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• W1978557938 abstract "The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage. The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage. Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated kinase; HSC, hepatic stellate cell(s); MMPs, matrix metalloproteinases; PI, phosphatidylinositol; AP-2, activator protein-2; SFIF, serum-free/insulin-free; PMSF, phenylmethylsulfonyl fluoride; TIMP, tissue inhibitor of metalloproteinase; mAb, monoclonal antibody; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; siRNA, small interfering RNA; MT1, membrane type 1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.1The abbreviations used are: HCV, hepatitis C virus; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated kinase; HSC, hepatic stellate cell(s); MMPs, matrix metalloproteinases; PI, phosphatidylinositol; AP-2, activator protein-2; SFIF, serum-free/insulin-free; PMSF, phenylmethylsulfonyl fluoride; TIMP, tissue inhibitor of metalloproteinase; mAb, monoclonal antibody; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; siRNA, small interfering RNA; MT1, membrane type 1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is the most common cause of chronic liver disease, leading to hepatic fibrosis and ultimately to cirrhosis (1Lauer G.M. Walker B.D. N. Engl. J. Med. 2001; 345: 41-52Crossref PubMed Scopus (2464) Google Scholar). The HCV major envelope E2 glycoprotein exposed on the surface of virions is likely to be involved in the interactions with the host and has been identified as responsible for binding of HCV to target cells (2Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Science. 1998; 282: 938-941Crossref PubMed Scopus (1776) Google Scholar). Truncated, secreted versions of E2 glycoprotein have been utilized as soluble mimics of viral particles in order to study virus-cell interactions (3Rosa D. Campagnoli S. Moretto C. Guenzi E. Cousens L. Chin M. Dong C. Weiner A.J. Lau J.Y. Choo Q.L. Chien D. Pileri P. Houghton M. Abrignani S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1759-1763Crossref PubMed Scopus (321) Google Scholar, 4Heile J.M. Fong Y.L. Rosa D. Berge R.K. Saletti G. Campagnoli S. Bensi G. Capo S. Coates S. Crawford K. Dong C. Wininger M. Bake R.G. Cousens L. Chien D. Ng P. Archangel P. Grandi G. Houghton M. Abrignani S. J. Virol. 2000; 74: 6885-6892Crossref PubMed Scopus (66) Google Scholar). In particular, E2 glycoprotein has been reported to bind to the major loop of human transmembrane molecule CD81, a member of tetraspanin protein superfamily, expressed on various cell types including hepatocytes and B lymphocytes, and, accordingly, CD81 has been proposed as a putative receptor for HCV (2Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Science. 1998; 282: 938-941Crossref PubMed Scopus (1776) Google Scholar). The most striking feature of CD81, as well as other tetraspanin molecules, is a propensity to associate with a wide variety of membrane proteins, acting as a molecular facilitator for downstream intracellular signaling (5Maecker H.T. Todd S.C. Levy S. FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (801) Google Scholar). The wide range of complexes into which CD81 assembles suggests that the association of CD81 with different partners may have important and diverse effects on different cell types (6Levy S. Todd S.C. Maecker H.T. Annu. Rev. Immunol. 1998; 16: 89-109Crossref PubMed Scopus (435) Google Scholar). Accordingly, binding of E2 glycoprotein to CD81 may be relevant for explaining the development and the persistence of HCV infection in the liver as well as in extrahepatic tissues (7Flint M. McKeating J.A. Rev. Med. Virol. 2000; 10: 101-117Crossref PubMed Scopus (71) Google Scholar). A preliminary study performed in our laboratory on sections of normal human liver indicates that CD81 is expressed at the cell surface of hepatocytes, as well as within sinusoidal structures (Fig. 1). HCV E2 binds different cell types present in liver tissue samples, although with different intensities, and this binding is prevented by preincubation of liver tissue samples with anti-CD81 antibodies (8Petracca R. Falugi F. Galli G. Norais N. Rosa D. Campagnoli S. Burgio V. Di Stasio E. Giardina B. Houghton M. Abrignani S. Grandi G. J. Virol. 2000; 74: 4824-4830Crossref PubMed Scopus (195) Google Scholar). In addition, we have recently reported that human HSC, liver-specific pericytes and key effectors of hepatic fibrogenesis, express high levels of CD81 protein at the cell surface (9Mazzocca A. Carloni V. Cappadona Sciammetta S. Cordella C. Pantaleo P. Caldini A. Gentilini P. Pinzani M. J. Hepatol. 2002; 37: 322-330Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). An essential step in the progression of HCV-related hepatic fibrogenesis is the degradation of normal liver extracellular matrix (ECM), mediated by increased expression of proteolytic enzymes, such as matrix metalloproteinases (MMPs). These enzymes are involved in the acute phases of liver injury as well as during chronic hepatic wound healing and fibrogenesis (10Arthur M.J.P. Am. J. Physiol. 2000; 279: G245-G249Crossref PubMed Google Scholar) and may contribute to the inflammatory and fibrogenic response to many stimuli (11Goetzl E.J. Banda M.J. Leppert D. J. Immunol. 1996; 156: 1-4PubMed Google Scholar, 12Takahara T. Furui K. Yata Y. Jin B. Zhang L.P. Nambu S. Sato H. Seiki M. Watanab A. Hepatology. 1997; 26: 1521-1529Crossref PubMed Scopus (171) Google Scholar), including viral proteins (13Johnston J.B. Jiang Y. van Marle G. Mayne M.B. Ni W. Holden J. McArthur J.C. Power C. J. Virol. 2000; 74: 7211-7220Crossref PubMed Scopus (86) Google Scholar). The activity of MMPs, secreted mainly by connective tissue cells as proenzymes, is controlled by transcriptional regulation, zymogen activation, and specific tissue inhibitors (TIMPs) (14Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3827) Google Scholar). MMP-2 (also known as gelatinase A or collagenase IV), the most relevant MMP component involved in remodeling of normal liver ECM during hepatic fibrogenesis, efficiently degrades collagen types IV and I, fibronectin, and laminin (15Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar). Secretion of active MMP-2 may be stimulated by several mediators, including proinflammatory cytokines, during the inflammatory phase of wound healing. Moreover, the same mediators are able to activate the proenzymatic form of MMP-2 by inducing a membrane type matrix metalloproteinase (MT1-MMP), which subsequently activates MMP-2 (16Han Y.P. Tuan T.L. Wu H. Hughes M. Garner W.L. J. Cell Sci. 2001; 114: 131-139PubMed Google Scholar). It is conceivable that activation of MMP-2 represents a key event in the wound healing process following acute or chronic tissue damage. Degradation of the normal ECM in areas of necrosis allows penetration of inflammatory cells exerting phagocytic and/or immunological activities. Along these lines, activation of MMP-2 can be viewed as a relevant proinflammatory event contributing to tissue injury. In the present study, we investigated the interaction of E2/CD81 in human HSC and the expression of MMP-2 following this interaction as a possible host cellular response to viral infection. Our results indicate that human HSC could represent a novel potential cellular target for HCV within the liver and show that binding of E2 to CD81 on HSC surface leads to an increased expression of MMP-2, mediated by ERK/MAPK activation followed by AP-2 transcriptional activity. Reagents—The HCV E2 protein used throughout this study was a clinical grade batch prepared by Chiron Co. (Emeryville, CA). Briefly, the protein was prepared from a Chinese hamster ovary cell line stably transfected with plasmid pCMVa120 (17Chapman B.S. Thayer R.M. Vincent K.A. Haigwood L. Nucleic Acids Res. 1991; 19: 3979-3986Crossref PubMed Scopus (276) Google Scholar) in which the E2 sequence from amino acids 384–715 was fused to the tissue plasminogen activator leader sequence. After cell disruption and debris removal by microfiltration, the protein was purified by three subsequent chromatographic steps: lectin affinity chromatography, hydroxyapatite chromatography, and ion exchange chromatography. As described elsewhere, all of the HCV envelope E2 proteins were ∼90% pure after purification (4Heile J.M. Fong Y.L. Rosa D. Berge R.K. Saletti G. Campagnoli S. Bensi G. Capo S. Coates S. Crawford K. Dong C. Wininger M. Bake R.G. Cousens L. Chien D. Ng P. Archangel P. Grandi G. Houghton M. Abrignani S. J. Virol. 2000; 74: 6885-6892Crossref PubMed Scopus (66) Google Scholar). The mAb 291 (IgG1) was obtained from mice immunized with Chinese hamster ovary/E2715 and screened for the ability to recognize E2 bound to target cells as described previously (4Heile J.M. Fong Y.L. Rosa D. Berge R.K. Saletti G. Campagnoli S. Bensi G. Capo S. Coates S. Crawford K. Dong C. Wininger M. Bake R.G. Cousens L. Chien D. Ng P. Archangel P. Grandi G. Houghton M. Abrignani S. J. Virol. 2000; 74: 6885-6892Crossref PubMed Scopus (66) Google Scholar). The antibodies against CD81, JS81, and 1.3.3.22 were purchased from Pharmingen (San Diego, CA) and from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. The rabbit polyclonal anti-MMP-2 (AB809) was purchased from Chemicon International (Temecula, CA). The Immobilon-P was purchased from Millipore Corp. (Bedford, MA). The ECL system was from Amersham Biosciences. Gelatin-Sepharose 4B was purchased from Amersham Biosciences. Protein concentration in samples was determined using the bicinchoninic acid kit from Sigma. PD-98059 was purchased from Calbiochem. Human Tissues and Immunohistochemistry—These experiments were conducted on frozen surgical sections of human liver as described in detail elsewhere (18Pinzani M. Milani S. Herbst H. DeFranco R. Grappone C. Gentilini A. Caligiuri A. Pellegrini G. Ngo D.V. Romanelli R.G. Gentilini P. Am. J. Pathol. 1996; 148: 785-800PubMed Google Scholar). Dried sections were sequentially incubated with the primary anti-CD81 mAb and, after washing, with the affinity-purified rabbit anti-mouse antibody. At the end of the incubation, sections were washed twice in TBS and then incubated with alkaline antialkaline phosphatase and developed. A nonimmune mouse IgG primary antibody was used as negative control. Isolation and Culture of Human HSC—Human HSC were isolated from wedge sections of normal human liver unsuitable for transplantation as previously reported (19Casini A. Pinzani M. Milani S. Grappone C. Galli G. Jezequel A.M. Schuppan D. Rotella C.M. Surrenti C. Gastroenterology. 1993; 105: 245-253Abstract Full Text PDF PubMed Google Scholar). Briefly, after a combined digestion with collagenase/Pronase, HSC were separated from other liver nonparenchymal cells by ultracentrifugation over gradients of stractan (Cellsep™ isotonic solution; Larex Inc., St. Paul, MN). Extensive characterization was performed as described elsewhere (19Casini A. Pinzani M. Milani S. Grappone C. Galli G. Jezequel A.M. Schuppan D. Rotella C.M. Surrenti C. Gastroenterology. 1993; 105: 245-253Abstract Full Text PDF PubMed Google Scholar). Cells were cultured on plastic culture dishes (Falcon; BD Biosciences) in Iscove's modified Dulbecco's medium supplemented with 0.6 units/ml insulin, 2.0 mmol/liter glutamine, 0.1 mmol/liter nonessential amino acids, 1.0 mmol/liter sodium pyruvate, antibiotic antifungal solution (all provided by Invitrogen), and 20% fetal bovine serum (Imperial Laboratories, Andover, UK). Experiments described in this study were performed on cells between first and third serial passages (1:3 split ratio) using three independent cell lines. At these stages of culture, cells show functional and ultrastructural features of fully activated HSC. In particular, analysis of cell surface markers indicates that, at these stages of culture in vitro, HSC present with a cell marker profile identical to the so-called “interface” myofibroblasts detected in liver tissue specimens at the border between “active” fibrotic septa and the parenchyma of the liver lobule (20Cassiman D. Roskams T. J. Hepatol. 2002; 37: 527-535Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In all experiments, confluent HSC were left for 24 h in Iscove's SFIF medium before the addition of the different stimuli. Flow Cytometry Analysis—For flow cytometry (fluorescence-activated cell sorting) analysis, cells were trypsinized, harvested, and washed with 2% fetal calf serum in Iscove's medium. E2 protein was bound to cells for 30 min on ice. Cells were washed and incubated with anti-E2 mAb for further 30 min on ice. After three washes, cells were resuspended in goat anti-mouse fluorescein isothiocyanate (1:40 dilution) and incubated for 30 min on ice. Cells were washed again, resuspended in serum-free medium, and fluorescence-analyzed using a FAC-Scan flow cytometer (Beckman Coulter, Inc., Fullerton, CA). Cells incubated with 1% BSA/PBS were used as negative controls. For CD81 analysis, cells were stained with anti-CD81 mAb (JS81) or with the isotype mouse IgG as control, washed, and incubated with an anti-mouse fluorescein isothiocyanate conjugate, washed again, and analyzed as described previously (9Mazzocca A. Carloni V. Cappadona Sciammetta S. Cordella C. Pantaleo P. Caldini A. Gentilini P. Pinzani M. J. Hepatol. 2002; 37: 322-330Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Immunofluorescence and Confocal Microscopy—For immunofluorescence analysis, cells were plated in Iscove's medium supplemented with 1% fetal calf serum on glass coverslips for 2–4 h. Once spread, cells were fixed for 5 min with 2% paraformaldehyde in PBS, pH 7.2. Coverslips were blocked for 1 h with 3% heat-inactivated BSA/PBS. Cells were then incubated with E2 diluted in 3% BSA-PBS and, after three washes, stained with anti-E2 mAb, incubated, and washed again. Staining was subsequently visualized with fluorescein isothiocyanate-goat anti-mouse antibody (Calbiochem) before the coverslips were mounted with anti-fading Vectashield (Vector Laboratories, Inc., Burlingame, CA). Immunofluorescence was examined by a series of optical sections, obtained with a confocal scanning laser microscope (MRC-1024; Bio-Rad), and images were captured, processed, and superimposed by using the LaserSharp software (Bio-Rad). An anti-CD81 mAb staining was included as a positive control. A solution of BSA/PBS 3% and a nonimmune mouse IgG were employed as a negative control for E2 and CD81, respectively. E2 Binding Assay—Binding of E2 to HSC was evaluated by a competition assay. HSC were incubated with increasing concentrations of anti-CD81 mAbs (JS81 and 1.3.3.22) or anti-HLA-I (clone W6/32; Sigma) as negative control and seeded onto 96-well E2 protein-coated plates for 1 h at 37 °C. To remove unbound cells, wells were then filled with PBS and gently washed three times. Adherent cells were fixed in 3% paraformaldehyde/PBS and then stained with 0.5% crystal violet in 20% methanol, 80% H2O. Wells were then washed with water to remove excess dye, cells were then solubilized in 1% SDS, and the amount of dye was quantified by using a Bio-Rad Multiscan plate reader at 595 nm. The specificity of E2 protein binding to CD81 on human HSC was further confirmed by the displacement of E2 from the binding to CD81 in the presence of the competitive anti-human CD81 mAb (JS81). For these experiments, HSC were preincubated with increasing concentrations of E2 protein (0.1, 1.0, 10.0, 50.0, 100 μg/ml) prior to plating onto 96-well plates coated with immobilized anti-CD81 mAb (JS81) for 1 h at 37 °C. Adherent cells were then washed, fixed, stained, and solubilized in 1% SDS, and the amount of dye was quantified as described above. Gelatinolytic Zymography—Conditioned media from the cell cultures were analyzed for gelatin degradation activity by SDS-PAGE under nonreducing conditions. The gel contained 5.8 mg/ml gelatin and 8% acrylamide. Electrophoresis was carried out at 4 °C. After a brief wash with water, the SDS in the acrylamide gel was extracted by incubation with 2% Triton X-100/PBS solution. Gelatinolytic activities were developed in a buffer containing 5 mm CaCl2, 150 mm NaCl, and 50 mm Tris at 37 °C for 16 h. The gelatinolytic activities were visualized by staining the gel with Coomassie Blue R-250. The amount of 66-kDa MMP-2 was determined using scanning densitometry with NIH Image analysis software. Western Blot—Samples for Western blot were prepared differently for conditioned media and for cells. Cell monolayers were extracted with buffer containing 1% Triton X-100, 150 mm NaCl, 50 mm Tris, pH 7.5, and 1 mm PMSF. Cell debris were removed by centrifugation at 14,000 × g. The supernatant was subjected to 8% SDS-PAGE. In the conditioned media, the MMP-2 enzyme was enriched by incubation of 500 ml of conditioned medium with 30 ml of gelatin-conjugated Sepharose 4B. The Sepharose was washed with buffer containing 400 mm NaCl, 50 mm Tris, pH 7.5, and the bound protein was eluted with SDS-sample buffer. All samples were resolved by reducing SDS-PAGE and transferred to Immobilon-P. Filters were blocked with 5% nonfat milk in TBS (150 mm NaCl, 50 mm Tris, pH 7.5) for 2 h at room temperature and then incubated overnight at 4 °C with individual antibodies in TBS plus 0.05% Tween 20 (TBS-T). The polyclonal anti-MMP-2 was used at 1:5000 dilution. Blots were incubated for 2 h with a horseradish peroxidase-conjugated goat anti-rabbit antibody at a 1:5000 dilution in TBS-T and visualized with enhanced chemiluminescence. In some experiments, cells were treated with a 1 μm concentration of the protein synthesis inhibitor cycloheximide (Sigma). For AP-2 blotting, a mouse mAb anti-human AP-2α (Santa Cruz Biotechnology) was used to detect AP-2 protein. Generation of Human CD81 Stable Cell Lines—NIH3T3 cells were maintained in complete medium (90% minimal essential medium, 10% (w/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine) (Invitrogen) at 37 °C in a 5% CO2 atmosphere, and cells at 50–60% confluence were used in transfection assays. Transfection into cells was done by the Lipofectamine™ method with the 2000 reagent according to the manufacturer's instructions (Invitrogen) with the use of 4 μg/60-mm dish of pcDNA 3.1-CD81 or 1.5 μg/35-mm dish of pcDNA 3.1-CD81 vector. The stably transfected cell clones were generated by culturing the cells in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and G418 (0.2 mg/ml). A stable clone expressing a high level of human CD81 (clone 42) was selected by flow cytometry analysis and used for the experiments. Immunoprecipitation and Lipid Kinase Assay—For immunoprecipitation, HSC monolayers were lysed in buffer containing 1% CHAPS, 20 mm Hepes (pH 7.5), 200 mm NaCl, 5 mm MgCl2, 200 μm Na3VO4, 2 mm NaF, 10 mm Na4P3O7, 2 mm PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and immunoprecipitates were prepared using protein G-Sepharose (Amersham Biosciences). After immunoprecipitation with anti-CD81 mAb (1.3.3.22), anti-E2 (mAb 291)-E2 protein or with nonimmune mouse IgG as a control, immune complexes were washed four times in lysis buffer and one time in 10 mm HEPES plus 5 mm MgCl2 before phosphoinositide kinase reactions were performed directly on beads. Briefly, the reaction mixture included 20 mm HEPES (pH 7.5), 10 mm MgCl2, 50 μm ATP (Amersham Biosciences), 0.3% Triton X-100, 10–15 μCi of [32P]ATP (ICN Biomedicals, Inc.), and 200 μg/ml sonicated l-α-phosphatidylinositol (Sigma) as a substrate. Adenosine (200 μm; Sigma) was employed in these experiments as an established in vitro inhibitor of type II PI 4-kinase reactions (21Graziani A. Ling L.E. Endemann G. Carpenter C.L. Cantley L.C. Biochem. J. 1992; 284: 39-45Crossref PubMed Scopus (34) Google Scholar). Reactions were carried out for 5 min at room temperature and stopped with 2 m HCl. Lipids were extracted with 1:1 (v/v) chloroform-methanol, and the organic layer was resolved by TLC on potassium oxalate-treated silica gel 60 silica plates (EM Science, Darmstadt, Germany). β-Emitting radioactivity corresponding to PIP was quantified using a Betascope 603 blot analyzer (Betagen, Waltham, MA). Lipid kinase activity is expressed as counts/min within a defined area representing the PI 4-32P. ERK Phosphorylation—Confluent cell monolayers on 100-mm dishes were growth-arrested in serum-free medium for 24 h prior to treatment with E2. Cells were washed twice with PBS, scraped, resuspended in lysis buffer (50 mm HEPES, 150 mm NaCl, 1% Triton X-100, 1 mm PMSF, 20 μg/ml aprotinin, 20 μg/ml leupeptin, and 20 μg/ml pepstatin) and clarified by centrifugation at 14,000 × g for 10 min. Thirty μg of protein/sample was separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blocked for 2 h at 25 °C with 5% nonfat milk in PBS buffer (20 mm Tris, 500 mm NaCl, and 0.01% Tween 20). The membrane was then incubated overnight at 4 °C with an appropriate dilution of anti-phospho-ERK or anti-ERK (New England Biolabs Inc.) polyclonal antibody at 4 °C overnight, washed, and followed by incubation for 2 h with a 1:2000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody. The immunoblot signal was visualized through enhanced chemiluminescence. Nuclear Extracts and Electrophoretic Mobility Shift Assay—For the nuclear extract preparation, HSC were grown in 100-mm dishes and incubated in serum-free medium for 24 h prior to exposure to E2 protein at different times. Cells were then washed with cold PBS, harvested by scraping, and pelleted. Cells were resuspended in 1 ml of buffer A (10 mm KCl, 20 mm HEPES, 1 mm MgCl2, 1 mm dithiothreitol, 0.4 mm PMSF, 1 mm sodium fluoride, 1 mm Na3VO4), incubated on ice for 10 min, and pelleted at 1000 × g for 10 min. Pellets were resuspended in 0.5 ml of buffer A plus 0.1% Nonidet P-40, incubated on ice for 10 min, and centrifuged at 3,000 × g for 10 min. The nuclear pellet was resuspended in 1 ml of buffer B (10 mm HEPES, 400 mm NaCl, 0.1 mm EDTA, 1 mm MgCl2, 1 mm dithiothreitol, 0.4 mm PMSF, 15% glycerol, 1 mm sodium fluoride, 1 mm Na3VO4) and incubated for 30 min at 4 °C with constant gentle mixing. Nuclei were then pelleted at 40,000 × g for 30 min, and extracts were dialyzed for 2 h at 4 °C against 1 liter of buffer C (20 mm HEPES, 200 mm KCl, 1 mm MgCl2, 0.1 mm EDTA, 1 mm dithiothreitol, 0.4 mm PMSF, 15% glycerol, 1 mm sodium fluoride, 1 mm Na3VO4). Extracts were cleared by centrifugation at 14,000 × g for 15 min at 4 °C. Protein concentrations were determined using a Bio-Rad protein assay. For electrophoretic mobility shift assay 4 μg of nuclear extract were mixed with 20 μg of poly(dI·dC) in 20 μl of a reaction buffer consisting of 25 mm HEPES, pH 7.5, 1.2 mm dithiothreitol, 4 mm MgCl2, and 150 mm NaCl as well as 5% glycerol, 0.005% bromphenol blue, and 0.05% Nonidet P-40 (Sigma). The mixture was incubated on ice for 15 min followed by the addition of 10 fmol of γ-32P-end-labeled AP-2 consensus binding sequence oligonucleotide (nucleotide sequence, 5′-GAT CGA ACT GAC CGC CCG CGG CCC GT-3′; Promega, Madison, WI). Incubation was continued for 30 min. The incubation mixture was subjected to electrophoresis on a 6% polyacrylamide gel in Tris-glycine buffer. The gels were dried, and autoradiography was performed at -70 °C with an intensifying screen. Bands were quantitated by laser densitometry (model 300S; Amersham Biosciences). Competition experiments were performed with a 200-fold excess of unlabeled AP-2 consensus binding sequence oligonucleotide. CD81 Silencing by Small RNA Interference—The siRNA sequence targeting human CD81 (GenBank™ accession number NM_004356) spans nucleotides 138–156 (target was 5′-ATCTGGAGCTGGGAGACAA-3′) and is specific for human CD81 based on BLAST search (NCBI data base). Sense siRNA sequence was 5′-AUCUGGAGCUGGGAGACAAdTdT-3′, and antisense was 5′-UUGUCUCCCAGCUCCAGAUd-TdT. A siRNA sequence corresponding to nucleotides 695–715 of the firefly luciferase (U31240) was used as a negative control. The siRNAs were chemically synthesized by Qiagen-Xeragon (Germantown, MD), and, for annealing, 40 μm siRNA single strands were incubated in the annealing buffer (100 mm potassium acetate, 30 mm HEPES-KOH, pH 7.4, 2 mm magnesium acetate) for 1 min at 90 °C followed by 1 h at 37 °C. For transfection, the Amaxa nucleofection technology (Amaxa; Koeln, Germany) was employed. HSC were resuspended in the nucleofector T solution, available as part of the Amaxa cell optimization kit, following the Amaxa guidelines for cell line transfection. Briefly, 100 μl of 2–5 × 106 cell suspension mixed with 2 μg of pmaxGFP vector (to evaluate the transfection efficiency) plus 1 μl of 40 μm CD81-siRNA or negative control was transferred to a cuvette and nucleofected with an Amaxa Nucleofector apparatus. Cells were transfected using the U-25 pulsing parameter and were immediately transferred into wells containing 37 °C prewarmed culture medium in 6-well plates. 72 h after transfection, the CD81 protein expression levels at the cell surface were analyzed on a BDLSRII cytofluorimeter, using the DIVA software (BD Biosciences). The area of positivity was determined using an isotype-matched control antibody. 104 events for each sample were acquired. Statistical Analysis—Results, relative to the number of experiments indicated, are expressed as mean ± S.D. Statistical analysis was performed by one-way analysis of variance and, when the F value was significant, by Duncan's test. Expression and Distribution of CD81 in Normal and Pathologic Human Liver Tissue—In agreement with a previous study exploring the binding of E2 in normal human liver (8Petracca R. Falugi F. Galli G. No" @default.
• W1978557938 created "2016-06-24" @default.
• W1978557938 creator A5011711495 @default.
• W1978557938 creator A5012623826 @default.
• W1978557938 creator A5035706661 @default.
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• W1978557938 date "2005-03-01" @default.
• W1978557938 modified "2023-10-12" @default.
• W1978557938 title "Binding of Hepatitis C Virus Envelope Protein E2 to CD81 Up-regulates Matrix Metalloproteinase-2 in Human Hepatic Stellate Cells" @default.
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