of some genes involved in HCV-related EMT, such as: HNF4, a central regulators of hepatocyte differentiation, E-Cadherin, SNAILs. Our data suggest that SHP1 results not only to be strictly connected to the pathogenesis of HCV-related liver steatosis, but also to its progression towards the liver transformation.