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Adult hepatocyte identity is constructed throughout embryonic development and fine-tuned after birth. A multinodular network of transcription factors, along with pre-mRNA splicing regulators, define the transcriptome, which encodes the proteins needed to perform the complex metabolic and secretory functions of the mature liver. Transient hepatocellular dedifferentiation can occur as part of the regenerative mechanisms triggered in response to acute liver injury. However, persistent downregulation of key identity genes is now accepted as a strong determinant of organ dysfunction in chronic liver disease, a major global health burden. Therefore, the identification of core transcription factors and splicing regulators that preserve hepatocellular phenotype, and a thorough understanding of how these networks become disrupted in diseased hepatocytes, is of high clinical relevance. In this context, we review the key players in liver differentiation and discuss in detail critical factors, such as HNF4α, whose impairment mediates the breakdown of liver function. Moreover, we present compelling experimental evidence demonstrating that restoration of core transcription factor expression in a chronically injured liver can reset hepatocellular identity, improve function and ameliorate structural abnormalities. The possibility of correcting the phenotype of severely damaged and malfunctional livers may reveal new therapeutic opportunities for individuals with cirrhosis and advanced liver disease.

El hígado es un órgano con un papel central en el organismo puesto que realiza numerosas funciones esenciales para mantener la homeostasis sistémica. A nivel histológico, el hígado presenta una arquitectura compleja y muy organizada, formada por diferentes poblaciones celulares que desarrollan funciones concretas y, en conjunto, permiten el correcto funcionamiento del órgano. Así, el hígado está formado por las células parenquimales que incluyen a los hepatocitos y a los colangiocitos, y las células no parenquimales, que engloban a las células estrelladas hepáticas (HSC, hepatic stellate cells), las células de Kupffer o las células endoteliales sinusoidales. La población celular más abundante son los hepatocitos, que constituyen alrededor de un 80 % de la masa hepática total, y son los que más contribuyen al desarrollo de la función hepática. Se trata de unas células epiteliales polarizadas, muy diferenciadas y quiescentes, aunque presentan una gran capacidad de regeneración en respuesta a estímulos nocivos 1–4 . Las células hepáticas se organizan en el hígado alrededor de una estructura anatómica peculiar y compleja, determinada por el patrón del flujo sanguíneo, y que es esencial para que se puedan llevar a cabo las funciones hepáticas. Brevemente, el hígado recibe sangre de la vena porta rica en nutrientes y productos de deshecho, y sangre de la arteria hepática rica en oxígeno, que fluye a través de una red de capilares sinusoidales entre las células hepáticas, hasta drenar en las venas centrales. Junto a las venas porta y las arterias hepáticas se encuentran los conductos biliares que conducen la bilis hasta la vesícula biliar y el intestino, y los tres conductos juntos forman la triada portal. La unidad funcional del hígado son los lobulillos hepáticos, que consisten en fragmentos poligonales de células hepáticas dispuestas en torno a una vena central y rodeadas de triadas portales. Los capilares sinusoidales conectan la vena central con los espacios porta y, en paralelo a ellos, se organizan los hepatocitos formando cordones gruesos unicelulares (Figura 1A). Tanto la vena porta como la arteria hepática suministran la sangre a los hepatocitos conforme fluye a través de la red de sinusoides hasta drenar en las venas centrales. En función de si los hepatocitos se localizan más cerca de la región periportal o más cerca de la región pericentral, presentan diferencias fenotípicas en cuanto a la expresión de enzimas metabólicas, por lo que realizan funciones metabólicas diferentes. Esta distribución diferencial de las funciones metabólicas y secretoras del hígado a lo largo del eje porto-central se conoce como zonación, y constituye un marcador clave de la maduración hepática (Figura 1B) 1–5.

La expresión génica es el proceso por el cual la información codificada en el DNA (ácido desoxirribonucleico) se transforma en productos funcionales, bien sean proteínas o moléculas de RNA (ácido ribonucleico) no codificante (ncRNA) incluidos los RNA de transferencia (tRNA) y ribosómico (rRNA) o múltiples tipos de RNAs reguladores como por ejemplo, los microRNAs (miRNAs), los RNAs pequeños de interferencia (siRNA) o los RNA no codificantes largos (lncRNAs) (Figura 1).La adecuada expresión espacio-temporal de los genes es un proceso dinámico y estrictamente controlado. De hecho, existen múltiples niveles de regulación de la expresión génica en los que participan distintos eventos moleculares interconectados: modulación de la accesibilidad a la cromatina, transcripción, corte y empalme (splicing), transporte y estabilidad del RNA mensajero (mRNA) y traducción, procesamiento, modificaciones postraduccionales y regulación de la estabilidad de las proteínas (Figura 1)4 . Todas las células de un organismo multicelular contienen el mismo material genético, sin embargo, gracias a la expresión génica diferencial y a su correcta regulación, cada célula, en cada momento, expresa un subconjunto específico de genes que definirán su identidad y su función.

Gastric cancer (GC) is one of the deadliest malignancies worldwide. Complex disease heterogeneity, late diagnosis, and suboptimal therapies result in the poor prognosis of patients. Besides genetic alterations and environmental factors, it has been demonstrated that alterations of the epigenetic machinery guide cancer onset and progression, representing a hallmark of gastric malignancies. Moreover, epigenetic mechanisms undergo an intricate crosstalk, and distinct epigenomic profiles can be shaped under different microenvironmental contexts. In this scenario, targeting epigenetic mechanisms could be an interesting therapeutic strategy to overcome gastric cancer heterogeneity, and the efforts conducted to date are delivering promising results. In this review, we summarize the key epigenetic events involved in gastric cancer development. We conclude with a discussion of new promising epigenetic strategies for gastric cancer treatment.

SLU7 (Splicing factor synergistic lethal with U5 snRNA 7) was first identified as a splicing factor necessary for the correct selection of 3 ' splice sites, strongly impacting on the diversity of gene transcripts in a cell. More recent studies have uncovered new and non-redundant roles of SLU7 as an integrative hub of different levels of gene expression regulation, including epigenetic DNA remodeling, modulation of transcription and protein stability. Here we review those findings, the multiple factors and mechanisms implicated as well as the cellular functions affected. For instance, SLU7 is essential to secure liver differentiation, genome integrity acting at different levels and a correct cell cycle progression. Accordingly, the aberrant expression of SLU7 could be associated with human diseases including cancer, although strikingly, it is an essential survival factor for cancer cells. Finally, we discuss the implications of SLU7 in pathophysiology, with particular emphasis on the progression of liver disease and its possible role as a therapeutic target in human cancer.

Polycystic liver disease (PLD) is a group of rare disorders that result from structural changes in the biliary tree development in the liver. In the present work, we studied alterations in molecular mechanisms and signaling pathways that might be responsible for these pathologies. We found that activation of the unfolded protein response, a process that occurs in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum, as well as the scarring of the liver tissue, contribute to the pathogenesis of PLD and the development of cancer. As a preclinical animal model we have used mutant mice of a specific signaling pathway, the c-Jun N-terminal kinase 1/2 (Jnk1/2). These mice resemble a perfect model for the study of PLD and early cancer development.

OBJECTIVE: To determine the role of enterokine FGF15/19 in adipose tissue thermogenic adaptations. METHODS: Circulating FGF19 and gene expression (qRT-PCR) levels were assessed in subcutaneous adipose tissue from obese human patients. Effects of experimentally increased FGF15 and FGF19 levels in vivo were determined in mice using adenoviral and adeno-associated vectors. Adipose tissues were characterized in FGF15-null mice under distinct cold-related thermogenic challenges. The analyses spanned metabolic profiling, tissue characterization, histology, gene expression, and immunoblot assays. RESULTS: In humans, FGF19 levels are directly associated with UCP1 gene expression in subcutaneous adipose tissue. Experimental increases in FGF15 or FGF19 induced white fat browning in mice as demonstrated by the appearance of multilocular beige cells and markers indicative of a beige phenotype, including increased UCP1 protein levels. Mice lacking FGF15 showed markedly impaired white adipose tissue browning and a mild reduction in parameters indicative of BAT activity in response to cold-induced environmental thermogenic challenges. This was concomitant with signs of altered systemic metabolism, such as reduced glucose tolerance and impaired cold-induced insulin sensitization. CONCLUSIONS: Enterokine FGF15/19 is a key factor required for adipose tissue plasticity in response to thermogenic adaptations.

Chronic liver diseases (CLD) represent a worldwide health problem. While CLDs may have diverse etiologies, a common pathogenic denominator is the presence of liver fibrosis. Cirrhosis, the end-stage of CLD, is characterized by extensive fibrosis and is markedly associated with the development of hepatocellular carcinoma. The most important event in hepatic fibrogenesis is the activation of hepatic stellate cells (HSC) following liver injury. Activated HSCs acquire a myofibroblast-like phenotype becoming proliferative, fibrogenic, and contractile cells. While transient activation of HSCs is part of the physiological mechanisms of tissue repair, protracted activation of a wound healing reaction leads to organ fibrosis. The phenotypic changes of activated HSCs involve epigenetic mechanisms mediated by non-coding RNAs (ncRNA) as well as by changes in DNA methylation and histone modifications. During CLD these epigenetic mechanisms become deregulated, with alterations in the expression and activity of epigenetic modulators. Here we provide an overview of the epigenetic alterations involved in fibrogenic HSCs transdifferentiation with particular focus on histones acetylation changes. We also discuss recent studies supporting the promising therapeutic potential of histone deacetylase inhibitors in liver fibrosis.

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated Mdr2-KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor β (TGFβ). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strategy.

Hemos estudiado el desconocido papel del factor de splicing SLU7 como mediador de la integridad genómica y regulador de la progresión en la mitosis durante el ciclo celular. Hemos demostrado que la disminución de la expresión de SLU7 en líneas celulares cancerosas de diferentes orígenes contribuye (1) a la formación de híbridos RNA:DNA (R-loops) asociados con la inducción de daño en el DNA y (2) a la parada en la mitosis asociada con la pérdida de cohesión entre las cromátidas hermanas. Ambos eventos están directamente relacionados con la inducción de inestabilidad genómica, característica fundamental de la carcinogénesis. Tanto in vitro como in vivo, hemos caracterizado los mecanismos moleculares que implican alteraciones del splicing y la generación de proteínas aberrantes de SRSF3 y la disminución de SRSF1 y sororina. Nuestros datos presentan a SLU7 como un guardián de la estabilidad genómica y la disminución de su expresión observada en el hígado enfermo de los pacientes podría estar participando en el proceso de hepatocarcinogénesis. Así mismo, en las células transformadas la dependencia de SLU7 para su supervivencia, lo presentan como una nueva diana terapéutica para el cáncer.

Hepatocellular carcinoma (HCC) is the most frequent primary liver tumor and a leading cause of cancer-related mortality. Unfortunately, the majority of HCC patients present with advanced disease, when locoregional curative strategies are no longer applicable. On the other hand, early, HCCs proved to be resistant to conventional chemotherapies, and drugs targeting specific growth factor signaling pathways tested over the past decade have not succeeded in clinical trials. The molecular heterogeneity of HCCs and the lack of biomarker-based patient stratification strategies may underlie the failure of most of these trials. HCCs usually develop on a background of chronic liver injury and regeneration, inflammation, and fibrosis, features that promote tumor hypervascularity, the other histologic hallmark of this neoplasia. Indeed, the fibrotic and immune microenvironment plays a key role in pathogenic angiogenesis and HCC development and progression. This tenet is supported not only by experimental evidence, but also by the fact that the only therapeutic agents showing clinical efficacy in advanced HCC are those directed toward the interaction of HCC with its microenvironment (ie, anti-angiogenic multikinase inhibitors such as sorafenib, and immune checkpoint inhibitors such as antibodies targeting programmed cell death receptor 1 and its ligand).

Previous studies showed that YAP1 is over-expressed in hepatocellular carcinoma (HCC). Here we observed higher expression of Yap1/Ctgf axis in dysplastic nodules and HCC chemically-induced in F344 rats, genetically susceptible to hepatocarcinogenesis, than in lesions induced in resistant BN rats. In BN rats, highest increase in Yap1- tyr357, p73 phosphorylation and Caspase 3 cleavage occurred. In human HCCs with poorer prognosis (< 3 years survival after partial liver resection, HCCP), levels of YAP1, CTGF, 14–3–3, and TEAD proteins, and YAP1-14-3-3 and YAP1-TEAD complexes were higher than in HCCs with better outcome (> 3 years survival; HCCB). In the latter, higher levels of phosphorylated YAP1-ser127, YAP1-tyr357 and p73, YAP1 ubiquitination, and Caspase 3 cleavage occurred. Expression of stemness markers NANOG, OCT-3/4, and CD133 were highest in HCCP and correlated with YAP1 and YAP1-TEAD levels. In HepG2, Huh7, and Hep3B cells, forced YAP1 over-expression led to stem cell markers expression and increased cell viability, whereas inhibition of YAP1 expression by specific siRNA, or transfection of mutant YAP1 which does not bind to TEAD, induced opposite alterations. These changes were associated, in Huh7 cells transfected with YAP1 or YAP1 siRNA, with stimulation or inhibition of cell migration and invasivity, respectively. Furthermore, transcriptome analysis showed that YAP1 transfection in Huh7 cells induces over-expression of genes involved in tumor stemness. In conclusion, Yap1 post-translational modifications favoring its ubiquitination and apoptosis characterize HCC with better prognosis, whereas conditions favoring the formation of YAP1-TEAD complexes are associated with aggressiveness and acquisition of stemness features by HCC cells.

Upon tissue injury, the liver mounts a potent reparative and regenerative response. This wound-healing response involves a transitory substitution of damaged hepatic parenchyma by extracellular matrix (ECM) that subsequently needs to be removed when liver tissue is regenerated. A role for proteases, including matrix metalloproteinases (MMPs), in this process is increasingly recognized, acting as regulatory molecules by the selective proteolytic activation of growth factors and cell surface receptors in addition to their ability to degrade ECM constituents. Moreover, MMPs also have been demonstrated to participate in cancer by promoting a protumorigenic microenvironment. We have evaluated the hepatic expression and function of MMP10 (stromelysin-2) in mouse models of liver wound-healing and regeneration, in human hepatocellular carcinoma (HCC) and in diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis. The role of MMP10 was evaluated by comparing the response of wild type (MMP10+/+) and MMP10-deficient (MMP10-/-) mice to the different experimental models. Mouse MMP10 hepatic expression was readily induced in different injury and regeneration models: two-thirds partial hepatectomy (PH), bile duct ligation (BDL) and carbon tetrachloride (CCl4) acute administration. MMP10 protein was detected in hepatocytes, cholangiocytes and macrophages. MMP10 was also induced in human and murine HCC tissues and cells. In cultured liver epithelial cells, MMP10 expression was additively induced by transforming growth factor-β and epidermal growth factor receptor ligands. TLR4 ligands also stimulated its expression in macrophages. Lack of MMP10 resulted in increased liver injury and fibrogenesis, impaired resolution of necrotic areas and defective turnover of fibrin(ogen) and fibronectin upon PH, BDL and CCl4 administration. On the other hand, in the DEN model, MMP10-/- mice showed less HCC incidence, smaller histological lesions, reduced tumor vascularization, and less lung metastases. Importantly, expression of the protumorigenic C-X-C chemokine receptor 4 (CXCR4) was reduced in those mice. A MMP10 overexpressing human HCC cell line was generated. These HCC cells had increased CXCR4 expression and showed enhanced migratory capacity, and the pharmacological inhibition of CXCR4 reduced MMP10-stimulated cell migration. MMP10 expression in HCC cells was induced by hypoxia and the CXCR4 ligand stroma-derived factor 1 (SDF1), through the ERK1/2 pathway, involving an activator protein 1 site in MMP10 gene promoter. Conclusions: MMP10 expression is induced during mouse liver injury and participates in the hepatic wound-healing response, where the profibrinolytic activity of MMP10 may be essential in this hepatoprotective role. In contrast, MMP10 contributes to HCC development, participating in tumor angiogenesis, growth, and dissemination. We identified a new reciprocal crosstalk between MMP10 and the CXCR4/SDF1 axis contributing to HCC progression and metastasis.

A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.

BACKGROUND & AIMS: Cell therapy has been used to attenuate liver injury. Here we evaluated whether genetic engineering of either bone marrow-derived mononuclear cells (MNC) or endothelial progenitor cells (EPC) many enhance their hepatoprotective properties. METHODS: Mice with ConA-induced hepatitis or with lethal fulminant hepatitis resulting from administration of an adenovirus encoding CD40L (AdCD40L) received an intra-splenic injection of saline or 2 × 10(6) unmodified MNC or EPC or the same cells transduced ex vivo with an adenovirus expressing luciferase (MNCLUC and EPCLUC) or encoding the hepatoprotective cytokine cardiotrophin-1 (CT-1) (MNCCT-1 and EPCCT-1). We analyzed the extent of liver damage, the intensity of inflammatory reaction, and animal survival. RESULTS: Luciferase immunohistochemistry showed that after injection into the spleen, the engineered cells migrated efficiently to the damaged liver. In mice with ConA hepatitis EPCCT-1, but not other forms of cell therapy, significantly decreased serum transaminases and induced more intense histological improvement than other treatments. This superior therapeutic effect was associated with upregulation of cytoprotective molecules including IGF-I and EGF, lower expression of proinflammatory cytokines, IL-1b and TNFα, and decreased granzyme B levels. In AdCD40L-induced lethal fulminant hepatitis, EPCCT-1 also exceeded other cell therapies in attenuating the expression of proinflammatory mediators and hepatic injury enabling 35.7% survival while mortality was 100% in the other treatment groups. CONCLUSIONS: Genetic engineering of EPC to overexpress CT-1 enhances the hepatoprotective properties of EPC and constitutes a therapy that deserves consideration for acute liver failure. Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

BACKGROUND: Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5'-methylthioadenosine (MTA) in Mdr2(-/-) mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. METHODOLOGY: MTA was administered daily by gavage to wild type and Mdr2(-/-) mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2(-/-) mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). PRINCIPAL FINDINGS: MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFβ2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. CONCLUSIONS/SIGNIFICANCE: Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis.

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.

Methionine (Met) metabolism involves the sequential formation of S-adenosylmethionine (SAM, the main biological methyl donor), S-adenosylhomocysteine (SAH) and homocysteine (Hcy). Hcy can be remethylated to Met or catabolized through the trans-sulfuration pathway. In mammals, as much as 48% of Met metabolism and up to 85% of all transmethylation reactions occur in the liver. These figures underscore the central role played by this organ in Met metabolism. Maintaining the homeostasis of this metabolic cycle has proved to be essential for the preservation of liver function up to the point of preventing its neoplastic transformation. However, an adequate hepatic metabolism of Met is not only important for the liver parenchymal cell. Evidence has accumulated over the past few years supporting the involvement of Met-derived metabolites in the triggering or attenuation of pathological processes with systemic implications. This is best illustrated by the fact that a deteriorated liver function has emerged as a major factor in the development of hyperhomocysteinemia. Elevated plasma levels of Hcy have been related to several disorders including cardiovascular and cerebrovascular diseases. On the other end, liver damage also leads to deficient SAM synthesis. Among the consequences of impaired SAM synthesis in liver tissue are the enhanced production of pro-inflammatory cytokines and mediators. In this review, we will address the mechanisms and consequences of abnormal Met metabolism in liver injury, the systemic implications of such impairment and finally the potential therapeutic interventions.

Systems for in vitro culture of Hepatitis C virus (HCV) are essential tools to analyse virus-cell interactions and to investigate relevant pathophysiological aspects of HCV infection. Although the HCV replicon methodology has increased our understanding of HCV biology, this system does not reproduce the natural infection. Recently, tupaia (Tupaia belangeri chinensis) hepatocytes have been utilized for in vitro culture of HCV. In the present work, primary tupaia hepatocytes infected in vitro with HCV were used to analyse the evolution of HCV quasispecies in infected cells and the ability of the virus to influence antiviral and proinflammatory responses in cells sustaining virus replication. The results confirmed the potential of tupaia hepatocytes as a model for HCV infection, although this system is limited by rapid loss of differentiated cell phenotype in culture. These findings revealed an extraordinary plasticity of HCV quasispecies, which underwent rapid evolution to tupaia-tropic variants as early as 24 h after infection. It was also shown that HCV could activate interferon-sensitive genes, albeit modestly in comparison with other viruses such as Semliki Forest virus. Importantly, HCV activated NF-kappaB in primary hepatocytes and upregulated NF-kappaB-responsive genes including the chemokines MCP-1 and CXCL2 (MIP-2). This effect may play a role in induction of the hepatic inflammatory reaction in vivo. In summary, HCV quasispecies adapt rapidly to the specific biology of the host and HCV stimulates a blunted interferon response while inducing a proinflammatory phenotype in the infected cell.

5'-methylthioadenosine (MTA) is a nucleoside generated from S-adenosylmethionine (AdoMet) during polyamine synthesis. Recent evidence indicates that AdoMet modulates in vivo the production of inflammatory mediators. We have evaluated the anti-inflammatory properties of MTA in bacterial lipopolysaccharide (LPS) challenged mice, murine macrophage RAW 264.7 cells, and isolated rat hepatocytes treated with pro-inflammatory cytokines. MTA administration completely prevented LPS-induced lethality. The life-sparing effect of MTA was accompanied by the suppression of circulating tumor necrosis factor-alpha (TNF-alpha), inducible NO synthase (iNOS) expression, and by the stimulation of IL-10 synthesis. These responses to MTA were also observed in LPS-treated RAW 264.7 cells. MTA prevented the transcriptional activation of iNOS by pro-inflammatory cytokines in isolated hepatocytes, and the induction of cyclooxygenase 2 (COX2) in RAW 264.7 cells. MTA inhibited the activation of p38 mitogen-activated protein kinase (MAPK), c-jun phosphorylation, inhibitor kappa B alpha (IkappaBalpha) degradation, and nuclear factor kappaB (NFkappaB) activation, all of which are signaling pathways related to the generation of inflammatory mediators. These effects were independent of the metabolic conversion of MTA into AdoMet and the potential interaction of MTA with the cAMP signaling pathway, central to the anti-inflammatory actions of its structural analog adenosine. In conclusion, these observations demonstrate novel immunomodulatory properties for MTA that may be of value in the management of inflammatory diseases.

Staphylococcus aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA/PNAG), the product of the ica operon. The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the production of S. aureus virulence factors. By screening a library of Tn917 insertions in a clinical S. aureus strain, we identified SarA as being essential for biofilm development. Non-polar mutations of sarA in four genetically unrelated S. aureus strains decreased PIA/PNAG production and completely impaired biofilm development, both in steady state and flow conditions via an agr-independent mechanism. Accordingly, real-time PCR showed that the mutation in the sarA gene resulted in downregulation of the ica operon transcription. We also demonstrated that complete deletion of sigmaB did not affect PIA/PNAG production and biofilm formation, although it slightly decreased ica operon transcription. Furthermore, the sarA-sigmaB double mutant showed a significant decrease of ica expression but an increase of PIA/PNAG production and biofilm formation compared to the sarA single mutant. We propose that SarA activates S. aureus development of biofilm by both enhancing the ica operon transcription and suppressing the transcription of either a protein involved in the turnover of PIA/PNAG or a repressor of its synthesis, whose expression would be sigmaB-dependent.