Intercellular Adhesion Molecule-1 (ICAM-1) Gene Expression in Human T Cells Is Regulated by Phosphotyrosyl Phosphatase Activity

INVOLVEMENT OF NF-kappa B, Ets, AND PALINDROMIC INTERFERON-gamma -RESPONSIVE ELEMENT-BINDING SITES*

Jocelyn RoyDagger, Marie Audette§, and Michel J. Tremblay||

From the Centre de Recherche en Infectiologie, § Centre de Recherche en Oncologie et Endocrinologie Moléculaire, Centre Hospitalier Universitaire de Québec, Pavillon CHUL and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec G1V 4G2, Canada

Received for publication, June 12, 2000, and in revised form, November 26, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa B-, Ets-, and pIgamma RE-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 with trans-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-kappa B-, Ets-, and STAT-1-dependent signaling pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-5). 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 (6-8).

In a normal immune response, the initial contact between T lymphocytes and antigen presenting cells is made possible through an interaction between adhesion molecules such as ICAM-1 and LFA-1 expressed on the surface of both T lymphocytes and antigen presenting cells. This interaction leads to the association between the T cell receptor-CD3 complex and antigenic peptides in the context of major histocompatibility complex class I and II molecules. If the latter interaction occurs, T cell receptor-CD3 receptors and major histocompatibility complex molecules transmit activation signals in both cell partners. One of the first reactions following such activation is an increase of adhesion strength stabilizing the association between T cells and antigen presenting cells. 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 (7, 8).

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-gamma -responsive element (pIgamma RE), NF-kappa B, Ets, C/EBP, AP-1-like, Sp1, and retinoic acid response elements (7-9). These numerous enhancer elements contained in the ICAM-1 promoter suggest a complex regulation that is still ill-defined in human T cells. In various cell types, signal transducers and activators of transcription (STAT) factors, and more specifically STAT-1 and STAT-3, can bind the ICAM-1 promoter pIgamma RE and are strongly involved in ICAM-1 gene expression (10-19). NF-kappa B has also been reported to play a pivotal role in ICAM-1 gene regulation where RelA (p65)/RelA, RelA/c-Rel, and RelA/NF-kappa B1 (p50) dimers can potently induce ICAM-1 expression in several cell types (20-25). Both JAK/STAT and NF-kappa B 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-kappa B, the Ets gene family of transcriptional factors is also involved in the regulation of ICAM-1 expression (26, 27). 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 (28).

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 (29). Recently, some reports have described the role of PTP in T cell signaling and T cell transduction (30-35), 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-gamma -mediated induction of ICAM-1 expression via nuclear translocation of STAT-1 proteins in human keratinocytes (17). 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 (36). 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-kappa B, Ets, and pIgamma RE-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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- 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 (37). 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.

Cells and Culture Conditions-- 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 (38) 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 (40).

Flow Cytometry-- 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).

Plasmids and Antibodies-- 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 kappa Bmut, pGL HindIII, 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) (41). The dominant negative Ikappa Balpha -expressing vector pCMV-Ikappa Balpha S32A/36A has been described previously (42) (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-Ikappa Balpha S32A/36A by deletion of the cDNA encoding for Ikappa Balpha S32A/36A with EcoRV/SmaI digestion.

Transient Transfection by DEAE-Dextran-- 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 mM CaCl2) 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).

Preparation of Nuclear Extracts-- 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 (43). 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 mM phenylmethylsulfonyl 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 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl 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-- 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 mM EDTA, 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 [gamma -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-kappa B-binding site (5'-GATTGCTTTAGCTTGGAAATTCCGGAGCTG-3'), the distal NF-kappa B-binding site (5'-AGGGAGCCCGGGGAGGATTCCTGGGCC-3'), the pIgamma RE (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-kappa B, pIgamma RE, 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ICAM-1 Expression Is Increased in Human T Lymphoid Cells and Primary Cells by the PTP Inhibitor bpV[pic]-- 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.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   Cytometric analyses of PMA/Iono- and bpV[pic]-dependent modulation of ICAM-1 surface expression on Jurkat, HUT 78, WE17/10 cells, and PBMCs. Cells were incubated with an antibody specific for ICAM-1 (clone RR1/1.1.1) in combination with a (R)-phycoerythrin-conjugated goat anti-mouse IgG (dotted lines). Controls consisted of cells incubated with an isotype-matched irrelevant monoclonal antibody (solid lines).

Transcriptional Regulation of the ICAM-1 Gene by bpV[pic] Compound-- 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-1 promoter (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. 2A). 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. 2B, 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 of ICAM-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. 2C, 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.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Dose-dependent and kinetic analyses of bpV[pic]-, PMA/Iono-, and sodium orthovanadate-mediated effect on ICAM-1 gene expression in human T cells. Jurkat cells were transiently transfected with pGL1.3 and were next stimulated for 8 h with increasing doses of bpV[pic] (1, 2.5, 5, 10, and 20 µM) (A) or for 24 h with Na3VO4 (12.5, 25, and 50 µM) (B). Cells were then lysed, and luciferase activity was monitored with a microplate luminometer. Transiently transfected Jurkat cells were either treated with bpV[pic] (10 µM) or PMA/Iono (20 ng/ml and 1 µM, respectively) for different times (2, 4, 6, 8, 24, and 48 h) prior to monitoring luciferase activity in cell lysates (C). Results shown are the means ± S.D. of four determinations. These results are representative of three independent experiments.

Identification of bpV[pic]-responsive Elements in the ICAM-1 Promoter in Human T Cells-- 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-1 promoter 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 and trans-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-kappa B in bpV-induced activation of ICAM-1 promoter transcription. NF-kappa B is a pleiotropic transcription factor complex that mediates the regulated expression of multiple immunomodulatory genes bearing cis-acting kappa B enhancer elements, including the kappa  light chain of immunoglobulins, cytokines, as well as known genes for some cell adhesion molecules including ICAM-1 (44). NF-kappa B has been postulated to play a key role in the cell type- and stimulus-specific regulation of ICAM-1 (7). Considering that the proximal NF-kappa B-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 (45, 46), 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-kappa B-binding site (i.e. pGL1.3 kappa Bmut). 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. 3A). However, mutation of the proximal NF-kappa B-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 increase). Therefore, it can be concluded that the proximal NF-kappa B-binding site is a critical DNA-regulatory element responsible for ICAM-1 induction in T cells which is seen upon treatment with the PTP-specific inhibitor bpV[pic].


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Involvement of NF-kappa B in bpV[pic]-mediated induction of ICAM-1 transcription. A, Jurkat cells were transiently transfected with pGL1.3 or pGL1.3 kappa Bmut and were next either left untreated or were treated with PMA/Iono (20 ng/ml and 1 µM, respectively) or bpV[pic] (10 µM). B, Jurkat cells were transiently co-transfected with pGL1.3 and either pCMV-EcoRV/SmaI (empty vector control) or pCMV-Ikappa Balpha S32A/36A. Next, cells were left untreated or were treated with PMA/Iono (20 ng/ml and 1 µM, respectively) or bpV[pic] (10 µM). The cells were lysed, and luciferase activity was monitored with a microplate luminometer. Results shown are the means ± S.D. of four determinations. These results are representative of three independent experiments.

Previous observations suggest that NF-kappa B complexes can interact with other transcription factors that are recognized to bind to the ICAM-1 promoter (47, 48). Thus, to address more completely the relative contribution of NF-kappa B in bpV-dependent activation of ICAM-1 transcription, Jurkat cells were co-transfected with pGL1.3 and a construct encoding for a dominant negative version of Ikappa Balpha mutated to alanine on both serine 32 and 36 residues (i.e. pCMV-Ikappa Balpha S32A/36A). The Ikappa Balpha repressor protein encoded by pCMV-Ikappa Balpha S32A/36A is sequestered in the cytoplasm and renders the NF-kappa B complex unable to translocate to the nucleus. The capacity of the used Ikappa Balpha repressor to abolish nuclear translocation and activation of NF-kappa B was initially tested by transient transfection of Jurkat cells with pCMV-Ikappa Balpha S32A/36A and pNF-kappa B-LUC, a vector made of five consensus binding sites for NF-kappa B (data not shown). Transfection of the empty promoter vector (i.e. pCMV-EcoRV/SmaI) had no effect on bpV[pic]-mediated reporter gene activity (Fig. 3B). However, when the mutated version of the repressor was instead used, incubation of the cells with bpV[pic] compound showed a marked reduction in ICAM-1 promoter activity (compare 35.0- and 16.0-fold increase). A more dramatic diminution of ICAM-1-driven reporter gene activity was seen following treatment with PMA/Iono combination (compare 7.8- and 1.0-fold induction). These data further confirmed the essential role played by NF-kappa B in bpV[pic]-induced ICAM-1 promoter activity.

The ICAM-1 promoter contains also a more distal consensus NF-kappa B-binding site. In an attempt to assess the putative implication of both domains in the noticed up-regulation of ICAM-1 transcription by bpV[pic] compound, the proximal and distal NF-kappa B-binding sites of the ICAM-1 promoter were labeled and used as probes for DNA mobility shift assays. Nuclear extracts from untreated, PMA/Iono-, and bpV[pic]-treated Jurkat cells were used in these experiments. Nuclear proteins were extracted after 60 min of treatment with either PMA/Iono or bpV[pic] since maximal NF-kappa B nuclear translocation was seen at this time (data not shown), an observation that is consistent with a previous report (49). As illustrated in Fig. 4, a complex was formed following treatment with both the tyrosine phosphatase-specific inhibitor bpV[pic] (Fig. 4A, compare lanes 2 and 1) and PMA/Iono combination (Fig. 4B, compare lanes 2 and 1) only when using the proximal NF-kappa B-binding site as a probe. Indeed, no such complex could be induced by either PMA/Iono or bpV[pic] treatment when the distal NF-kappa B-binding site was used as a probe. The complex was competed away by the addition of a 100-fold molar excess of cold NF-kappa B oligonucleotide (lanes 5). However, complex formation was not affected by a nonspecific oligonucleotide (Oct-2A) (lanes 6), demonstrating the specificity of the signal. To identify the NF-kappa B proteins involved in complex formation, antibodies against two of the most prominent NF-kappa B isoforms (i.e. p50 and p65) were incubated with nuclear extracts from PMA/Iono- and bpV[pic]-treated Jurkat cells before the addition of labeled probes. Antibody against p50 significantly decreased the complex formation and a supershift (lanes 3), whereas anti-p65 antibody caused a partial supershift (lanes 4). It is thus clear that the protein complex bound to the proximal NF-kappa B-binding site of the ICAM-1 promoter is composed of both p50 and p65 subunits. These results clearly indicate that bpV[pic] compound is mediating nuclear translocation of NF-kappa B, and such a finding is perfectly in line with our previous transcriptional studies (Figs. 2 and 3).


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 4.   bpV[pic] induces NF-kappa B complexes that bind to the proximal NF-kappa B-binding site of the ICAM-1 promoter. Labeled proximal (prox) and distal (dist) NF-kappa B-binding sites of the ICAM-1 promoter were incubated with nuclear extracts from Jurkat cells either left untreated (lanes 1 and 7) or treated for 60 min with bpV[pic] (A, lanes 2-6 and 8-12) or PMA/Iono combination (B, lanes 2-6 and 8-12). Binding specificity was tested by adding a 100-fold molar excess of either cognate NF-kappa B oligonucleotide (lanes 5 and 11) or a nonspecific (non-spec) probe (Oct-2A) (lanes 6 and 12). For gel supershift assays, nuclear extracts were also incubated with antibody against p50 and p65 isoforms. These results are representative of three independent experiments.

Our previous results indicated that a point mutation in the proximal NF-kappa B-binding site or the use of a dominant negative form of the Ikappa Balpha repressor did not completely eliminate the ability of the full-length ICAM-1 promoter to respond to bpV[pic] (Fig. 3, A and B). Thus, it suggests that NF-kappa B-independent signal transduction pathway(s) might be involved as well. Previous studies have identified two functional Ets-binding sites in the ICAM-1 proximal promoter (26, 27). It should be emphasized that the role of these two transcription regulatory elements in human T cells with respect to the transcriptional regulation of the ICAM-1 gene remains to be defined. Cells were then transfected in a transient fashion with a luciferase-encoding vector driven by the first 176 nucleotides of the ICAM-1 promoter region (pGLE WT). This region of the ICAM-1 promoter harbors two Ets-binding sites (positions -138 and -158 relative to the start of transcription) in addition to the pIgamma RE, Sp1- and AP-2-binding sites, and a TATA box. Moreover, cells were also transfected with molecular constructs bearing point mutations either at each (pGLE -138mut and pGLE -158mut) or both (pGLE -138/-158mut) Ets-binding sites. Jurkat cells were next either left untreated or were treated with bpV[pic] for 8 h and PMA/Iono for 24 h. A comparable level of bpV[pic]- and PMA/Iono-mediated ICAM-1 induction was observed when using wild-type and mutated versions of the ICAM-1 promoter carrying single point mutation in either -138 or -158 Ets-binding site (Fig. 5). Interestingly, when both Ets-binding sites were mutated, the induction by bpV[pic] was severely reduced (31.2-fold versus 14.2-fold increase). It should be noted that PMA/Iono-mediated induction of ICAM-1 transcription is not markedly affected by such mutations. To determine whether these two sequence motifs can function as protein-binding sites, the responsive region composed of both -138 and -158 wild-type Ets-binding sites was labeled and used as a probe for mobility shift assays. First, we noticed that there was a constitutive nuclear translocation of Ets in Jurkat cells that was not modulated by a treatment with bpV[pic] or PMA/Iono (Fig. 6, compare lanes 1 and 2). The Ets complex was competed away by addition of a 100-fold molar excess of cold Ets-binding sites (lane 3) and not by a nonspecific oligonucleotide (Oct-2A) (lane 4). No signal was detected when using a probe containing mutations at the two Ets-binding sites (lanes 5-8). Altogether results from transient transfection experiments and mobility shift assays demonstrated the importance of Ets-binding sites in bpV[pic]-mediated ICAM-1 gene expression, although the formed migrating complex with the Ets probe is not modulated by bpV[pic] treatment.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   bpV[pic]-mediated up-regulation of ICAM-1 transcription necessitates Ets-binding sites. Jurkat cells were transiently transfected with pGLE WT, pGLE -138mut, pGLE -158mut, and pGLE -138/-158 mut before being either left untreated or treated with PMA/Iono (20 ng/ml and 1 µM, respectively) or bpV[pic] (10 µM). Next, cells were then lysed, and luciferase activity was monitored with a microplate luminometer. Results shown are the means ± S.D. of four determinations. These results are representative of three independent experiments.


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 6.   Constitutive expression of transcription factors that bind to the Ets-binding sites in the ICAM-1 promoter. Labeled Ets-binding sites of the ICAM-1 promoter were incubated with nuclear extracts from Jurkat cells either left untreated (lanes 1 and 5) or treated for 60 min with bpV[pic] (lanes 2 and 6) or PMA/Iono combination (lanes 2 and 6). Binding specificity was tested by adding a 100-fold molar excess of a probe constituted of either the cognate -138/-158 WT Ets oligonucleotide (lanes 3 and 7) or a nonspecific (non-spec) probe (Oct-2A) (lanes 4 and 8). These results are representative of three independent experiments.

The proximity of pIgamma RE (i.e. about 100 bp upstream of the translation initiation site) to the Ets-binding sites prompted us to investigate the functional importance of the ICAM-1 pIgamma RE palindromic STAT-binding site in the transcriptional regulation of the ICAM-1 gene by the potent tyrosine phosphatase inhibitor bpV[pic]. Cells were transiently transfected with the ICAM-1 WT reporter construct (pGL1.3), a 5' deletion mutant of pGL1.3 that contains 277 bp of the ICAM-1 promoter (pGL1.3 HindIII) or a vector carrying a site-specific mutation in pIgamma RE (pGL1.3 HindIII IRE mut). It is clear that the first 277-bp region of the ICAM-1 5'-flanking sequence is as efficient as the full-length ICAM-1 promoter to drive the expression of a reporter gene in response to bpV[pic] and PMA/Iono treatment (Fig. 7). A significant decrease in the induction ratio with bpV[pic] was seen with the site-specific mutant plasmid pGL1.3 HindIII IRE mut as compared with the 5' deletion mutant pGL1.3 HindIII (compare 25.3- and 5.4-fold induction). We next defined by EMSA whether treatment of Jurkat cells with bpV[pic] induced the specific binding of transcription factors to the ICAM-1 pIgamma RE site. Results from Fig. 8 indicate that bpV[pic] (compare lane 3 with lane 1) but not PMA/Iono (compare lanes 9 and 10 with 8) treatment leads to a DNA-protein complex of increased intensity. The complex formation was competed away by the addition of a 100-fold molar excess of an unlabeled pIgamma RE probe (lane 6) but not by a nonspecific oligonucleotide (Oct-2A) (lane 7).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 7.   pIgamma RE is important in the ICAM-1 promoter to confer responsiveness to bpV[pic] treatment. Jurkat cells were transiently transfected with pGL1.3, pGL 1.3 HindIII, or pGL HindIII IRE mut. Next, cells were either left untreated or were treated with PMA/Iono (20 ng/ml and 1 µM, respectively) or bpV[pic] (10 µM) and lysed, and luciferase activity was monitored with a microplate luminometer. Results shown are the means ± S.D. of four determinations. These results are representative of three independent experiments.


View larger version (99K):
[in this window]
[in a new window]
 
Fig. 8.   Treatment with bpV[pic] leads to nuclear translocation of STAT-1 that binds to the pIgamma RE element in the ICAM-1 promoter. Labeled pIgamma RE oligonucleotide was incubated with nuclear extracts from Jurkat cells either left untreated (lanes 1 and 8) or treated for 60 and 120 min with bpV[pic] (lanes 2 and 3) or PMA/Iono combination (lanes 9 and 10). Binding specificity was tested by adding a 100-fold molar excess of a probe composed of either the cognate pIgamma RE oligonucleotide (lanes 6 and 13) or a nonspecific (non-spec) probe (Oct-2A) (lanes 7 and 14). For gel supershift assays, nuclear extracts were also incubated with antibody specific either for STAT-1 (lanes 4 and 11) or STAT-3 (lanes 5 and 12). These results are representative of three independent experiments.

It has been previously demonstrated that IFN-gamma induces STAT-1, whereas IL-6 mediates binding of both STAT-1 and STAT-3 transcriptional factors to the pIgamma RE element in the ICAM-1 promoter (17, 50). Thus, we defined whether the bpV[pic]-induced complex is constituted of either STAT-1, STAT-3, or both by performing supershift analyses. To this end, nuclear proteins were extracted from untreated and bpV[pic]-treated Jurkat cells (60 and 120 min) before incubation with anti-STAT-1 or anti-STAT-3 antibody and the radiolabeled ICAM-1 pIgamma RE. Antibody against STAT-1 diminished the binding and caused a partial supershift of the bpV[pic]-induced complex (compare lane 4 and 3), whereas anti-STAT-3 antibody did not affect the complex formation mediated by bpV[pic] treatment (compare lanes 5 and 3). These results suggest that bpV[pic] results in the formation of a DNA-protein complex constituted of ICAM-1 pIgamma RE site and STAT-1 transcription factor.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ICAM-1 gene expression is regulated by numerous inducing (e.g. TNF-alpha , IFN-gamma , PMA, and IL-1) or inhibitory factors (e.g. IL-4, IL-10, and glucocorticoids). The architecture of the ICAM-1 promoter is complex and is thus regulated by an interplay between different transcription factors such as STAT, NF-kappa B, Ets, C/EBP, and Sp1 as demonstrated in various cell types including acute myeloid leukemia blast cells, B lymphocytes, endothelial cells, and epithelial cells (7, 8). Previous studies reported that protein phosphorylation could be involved in ICAM-1 gene expression. In human monocytic U937 and human lymphocytic Jurkat cell lines, okadaic acid and calyculin A (Ser/Thr phosphatases inhibitors) promote ICAM-1 and LFA-1-mediated homotypic aggregation (36). In addition, phenylarsine oxide, pervanadate, and diamide (PTP inhibitors) have been shown to block the TNF-induced endothelial cell surface adhesion molecules (ICAM-1, VCAM-1, and ECAM-1) (45). Another study reported that pervanadate mimics the IFN-gamma induction of ICAM-1 gene expression (17). Although central to many different pathways in T cells, tyrosine phosphorylation events per se have not been directly investigated in the regulation of ICAM-1 gene expression. In this study, we have thus analyzed the role of tyrosine phosphorylation events in transcriptional regulation of the ICAM-1 promoter in human T cells. The bpV[pic] compound, previously characterized as one of the most potent PTP inhibitors, was used in this study to break the equilibrium between PTP and protein-tyrosine kinases and therefore to increase intracellular phosphotyrosine levels.

Our results first demonstrated that bpV[pic] was effective in inducing ICAM-1 surface expression. Flow cytometry analyses showed that bpV[pic] compound was able to increase both the number of ICAM-1-expressing cells and the mean fluorescence intensity. The physiological significance of the current work was provided by the observation that bpV[pic] treatment leads to the induction of ICAM-1 expression not only in established T cell lines (i.e. Jurkat, HUT 78, and WE17/10) but also in primary human PBMCs. Further experiments revealed that the up-regulatory effects of bpV[pic] on ICAM-1 transcriptional activity are far superior than those of sodium orthovanadate (Na3VO4), another described powerful PTP inhibitor. Interestingly, such results are consistent with a previous report showing that bpV molecules are more potent inducers of human immunodeficiency virus promoter activity than Na3VO4 (49). These observations suggest that the inhibitory effects of bpV[pic] on intracellular PTP may be distinct from those of sodium orthovanadate, in terms of potency and/or substrate specificity. Further studies are needed to shed light on this matter.

Data from several reports have clearly indicated that the transcription factor NF-kappa B is playing a central role in the induction of ICAM-1 (25, 46-48) that is seen following treatment with PMA, TNF-alpha , IL-1, and lipopolysaccharide (20, 24, 51). This family of transcriptional factors has the ability to interact with other transcriptional factors such as Fos/Jun, C/EBP, and Sp1 (52-54). Our results are perfectly in line with these previous observations since we defined that NF-kappa B is a second messenger actively participating in bpV[pic]-mediated expression of ICAM-1 in human T cells. Although Imbert and colleagues (55) have reported that the PTP inhibitor pervanadate can activate NF-kappa B via tyrosine phosphorylation of Ikappa Balpha with no concomitant proteolytic degradation of Ikappa Balpha , our experiments performed with a dominant negative form of Ikappa Balpha lead us to propose that bpV[pic]-dependent nuclear translocation and activation of NF-kappa B is most likely associated with phosphorylation on both serine residues 32 and 36 of Ikappa Balpha . This series of events is known to be necessary to ensure dissociation of Ikappa Balpha from NF-kappa B through ubiquitination of Ikappa Balpha and its final degradation by the proteasome. Our data indicate also that bpV[pic]-mediated activation of ICAM-1 gene expression is dependent on both p50 and p65 NF-kappa B/Rel family members. These findings are consistent with previous reports indicating that the ICAM-1 kappa B site binds p50 and p65 (20, 48). Although we provide conclusive evidence of the intimate interplay between the phosphotyrosine level and NF-kappa B-dependent induction of ICAM-1 expression in T cells, the various putative protein-protein interactions between NF-kappa B complexes and other transcription factors binding to the ICAM-1 promoter (e.g. AP-1 and C/EBP) remain to be explored.

The nuclear phosphoproteins Ets have been reported to be involved in activation of the ICAM-1 promoter in various cell types including rabbit kidney carcinoma, human choriocarcinoma, and endothelial cell lines (26, 27). To evaluate the implication of Ets transcriptional factors in bpV-mediated activation of ICAM-1, we transiently transfected Jurkat cells either with pGLE, pGLE -138mut, pGLE -158mut, or pGLE -138/-158mut. Our results demonstrated that no significant change occurred following a point mutation in either the -138 (pGLE -138mut) or -158 (pGLE -158mut) Ets-binding sites when compared with a vector carrying the wild-type Ets-binding sites (pGLE WT). However, when both Ets-binding sites were mutated, the bpV[pic]-induced up-regulation of ICAM-1 promoter-driven luciferase activity was significantly decreased. Our results are contrasting with previous data indicating that a point mutation in the -158 Ets-binding site strongly diminished the ICAM-1 promoter activity (27). In this report, it was demonstrated that expression vectors encoding for Ets-2 and ERM significantly up-regulate ICAM-1 transcription in rabbit kidney carcinoma RK13 cells, and the ERM-mediated activation of ICAM-1 transcription was strongly diminished when using a mutated -158 Ets-binding site. In addition, a mutated version of the -138 Ets-binding site did not change the ERM-mediated induction of ICAM-1 gene expression. There are at least two possibilities that could explain such a discrepancy. First, experiments conducted by de Launoit and co-workers (27) were performed using established cell lines different from the one used in the current study. Second, in contrast to their work that is based on overexpression of Ets-2 and ERM, we could not detect any positive modulation of Ets nuclear translocation upon bpV[pic] treatment. The observation that both Ets-binding sites in the ICAM-1 promoter are important for bpV[pic]-mediated induction of ICAM-1 transcription despite an absence of Ets activation is suggestive of a functional interaction between binding sites for Ets and another transcription factor. This possibility is supported by a previous study demonstrating that the H2O2-responsive element in the ICAM-1 promoter is constituted of the Ets- and AP-1-binding sites (26). Similarly, a synergic activation of ICAM-1 by retinoic acid and TNF-alpha is due to a functional cooperation between retinoic acid response elements and NF-kappa B-binding sites (56). Further studies are warranted to identify the binding site(s) in the ICAM-1 promoter that functionally cooperate with Ets-binding sites to form the bpV[pic]-responsive elements.

In addition to binding sites for NF-kappa B and Ets transcription factors, our results indicate that the JAK/STAT signaling pathway is also implicated in the induction of ICAM-1 expression in human T cells that is seen upon treatment with the potent PTP inhibitor bpV compound. Indeed, a point mutation in the IFN-gamma -responsive element (i.e. pIgamma RE) markedly reduced bpV[pic]-mediated up-regulation of ICAM-1 promoter activity. We also demonstrated that bpV[pic] treatment was resulting in nuclear translocation of STAT-1. Our results are consistent with a previous report demonstrating that the PTP inhibitor pervanadate activates the protein-tyrosine kinases JAKs which will in turn induce tyrosine phosphorylation of STAT-1, STAT-3, and STAT-6 (57). Of interest for the present study, STAT-1 has been recently shown to be inactivated by a nuclear PTP (58). Therefore, it can be proposed that the PTP inhibitor bpV[pic] could result in phosphorylation of STAT-1 in the cytoplasm, translocation to the nucleus, and then the maintenance for a longer period of a high level of STAT-1 in an active phosphorylated state.

In conclusion, our findings indicate that ICAM-1 gene expression in human T cells is under the control of constitutive PTP activity, which serves to maintain ICAM-1 expression at a basal level. A more detailed understanding of the transcription factors involved in bpV[pic]-mediated activation of ICAM-1 expression will be needed to determine how the various cooperative protein-protein interactions regulate the transcriptional activation of the ICAM-1 gene in human T cells.

    ACKNOWLEDGEMENTS

We thank Dr. Benoit Barbeau for critical reading of the manuscript; Dr. M. Dufour for technical assistance in flow cytometry studies; Drs. T. P. Parks and Y. de Launoit for the reporter vectors of the ICAM-1 promoter (i.e. pGL1.3, pGL1.3 kappa Bmut, pGL HindIII, pGL HindIII IRE mut, pGLE, pGLE -138mut, pGLE -158mut, and pGLE -138/-158mut); Dr. W. C. Greene for pCMV-Ikappa Balpha S32A/S36A; Dr. R. Rothlein for RR1/1.1.1 antibody (anti-ICAM-1); and Dr. N. Rice for antisera against NF-kappa B proteins.

    FOOTNOTES

* This work was supported in part by Grant HOP-15575 from the Canadian Institutes of Health Research HIV/AIDS Research Program (to M. J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Submitted in partial fulfillment for the M.Sc. degree from the Microbiology-Immunology Program, Faculty of Medicine, Laval University.

Recipient of a Chercheur National award from the Fonds de la Recherche en Santé du Québec.

|| Holds a Canada Research Chair in Human Immuno-Retrovirology and recipient of Canadian Institutes of Health Research Investigator award. To whom correspondence should be addressed: Laboratoire d'Immuno-Rétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Blvd. Laurier, Ste-Foy, Québec G1V 4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2212; E-mail: Michel.J.Tremblay@crchul.ulaval.ca.

Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M005067200

    ABBREVIATIONS

The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; PTP, protein tyrosine phosphatases; bpV, bis-peroxovanadium; bpV[pic], bis-peroxovanadium compound carrying the picolinic acid as an auxillary ligand; pIgamma RE, palindromic interferon-gamma -responsive element; STAT, signal transducers and activators of transcription; PMA, phorbol 12-myristate 13-acetate; Iono, ionomycin; FBS, fetal bovine serum; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; WT, wild type; dsDNA, double-stranded DNA; mut, mutant; IL, interleukin; IFN, interferon; TNF-alpha , tumor necrosis factor-alpha ; bp, base pair.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Marlin, S. D., and Springer, T. A. (1987) Cell 51, 813-819[Medline] [Order article via Infotrieve]
2. Staunton, D. E., Merluzzi, V. J., Rothlein, R., Barton, R., Marlin, S. D., and Springer, T. A. (1989) Cell 56, 849-853[Medline] [Order article via Infotrieve]
3. Greve, J. M., Davis, G., Meyer, A. M., Forte, C. P., Yost, S. C., Marlor, C. W., Kamarck, M. E., and McClelland, A. (1989) Cell 56, 839-847[Medline] [Order article via Infotrieve]
4. Berendt, A. R., Simmons, D. L., Tansey, J., Newbold, C. I., and Marsh, K. (1989) Nature 341, 57-59[CrossRef][Medline] [Order article via Infotrieve]
5. Diamond, M. S., Staunton, D. E., de Fougerolles, A. R., Stacker, S. A., Garcia-Aguilar, J., Hibbs, M. L., and Springer, T. A. (1990) J. Cell Biol. 111, 3129-3139[Abstract]
6. Noraz, N., Verrier, B., Fraisier, C., and Desgranges, C. (1995) AIDS Res. Hum. Retroviruses 11, 145-154[Medline] [Order article via Infotrieve]
7. van de Stolpe, A., and van der Saag, P. T. (1996) J. Mol. Med. 74, 13-33[Medline] [Order article via Infotrieve]
8. Roebuck, K. A., and Finnegan, A. (1999) J. Leukocyte Biol. 66, 876-888[Abstract]
9. Stratowa, C., and Audette, M. (1995) Immunobiology 193, 293-304[Medline] [Order article via Infotrieve]
10. Coccia, E. M., Del Russo, N. D., Stellacci, E., Testa, U., Marziali, G., and Battistini, A. (1999) Int. Immunol. 11, 1075-1083[Abstract/Free Full Text]
11. Song, S., Ling-Hu, H., Roebuck, K. A., Rabbi, M. F., Donnelly, R. P., and Finnegan, A. (1997) Blood 89, 4461-4469[Abstract/Free Full Text]
12. Sampath, D., Castro, M., Look, D. C., and Holtzmann, M. J. (1999) J. Clin. Invest. 103, 1353-1361[Abstract/Free Full Text]
13. Li, W., Nagineni, C. N., Hooks, J. J., Chepelinsky, A. B., and Egwuagu, C. E. (1999) Invest. Ophthalmol. Vis. Sci. 40, 976-982[Abstract]
14. Lee, S. J., Park, J. Y., Hou, J., and Benveniste, E. N. (1999) Glia 25, 21-32[CrossRef][Medline] [Order article via Infotrieve]
15. Cantwell, C. A., Sterneck, E., and Johnson, P. F. (1998) Mol. Cell. Biol. 18, 2108-2117[Abstract/Free Full Text]
16. Wu, A. J., Chen, Z. J., Kan, E. C., and Baum, B. J. (1997) J. Cell. Physiol. 173, 110-114[CrossRef][Medline] [Order article via Infotrieve]
17. Duff, J. L., Quinlan, K. L., Paxton, L. L., Naik, S. M., and Caughman, S. W. (1997) J. Invest. Dermatol. 108, 295-301[Abstract]
18. Naik, S. M., Shibagaki, N., Li, L. J., Quinlan, K. L., Paxton, L. L., and Caughman, S. W. (1997) J. Biol. Chem. 272, 1283-1290[Abstract/Free Full Text]
19. van der Bruggen, T., Caldenhoven, E., Kanters, D., Coffer, P., Raaijmakers, J. A., Lammers, J. W., and Koenderman, L. (1995) Blood 85, 1442-1448[Abstract/Free Full Text]
20. Ledebur, H. C., and Parks, T. P. (1995) J. Biol. Chem. 270, 933-943[Abstract/Free Full Text]
21. Parry, G. C., and Mackman, N. (1994) J. Biol. Chem. 269, 20823-20825[Abstract/Free Full Text]
22. Jahnke, A., and Johnson, J. P. (1994) FEBS Lett. 354, 220-226[CrossRef][Medline] [Order article via Infotrieve]
23. Hou, J., Baichwal, V., and Cao, Z. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11641-11645[Abstract/Free Full Text]
24. van de Stolpe, A., Caldenhoven, E., Stade, B. G., Koenderman, L., Raaijmakers, J. A., Johnson, J. P., and van der Saag, P. T. (1994) J. Biol. Chem. 269, 6185-6192[Abstract/Free Full Text]
25. Aoudjit, F., Brochu, N., Bélanger, B., Stratowa, C., Hiscott, J., and Audette, M. (1997) Cell Growth Differ. 8, 335-342[Abstract]
26. Roebuck, K. A., Rahman, A., Lakshminarayanan, V., Janakidevi, K., and Malik, A. B. (1995) J. Biol. Chem. 270, 18966-18974[Abstract/Free Full Text]
27. de Launoit, Y., Audette, M., Pelczar, H., Plaza, S., and Baert, J.-L. (1998) Oncogene 16, 2065-2073[CrossRef][Medline] [Order article via Infotrieve]
28. Wasylyk, B., Hagman, J., and Gutierrez-Hartmann, A. (1998) Trends Biochem. Sci. 23, 213-216[CrossRef][Medline] [Order article via Infotrieve]
29. Germain, R. N., and Stefanova, I. (1999) Annu. Rev. Immunol. 17, 467-522[CrossRef][Medline] [Order article via Infotrieve]
30. Chan, A. C., Desai, D. M., and Weiss, A. (1994) Annu. Rev. Immunol. 12, 555-592[CrossRef][Medline] [Order article via Infotrieve]
31. Olivero, S., Bléry, M., and Vivier, É. (1998) Méd./Sci. 14, 262-268
32. Streuli, M. (1996) Curr. Opin. Cell Biol. 8, 182-188[CrossRef][Medline] [Order article via Infotrieve]
33. Neel, G. B., and Tonks, K. N. (1997) Curr. Opin. Cell Biol. 9, 193-204[CrossRef][Medline] [Order article via Infotrieve]
34. Neel, B. G. (1997) Curr. Opin. Immunol. 9, 405-420[CrossRef][Medline] [Order article via Infotrieve]
35. Weiss, A., and Littman, D. R. (1994) Cell 76, 263-274[Medline] [Order article via Infotrieve]
36. Weeks, B. S., and Iuorno, J. (1996) Biochem. Biophys. Res. Commun. 226, 82-87[CrossRef][Medline] [Order article via Infotrieve]
37. Posner, 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., and Shaver, A. (1994) J. Biol. Chem. 269, 4596-4604[Abstract/Free Full Text]
38. Willard-Gallo, K. E., van de Keere, F., and Kettmann, R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6713-6717[Abstract]
39. Gazdar, A. F., Carney, D. N., Bunn, P. A., Russell, E. K., Jaffe, E. S., Schechter, G. P., and Guccion, J. G. (1980) Blood 55, 409-417[Medline] [Order article via Infotrieve]
40. Lahm, H. W., and Stein, S. (1985) J. Chromatogr. 326, 357-361[CrossRef][Medline] [Order article via Infotrieve]
41. Rothlein, R., Dustin, M. L., Marlin, S. D., and Springer, T. A. (1986) J. Immunol. 137, 1270-1274[Abstract/Free Full Text]
42. Sun, S.-C., Elwood, J., and Greene, W. C. (1996) Mol. Cell. Biol. 16, 1058-1065[Abstract]
43. Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499-2500[Medline] [Order article via Infotrieve]
44. Siebenlist, U., Franzoso, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, 405-455[CrossRef]
45. Dhawan, S., Singh, S., and Aggarwal, B. B. (1997) Eur. J. Immunol. 27, 2172-2179[Medline] [Order article via Infotrieve]
46. Müller, S., Kammerbauer, C., Simons, U., Shibagaki, N., Li, L.-J., Caughman, S. W., and Degitz, K. (1995) J. Invest. Dermatol. 104, 970-975[Abstract]
47. Collins, T., Read, M. A., Neish, A. S., Whitley, M. Z., Thanos, D., and Maniatis, T. (1995) FASEB J. 9, 899-909[Abstract/Free Full Text]
48. Wissink, S., van de Stolpe, A., Caldenhoven, E., Koenderman, L., and van der Saag, P. (1997) Immunobiology 198, 50-64[Medline] [Order article via Infotrieve]
49. Barbeau, B., Bernier, R., Dumais, N., Briand, G., Olivier, M., Faure, R., Posner, B. I., and Tremblay, M. (1997) J. Biol. Chem. 272, 12968-12977[Abstract/Free Full Text]
50. Caldenhoven, E., Coffer, P., Yuan, J., van de Stolpe, A., Horn, F., Kruijer, W., and van der Saag, P. T. (1994) J. Biol. Chem. 269, 21146-21154[Abstract/Free Full Text]
51. Eck, S. L., Perkins, N. D., Carr, D. P., and Nabel, G. J. (1993) Mol. Cell. Biol. 13, 6530-6536[Abstract]
52. Perkins, N. D., Edwards, N. L., Duckett, C. S., Agranoff, A. B., Schmid, R. M., and Nabel, G. J. (1993) EMBO J. 12, 3551-3558[Abstract]
53. Stein, B., Cogswell, P. C., and Baldwin, A. S. J. (1993) Mol. Cell. Biol. 13, 3964-3974[Abstract]
54. Stein, B., Baldwin, A. S. J., Ballard, D. W., Green, W. C., Angel, P., and Herlich, P. (1993) EMBO J. 12, 3879-3891[Abstract]
55. Imbert, V., Rupec, R. A., Livolsi, A., Pahl, H. L., Traenckner, E. B., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeurle, P. A., and Peyron, J. F. (1996) Cell 86, 787-798[Medline] [Order article via Infotrieve]
56. Chadwick, C. C., Shaw, L. J., and Winneker, R. C. (1998) Exp. Cell Res. 239, 423-429[CrossRef][Medline] [Order article via Infotrieve]
57. Haque, S. J., Wu, Q., Kammer, W., Friedrich, K., Smith, J. M., Kerr, I. M., Stark, G. R., and Williams, B. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8563-8568[Abstract/Free Full Text]
58. Haspel, R. L., and Darnell, J. E., Jr. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10188-10193[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.