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• W1567250782 abstract "Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-κB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin κB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIκBαSR) that constitutively sequesters IκB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated κB translocation and E-selectin up-regulation. Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-κB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin κB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIκBαSR) that constitutively sequesters IκB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated κB translocation and E-selectin up-regulation. Inflammation-mediated tissue destruction results from recruitment and extravasation of inflammatory cells at some tissue site, usually because of injury or infection. Host-pathogen interactions trigger the inflammatory response, and the balance between the pro- and anti-inflammatory forces will determine the outcome of the infection. The vascular endothelium is an essential component of this balance and contributes to the separation between vascular and interstitial spaces. At areas of inflammation, neutrophils are recruited by a combination of adhesion molecule up-regulation and chemokine secretion. The leukocytes in turn activate their own inflammatory programs of increased reactive oxygen species generation and cytokine secretion. Endothelial cells respond in kind by becoming further activated. Accordingly, diseases that affect the endothelium result in marked vasculopathies and failure of the endothelial barrier. In HIV 1The abbreviations used are: HIVhuman immunodeficiency virusLTRlong terminal repeatPBSphosphate-buffered salineDTTdithiothreitolFACSfluorescence-activated cell sorterTARtrans-activation response elementTNFtumor necrosis factorILinterleukinIPTGisopropyl-1-thio-β-d-galactopyranosideGSTglutathioneS- transferaseHUVEChuman umbilical vein endothelial cellsEMSAelectrophoretic mobility shift assayGFPgreen fluorescent protein1The abbreviations used are: HIVhuman immunodeficiency virusLTRlong terminal repeatPBSphosphate-buffered salineDTTdithiothreitolFACSfluorescence-activated cell sorterTARtrans-activation response elementTNFtumor necrosis factorILinterleukinIPTGisopropyl-1-thio-β-d-galactopyranosideGSTglutathioneS- transferaseHUVEChuman umbilical vein endothelial cellsEMSAelectrophoretic mobility shift assayGFPgreen fluorescent protein infection there is a diffuse endothelial cell involvement, not often recognized, that includes increased susceptibility to inflammatory cytokines, increased adhesion molecule expression, and increased neutrophil and mononuclear cell adhesion. Cardiovascular complications, such as myocarditis with infiltrating neutrophils and mononuclear cells are often observed, even in the absence of infectious pathogens (1.Anderson D.W. Virmani R. Reilly J.M. O'Leary T. Cunnion R.E. Robinowitz M. Macher A.M. Punja U. Villaflor S.T. Parrillo J.E. Roberts W.C. J. Am. Coll. Cardiol. 1988; 11: 792-799Crossref PubMed Scopus (190) Google Scholar, 2.Kaul S. Fishbein M.C. Siegel R.J. Am. Heart J. 1991; 122: 535-544Crossref PubMed Scopus (131) Google Scholar). Even though HIV may enter endothelial cells via transcytosis (3.Gujuluva C. Burns A.R. Pushkarsky T. Popik W. Berger O. Bukrinsky M. Graves M.C. Fiala M. Mol. Med. 2001; 7: 169-176Crossref PubMed Google Scholar), productive infection is very hard to achieve unless the cells are proliferating in the presence of cytokines (4.Conaldi P.G. Serra C. Dolei A. Basolo F. Falcone V. Mariani G. Speziale P. Toniolo A. J. Med. Virol. 1995; 47: 355-363Crossref PubMed Scopus (57) Google Scholar). On the other hand, transgenic mice carrying a replication-defective provirus develop smooth muscle hypertrophy as well as adventitial infiltration by T-lymphocytes. This vascular remodeling leads to narrowing of the blood vessels of different sizes with the resultant ischemia in organs such as brain, heart, kidney, pancreas, and spleen (5.Tinkle B.T. Ngo L. Luciw P.A. Maciag T. Jay G. J. Virol. 1997; 71: 4809-4814Crossref PubMed Google Scholar). In view of these observations, the question remains as to what is causing this inflammation and vasculopathy when there is no obvious infectious or opportunistic pathogen. human immunodeficiency virus long terminal repeat phosphate-buffered saline dithiothreitol fluorescence-activated cell sorter trans-activation response element tumor necrosis factor interleukin isopropyl-1-thio-β-d-galactopyranoside glutathioneS- transferase human umbilical vein endothelial cells electrophoretic mobility shift assay green fluorescent protein human immunodeficiency virus long terminal repeat phosphate-buffered saline dithiothreitol fluorescence-activated cell sorter trans-activation response element tumor necrosis factor interleukin isopropyl-1-thio-β-d-galactopyranoside glutathioneS- transferase human umbilical vein endothelial cells electrophoretic mobility shift assay green fluorescent protein The HIV-1 Tat protein is an early viral protein of 101 amino acids when isolated from primary HIV isolates, or of 86 amino acids when isolated from the laboratory strain HBX2, and which still retains full activity (6.Song C.Z. Loewenstein P.M. Green M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9357-9361Crossref PubMed Scopus (20) Google Scholar). Expression of Tat is critical for productive HIV infection. Thetat gene consists of 2 coding exons. The first one, which encodes 72 amino acids, is sufficient to activate HIV LTR-mediated gene expression in co-transfection promoter-reporter assays (7.Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). However, in the context of viral infection, where the integrated provirus is subject to chromatin influences, the second exon is required for trans activation of the LTR (8.Jeang K.T. Berkhout B. Dropulic B. J. Biol. Chem. 1993; 268: 24940-24949Abstract Full Text PDF PubMed Google Scholar). Exon 1 contains the cysteine-richtrans-activation domain, the core, and an arginine-rich motif that is responsible for Tat-TAR RNA interactions (9.Barillari G. Gendelman R. Gallo R.C. Ensoli B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7941-7945Crossref PubMed Scopus (340) Google Scholar, 10.Brake D.A. Debouck C. Biesecker G. J. Cell Biol. 1990; 111: 1275-1281Crossref PubMed Scopus (164) Google Scholar, 11.Taube R. Fujinaga K. Wimmer J. Barboric M. Peterlin B.M. Virology. 1999; 264: 245-253Crossref PubMed Scopus (116) Google Scholar). The arginine-rich motif acts as a nuclear localization signal as well (12.Endo S. Kubota S. Siomi H. Adachi A. Oroszlan S. Maki M. Hatanaka M. Virus Genes. 1989; 3: 99-110Crossref PubMed Scopus (54) Google Scholar, 13.Siomi H. Shida H. Maki M. Hatanaka M. J. Virol. 1990; 64: 1803-1807Crossref PubMed Google Scholar) and is responsible for NF-κB activation in HeLa cells (14.Demarchi F. Gutierrez M.I. Giacca M. J. Virol. 1999; 73: 7080-7086Crossref PubMed Google Scholar). The second exon codes for the remaining amino acids. Tat acts by binding to a region located at the 5′-end of the viral transcript. This region, called TAR, forms a stable stem-loop structure having a high affinity for Tat (7.Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 11.Taube R. Fujinaga K. Wimmer J. Barboric M. Peterlin B.M. Virology. 1999; 264: 245-253Crossref PubMed Scopus (116) Google Scholar). The NH2-terminaltrans-activation domain, when tethered to TAR, strongly interacts with cyclin T1, which is a component of transcription elongation factor β (7.Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 15.Ping Y.H. Rana T.M. J. Biol. Chem. 1999; 274: 7399-7404Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The cyclin-dependent kinase 9 that is part of this complex then phosphorylates the carboxyl-terminal domain of RNA polymerase II. This phosphorylation facilitates the elongation step by preventing premature termination and ensures production of the full-length viral transcript (16.Okamoto H. Sheline C.T. Corden J.L. Jones K.A. Peterlin B.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11575-11579Crossref PubMed Scopus (89) Google Scholar, 17.Jones K.A. Genes Dev. 1997; 11: 2593-2599Crossref PubMed Scopus (195) Google Scholar, 18.Chun R.F. Jeang K.T. J. Biol. Chem. 1996; 271: 27888-27894Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The end result is a significant increase in the production of viral proteins, essential for productive infection. Tat can be secreted from infected cells and circulates in the bloodstream of infected individuals. From the circulation, Tat enters uninfected cells and once internalized, it alters cellular physiology by positively or negatively affecting gene expression. For example, Tat activates transforming growth factor-β expression in human chondrocytes (19.Lotz M. Clarklewis I. Ganu V. J. Cell Biol. 1994; 124: 365-371Crossref PubMed Scopus (77) Google Scholar), TNF expression in mononuclear cells (20.Buonaguro L. Buonaguro F.M. Giraldo G. Ensoli B. J. Virol. 1994; 68: 2677-2682Crossref PubMed Google Scholar), IL-8 secretion in endothelial cells (21.Hofman F.M. Chen P. Incardona F. Zidovetzki R. Hinton D.R. J. Neuroimmunol. 1999; 94: 28-39Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and T cell lines following CD3- and CD28-mediated co-stimulation (22.Ott M. Lovett J.L. Mueller L. Verdin E. J. Immunol. 1998; 160: 2872-2880PubMed Google Scholar). Tat increases IL-1β production in monocytic cells, and IL-6 protein and mRNA in astrocytes, both effects independent of TNF-α production (23.Nath A. Conant K. Chen P. Scott C. Major E.O. J. Biol. Chem. 1999; 274: 17098-17102Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Tat also increases FAS and FAS-ligand transcription, presumably via NF-κB (24.Li-Weber M. Laur O. Dern K. Krammer P.H. Eur. J. Immunol. 2000; 30: 661-670Crossref PubMed Scopus (85) Google Scholar), and induces IL-10 in peripheral blood monocytes (25.Badou A. Bennasser Y. Moreau M. Leclerc C. Benkirane M. Bahraoui E. J. Virol. 2000; 74: 10551-10562Crossref PubMed Scopus (98) Google Scholar). Microglia exposed to Tat induce nitric-oxide synthase and NO production, and this induction is also dependent upon NF-κB (26.Polazzi E. Levi G. Minghetti L. J. Neuropathol. Exp. Neurol. 1999; 58: 825-831Crossref PubMed Scopus (66) Google Scholar). On the other hand, negative effects include repression of major histocompatibility class-I gene promoter activity (27.Howcroft T.K. Strebel K. Martin M.A. Singer D.S. Science. 1993; 260: 1320-1322Crossref PubMed Scopus (200) Google Scholar) and of the important antioxidant enzyme maganese-superoxide dismutase (28.Flores S.C. Marecki J.C. Harper K.P. Bose S.K. Nelson S.K. McCord J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7632-7636Crossref PubMed Scopus (207) Google Scholar). Interestingly, a deregulation of cytokine expression and/or secretion is a hallmark of HIV infection (29.Buonaguro L. Barillari G. Chang H.K. Bohan C.A. Kao V. Morgan R. Gallo R.C. Ensoli B. J. Virol. 1992; 66: 7159-7167Crossref PubMed Google Scholar, 30.Poli G. Biswas P. Fauci A.S. Antiviral Res. 1994; 24: 221-233Crossref PubMed Scopus (71) Google Scholar, 31.Cox R.A. Anders G.T. Cappelli P.J. Johnson J.E. Blanton H.M. Seaworth B.J. Treasure R.L. AIDS Res. Hum. Retroviruses. 1990; 6: 431-441Crossref PubMed Scopus (32) Google Scholar, 32.Denis M. Ghadirian E. AIDS Res. Hum. Retroviruses. 1994; 10: 1619-1627Crossref PubMed Scopus (94) Google Scholar). These studies indicate that Tat may be having some of its positive transcriptional effects through NF-κB activation. Indeed, Tat activates the transcription factor NF-κB in HeLa cells. This activation is mediated by PKR, the double-stranded RNA-dependent protein kinase, which phosphorylates IκB leading to its degradation (14.Demarchi F. Gutierrez M.I. Giacca M. J. Virol. 1999; 73: 7080-7086Crossref PubMed Google Scholar). Subsequent NF-κB translocation into the nucleus increases expression of a cascade of inflammatory genes. Tat introduced via liposomes results in nuclear translocation of NF-κB (34.Demarchi F. d'Adda di Fagagna F. Falaschi A. Giacca M. J. Virol. 1996; 70: 4427-4437Crossref PubMed Google Scholar) and induction of CD69 gene transcription in an erythroleukemia cell line (35.Blazquez M.V. Macho A. Ortiz C. Lucena C. Lopez-Cabrera M. Sanchez-Madrid F. Munoz E. AIDS Res. Hum. Retroviruses. 1999; 15: 1209-1218Crossref PubMed Scopus (22) Google Scholar) and interleukin-8 secretion in a T-cell line (22.Ott M. Lovett J.L. Mueller L. Verdin E. J. Immunol. 1998; 160: 2872-2880PubMed Google Scholar). Extracellular Tat is associated with an increase in both NF-κB binding and protein kinase C activity in primary fetal human astrocytes (36.Conant K. Ma M. Nath A. Major E.O. J. Virol. 1996; 70: 1384-1389Crossref PubMed Google Scholar). On the other hand, Tat does not activate the NF-κB responsive reporter construct, (PRDII)4-CAT, but can synergize with NF-κB in the activation of both HIV-derived and non-HIV-derived promoters (37.Kelly G.D. Morris C.B. Offermann M.K. Virology. 1999; 263: 128-138Crossref PubMed Scopus (7) Google Scholar). Tat induces matrix metalloproteinase-9 in monocytes through protein-tyrosine phosphatase-mediated activation of NF-κB (38.Kumar A. Dhawan S. Mukhopadhyay A. Aggarwal B.B. FEBS Lett. 1999; 462: 140-144Crossref PubMed Scopus (63) Google Scholar). In Jurkat T cells, Tat-mediated activation of NF-κB is dependent upon activation of the T cell-specific tyrosine kinase p56lck (33.Manna S.K. Aggarwal B.B. J. Immunol. 2000; 164: 5156-5166Crossref PubMed Scopus (46) Google Scholar). Thus, Tat-mediated NF-κB activation may be via multiple signal transduction pathways. The adhesion molecule E-selectin is induced at inflammatory sites where it exhibits restricted and tightly regulated expression in endothelial cells. E-selectin up-regulation is accomplished via increased cytokines such as TNF, and mediated in part by two closely apposed NF-κB sites (39.Schindler U. Baichwal V.R. Mol. Cell. Biol. 1994; 14: 5820-5831Crossref PubMed Google Scholar). Early studies demonstrated that as part of the immune activation seen in AIDS patients, soluble E-selectin levels were elevated (40.Seigneur M. Constans J. Blann A. Renard M. Pellegrin J.L. Amiral J. Boisseau M. Conri C. Thromb. Haemostasis. 1997; 77: 646-649Crossref PubMed Scopus (82) Google Scholar,41.Sfikakis P.P. Tzavara V. Sipsas N. Kosmopoulou O. Sfikakis P. Kordossis T. Infection. 1995; 23: 207-211Crossref PubMed Scopus (14) Google Scholar). Furthermore, Tat increases E-selectin expression in human umbilical vein endothelial cells (42.Dhawan S. Puri R.K. Kumar A. Duplan H. Masson J.M. Aggarwal B.B. Blood. 1997; 90: 1535-1544PubMed Google Scholar). Because endothelial cell adhesiveness for neutrophils consists of an interplay between adhesion molecules and circulating neutrophils, and since Tat has been demonstrated to increase E-selectin expression in normal endothelial cells and activate NF-κB in some cells, we examined whether the Tat-mediated increases in E-selectin expression require NF-κB. Here we demonstrate for the first time that this Tat-mediated up-regulation of E-selectin requires NF-κB and the synthesis of new macromolecules. Furthermore, consistent with previous reports of Tat-mediated NF-κB activation in HeLa cells, we demonstrate that the basic domain of Tat is necessary for this induction. Isopropyl-1-thio-β-d-galactopyranoside (IPTG), antibiotics, cycloheximide, actinomycin D, cell dissociation buffer, IGEPAL (Nonidet P-40), salts, bovine heart cAMP-dependent protein kinase, and buffers were purchased from Sigma. GSH-Sepharose and ECL chemiluminescence kit were from Amersham Bioscience; tumor necrosis factor-α (TNF) used for all the present studies was obtained from Pepro Tech, Inc.; fluorescein isothiocyanate-conjugated Cd62E (clone 1.2B6) was obtained from Research Diagnostics Inc.; antibodies against p65, p52, RelB, Sp3, and actin were obtained from Santa Cruz Biotechnology; antibodies against cRel were obtained from Rockland, and p50 from Geneka Biotechnology Inc. Endothelial cell culture medium was EGM-2 (EGM-2 Bullet kit, BioWhittaker, San Diego, CA), supplemented with 2% fetal calf serum. Serum-free Opti-MEM and neomycin were purchased from Invitrogen (Gaithersburg, MD). Bradford reagent for protein determination was purchased from Bio-Rad. The Tat antibody was kindly provided by the AIDS Reference and Research Reagent Program and was originally contributed by Dr. Bryan Cullen (43.Hauber J. Perkins A. Heimer E.P. Cullen B.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6364-6368Crossref PubMed Scopus (147) Google Scholar). Superfect reagent for transfection was purchased from Qiagen and used according to manufacturer's specifications. Plasmid GST-Tat (GST-Tat 1 86R TK) contains the 2-exon 86-amino acid wild-type tat gene cloned into pGEX2TK (Amersham Bioscience); pGST-Tat 1 48Δ TK is truncated after amino acid 48 and contains a functional activation domain; pGST-Tat 1 48Δ C22G contains a truncation after amino acid 48, and a non-functional activation domain because there is a point mutation at cysteine 22. All of these GST fusion vectors were obtained from the NIH AIDS Research and Reference Reagent Program and contributed by Dr. Andrew P. Rice. Parental vector pGEX-2TK was purchased from Amersham Bioscience. This vector is designed for inducible expression of genes as fusions with the Schistosoma japonicum glutathioneS-transferase (GST). Because the fusion constructs are under the control of the tac promoter, induction with IPTG leads to high levels of expression. Affinity chromatography with GSH-Sepharose followed by thrombin cleavage is used to purify the protein of interest. Plasmid pMT/V5-His C (Invitrogen) is aDrosophila expression vector that carries the fly metallothionein promoter, which allows for CuSO4 induction of cloned genes. The HIV-1 86-amino acid Tat was cloned into the multiple cloning site of this vector to generate the plasmid pMT-tat. 2A. E. Casullo, N. C. Flores, L. Uyetake, A. Cota-Gomez, and S. C. Flores, manuscript in preparation. Plasmid pELAM-Luc (pE-Luc), kindly provided by Dr. Tom McIntyre, contains sequences 840 base pairs upstream of the transcriptional start site of the human E-selectin gene driving firefly luciferase transcription. Control plasmid pGL3B containing the promoterless luciferase gene was obtained from Promega Biotechnology. Plasmid pCoHygro, obtained from Invitrogen, containing a hygromycin resistance gene under the control of the Drosophila copia promoter, was used for the generation of stable S2-tat cells. Plasmid pHOOK-3 (Invitrogen), an expression vector carrying the cytomegalovirus promoter, was used to create a pHOOK-tat expression vector for transient transfections of mammalian cells. HUVEC were obtained from BioWhittaker and maintained in EGM-2. Cultures were maintained at 37 °C in a 6.5% CO2 humidified atmosphere. Adhesion molecule induction via NF-κB declines as passage number increases and is sensitive to growth state, therefore, expanded cells were used at passages 2–3 and cells at 3 days postconfluence were used for all the experiments. HeLa and HeLa-tat cells were obtained through the NIH AIDS Research and Reference Reagent Program and contributed by Drs. W. Haseltine and E. Terwilliger. These cells were cultured in Opti-MEM with 3.75% fetal calf serum in the absence of antibiotics. The HeLa-tat cells were grown in the presence of 800 μg/ml neomycin. Schneider 2 (S2) Drosophila cells were purchased from Invitrogen and cultured in complete DrosophilaExpression System medium according to the manufacturer's instructions. For transfection and selection of stable S2-tat cells, S2 cells were grown in 6-well culture dishes to a density of 1 × 106 cells, collected, washed, and co-transfected with pCoHygro/pmT-tat via Ca3PO4. Cells were plated and allowed to recover for 24 h in complete Drosophila Expression System medium. Stable clones were selected by growth in 300 μg/ml hygromycin for at least 2 months. Transgene expression was induced by 24 h incubation in the presence of 500 μmCuSO4. HeLa or HeLa-tat cells were seeded at a density of 5 × 105 cells/well on 6-well tissue culture dishes and grown until 60% confluence. Cells were washed with sterile PBS and incubated in the presence of 2 μg of pE-Luc or pGL3B in Superfect reagent for 18 h. Cells were lysed in passive lysis buffer (Promega luciferase transfection kit) according to the manufacturer, lysate collected, and total protein determined. Aliquots of 20 μl were assayed for light emission with a plate-reader luminometer. In a separate series of experiments, HUVEC were seeded in 6-well tissue culture dishes and grown to 60% confluence. Transfection and luciferase assays were performed as described above, except that the cells were co-transfected with equimolar amounts of either pHOOK-3/pE-Luc or pHOOK-tat/pE-Luc. For nuclear and cytoplasmic protein extraction, confluent HUVEC cultures grown on 10-cm tissue culture dishes were washed with sterile PBS and fed 5 ml of serum-free Opti-MEM containing 20 ng/ml recombinant human TNF, 500 ng/ml recombinant Tat, or the indicated control medium for 1 h. After addition of 5 ml of EGM-2 containing 2% fetal calf serum, cells were incubated for the indicated times. Therefore, no serum was present during the first hour of incubation, and only 1% during the remainder. Cells were harvested by rinsing twice with PBS (calcium and magnesium-free), followed by incubation in a non-enzymatic cell dissociation buffer for 5 min at 37 °C. Cells were detached by scraping, transferred to microcentrifuge tubes, and pelleted at 5,000 × g for 1 min. The supernatant was removed, cells resuspended in 1 ml of cold hypotonic buffer (10 mmHEPES, 1.5 mm MgCl2, and 10 mm KCl adjusted to pH 7.9 with KOH) containing 0.2 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 0.3 μg/ml leupeptin, and 0.5 mm DTT, and washed by centrifugation at 2,000 × g for 5 min at 4 °C. The pellet was resuspended in 5 times the packed cell volume of cold hypotonic buffer with protease inhibitors as described above and allowed to swell on ice for 20 min. IGEPAL was then added to 0.1% and the swollen cells incubated an additional 5 min on ice followed by homogenization with 20 strokes of a microtube pestle homogenizer (Fisher Scientific). Cell lysis and nuclear integrity were verified by trypan blue exclusion analysis. Nuclei were pelleted at 13,000 × g for 5 min at 4 °C. The supernatant containing the cytoplasmic fraction was transferred to another tube and stored at −80 °C until further use. The nuclei were resuspended in 3–4 times the packed nuclear volumes of cold hypotonic buffer with protease inhibitors as above, washed once by centrifugation at 13,000 × g at 4 °C, and resuspended gently in one-half packed nuclear volume of cold low salt buffer (20 mm HEPES, 0.2 mm EDTA, 25% glycerol, 1.5 mm MgCl2, 20 mm KCl, pH 7.9). One-quarter packed nuclear volume of cold hypertonic buffer (10 mm HEPES, 0.1 mm EDTA, 50 mmKCl, 300 mm NaCl, 10% glycerol, 1.5 mmMgCl2 at pH 7.9) was added in a dropwise manner to prevent lysis of the nuclei. Nuclear proteins were extracted at 4 °C for 30 min, with a gently rotating motion. The nuclear protein extract was clarified by centrifugation at 13,000 × g for 20 min at 4 °C. Nuclear and cytoplasmic protein concentrations were determined by the Bradford reagent microassay protocol using bovine serum albumin as a standard. To assess the purity of the fractions and to test our cell fractionation technique, 15 μg of nuclear or cytoplasmic proteins extracted from HUVEC, HeLa, or HeLa-tatcells were immunoblotted with actin or Sp3 antibodies. Nuclear proteins were diluted in a 2:1 mixture of low salt:hypertonic buffer to a concentration of 0.75 μg/μl and 2.5 μg of this protein were used in the binding reaction. This procedure ensured that the salt as well as the protein concentration was the same in all the reactions. The oligonucleotide used contained the human E-selectin κB site 3 (39.Schindler U. Baichwal V.R. Mol. Cell. Biol. 1994; 14: 5820-5831Crossref PubMed Google Scholar) flanked by additional E-selectin-specific sequences: 5′- GCCATTGGGGATTTCCTCTTT-3′ (κB site underlined). Complementary oligos (Invitrogen) were annealed and end-labeled with [γ-32P]ATP and T4 polynucleotide kinase (Promega). Labeled oligos were separated from unincorporated nucleotides using a Sephadex G-25 spin column (5 Prime → 3 Prime). The labeled probe was diluted to 8–12 fmol/μl (∼10,000 cpm) and 1 μl/binding reaction was used. The binding reaction also contained 4% glycerol, 1 mm MgCl2, 0.5 mm EDTA, 0.5 mm DTT, 50 mm NaCl, 10 mm, Tris-HCl, pH 7.5, 0.05 mg/ml poly(dI-dC)-poly(dI-dC), and 2.5 μg of nuclear protein (3.3 μl of 0.75 μg/ml dilution) in a final volume of 9 μl. For competition studies, 50-fold molar excess of cold competitor κB or an irrelevant oligonucleotide was included. The reaction was mixed by gentle rocking and incubated at room temperature for 20 min. After binding, 1 μl of 10 × EMSA buffer (250 mm Tris-HCl, pH 7.5, 0.2% bromphenol blue, and 40% glycerol) was added to each reaction and loaded onto a 4% polyacrylamide gel (40:1 acrylamide:bis-acrylamide) containing 1 × TGE buffer (25 mm Tris-HCl, pH 7.5, 190 mmglycine, 1 mm EDTA, pH 8.3) and 10% glycerol. The protein complexes were separated at 12 mA/gel for ∼2 h. Gels were driedin vacuo and exposed to Bio-Max X-Ray (Kodak) film. Multiple exposures of the film were obtained to ensure that the signal was within the linear range of the film. After 20 min incubation of the oligonucleotide and the nuclear protein extract, 1 μg of antibodies against p50, p65, RelB, cRel, or p52 was added and the mixture incubated for an additional 15 min before electrophoresis. The HIV-1 Tat protein was purified in our laboratory as described (44.Herrmann C.H. Rice A.P. J. Virol. 1995; 69: 1612-1620Crossref PubMed Google Scholar), using affinity chromatography. The truncated Tat mutants and the parental GST were purified using the same method. Expression from these vectors results in GST-fusion proteins, linked by a thrombin-sensitive peptide. Host Escherichia coli cells were grown toA 590 = 0.5–0.7 in 500 ml of culture volume. Induction of fusion proteins was achieved by growth in the presence of 0.1 mm IPTG for 2.5 h. Cells were collected by centrifugation at 14,000 × g, the pellet was resuspended in 4 ml of EBC-DTT buffer (50 mm Tris-HCl, pH 8.0, 120 mm NaCl, 0.1% IGEPAL, and 5 mm DTT) and sonicated for 1 min twice, with a 1-min incubation on ice between sonications. The homogenate was clarified by centrifugation in microtubes at 13,000 × g for 15 min at 4 °C. The supernatant was collected and frozen at −80 °C. For GST-Tat purification, all of the buffers were degassed to minimize the exposure of Tat to oxygen. This treatment consistently gave biologically active Tat preparations. Glutathione-Sepharose beads (GSH-Sepharose, Amersham Bioscience) were equilibra" @default.
• W1567250782 created "2016-06-24" @default.
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• W1567250782 creator A5012372946 @default.
• W1567250782 creator A5025879783 @default.
• W1567250782 creator A5027516939 @default.
• W1567250782 creator A5030334470 @default.
• W1567250782 creator A5039215464 @default.
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• W1567250782 date "2002-04-01" @default.
• W1567250782 modified "2023-09-29" @default.
• W1567250782 title "The Human Immunodeficiency Virus-1 Tat Protein Activates Human Umbilical Vein Endothelial Cell E-selectin Expression via an NF-κB-dependent Mechanism" @default.
• W1567250782 cites W146196984 @default.
• W1567250782 cites W1488249325 @default.
• W1567250782 cites W1517171683 @default.
• W1567250782 cites W1522804001 @default.
• W1567250782 cites W1532792896 @default.
• W1567250782 cites W1547148847 @default.
• W1567250782 cites W1551937780 @default.
• W1567250782 cites W1552647437 @default.
• W1567250782 cites W1560943379 @default.
• W1567250782 cites W1569373357 @default.
• W1567250782 cites W1597888074 @default.
• W1567250782 cites W1604626968 @default.
• W1567250782 cites W1656832641 @default.
• W1567250782 cites W1693158227 @default.
• W1567250782 cites W1821435655 @default.
• W1567250782 cites W1870780415 @default.
• W1567250782 cites W1896714280 @default.
• W1567250782 cites W1914107766 @default.
• W1567250782 cites W1962437344 @default.
• W1567250782 cites W1966700067 @default.
• W1567250782 cites W1968264901 @default.
• W1567250782 cites W1971592398 @default.
• W1567250782 cites W1972691106 @default.
• W1567250782 cites W1992320411 @default.
• W1567250782 cites W1999401115 @default.
• W1567250782 cites W2001582257 @default.
• W1567250782 cites W2010358244 @default.
• W1567250782 cites W2011477764 @default.
• W1567250782 cites W2020675675 @default.
• W1567250782 cites W2021418970 @default.
• W1567250782 cites W2023674814 @default.
• W1567250782 cites W2025696302 @default.
• W1567250782 cites W2026075546 @default.
• W1567250782 cites W2026675906 @default.
• W1567250782 cites W2031286781 @default.
• W1567250782 cites W2041745835 @default.
• W1567250782 cites W2045851101 @default.
• W1567250782 cites W2047286220 @default.
• W1567250782 cites W2048404380 @default.
• W1567250782 cites W2056329949 @default.
• W1567250782 cites W2057905142 @default.
• W1567250782 cites W2057973294 @default.
• W1567250782 cites W2062862354 @default.
• W1567250782 cites W2063393407 @default.
• W1567250782 cites W2066391286 @default.
• W1567250782 cites W2066534222 @default.
• W1567250782 cites W2067294390 @default.
• W1567250782 cites W2067592236 @default.
• W1567250782 cites W2069869908 @default.
• W1567250782 cites W2073539437 @default.
• W1567250782 cites W2073994420 @default.
• W1567250782 cites W2076100163 @default.
• W1567250782 cites W2079335891 @default.
• W1567250782 cites W2086161207 @default.
• W1567250782 cites W2087281335 @default.
• W1567250782 cites W2088430031 @default.
• W1567250782 cites W2094431347 @default.
• W1567250782 cites W2095291302 @default.
• W1567250782 cites W2098166246 @default.
• W1567250782 cites W2108481498 @default.
• W1567250782 cites W2110097921 @default.
• W1567250782 cites W2138012613 @default.
• W1567250782 cites W2139319526 @default.
• W1567250782 cites W2150805739 @default.
• W1567250782 cites W2158566329 @default.
• W1567250782 cites W2164021685 @default.
• W1567250782 cites W2172185515 @default.
• W1567250782 cites W2204123985 @default.
• W1567250782 cites W2415192714 @default.
• W1567250782 cites W4245636198 @default.
• W1567250782 cites W4313333734 @default.
• W1567250782 cites W57150681 @default.
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