Ignacio Benedicto

The Tight Junction-Associated Protein Occludin Is Required for a Postbinding Step in Hepatitis C Virus Entry and Infection

ABSTRACT The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.

Hepatitis C virus envelope components alter localization of hepatocyte tight junction–associated proteins and promote occludin retention in the endoplasmic reticulum

Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ‐associated proteins occludin, claudin‐1, and Zonula Occludens protein‐1 (ZO‐1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ‐associated function, measured as FITC‐dextran paracellular permeability, of genomic replicon‐containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon‐α (IFN‐α) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ‐associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ‐associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co‐localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co‐immunoprecipitation and pull‐down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot‐like accumulation of occludin in infected Huh7 cells. Conclusion: We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ‐associated proteins, which may provide new insights for HCV‐related pathogenesis. (HEPATOLOGY 2008.)

Epithelial-to-mesenchymal transition of peritoneal mesothelial cells is regulated by an ERK/NF-κB/Snail1 pathway

SUMMARY Epithelial-to-mesenchymal transition (EMT) occurs in fibrotic diseases affecting the kidney, liver and lung, and in the peritoneum of patients undergoing peritoneal dialysis. EMT in the peritoneum is linked to peritoneal membrane dysfunction, and its establishment limits the effectiveness of peritoneal dialysis. The molecular regulation of EMT in the peritoneum is thus of interest from basic and clinical perspectives. Treatment of primary human mesothelial cells (MCs) with effluent from patients undergoing peritoneal dialysis induced a genuine EMT, characterized by downregulated E-cadherin and cytokeratin expression, cell scattering, and spindle-like morphology. This EMT was replicated by co-stimulation with transforming growth factor (TGF)-β1 and interleukin (IL)-1β. Retroviral overexpression of a mutant inhibitor of kappaB (IκB) demonstrated that NF-κB activation is required for E-cadherin and cytokeratin downregulation during EMT. Pre-treatment with the MAP kinase kinase (MEK)-1/2 inhibitor U0126 showed that cytokine-triggered NF-κB nuclear translocation and transcriptional activity are mediated by activation of extracellular regulated kinase (ERK). Cytokine-mediated induction of mRNA expression of the transcription factor Snail1, a repressor of E-cadherin expression and a potent inducer of EMT, was prevented by blockade of ERK or NF-κB. Finally, blockade of ERK/NF-κB signaling in ex vivo MCs that were cultured from peritoneal dialysis effluents reverted cells to an epithelioid morphology, upregulated E-cadherin and cytokeratin expression, and downregulated Snail1 expression. Modulation of the ERK/NF-κB/Snail1 pathway may provide a means of counteracting the progressive structural and functional deterioration of the peritoneal membrane during peritoneal dialysis.

p38 maintains E-cadherin expression by modulating TAK1-NF-kappa B during epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells is a pathological process that occurs during peritoneal dialysis. EMT leads to peritoneal fibrosis, ultrafiltration failure and eventually to the discontinuation of therapy. Signaling pathways involved in mesothelial EMT are thus of great interest, but are mostly unknown. We used primary mesothelial cells from human omentum to analyze the role of the p38 MAPK signaling pathway in the induction of EMT. The use of specific inhibitors, a dominant-negative p38 mutant and lentiviral silencing of p38α demonstrated that p38 promotes E-cadherin expression both in untreated cells and in cells co-stimulated with the EMT-inducing stimuli transforming growth factor (TGF)-β1 and interleukin (IL)-1β. p38 inhibition also led to disorganization and downregulation of cytokeratin filaments and zonula occludens (ZO)-1, whereas expression of vimentin was increased. Analysis of transcription factors that repress E-cadherin expression showed that p38 blockade inhibited expression of Snail1 while increasing expression of Twist. Nuclear translocation and transcriptional activity of p65 NF-κB, an important inducer of EMT, was increased by p38 inhibition. Moreover, p38 inhibition increased the phosphorylation of TGF-β-activated kinase 1 (TAK1), NF-κB and IκBα. The effect of p38 inhibition on E-cadherin expression was rescued by modulating the TAK1–NF-κB pathway. Our results demonstrate that p38 maintains E-cadherin expression by suppressing TAK1–NF-κB signaling, thus impeding the induction of EMT in human primary mesothelial cells. This represents a novel role of p38 as a brake or ‘gatekeeper’ of EMT induction by maintaining E-cadherin levels.

Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle

Hepatocytes are highly polarized cells where intercellular junctions, including tight junctions (TJs), determine the polarity. Recently, the TJ-associated proteins claudin-1 and occludin have been implicated in hepatitis C virus (HCV) entry and spread. Nevertheless, cell line-based experimental systems that exhibit hepatocyte-like polarity and permit robust infection and virion production are not currently available. Thus, we sought to determine whether cell line-based, Matrigel-embedded cultures could be used to study hepatitis C virus (HCV) infection and virion production in a context of hepatocyte-like polarized cells. In contrast to standard bidimensional cultures, Matrigel-cultured Huh-7 cells adopted hepatocyte polarization features forming a continuous network of functional proto-bile canaliculi structures. These 3D cultures supported HCV infection by JFH-1 virus and produced infective viral particles which shifted towards lower densities with higher associated specific infectivity. In conclusion, our findings describe a novel use of Matrigel to study the entire HCV cycle in a more relevant context.

A Polypodium leucotomos extract inhibits solar‐simulated radiation‐induced TNF‐α and iNOS expression, transcriptional activation and apoptosis

Abstract:  In this report, we have examined the molecular basis of the photoprotective effect of a hydrophilic extract of the fern Polypodium leucotomos (PL) in vitro, using a solar simulator as the source of UV radiation (SSR). We found that pretreatment of human keratinocytes with PL inhibited SSR‐mediated increase of tumor necrosis factor (TNF)‐α and also abrogated nitric oxide (NO) production. Consistent with this, PL blocked the induction of inducible nitric oxide synthase (iNOS) elicited by SSR. In addition, PL inhibited the SSR‐mediated transcriptional activation of NF‐κB and AP1. Finally, we demonstrated that pretreatment with PL exerted a cytoprotective effect against SSR‐induced damage, resulting in increased cell survival. Together, these data postulate a multifactor mechanism of protection not exclusively reliant on the antioxidant capability of PL, and strengthen the basic knowledge on the photoprotective effect of this botanical agent.

Liver X receptor α-mediated regulation of lipogenesis by core and NS5A proteins contributes to HCV-induced liver steatosis and HCV replication.

Molecular mechanisms contributing to hepatitis C virus (HCV)-associated steatosis are not well established, although HCV gene expression has been shown to alter host cell cholesterol/lipid metabolism. As liver X receptors (LXRs) play a role as key modulators of metabolism signaling in the development of steatosis, we aimed to investigate in an HCV in vitro model the effect of HCV NS5A protein, core protein, and viral replication on the intracellular lipid accumulation and the LXRα-regulated expression of lipogenic genes. The effects of LXRα siRNA or agonist GW3965 treatment on lipogenesis and HCV replication capacity in our HCV replicon system were also examined. NS5A- and core-expressing cells and replicon-containing cells exhibited an increase of lipid accumulation by inducing the gene expression and the transcriptional activity of LXRα, and leading to an increased expression of its lipogenic target genes sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor-γ, and fatty acid synthase. Transcriptional induction by NS5A protein, core protein, and viral replication occurred via LXR response element activation in the lipogenic gene promoter. No physical association between HCV proteins and LXRα was observed, whereas NS5A and core proteins indirectly upregulated LXRα through the phosphatidylinositol 3-kinase pathway. Finally, it was found that LXRα knockdown or agonist-mediated LXRα induction directly regulated HCV-induced lipogenesis and HCV replication efficiency in replicon-containing cells. Combined, our data suggest that LXRα-mediated regulation of lipogenesis by core and NS5A proteins may contribute to HCV-induced liver steatosis and to the efficient replication of HCV.

Inhibition of Transforming Growth Factor-Activated Kinase 1 (TAK1) Blocks and Reverses Epithelial to Mesenchymal Transition of Mesothelial Cells

Peritoneal fibrosis is a frequent complication of peritoneal dialysis following repeated low grade inflammatory and pro-fibrotic insults. This pathological process may lead to ultrafiltration failure and eventually to the discontinuing of the therapy. Fibrosis is linked to epithelial to mesenchymal transition (EMT) of the peritoneal mesothelial cells, which acquire invasive and fibrogenic abilities. Here, we analyzed the role of the transforming growth factor-activated kinase-1 (TAK1) in the EMT of primary mesothelial cells from human peritoneum. The inhibition of TAK1 in mesenchymal-like mesothelial cells from the effluents of patients undergoing peritoneal dialysis led to the reacquisition of the apical to basolateral polarity, to increased expression of epithelial and to down-regulation of mesenchymal markers. TAK1 inhibition also resulted in decreased migratory/invasive abilities of effluent-derived mesothelial cells. Simultaneous inhibition of ERK1/2 and TAK1 pathways did not lead to an additive effect in the reacquisition of the epithelial phenotype. Inhibition of TAK1 also blocked EMT in vitro and reduced the levels of PAI-1, which is involved in fibrosis and invasion. Analysis of signalling pathways downstream of TAK1 involved in EMT induction, showed that TAK1 inhibition reduced the transcriptional activity of NF-κB and Smad3, as well as the phosphorylation of c-jun, while enhancing Smad1–5–8 activity. These results demonstrate that TAK1 is a cross-point in a network including different pro-EMT transcription factors, such as NF-κB, Snail, AP-1 and Smads. The identification of TAK1 as a main biochemical mediator of EMT and fibrosis in mesothelial cells from human peritoneum and the study of signalling pathways induced by its activity may be relevant in the design of new therapies aimed to counteract peritoneal fibrosis.

Caveolin-1 deficiency induces a MEK-ERK1/2-Snail-1-dependent epithelial–mesenchymal transition and fibrosis during peritoneal dialysis

Peritoneal dialysis (PD) is a form of renal replacement therapy whose repeated use can alter dialytic function through induction of epithelial–mesenchymal transition (EMT) and fibrosis, eventually leading to PD discontinuation. The peritoneum from Cav1−/− mice showed increased EMT, thickness, and fibrosis. Exposure of Cav1−/− mice to PD fluids further increased peritoneal membrane thickness, altered permeability, and increased the number of FSP‐1/cytokeratin‐positive cells invading the sub‐mesothelial stroma. High‐throughput quantitative proteomics revealed increased abundance of collagens, FN, and laminin, as well as proteins related to TGF‐β activity in matrices derived from Cav1−/− cells. Lack of Cav1 was associated with hyperactivation of a MEK‐ERK1/2‐Snail‐1 pathway that regulated the Smad2‐3/Smad1‐5‐8 balance. Pharmacological blockade of MEK rescued E‐cadherin and ZO‐1 inter‐cellular junction localization, reduced fibrosis, and restored peritoneal function in Cav1−/− mice. Moreover, treatment of human PD‐patient‐derived MCs with drugs increasing Cav1 levels, as well as ectopic Cav1 expression, induced re‐acquisition of epithelial features. This study demonstrates a pivotal role of Cav1 in the balance of epithelial versus mesenchymal state and suggests targets for the prevention of fibrosis during PD.

Expression of pituitary tumor–transforming gene 1 (PTTG1)/securin in hepatitis B virus (HBV)‐associated liver diseases: Evidence for an HBV X protein–mediated inhibition of PTTG1 ubiquitination and degradation

Chronic infection with hepatitis B virus (HBV) is strongly associated with hepatocellular carcinoma (HCC), and the viral HBx protein plays a crucial role in the pathogenesis of liver tumors. Because the protooncogene pituitary tumor–transforming gene 1 (PTTG1) is overexpressed in HCC, we investigated the regulation of this protein by HBx. We analyzed PTTG1 expression levels in liver biopsies from patients chronically infected with HBV, presenting different disease stages, and from HBx transgenic mice. PTTG1 was undetectable in biopsies from chronic hepatitis B patients or from normal mouse livers. In contrast, hyperplastic livers from transgenic mice and biopsies from patients with cirrhosis, presented PTTG1 expression which was found mainly in HBx‐expressing hepatocytes. PTTG1 staining was further increased in HCC specimens. Experiments in vitro revealed that HBx induced a marked accumulation of PTTG1 protein without affecting its messenger RNA levels. HBx expression promoted the inhibition of PTTG1 ubiquitination, which in turn impaired its degradation by the proteasome. Glutathione S‐transferase pull‐down and co‐immunoprecipitation experiments demonstrated that the interaction between PTTG1 and the Skp1–Cul1–F‐box ubiquitin ligase complex (SCF) was partially disrupted, possibly through a mechanism involving protein–protein interactions of HBx with PTTG1 and/or SCF. Furthermore, confocal analysis revealed that HBx colocalized with PTTG1 and Cul1. We propose that HBx promotes an abnormal accumulation of PTTG1, which may provide new insights into the molecular mechanisms of HBV‐related pathogenesis of progressive liver disease leading to HCC development. (HEPATOLOGY 2010;51:777–787.)