Despite clear evidence that exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and that exosomal RNA cargo selection is deregulated in pathological conditions, the mechanisms controlling specific RNA sorting into extracellular vesicles are still poorly understood. Here, we identified the RNA binding protein SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein; also known as hnRNP-Q or NSAP1) as a component of the hepatocyte exosomal miRNA sorting machinery. SYNCRIP knockdown impairs sorting of miRNAs in exosomes. Furthermore, SYNCRIP directly binds to specific miRNAs enriched in exosomes sharing a common extra-seed sequence (hEXO motif). The hEXO motif has a role in the regulation of miRNA localization, since embedment of this motif into a poorly exported miRNA enhances its loading into exosomes. This evidence provides insights into the mechanisms of miRNA exosomal sorting process. Moreover, these findings open the way for the possible selective modification of the miRNAs exosomal cargo.

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 4a (HNF4a) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4a 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 HNF4a 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 HNF4a. HNF4a, in cooperation with its target HNF1a, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4a-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 HNF4a in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4a activator and repressor functions are necessary for the identity of hepatocytes. (HEPATOLOGY 2011;53:2063-2074 E pithelial-to-mesenchymal transition (EMT) is the process by which polarized cells of the epithelium lose cell-cell connections and acquire the mesenchymal characteristics of motility and invasiveness. The reverse process, mesenchymal-to-epithelial transition (MET), often occurs at a site secondary to the original EMT population. The dynamic EMT/MET processes are essential for embryonic development and wound repair and initiate the pathological states of fibrosis and metastatic cancer.

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.

Our data show a direct and hitherto unknown convergence of the canonical Wnt signaling on the HNF4alpha-driven transcription providing evidences of a mechanism controlling liver zonated gene expression.

The ErbB-4 receptors are unique in the EGFR/ErbB family for the ability to associate with WW domain-containing proteins. To identify new ligands of the cytoplasmic tail of ErbB-4, we panned a brain cDNA phage library with ErbB-4 peptides containing sequence motifs corresponding to putative docking sites for class-I WW domains. This approach led to identification of AIP4/Itch, a member of the Nedd4-like family of E3 ubiquitin protein ligases, as a protein that specifically interacts with and ubiquitinates ErbB-4 in vivo. Interaction with the ErbB-4 receptors occurs via the WW domains of AIP4/Itch. Functional analyses demonstrate that AIP4/Itch is recruited to the ErbB-4 receptor to promote its polyubiquitination and degradation, thereby regulating stability of the receptor and access of receptor intracellular domains to the nuclear compartment. These findings expand our understanding of the mechanisms contributing to the integrity of the ErbB signaling network and mechanistically link the cellular ubiquitination pathway of AIP4/Itch to the ErbB-4 receptor.

Epithelial-to-mesenchymal transition (EMT) and the reverse process mesenchymal-to-epithelial transition (MET) are events involved in development, wound healing and stem cell behaviour and contribute pathologically to cancer progression. The identification of the molecular mechanisms underlying these phenotypic conversions in hepatocytes are fundamental to design specific therapeutic strategies aimed at optimising liver repair. The role of autophagy in EMT/MET processes of hepatocytes was investigated in liver-specific autophagy-deficient mice (Alb-Cre;ATG7fl/fl) and using the nontumorigenic immortalised hepatocytes cell line MMH. Autophagy deficiency in vivo reduces epithelial markers' expression and increases the levels of mesenchymal markers. These alterations are associated with an increased protein level of the EMT master regulator Snail, without transcriptional induction. Interestingly, we found that autophagy degrades Snail in a p62/SQSTM1 (Sequestosome-1)-dependent manner. Moreover, accordingly to a pro-epithelial function, we observed that autophagy stimulation strongly affects EMT progression, whereas it is necessary for MET. Finally, we found that the EMT induced by TGFβ affects the autophagy flux, indicating that these processes regulate each other. Overall, we found that autophagy regulates the phenotype plasticity of hepatocytes promoting their epithelial identity through the inhibition of the mesenchymal programme.

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.

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 minicircuitry 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 HNF4a, directly represses the expression of the epithelial microRNAs (miRs)-200c and -34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4a, previously identified as a transcriptional repressor of Cellular differentiation implies an orchestrated sequence of events guiding stem cells/precursors toward specialized cell types based on the contemporary and strictly correlated phenomena of loss of stemness and acquisition of histotypic markers and functions. The homeostasis of the stem cell compartment requires mechanisms actively counteracting differentiation; 1 similarly, the maintenance of the differentiated state involves a stable repression of elements capable to induce morphological transition and dedifferentiation. 2 The observation that a number of stem cells are restricted to a specific differentiation fate suggests that elements pivotal for the coordinated execution of the opposite processes could be tissue-specific. Considering that stem cell compartments are rare and give rise to a heterogeneous cellular population capable to reversibly shift among different states, 3 the availability of a stable stem cell line executing specific differentiation programs discloses an unique possibility to investigate mechanisms regulating alternative cellular choices. A simple and direct molecular mini-circuitry of master elements of mutually exclusive biological processes, also able to reciprocally influence their own expression, may provide the theoretically best device to trigger such complex phenomena.We previously characterized a number of stable liver stem cell lines named RLSCs (from resident liver stem cells) that spontaneously acquire an epithelial morphology and differentiate into hepatocytes (named RLSCdH from RLSC-derived hepatocytes). Notably, RLSCs were also proved to recapitulate the hepatocyte post-differentiation patterning defined as 'zonation': their spontaneous differentiation, in fact, generates periportal hepatocytes that may be induced to switch into perivenular hepatocytes by means of the convergence of Wnt signaling on the HNF4a-driven transcription. 4 Furthermore, we identified a simple cross-regulatory circuitry between HNF4a (master regulator of hepatocyte differentiation) and Snail (master regulator of the epithelial-to-mesenchymal transition, EMT), whose expression is mutually exclusive because of their direct reciprocal transcriptional repression. 2,5 These findings, relevant for the comprehension of the EMT and of the reverse process mesenchymal-...