Laura Amicone

Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes.

Hepatocyte growth factor induces proliferation, motility and differentiation of epithelial cells through the tyrosine kinase receptor encoded by the MET proto‐oncogene. The cytoplasmic portion of Met (referred to as cyto‐Met) is activated but only weakly transforming. In order to determine the effect of activated Met on hepatocytes, we have targeted truncated Met expression to the liver by incorporating the cDNA into a vector carrying the entire human a‐1‐antitrypsin transcriptional unit. Transgenic expression in the liver of truncated human Met, containing the regulatory and the catalytic cytoplasmic domains, renders hepatocytes constitutively resistant to apoptosis and reproducibly permits immortalization. The emerging stable cell lines are not transformed and maintain a highly differentiated phenotype judged by the retention of epithelial cell polarity and the expression of hepatocyte‐enriched transcription factors as well as hepatic products.

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;)

Hepatitis C virus production requires apolipoprotein A-I and affects its association with nascent low-density lipoproteins

Background/aims The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. Methods A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naïve HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. Results A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. Conclusions This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy.

Human Haemato-Endothelial Precursors: Cord Blood CD34+ Cells Produce Haemogenic Endothelium

Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144−), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45−) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.

Searching for DNA-protein interactions by lambda phage display

We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.

An epistatic mini-circuitry between the transcription factors Snail and HNF4α controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs.

Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular mini-circuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4α, directly represses the expression of the epithelial microRNAs (miRs)-200c and -34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4α, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and -200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4α and miRs-200a, b, c and -34a as epistatic elements controlling hepatic stem cell maintenance/differentiation.

Molecular mechanisms controlling the phenotype and the EMT/MET dynamics of hepatocyte

The complex spatial and paracrine relationships between the various liver histotypes are essential for proper functioning of the hepatic parenchymal cells. Only within a correct tissue organization, in fact, they stably maintain their identity and differentiated phenotype. The loss of histotype identity, which invariably occurs in the primary hepatocytes in culture, or in vivo in particular pathological conditions (fibrosis and tumours), is mainly because of the phenomenon of epithelial‐to‐mesenchymal transition (EMT). The EMT process, that occurs in the many epithelial cells, appears to be driven by a number of general, non‐tissue‐specific, master transcriptional regulators. The reverse process, the mesenchymal‐to‐epithelial transition (MET), as yet much less characterized at a molecular level, restores specific epithelial identities, and thus must include tissue‐specific master elements. In this review, we will summarize the so far unveiled events of EMT/MET occurring in liver cells. In particular, we will focus on hepatocyte and describe the pivotal role in the control of EMT/MET dynamics exerted by a tissue‐specific molecular mini‐circuitry. Recent evidence, indeed, highlighted as two transcriptional factors, the master gene of EMT Snail, and the master gene of hepatocyte differentiation HNF4α, exhorting a direct reciprocal repression, act as pivotal elements in determining opposite cellular outcomes. The different balances between these two master regulators, further integrated by specific microRNAs, in fact, were found responsible for the EMT/METs dynamics as well as for the preservation of both hepatocyte and stem/precursor cells identity and differentiation. Overall, these findings impact the maintenance of stem cells and differentiated cells both in in vivo EMT/MET physio‐pathological processes as well as in culture.

Isolation and characterization of a murine resident liver stem cell

Increasing evidence provides support that mammalian liver contains stem/progenitor cells, but their molecular phenotype, embryological derivation, biology and their role in liver cell turnover and regeneration remain to be further clarified. In this study, we report the isolation, characterization and reproducible establishment in line of a resident liver stem cell (RLSC) with immunophenotype and differentiative potentiality distinct from other previously described liver precursor/stem cells. RLSCs, derived from fetal and neonatal murine livers as well as from immortalized hepatocytic MMH lines and established in lines, are Sca+, CD34−, CD45−, α-fetoprotein+ and albumin−. This molecular phenotype suggests a non-hematopoietic origin. RLSC transcriptional profile, defined by microArray technology, highlighted the expression of a broad spectrum of ‘plasticity-related genes’ and ‘developmental genes’ suggesting a multi-differentiative potentiality. Indeed, RLSCs spontaneously differentiate into hepatocytes and cholangiocytes and, when cultured in appropriate conditions, into mesenchymal and neuro-ectodermal cell lineages such as osteoblasts/osteocytes, chondrocytes, astrocytes and neural cells. RLSC capability to spontaneously differentiate into hepatocytes, the lack of albumin expression and the broad differentiative potentiality locate them in a pre-hepatoblast/liver precursor cells hierarchical position. In conclusion, RLSCs may provide a useful tool to improve liver stem cell knowledge and to assess new therapeutic approaches for liver diseases.

OXIDATION AFFECTS THE REGULATION OF HEPATIC LIPID SYNTHESIS BY CHYLOMICRON REMNANTS

The effects of native and oxidized chylomicron remnants on lipid synthesis in normal and oxidatively stressed liver cells were investigated using MET murine hepatocytes (MMH cells), a nontransformed mouse hepatocyte cell line that maintains a highly differentiated hepatic phenotype in culture. Lipid synthesis was determined by measuring the incorporation of [(3)H]oleate into cholesteryl ester, triacylglycerol, and phospholipid by the cells. The formation of cholesteryl ester and phospholipid was decreased by chylomicron remnants in a dose-dependent manner, while triacylglycerol synthesis was increased. Exposure of MMH cells to mild oxidative stress by incubation with CuSO(4) (2.5 microM) for 24 h led to significantly increased incorporation of [(3)H]oleate into triacylglycerol and phospholipid, but not cholesteryl ester, in the absence of chylomicron remnants. In the presence of the lipoproteins, however, similar effects to those found in untreated cells were observed. Oxidatively modified chylomicron remnants prepared by incubation with CuSO(4) (10 microM, 18 h, 37 degrees C) did not influence cholesteryl ester or phospholipid synthesis in MMH cells, but had a similar effect to that found with native remnants on triacylglycerol synthesis. These findings show that hepatic lipid metabolism is altered by exposure to mild oxidative stress and by lipids from the diet delivered to the liver in chylomicron remnants, and these effects may play a role in the development of atherosclerosis.