ABSTRACT
Epstein-Barr virus (EBV) establishes a latent infection in greater than 90% of the world's adult population and associates with various tumors. EBV primarily infects epithelial cells and B cell in vivo. Mechanism of EBV infection in B cells is known to involve binding of EBV glycoprotein gp350 to CD21 on B cell surface. Epithelial cells are infected with EBV even though most of epithelial cells do not express CD21. Recently, integrin alphavbeta5, alphavbeta6 and alphavbeta8 on epithelial cells were reported to facilitate EBV infection by interacting with gHgL complex. We examined the expression profile of integrins known to be expressed on epithelial cells. Integrin alphavbeta5 and alphavbeta6, but not alphavbeta8 were detected in a gastric epithelial cell line, AGS. We then tested whether siRNAs specific to beta6 can inhibit EBV infection of epithelial cells. One among the four tested siRNAs significantly reduced beta6 expression and suppressed transfer infection of EBV to AGS cells. Our data suggest that siRNAs to integrins might be useful to control EBV infection to epithelial cells.
Subject(s)
Adult , Humans , B-Lymphocytes , Epithelial Cells , Epstein-Barr Virus Infections , Glycoproteins , Herpesvirus 4, Human , Integrin beta Chains , Integrins , Receptors, Vitronectin , RNA, Small InterferingABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro.</p><p><b>METHODS</b>The expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture.</p><p><b>RESULTS</b>The expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5.</p><p><b>CONCLUSION</b>alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.</p>
Subject(s)
Humans , Antibodies , Allergy and Immunology , Cell Adhesion , Cell Hypoxia , Cell Line, Tumor , Endothelial Cells , Cell Biology , Metabolism , Integrin alphaVbeta3 , Genetics , Allergy and Immunology , Metabolism , Intercellular Adhesion Molecule-1 , Allergy and Immunology , Metabolism , Ligands , Neoplasms , Metabolism , Pathology , RNA Interference , RNA, Messenger , Metabolism , RNA, Small Interfering , Pharmacology , Receptors, Vitronectin , Genetics , Allergy and Immunology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To analyze the phenotypic and functional characteristics of endothelial (T3A) cells derived from human hepatocellular cell carcinoma.</p><p><b>METHODS</b>Endothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking von Willebrand Factor (vWF), CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypic characteristics of T3A cells were analyzed by fluorescence-activated cell sorter (FACS) and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity was evaluated by 3-(4, 5-dimethythiazolyl) -2, -diphenyl-2H-tetrazolium-bromide (MTT) assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The isolated T3A cells exhibited classic "spindle-shape" morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express alphav3, alphavbeta5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells (LSEC). In response to TNFalpha, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor (TF), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and urine plasminogen activator (u-PA) than LSEC in response to TNFalpha.</p><p><b>CONCLUSION</b>Tumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.</p>
Subject(s)
Humans , Antigens, CD34 , Metabolism , Carcinoma, Hepatocellular , Genetics , Metabolism , Pathology , Cell Proliferation , Cell Shape , Cells, Cultured , Endothelial Cells , Metabolism , Pathology , Gene Expression , Integrin alphaVbeta3 , Metabolism , Integrins , Metabolism , Intercellular Adhesion Molecule-1 , Metabolism , Lipoproteins, LDL , Metabolism , Liver Neoplasms , Genetics , Metabolism , Pathology , Lung , Metabolism , Pathology , Matrix Metalloproteinase 2 , Metabolism , Microscopy, Electron, Scanning , Neovascularization, Pathologic , Metabolism , Pathology , Phenotype , Plasminogen Activator Inhibitor 1 , Metabolism , Platelet Endothelial Cell Adhesion Molecule-1 , Metabolism , Receptors, Tumor Necrosis Factor, Type I , Metabolism , Receptors, Vitronectin , Metabolism , Tissue Plasminogen Activator , Metabolism , Tumor Cells, Cultured , Tumor Necrosis Factor Decoy Receptors , Metabolism , Tumor Necrosis Factor-alpha , Pharmacology , von Willebrand Factor , MetabolismABSTRACT
Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.
Subject(s)
Humans , Animals , src-Family Kinases/antagonists & inhibitors , Transforming Growth Factor beta/genetics , Signal Transduction/physiology , Receptors, Vitronectin/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Paxillin/metabolism , Myocytes, Smooth Muscle/drug effects , Muscle, Smooth, Vascular/cytology , Morpholines/pharmacology , Molecular Sequence Data , Integrins/genetics , Flavonoids/pharmacology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Matrix Proteins/genetics , Enzyme Inhibitors/pharmacology , Chromones/pharmacology , Cells, Cultured , Cell Movement/physiology , Cell Adhesion/physiology , Amino Acid Sequence , Amino Acid Motifs/genetics , Phosphatidylinositol 3-Kinase/antagonists & inhibitorsABSTRACT
Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Recently described evidence that Thy-1 interacts with beta 3 integrin on astrocytes will be discussed. Thy-1 binding to beta 3 integrin triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment and spreading. Thy-1 has been reported to modulate neurite outgrowth by triggering a cellular response in neurons. However, our data indicate that Thy-1 can also initiate signaling events that promote adhesion of adjacent astrocytes to the underlying surface. Preliminary results suggest that morphological changes observed in the actin cytoskeleton of astrocytes as a consequence of Thy-1 binding is mediated by small GTPases from the Rho family. Our findings argue that Thy-1 functions in a bimodal fashion, as a receptor on neuronal cells and as a ligand for beta 3 integrin receptor on astrocytes. Since Thy-1 is implicated in the inhibition of neurite outgrowth, signaling events in astrocytes are likely to play an important role in this process