Intercellular Adhesion Molecule-1 (ICAM-1) Gene Expression in Human T Cells Is Regulated by Phosphotyrosyl Phosphatase Activity
2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês
10.1074/jbc.m005067200
ISSN1083-351X
AutoresJocelyn Roy, Marie Audette, Michel J. Tremblay,
Tópico(s)Protein Tyrosine Phosphatases
ResumoIntercellular adhesion molecule-1 (ICAM-1) plays an important role in adhesion phenomena involved in the immune response. The strength of adhesion has been shown to be modulated by changes in ICAM-1 gene expression. In T cells, signaling pathways are intimately regulated by an equilibrium between protein-tyrosine kinases and protein tyrosine phosphatases (PTP). The use of bis-peroxovanadium (bpV) compounds, a class of potent PTP inhibitors, enabled us to investigate the involvement of phosphotyrosyl phosphatases in the regulation of ICAM-1 gene expression in human T cells. Here, we demonstrate for the first time that inhibition of PTP results in an increase of ICAM-1 surface expression on both human T lymphoid and primary mononuclear cells. The crucial role played by the NF-κB-, Ets-, and pIγRE-binding sites in bpV[pic]-mediated activation of ICAM-1 was demonstrated using various 5′ deletion and site-specific mutants of the ICAM-1 gene promoter driving the luciferase reporter gene. Co-transfection experiments withtrans-dominant mutants and electrophoretic mobility shift assays confirmed the importance of constitutive and inducible transcription factors that bind to specific responsive elements in bpV-dependent up-regulation of ICAM-1 surface expression. Altogether, these observations suggest that expression of ICAM-1 in human T cells is regulated by phosphotyrosyl phosphatase activity through NF-κB-, Ets-, and STAT-1-dependent signaling pathways. Intercellular adhesion molecule-1 (ICAM-1) plays an important role in adhesion phenomena involved in the immune response. The strength of adhesion has been shown to be modulated by changes in ICAM-1 gene expression. In T cells, signaling pathways are intimately regulated by an equilibrium between protein-tyrosine kinases and protein tyrosine phosphatases (PTP). The use of bis-peroxovanadium (bpV) compounds, a class of potent PTP inhibitors, enabled us to investigate the involvement of phosphotyrosyl phosphatases in the regulation of ICAM-1 gene expression in human T cells. Here, we demonstrate for the first time that inhibition of PTP results in an increase of ICAM-1 surface expression on both human T lymphoid and primary mononuclear cells. The crucial role played by the NF-κB-, Ets-, and pIγRE-binding sites in bpV[pic]-mediated activation of ICAM-1 was demonstrated using various 5′ deletion and site-specific mutants of the ICAM-1 gene promoter driving the luciferase reporter gene. Co-transfection experiments withtrans-dominant mutants and electrophoretic mobility shift assays confirmed the importance of constitutive and inducible transcription factors that bind to specific responsive elements in bpV-dependent up-regulation of ICAM-1 surface expression. Altogether, these observations suggest that expression of ICAM-1 in human T cells is regulated by phosphotyrosyl phosphatase activity through NF-κB-, Ets-, and STAT-1-dependent signaling pathways. intercellular adhesion molecule-1 protein tyrosine phosphatases bis-peroxovanadium bis-peroxovanadium compound carrying the picolinic acid as an auxillary ligand palindromic interferon-γ-responsive element signal transducers and activators of transcription phorbol 12-myristate 13-acetate ionomycin fetal bovine serum peripheral blood mononuclear cells phosphate-buffered saline wild type double-stranded DNA mutant interleukin interferon tumor necrosis factor-α base pair Intercellular adhesion molecule-1 (ICAM-1)1 is an inducible cell surface glycoprotein belonging to the immunoglobulin supergene family that shows a molecular mass ranging from 76 to 114 kDa depending on the degree of glycosylation. Its cognate ligands include the membrane-bound integrin receptor LFA-1, Mac-1 on leukocytes, the soluble molecule fibrinogen, rhinoviruses, and Plasmodium falciparum malaria-infected erythrocytes (1Marlin S.D. Springer T.A. Cell. 1987; 51: 813-819Abstract Full Text PDF PubMed Scopus (1393) Google Scholar, 2Staunton D.E. Merluzzi V.J. Rothlein R. Barton R. Marlin S.D. Springer T.A. Cell. 1989; 56: 849-853Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 3Greve J.M. Davis G. Meyer A.M. Forte C.P. Yost S.C. Marlor C.W. Kamarck M.E. McClelland A. Cell. 1989; 56: 839-847Abstract Full Text PDF PubMed Scopus (857) Google Scholar, 4Berendt A.R. Simmons D.L. Tansey J. Newbold C.I. Marsh K. Nature. 1989; 341: 57-59Crossref PubMed Scopus (570) Google Scholar, 5Diamond M.S. Staunton D.E. de Fougerolles A.R. Stacker S.A. Garcia-Aguilar J. Hibbs M.L. Springer T.A. J. Cell Biol. 1990; 111: 3129-3139Crossref PubMed Scopus (769) Google Scholar). Within the immune system, ICAM-1 is expressed on cells of the monocyte-macrophage lineage, B lymphocytes, plasma cells, and on both memory and activated T lymphocytes. The association between ICAM-1 and the activated form of the LFA-1 counter-receptor has many important roles in adhesion phenomena involved in the immune system. Its basic function is the induction of a specific and reversible cell-cell adhesion that enables intercellular communication, T cell-mediated defense mechanism, and inflammatory response. In addition, ICAM-1 is also involved in leukocyte-endothelial cell interaction, cell differentiation, and in many pathological complications such as acquired immunodeficiency syndrome, malignancies of both myeloid and lymphoid origin, and allergic asthma (6Noraz N. Verrier B. Fraisier C. Desgranges C. AIDS Res. Hum. Retroviruses. 1995; 11: 145-154Crossref PubMed Scopus (23) Google Scholar, 7van de Stolpe A. van der Saag P.T. J. Mol. 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Additional links occur between other molecules expressed on both cell surfaces that are required for completing adhesion and cell signaling and consequently determining the following response. It ensues that a dysfunction in ICAM-1 gene expression results in an immunological impairment or a physiopathological situation (7van de Stolpe A. van der Saag P.T. J. Mol. Med. 1996; 74: 13-33Crossref PubMed Scopus (648) Google Scholar,8Roebuck K.A. Finnegan A. J. Leukocyte Biol. 1999; 66: 876-888Crossref PubMed Scopus (462) Google Scholar). The regulation of ICAM-1 gene expression occurs primarily at the level of transcription and is cell type-specific. This phenomenon involves different signaling pathways and several enhancer elements such as palindromic interferon-γ-responsive element (pIγRE), NF-κB, Ets, C/EBP, AP-1-like, Sp1, and retinoic acid response elements (7van de Stolpe A. van der Saag P.T. J. Mol. Med. 1996; 74: 13-33Crossref PubMed Scopus (648) Google Scholar, 8Roebuck K.A. 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NF-κB has also been reported to play a pivotal role inICAM-1 gene regulation where RelA (p65)/RelA, RelA/c-Rel, and RelA/NF-κB1 (p50) dimers can potently induce ICAM-1 expression in several cell types (20Ledebur H.C. Parks T.P. J. Biol. Chem. 1995; 270: 933-943Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 21Parry G.C. Mackman N. J. Biol. Chem. 1994; 269: 20823-20825Abstract Full Text PDF PubMed Google Scholar, 22Jahnke A. Johnson J.P. FEBS Lett. 1994; 354: 220-226Crossref PubMed Scopus (102) Google Scholar, 23Hou J. Baichwal V. Cao Z. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11641-11645Crossref PubMed Scopus (214) Google Scholar, 24van de Stolpe A. Caldenhoven E. Stade B.G. Koenderman L. Raaijmakers J.A. Johnson J.P. van der Saag P.T. J. Biol. Chem. 1994; 269: 6185-6192Abstract Full Text PDF PubMed Google Scholar, 25Aoudjit F. Brochu N. Bélanger B. Stratowa C. Hiscott J. Audette M. Cell Growth Differ. 1997; 8: 335-342PubMed Google Scholar). Both JAK/STAT and NF-κB pathways have been shown to be modulated by phosphorylation events that lead to their translocation into the nucleus. In addition to JAK/STAT and NF-κB, the Ets gene family of transcriptional factors is also involved in the regulation of ICAM-1 expression (26Roebuck K.A. Rahman A. Lakshminarayanan V. Janakidevi K. Malik A.B. J. Biol. Chem. 1995; 270: 18966-18974Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 27de Launoit Y. Audette M. Pelczar H. Plaza S. Baert J.-L. Oncogene. 1998; 16: 2065-2073Crossref PubMed Scopus (57) Google Scholar). The control of highly diverse sets of genes by Ets proteins involves their own regulation at different levels which include, among others, specific phosphorylation events mediated by the Ras-MAPK pathway in response to extracellular signals (28Wasylyk B. Hagman J. Gutierrez-Hartmann A. Trends Biochem. Sci. 1998; 23: 213-216Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). In T cells, the expression of many genes is tightly regulated by an equilibrium between two sets of enzymes with distinct properties, the protein-tyrosine kinases and protein tyrosine phosphatases (PTP). The role of protein-tyrosine kinases in T cell gene expression has been well documented (29Germain R.N. Stefanova I. Annu. Rev. Immunol. 1999; 17: 467-522Crossref PubMed Scopus (366) Google Scholar). Recently, some reports have described the role of PTP in T cell signaling and T cell transduction (30Chan A.C. Desai D.M. Weiss A. Annu. Rev. Immunol. 1994; 12: 555-592Crossref PubMed Scopus (497) Google Scholar, 31Olivero S. Bléry M. Vivier É. Méd./Sci. 1998; 14: 262-268Google Scholar, 32Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (164) Google Scholar, 33Neel G.B. Tonks K.N. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (731) Google Scholar, 34Neel B.G. Curr. Opin. Immunol. 1997; 9: 405-420Crossref PubMed Scopus (139) Google Scholar, 35Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1941) Google Scholar), but the involvement of PTP in the regulation of ICAM-1 gene expression in T cells is unclear. Of interest is the observation that the PTP inhibitor pervanadate can mimic IFN-γ-mediated induction of ICAM-1 expression via nuclear translocation of STAT-1 proteins in human keratinocytes (17Duff J.L. Quinlan K.L. Paxton L.L. Naik S.M. Caughman S.W. J. Invest. Dermatol. 1997; 108: 295-301Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Moreover, calyculin A and okadaic acid, two phosphoseryl/threonyl phosphatase inhibitors, induce an ICAM-1/LFA-1-dependant homotypic aggregation of both Jurkat and U937 cells (36Weeks B.S. Iuorno J. Biochem. Biophys. Res. Commun. 1996; 226: 82-87Crossref PubMed Scopus (14) Google Scholar). However, the mechanisms leading to this ICAM-1/LFA-1 aggregation have not been defined. Altogether, these reports suggest that both PTP and phosphoseryl/threonyl phosphatases are involved in ICAM-1 expression. The primary objective of the present work was to investigate the role of PTP in the regulation of ICAM-1 gene expression in human T cells. We show here that treatment of primary human peripheral blood mononuclear cells and the human leukemic T cell lines Jurkat, HUT 78, and WE17/10 with the bis-peroxovanadium compound bpV[pic], a strong inhibitor of PTP, results in the induction of ICAM-1 surface expression. Further experiments revealed that NF-κB, Ets, and pIγRE-binding sites are important sequence motifs in bpV[pic]-mediated up-regulation of ICAM-1 expression. These results suggest that ICAM-1 is regulated in human T cells by PTP activity. Phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono) were purchased from Sigma and Calbiochem, respectively. Sodium orthovanadate (Sigma) was freshly dissolved before its use in 10 mm HEPES, pH 7.4. The bpV[pic] compound was prepared as described previously (37Posner B.I. Faure R. Burgess J.W. Bevan A.P. Lachance D. Zhang-Sun G. Fantus I.G. Ng J.B. Hall D.A. Soo Lum B.S. Shaver A. J. Biol. Chem. 1994; 269: 4596-4604Abstract Full Text PDF PubMed Google Scholar). Briefly, V2O5 was dissolved in an aqueous KOH solution and then mixed with 30% H2O2 and an ancillary ligand (picolinic acid anion in this study hence bpV[pic]) in addition to the ethanol for optimal precipitation. Characterization of bpV[pic] was carried out by infrared 1H NMR and vanadium 51 (51V) NMR spectroscopy. Stock solutions of bpV[pic] (1 mm in phosphate-buffered saline, pH 7.4) were kept at −85 °C into small aliquots until used. The parental lymphoid T cell line Jurkat (clone E6.1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Jurkat cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.22% NaHCO3, in a 5% CO2-humidified atmosphere. The human IL-2-dependent T lymphoblastoid cell line WE17/10 (38Willard-Gallo K.E. van de Keere F. Kettmann R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6713-6717Crossref PubMed Scopus (36) Google Scholar) and the human cutaneous T lymphoma cell line HUT 78 (39) were provided by the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health (Bethesda), and were maintained in complete culture medium in the presence of recombinant human IL-2 (50 units/ml) for WE17/10. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation and were cultured in complete culture medium in the presence of phytohemagglutinin (Sigma) (3 μg/ml) and recombinant human IL-2 (30 units/ml) for 3 days at 37 °C. Such cells were left untreated in complete culture medium containing 20% heat-inactivated FBS for 3 days prior to treatment with either bpV[pic] or PMA/Iono. The following reagent was obtained through the AIDS Research and Reference Reagent Program: recombinant human interleukin-2 from Maurice Gately, Hoffmann-La Roche (40Lahm H.W. Stein S. J. Chromatogr. 1985; 326: 357-361Crossref PubMed Scopus (107) Google Scholar). Cell surface expression of ICAM-1 was evaluated by flow cytometry as follows. Jurkat, HUT 78, WE17/10 cells, and PBMCs (1 × 106) were washed once in phosphate-buffered saline containing 2% FBS (PBSA). Cells were then resuspended in 100 μl of PBSA to which was added 1 μg of monoclonal anti-ICAM-1 antibody (clone RR1/1.1.1), vortexed gently, and incubated for 30 min on ice. Cells were subsequently washed with PBS containing 2% FBS and resuspended in 100 μl of PBS containing (R)-phycoerythrin-conjugated goat anti-mouse IgG (0.5 μg total) and further incubated for 30 min on ice. Cells were finally centrifuged and resuspended in 1% paraformaldehyde in PBS before being analyzed by flow cytometry (EPICS XL, Coulter Corp., Miami, FL). Reporter plasmids of the ICAM-1 5′-regulatory element and mutants used in these experiments are cloned upstream from the firefly luciferase gene. pGL1.3, pGL1.3 κBmut, pGLHindIII, and pGL HindIII IRE mut were provided by Dr. T. P. Parks (Boehringer Ingelheim, Ridgefiel, CN), and pGLE WT, pGLE −138mut, pGLE −158mut, and pGLE −138/−158mut were kindly supplied by Dr. Y. de Launoit (Institut Pasteur, Lille, France). Anti-STAT-1, anti-STAT-3, and anti-p50 antibodies were purchased from Santa Cruz Biotechnology. Dr. N. Rice (NCI, Frederick, MD) kindly provided the polyclonal anti-p65 antibodies. Dr. Rothlein (Boehringer Ingelheim, Ridgefield, CN) provided the anti-ICAM-1 antibody RR1/1.1.1 (anti-CD54) (41Rothlein R. Dustin M.L. Marlin S.D. Springer T.A. J. Immunol. 1986; 137: 1270-1274PubMed Google Scholar). The dominant negative IκBα-expressing vector pCMV-IκBα S32A/36A has been described previously (42Sun S.-C. Elwood J. Greene W.C. Mol. Cell. Biol. 1996; 16: 1058-1065Crossref PubMed Google Scholar) (a kind gift from Dr. W. C. Greene, The Gladstone Institutes, San Francisco). The DNA filler pCMV-EcoRV/SmaI was constructed from the expressing vector pCMV-IκBα S32A/36A by deletion of the cDNA encoding for IκBα S32A/36A with EcoRV/SmaI digestion. Jurkat cells (5 × 106) were first washed once in TS buffer (137 mm NaCl, 25 mm Tris-HCl, pH 7.4, 5 mm KCl, 0.6 mm NaHPO4, 0.5 mm MgCl2, and 0.7 mmCaCl2) and resuspended in 0.5 ml of TS containing 15 μg of the indicated plasmids and 500 μg/ml DEAE-dextran (final concentration). The cell/TS/plasmid/DEAE-dextran mixture was incubated for 25 min at room temperature. Thereafter, cells were diluted at a concentration of 1 × 106 per ml using complete culture medium supplemented with 100 μm chloroquine (Sigma). After 45 min of incubation at 37 °C, cells were centrifuged, washed once, resuspended in complete culture medium, and incubated at 37 °C for 24 h. Transiently transfected cells were seeded at a density of 105 cells per well (100 μl) in 96-well flat-bottom plates. In most experiments, cells were left untreated or were either treated with bpV[pic], sodium orthovanadate, or PMA/Iono in a final volume of 200 μl for a period of 8 h for bpV[pic] or 24 h for PMA/Iono and sodium orthovanadate. Cells were then lysed, and luciferase activity was monitored with a microplate luminometer (MLX; Dynex Technologies, Chantilly, VA). Jurkat cells were either left untreated or were incubated for different times at 37 °C with bpV[pic] (10 μm) or PMA/Iono (20 ng/ml and 1 μm, respectively). Incubation of Jurkat cells with the various stimulating agents was terminated by the addition of ice-cold PBS, and nuclear extracts were prepared according to the microscale preparation protocol (43Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499-2500Crossref PubMed Scopus (2207) Google Scholar). In brief, sedimented cells were resuspended in 400 μl of cold buffer A (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, and 0.5 mmphenylmethylsulfonyl fluoride). After 15 min on ice, 25 μl of 10% Nonidet P-40 was added. The lysate was vortexed for 10 s, and samples were centrifuged for 30 s at 12,000 × g. The supernatant fraction was discarded, and the cell pellet was resuspended in 100 μl of cold buffer B (20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mmEGTA, 1 mm dithiothreitol, and 1 mmphenylmethylsulfonyl fluoride). Cells were then rocked vigorously at 4 °C for 15 min. Cellular debris were removed by centrifugation at 12,000 × g for 5 min at 4 °C, and the supernatant fraction was stored at −70 °C until used. Electrophoretic mobility shift assay was performed with 10 μg of nuclear extracts. Protein concentrations were determined by the bicinchoninic assay with a commercial protein assay reagent (Pierce). Nuclear extracts were incubated for 30 min at room temperature in 15 μl of buffer C (100 mm HEPES, pH 7.9, 40% glycerol, 10% Ficoll, 250 mm KCl, 10 mm dithiothreitol, 5 mmEDTA, 250 mm NaCl, 2 μg of poly(dI-dC), 10 μg of nuclease-free bovine serum albumin (fraction V) containing 0.8 ng of radiolabeled-labeled double-stranded DNA (dsDNA) oligonucleotide. Double-stranded DNA (100 ng) was labeled with [γ-32P]ATP and T4 polynucleotide kinase in a kinase buffer (New England Biolabs, Beverly, MA). This mixture was incubated for 20 min at room temperature, and the reaction was stopped with 5 μl of 0.2 m EDTA. The labeled oligonucleotide was extracted with phenol/chloroform and passed through a G-50 spin column. The dsDNA oligonucleotides, which were used as probes or as competitors, contained either the nonspecific probe Oct-2A (5′-GGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3′), the proximal NF-κB-binding site (5′-GATTGCTTTAGCTTGGAAATTCCGGAGCTG-3′), the distal NF-κB-binding site (5′-AGGGAGCCCGGGGAGGATTCCTGGGCC-3′), the pIγRE (5′-AAGGCGGAGGTTTCCGGGAAAGCAGCACC-3′), the wild-type −138/−158 Ets-binding sites (5′-CTGTCAGTCCGGAAATAACTGCAGCATTTGTTCCGGAGGGGAAG-3′), or the −138/−158-mutated Ets-binding sites (5′-CTGTCAGTCCCCAAATAACTGCAGCATTTGTTGGGGAGGGGAAG-3′) of the ICAM-1 5′-regulatory element. DNA-probe complexes were resolved from free labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels containing 50 mm Tris-HCl, pH 8.5, 200 mm glycine, and 1 mm EDTA. The gels were subsequently dried and autoradiographed. Cold competitor assays were carried out by adding a 100-fold molar excess of homologous unlabeled dsDNA proximal or distal NF-κB, pIγRE, or Ets oligonucleotides simultaneously with the labeled probe. Supershift assays were performed by preincubation of nuclear extracts with 1 μl of specific antibodies in the presence of all the components of the binding reaction described above for 30 min at 4 °C. Given that intracellular tyrosine phosphorylation levels are crucial in the regulation of numerous genes, we investigated the effect of the PTP-specific inhibitor bpV[pic] on ICAM-1 protein expression in the human leukemic T cell line Jurkat and also in primary cells (i.e. PBMCs). In this set of experiments, cells were treated either with the PMA/Iono combination (as a control) or bpV[pic] compound, and the percentage of ICAM-1-expressing cells as well as the mean fluorescence intensity (indicative of the number of molecules per single cell shown on a logarithmic scale) were defined by flow cytometry analyses with the use of an antibody specific for ICAM-1 (clone RR1/1.1.1). As depicted in Fig. 1,A and D, ICAM-1 is constitutively expressed on both Jurkat cells and PBMCs. PMA/Iono treatment resulted in a slight increase ICAM-1 expression on Jurkat cells, whereas a much greater induction of ICAM-1 protein was mediated by these stimuli in primary cells. Interestingly, treatment with the tyrosine phosphatase-specific inhibitor bpV[pic] resulted in a much greater up-regulation of ICAM-1 protein expression on Jurkat leukemic T cells than PMA/Iono. Inhibition of PTP by the specific inhibitor bpV[pic] also leads to a marked induction of ICAM-1 expression in PBMCs. Cell viability was not affected by PMA/Iono and bpV[pic] treatments as monitored by performing in parallel MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays (data not shown). These data represent the first demonstration that PTPs are implicated in ICAM-1 gene expression in human T cells. It should be noted that we have made similar observations using HUT 78, another human T cell lymphoma line, and WE17/10, an IL-2-dependent T cell receptor/CD4-expressing cell line established from the blood cells of a patient with T cell lymphoma (Fig. 1, B and C, respectively). This last series of experiments indicate that the noticed bpV[pic]-mediated induction of ICAM-1 gene expression is not an epiphenomenon since it is observed in several human T cell lines. It is well documented that ICAM-1 gene expression is primarily regulated at the transcriptional level. In an attempt to study the effect of bpV[pic] on ICAM-1 transcription, a dose-response experiment was initially carried out using increasing concentrations of this PTP inhibitor. To this end, Jurkat cells were transiently transfected with a reporter construct made of the luciferase gene placed downstream of the entire ICAM-1promoter (i.e. pGL1.3). Next, cells harboring the ICAM-1-luciferase vector were either left untreated or were treated for 8 h with the indicated bpV[pic] concentrations (Fig.2 A). A dose-dependent increase of ICAM-1 promoter activity in Jurkat cells transiently transfected with pGL1.3 was observed when using concentrations of bpV[pic] ranging from 1 to 10 μm (1.2–42.9-fold increase). A slight decrease of ICAM-1-driven luciferase activity was detected at the highest concentration tested (i.e. 20 μm) (34.0-fold increase), which could be due to cell toxicity. Subsequent experiments were thus conducted using bpV[pic] at a maximal concentration of 10 μm. Sodium orthovanadate (Na3VO4), a commonly used PTP inhibitor, was similarly tested in this series of investigations. As shown in Fig.2 B, a weak increase in ICAM-1-driven luciferase activity was obtained with concentrations of Na3VO4 ranging from 12.5 to 50 μm (1.1–1.5-fold induction). Therefore, these data suggest that bpV[pic] is a much more potent activator ofICAM-1 promoter transcription than the other PTP inhibitor tested, i.e. sodium orthovanadate. Kinetic analyses were also performed to define the appropriate incubation time to reach optimal bpV[pic]- and PMA/Iono-mediated activation of ICAM-1 transcription. As shown in Fig. 2 C, bpV[pic] was found to be markedly more potent than PMA/Iono combination with respect to activation of ICAM-1 transcription. Moreover, maximal activation of ICAM-1-dependent luciferase activity was reached after 8 h following bpV[pic] treatment (22.6-fold increase), whereas the highest induction of ICAM-1 transcription was seen following 24 h of treatment with PMA/Iono (6.7-fold increase). These time points were thus used for the following series of investigations. By having demonstrated that bpV[pic] compound acts as a potent inducer of ICAM-1 transcription in human T cells, we next characterized the cis-regulatory element(s) located within the 5′-flanking sequences of the ICAM-1promoter that confers responsiveness to this tyrosine phosphatase-specific inhibitor. This goal was achieved using a series of ICAM-1 reporter constructs carrying either deletions or point mutations in the 5′ region of the promoter andtrans-dominant negative mutants of some specific transcription factors. Each of these molecular constructs was transiently transfected into Jurkat cells, and the luciferase activities of control, PMA/Iono, and bpV[pic]-treated cells were determined. We initially tested the involvement of the mammalian ubiquitous transcription factor NF-κB in bpV-induced activation ofICAM-1 promoter transcription. NF-κB is a pleiotropic transcription factor complex that mediates the regulated expression of multiple immunomodulatory genes bearing cis-acting κB enhancer elements, including the κ light chain of immunoglobulins, cytokines, as well as known genes for some cell adhesion molecules including ICAM-1 (44Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2003) Google Scholar). NF-κB has been postulated to play a key role in the cell type- and stimulus-specific regulation of ICAM-1 (7van de Stolpe A. van der Saag P.T. J. Mol. Med. 1996; 74: 13-33Crossref PubMed Scopus (648) Google Scholar). Considering that the proximal NF-κB-binding site located about 200 bp upstream of the translation initiation site has been demonstrated to be particularly important for the induction of ICAM-1 transcription (45Dhawan S. Singh S. Aggarwal B.B. Eur. J. Immunol. 1997; 27: 2172-2179Crossref PubMed Scopus (52) Google Scholar,46Müller S. Kammerbauer C. Simons U. Shibagaki N. Li L.-J. Caughman S.W. Degitz K. J. Invest. Dermatol. 1995; 104: 970-975Abstract Full Text PDF PubMed Scopus (38) Google Scholar), we used pGL1.3 and a luciferase-encoding vector constituted of the full-length ICAM-1 promoter bearing a point mutation in the most proximal NF-κB-binding site (i.e. pGL1.3 κBmut). Cells were then either left untreated or were treated with bpV[pic] for 8 h and PMA/Iono for 24 h. Again, high levels of ICAM-1 induction were observed with the reporter construct containing the complete ICAM-1 promoter (Fig.3 A). However, mutation of the proximal NF-κB-binding site resulted in a significant decrease in the induction ratio in response to both bpV[pic] (compare 20.3- and 4.4-fold induction) and PMA/Iono treatment (compare 8.9- and 5.1-fold inc
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