Enrique Lara-Pezzi

The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression

Hepatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell-dependent (NF-AT-dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.

Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation.

Increased nitric oxide (NO) production may contribute to the pathological changes featuring in some inflammatory diseases, but the role of NO in chronic viral hepatitis is still unknown. We compared the inducible NO synthase (NOS2) expression in the liver of patients with chronic viral hepatitis with that of both nonviral liver disease and histologically normal liver. NOS2 expression was assessed by immunohistochemical and in situ hybridization studies of liver biopsy sections. An intense hepatocellular NOS2 reactivity was detected in chronic viral hepatitis, whereas it was weakly or not observed in nonviral liver disease or normal liver, respectively. In addition, we determined whether the hepatitis B virus (HBV) might regulate the synthesis of this enzyme. NOS2 mRNA and protein levels as well as enzyme activity were assessed in cytokine-stimulated HBV-transfected and untransfected hepatoma cells. Transfection with either HBV genome or HBV X gene resulted in induction of NOS2 mRNA expression, and the maximal induction of this transcript and NO production was observed in cytokine-stimulated HBV-transfected cells. These results indicate that hepatotropic viral infections are able to upregulate the NOS2 gene expression in human hepatocytes, suggesting that NO may mediate important pathogenic events in the course of chronic viral hepatitis.

Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis

Peritoneal fibrosis is one of the most common morphological changes observed in continuous ambulatory peritoneal dialysis (CAPD) patients. Both resident fibroblasts and new fibroblast-like cells derived from the mesothelium by epithelial-to-mesenchymal transition are the main cells involved fibrogenesis. In order to establish markers of peritoneal impairment and pathogenic clues to explain the fibrogenic process, we conducted an immunohistochemical study focused on peritoneal fibroblasts. Parietal peritoneal biopsies were collected from four patient groups: normal controls (n=15), non-CAPD uremic patients (n=17), uremic patients on CAPD (n=27) and non-renal patients with inguinal hernia (n=12). To study myofibroblastic conversion of mesothelial cells, α-smooth muscle actin (SMA), desmin, cytokeratins and E-cadherin were analyzed. The expression of CD34 by fibroblasts was also analyzed. Fibroblasts from controls and non-CAPD uremic patients showed expression of CD34, but no myofibroblastic or mesothelial markers. The opposite pattern was present during CAPD-related fibrosis. Expression of cytokeratins and E-cadherin by fibroblast-like cells and α-SMA by mesothelial and stromal cells supports that mesothelial-to-myofibroblast transition occurs during CAPD. Loss of CD34 expression correlated with the degree of peritoneal fibrosis. The immunophenotype of fibroblasts varies during the progression of fibrosis. Myofibroblasts seem to derive from both activation of resident fibroblasts and local conversion of mesothelial cells.

Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding.

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.

The hepatitis B virus X protein activates nuclear factor of activated T cells (NF-AT) by a cyclosporin A-sensitive pathway.

The X gene product of the human hepatitis B virus (HBx) is a transcriptional activator of various viral and cellular genes. We recently have determined that the production of tumor necrosis factor‐α (TNF‐α) by HBV‐infected hepatocytes is transcriptionally up‐regulated by HBx, involving nuclear factor of activated T cells (NF‐AT)‐dependent activation of the TNF‐α gene promoter. Here we show that HBx activates NF‐AT by a cyclosporin A‐sensitive mechanism involving dephosphorylation and nuclear translocation of the transcription factor. Luciferase gene expression assays demonstrated that HBx transactivates transcription through NF‐AT‐binding sites and activates a Gal4–NF‐AT chimeric protein. DNA–protein interaction assays revealed that HBx induces the formation of NF‐AT‐containing DNA‐binding complexes. Immunofluorescence analysis demonstrated that HBx induces the nuclear translocation of NF‐AT, which can be blocked by the immunosuppressive drug cyclosporin A. Furthermore, immunoblot analysis showed that the HBx‐induced activation and translocation of NF‐AT are associated with its dephosphorylation. Thus, HBx may play a relevant role in the intrahepatic inflammatory processes by inducing locally the expression of cytokines that are regulated by NF‐AT.

The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner

Chronic hepatitis B virus infection is strongly associated with the development of hepatocellular carcinoma (HCC). Epithelial tumors are frequently characterized by loss of cadherin expression or function. Cadherin-dependent adhesion prevents the acquisition of a migratory and invasive phenotype, and loss of its function is itself enough for the progression from adenoma to carcinoma. The HBx protein of hepatitis B virus is thought to contribute to the development of the carcinoma, however, its role in the oncogenic and metastatic processes is far from being fully understood. We report herein the ability of HBx to disrupt intercellular adhesion in three different cell lines stably transfected with an inducible HBx expression vector. The linkage between the actin cytoskeleton and cadherin complex, which is essential for its function, is disrupted in the presence of HBx, as indicated by detergent solubility and immunoprecipitation experiments. In addition, β-catenin was tyrosine phosphorylated in HBx-expressing cells. Inhibition of the src family of tyrosine kinases resulted in the prevention of the disruption of adherens junctions. These results suggest that HBx is able to disrupt intercellular adhesion in a src-dependent manner, and provide a novel mechanism by which HBx may contribute to the development of HCC.

Effect of the hepatitis B virus HBx protein on integrin-mediated adhesion to and migration on extracellular matrix

BACKGROUND/AIMS The hepatitis B virus HBx protein is associated with the development of hepatocellular carcinoma (HCC). However, its possible contribution to tumor spreading has not been explored. The migration of tumor cells through the extracellular matrix (ECM) represents a crucial step in tumor metastasis. Our aim was to study the effect of HBx on the integrin-mediated cell-ECM interaction, and its possible consequences for cell migration. METHODS Cell-ECM interaction was evaluated by static adhesion experiments, using blocking and stimulating anti-beta1 integrin mAbs. ECM receptor expression was analyzed by flow cytometry. The cellular distribution of the activated beta1 integrin subunit was determined by immunofluorescence analysis, and cell motility was determined by wound-healing assays. RESULTS HBx-bearing cells showed decreased adhesion to fibronectin, which correlated with a decreased expression of the alpha5 integrin subunit. The activated beta1 subunit was redistributed to the tips of pseudopodial protrusions of HBx-bearing cells, whereas it was evenly localized in the control cells. HBx-induced cell migration was abrogated by irreversible stimulation of beta1 integrins. CONCLUSIONS These results suggest that HBx might play a role in tumor spreading by modulating the adhesion-deadhesion balance of the cells in the primary tumor site and favoring integrin-mediated cell migration.

Cyclooxygenase-2 Mediates Dialysate-Induced Alterations of the Peritoneal Membrane

During peritoneal dialysis (PD), exposure of the peritoneal membrane to nonphysiologic solutions causes inflammation, ultimately leading to altered structure and function. Myofibroblasts, one of the cell types that contribute to dysfunction of the peritoneal membrane, can originate from mesothelial cells (MCs) by epithelial-to-mesenchymal transition (EMT), a process that has been associated with an increased rate of peritoneal transport. Because cyclooxygenase-2 (COX-2) is induced by inflammation, we studied the role of COX-2 in the deterioration of the peritoneal membrane. We observed that nonepithelioid MCs found in peritoneal effluent expressed higher levels of COX-2 than epithelioid MCs. The mass transfer coefficient for creatinine correlated with MC phenotype and with COX-2 levels. Although COX-2 was upregulated during EMT of MCs in vitro, COX-2 inhibition did not prevent EMT. In a mouse model of PD, however, COX-2 inhibition with Celecoxib resulted in reduced fibrosis and in partial recovery of ultrafiltration, outcomes that were associated with a reduction of inflammatory cells. Furthermore, PD fluid with a low content of glucose degradation products did not induce EMT or COX-2; the peritoneal membranes of mice treated with this fluid showed less worsening than mice exposed to standard fluid. In conclusion, upregulation of COX-2 during EMT may mediate peritoneal inflammation, suggesting COX-2 inhibition as a potential strategy to ameliorate peritoneal deterioration in PD patients.

MouBeAT: A New and Open Toolbox for Guided Analysis of Behavioral Tests in Mice

Animal behavioral tests are essential to understand the bases of neurologic and psychological disorders, which can be evaluated by different methodological and experimental models. However, the quantification of behavioral tests results is limited by the considerable amount of time needed for manual evaluation and the high costs of automated analysis software. To overcome these limitations, we describe here a new, open source toolbox for ImageJ, called Mouse Behavioral Analysis Toolbox (MouBeAT), designed to analyze different behavioral tests in rodents semi-automatically. These tests include Open Field (OF), Elevated Plus Maze (EPM), Y-maze (YM) test and Morris Water Maze (MWM). MouBeAT showed a high correlation with manual evaluation in all the parameters analyzed for all the behavioral tests, reinforcing its value as an accurate analysis tool. This new tool is freely available online.