Intercellular Adhesion Molecule-1 (ICAM-1) gene expression in

Intercellular Adhesion Molecule-1 (ICAM-1) gene expression in human

T cells is regulated by phosphotyrosyl phosphatase activity:

involvement of NF- B, Ets, and pI RE-binding sites.

Jocelyn Roy, Marie Audette#, and Michel J. Tremblay*

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), Canada G1V 4G2

Running title: PTP activity regulates ICAM-1 gene expression.

Key words: ICAM-1 gene expression, protein tyrosine phosphatases, peroxovanadium

*Corresponding author. Mailing address:

Laboratoire d'Immuno-Rétrovirologie Humaine

Centre de Recherche en Infectiologie, RC709

Centre Hospitalier Universitaire de Québec, Pavillon CHUL

2705 boul. Laurier, Ste-Foy (Québec), Canada G1V 4G2

Phone: (418) 654-2705. Fax: (418) 654-2212

E-mail:[email protected]

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

JBC Papers in Press. Published on January 29, 2001 as Manuscript M005067200 by guest on February 20, 2018

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Abstract

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 (PTK) 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 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 upregulation 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.

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Introduction

Intercellular adhesion molecule-1 (ICAM-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 counterreceptor 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 (APCs) is made possible through an interaction between adhesion molecules such


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as ICAM-1 and LFA-1 expressed on the surface of both T lymphocytes and APCs. This

interaction leads to the association between the T-cell receptor (TCR)/CD3 complex and

antigenic peptides in the context of major histocompatibility complex (MHC) class I and II

molecules. If the latter interaction occurs, TCR/CD3 receptors and MHC 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 APCs. 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-γ responsive element (pIγRE), NF-κB, Ets,

C/EBP, AP-1-like, Sp1, and retinoic acid response elements (RARE) (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

pIγRE and are strongly involved in ICAM-1 gene expression (10-19). NF-κB has also been

reported to play a pivotal role in ICAM-1 gene regulation where RelA (p65)/RelA, RelA/c-Rel,


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and RelA/NF-κB1 (p50) dimers can potently induce ICAM-1 expression in several cell types (20-

25). Both JAK/STAT and NF-κB pathways have been shown to be modulated by

phosphorylation events which 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 (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 (PTK) and protein tyrosine

phosphatases (PTP). The role of PTK 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-γ-

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


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aggregation has 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 leukaemic 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 upregulation of

ICAM-1 expression. These results suggest that ICAM-1 is regulated in human T cells by PTP

activity.


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Experimental procedures

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 –85oC 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, Rockville, MD). 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,


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National Institute of Allergy and Infectious Diseases (Rockville, Md.) and were maintained in

complete culture medium in the presence of rhIL-2 (50 U/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 (PHA-P from Sigma, St. Louis, Mo.) (3 µg/ml) and recombinant human IL-2

(rhIL-2) (30 U/ml) for 3 days at 37oC. 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, Hoffman-La Roche

Inc.(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 x 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-PE-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%


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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

κBmut, pGL Hind III, and pGL HindIII IRE mut were provided by Dr. T.P. Parks (Boehringer

Ingelheim, Ridgefiel, Conn.), whereas pGLE WT, pGLE -138mut, pGLE -158mut, and pGLE

–138/–158mut were kindly supplied by Dr. Y. de Launoit (Institut Pasteur, Lille). Anti-STAT-1,

anti-STAT-3, and anti-p50 antibodies were purchased from Santa Cruz Biotechnology

(California). Dr N. Rice (NCI, Frederick, MD) kindly provided polyclonal anti-p65 antibodies.

Dr. R provided the anti-ICAM-1 antibody RR1/1.1.1 (anti-CD54). Rothlein (Boehringer

Ingelheim, Ridgefiel, Conn.) (41). The dominant negative IκBα expressing vector pCMV-IκBα

S32A/36A has been previously described (42) (a kind gift from Dr. W.C. Greene, The Gladstone

Institutes, San Francisco). The DNA filler pCMV-EcoRV/Sma1 has been constructed from the

expressing vector pCMV-IκBα S32A/36A by deletion of the cDNA encoding for IκBα

S32A/36A with EcoRV/Sma1 digestion.

Transient Transfection by DEAE-dextran. Jurkat cells (5 x 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,


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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 x 106 per ml using complete culture medium supplemented with 100 µM chloroquine (Sigma).

After 45 min of incubation at 37oC, cells were centrifuged, washed once, resuspended in

complete culture medium, and incubated at 37oC 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 time periods at 37oC 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 NP-40 were added.


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The lysate was vortexed for 10 s and samples were centrifuged for 30 s at 12,000 x 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 4oC for 15 min.

Cellular debris were removed by centrifugation at 12,000 x g for 5 min at 4oC and the supernatant

fraction was stored at -70oC 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 radiolabelled-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 was used as probes or

as competitors, contained either the non-specific probe Oct-2A (5'-


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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 oC.


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Results

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 leukaemic 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. 1A and 1D, 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, while 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 upregulation of ICAM-1

protein expression on Jurkat leukaemic 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] treatment as monitored by performing in

parallel MTS assays (data not shown). These data represent the first demonstration that PTP are


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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 TCR/CD4-expressing cell line established from the blood cells of a patient with T cell

lymphoma (Fig. 1B and 1C, 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.

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 to 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


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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-fold to 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 period 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) while 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.

Identification of bpV[pic]-responsive elements in the ICAM-1 promoter in human T cells.

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


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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 were 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 of ICAM-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 (44). NF-κB 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-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 (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-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. 3A).

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-fold and 4.4-fold induction) and


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PMA/Iono treatment (compare 8.9-fold and 5.1-fold increase). Therefore, it can be concluded that

the proximal NF-κB 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].

Previous observations suggest that NF-κB complexes can interact with other transcription

factors that are recognized to bind to the ICAM-1 promoter (47,48). Thus, to more fully address

the relative contribution of NF-κB in bpV-dependent activation of ICAM-1 transcription, Jurkat

cells were cotransfected with pGL1.3 and a construct encoding for a dominant negative version of

IκBα mutated to alanine on both serine 32 and 36 residues (i.e. pCMV-IκBα S32A/36A). The

IκBα repressor protein encoded by pCMV-IκBα S32A/36A is sequestered in the cytoplasm and

renders the NF-κB complex unable to translocate to the nucleus. The capacity of the used IκBα

repressor to abolish nuclear translocation and activation of NF-κB was initially tested by transient

transfection of Jurkat cells with pCMV-IκBα S32A/36A and pNF-κB-LUC, a vector made of

five consensus binding sites for NF-κB (data not shown). Transfection of the empty promoter

vector (i.e. pCMV-EcoRV/Sma1) 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-fold 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-


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fold and 1.0-fold induction). These data further confirmed the essential role played by NF-κB in

bpV[pic]-induced ICAM-1 promoter activity.

The ICAM-1 promoter contains also a more distal consensus NF-κB binding site. In an

attempt to assess the putative implication of both domains in the noticed upregulation of ICAM-1

transcription by bpV[pic] compound, the proximal and distal NF-κB-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-κB nuclear translocation was seen at this time period (data not shown), an

observation which 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-κB-binding site as a probe. Indeed, no such complex could be

induced by either PMA/Iono or bpV[pic] treatment when the distal NF-κB-binding site was used

as a probe. The complex was competed away by the addition of a 100 molar excess of cold NF-

κB oligonucleotide (lanes 5). However, complex formation was not affected by a non-specific

oligonucleotide (Oct-2A) (lanes 6), demonstrating the specificity of the signal. To identify the

NF-κB proteins involved in complex formation, antibodies against two of the most prominent

NF-κB isoforms (i.e. p50 and p65) were incubated with nuclear extracts from PMA/Iono- and


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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-κB-binding site of the ICAM-1 promoter is composed of both p50 and p65

subunits. These results clearly indicate that bpV compound is mediating nuclear translocation of

NF-κB and such finding is perfectly in line with our previous transcriptional studies (Figs 2 and

3).

Our previous results indicated that a point mutation in the proximal NF-κB-binding site or

the use of a dominant negative form of the IκΒα repressor did not completely eliminate the

ability of the full-length ICAM-1 promoter to respond to bpV[pic] (Figs 3A and 3B). Thus, it

suggests that NF-κB-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 pIγRE, Sp1- and AP-2-binding sites, and a

TATA box. Moreover, cells were also transfected with molecular constructs bearing point


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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

which 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 molar excess of cold Ets-

binding sites (lane 3) and not by a non-specific oligonucleotide (Oct-2A) (lane 4). No signal was

detected when using a probe containing mutations at the two Ets-binding sites (lanes 5 to 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.


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The proximity of pIγRE (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 pIγRE

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 pIγRE (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 to the 5' deletion mutant pGL1.3 HindIII (compare 25.3-fold 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 pIγRE 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 molar excess of an unlabeled pIγRE probe (lane 6) but

not by a non-specific oligonucleotide (Oct-2A) (lane 7).

It has been previously demonstrated that IFN-γ induces STAT-1, whereas IL-6 mediates

binding of both STAT-1 and STAT-3 transcriptional factors to the pIγRE element in the ICAM-1


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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 pIγRE.

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

pIγRE site and STAT-1 transcription factor.


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Discussion

ICAM-1 gene expression is regulated by numerous inducing (e.g. TNF-α, IFNγ, PMA, IL-

1) or inhibitory factors (e.g. IL-4, IL-10, 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-κB, 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 (PAO), 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-γ

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


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used in this study to break the equilibrium between PTP and PTK, 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 is 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 bpV[pic]’s

upregulatory effects on ICAM-1 transcriptional activity are far more 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 HIV-1 promter activity than Na3VO4 (49). These observations suggest that bpV[pic]’s

inhibitory effects 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-κB is

playing a central role in the induction of ICAM-1 (25,46-48) that is seen following treatment with

PMA, TNF-α, IL-1, and LPS (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


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are perfectly in line with these previous observations since we defined that NF-κB is a second

messenger actively participating in bpV[pic]-mediated expression of ICAM-1 in human T cells.

Although Imbert and colleagues have reported that the PTP inhibitor pervanadate can activate

NF-κB via tyrosine phosphorylation of IκBα with no concomitant proteolytic degradation of

IκBα (55), our experiments performed with a dominant negative form of IκBα leads us to

propose that bpV[pic]-dependent nuclear translocation and activation of NF-κB is most likely

associated with phosphorylation on both serine residues 32 and 36 of IκBα. This series of events

is known to be necessary to ensue dissociation of IκBα from NF-κB through ubiquitination of

IκBα 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-κB/Rel family

members. These findings are consistent with previous reports indicating that the ICAM-1 κB site

binds p50 and p65 (20,48). Although we provide conclusive evidence of the intimate interplay

between the phosphotyrosine level and NF-κB-dependent induction of ICAM-1 expression in T

cells, the various putative protein-protein interactions between NF-κB 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


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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 to a vector carrying

the wild type Ets-binding sites (pGLE WT). However, when both Ets-binding sites were mutated,

the bpV[pic]-induced upregulation 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

upregulate 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 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


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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-α is due to a functional cooperation between RARE and NF-κB 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-κB 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-γ responsive element (i.e. pIγRE) markedly reduced bpV[pic]-mediated

upregulation 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 phopshorylation 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 time of a high level of STAT-1 in an active phosphorylated

state.


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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.


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Acknowledgments

The authors thank 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

κBmut, pGL Hind III, pGL HindIII IRE mut, pGLE, pGLE -138mut, pGLE -158mut, and pGLE

–138/–158mut), Dr. W.C. Greene for pCMV-IκBα S32A/36A, Dr. R. Rothlein for RR1/1.1.1

antibody (anti-ICAM-1), and Dr. N. Rice for antisera against NF-κB proteins. This study was

supported by a grant to M.J.T. from the Canadian Institutes of Health Research (CIHR)

HIV/AIDS Research Program (Grant #HOP-15575). This work was performed by J.R. in partial

fulfillment of the M.Sc. degree at the Faculty of Graduate Studies, Department of Medical

Biology, Faculty of Medicine, Laval University. M.J.T. holds a CIHR Investigator (HIV/AIDS)

Award and M.A. is the recipient of a ‘Chercheur National’ Award from the Fonds de la

Recherche en Santé du Québec.


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Figure legends

Figure 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).

Figure 2. Dose-dependent and kinetic analyses of bpV[pic]-, PMA/Iono-, and sodium

orthovanadate (Na3VO4)-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) (panel A) or for 24 h with Na3VO4 (12.5,

25, and 50 µM) (panel 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 time periods (2, 4, 6, 8, 24,

and 48 h) prior to monitoring luciferase activity in cell lysates (panel C). Results shown are the

means +S.D. of four determinations. These results are representative of three independent

experiments.


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Figure 3. Involvement of NF- B in bpV[pic]-mediated induction of ICAM-1 transcription.

A) Jurkat cells were transiently transfected with pGL1.3 or pGL1.3 κBmut 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 cotransfected with pGL1.3 and either pCMV-EcoRV/Sma1

(empty vector control) or pCMV-IκBα 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.

Figure 4. bpV[pic] induces NF- B complexes that bind to the proximal NF- B-binding site

of the ICAM-1 promoter. Labeled proximal and distal NF-κB 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] (panel A; lanes 2 and 8) or PMA/Iono combination (panel

B; lanes 2 and 8). Binding specificity was tested by adding a 100-fold molar excess of either

cognate NF-κB oligonucleotide (lanes 5 and 11) or a non-specific 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.


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Figure 5. bpV[pic]-mediated upregulation 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.

Figure 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 non-specific probe (Oct-2A) (lanes 4 and 8). These

results are representative of three independent experiments.

Figure 7. pI RE is important in the ICAM-1 promoter to confer responsiveness to bpV[pic]

treatment. Jurkat 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), lysed, and luciferase activity was monitored with a microplate


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39

luminometer. Results shown are the means +S.D. of four determinations. These results are

representative of three independent experiments.

Figure 8. Treatment with bpV[pic] leads to nuclear translocation of STAT-1 that binds to

the pI RE element in the ICAM-1 promoter. Labeled pIγRE 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 constituted of either the

cognate pIγRE oligonucleotide (lanes 6 and 13) or a non-specific 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.


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Luciferase

activity(R

LU)

Hours after treatment

0

10

20

30

40

50

60

bpV

PMA/Iono

C

2 4 6 8 24 48

bpV[pic] (µM):

0

50

100

150

Luciferase

activity(R

LU)

1 2.5 5 10 20

A

0

2.5

3

3.5

4

4.5

5

Luciferase

activity(R

LU)

Na3VO4 (µM): 12.5 25 50

B

0


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Luciferase activity (RLU)A

pGL1.3

pGL1.3 kBmut

B

pGL1.3 +pCMV-EcoRV/Sma1

pGL1.3 +pCMV-IkBa S32A/36A

0 25

50

75

100 0 10

20

30

40

Untrea

ted8h

PMA/Io

no

Untrea

ted24h

bpV[pic]

Untrea

ted8h

PMA/Io

no

Untrea

ted24h

bpV[pic]

Luciferase activity (RLU)

by guest on February 20, 2018 http://www.jbc.org/ Downloaded from

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Free p

rob

e

NF

-κB

12

34

56

78

910

1112

Untreated

60 min

100 X spec.

100 X non-spec.

Anti-p50

Anti-p65

Untreated

60 min

100 X spec.

100 X non-spec.

Anti-p50

Anti-p65

NF

-κB p

roxN

F-κB

dist


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Free p

rob

e

NF

-κB

12

34

56

78

910

1112

Untreated

60 min

100 X spec.

100 X non-spec.

Anti-p50

Anti-p65

Untreated

60 min

100 X spec.

100 X non-spec.

Anti-p50

Anti-p65

NF

-κB p

roxN

F-κB

dist


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-176

-158

Ets

Ets

-1 38

-176

-176

Luciferase

-176

pIgRE

pGLE -138mut

pGLE -158mut

pGLE -138/-158mut

pGLE WT

0 10 20 30 40 50

PMA/Iono

Untreated 24 h

bpV[pic]

Untreated 8 h



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Ets

Ets

bp

V[p

ic]

PM

A/Io

no

12

34

56

78

Untreated

60 min

100 X spec.

100 X non-spec.

Untreated

60 min

100 X spec.

100 X non-spec.

-138/-158WT

-138/-158mut


http://www.jbc.org/

-2 7 7

- 2 7 7

pGL 1.3 Hind III IRE mut

pGL 1.3 Hind III

pGL 1.3-1 353

TATA

LuciferaseNF-kB

pIgRE


0 100755025

Untreated 8 h

PMA/Iono

bpV[pic]

Untreated 24 h


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Free p

rob

e

STA

T-1

12

34

56

78

910

1112

Untreated

60 min

120 min

Anti-STAT-1

Anti-STAT-3

100 X spec.

100 X non-spec.

Untreated

60 min

120 min

Anti-STAT-1

Anti-STAT-3

13 100 X spec.

14 100 X non-spec.

bp

V[p

ic]P

MA

/Ion

o


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Jocelyn Roy, Marie Audette and Michel J. TremblayRE-binding sites

γB, Ets, and pIκregulated by phosphotyrosyl phosphatase activity: involvement of NF-Intercellular adhesion molecule-1 (ICAM-1) gene expression in human T cells is

published online January 29, 2001J. Biol. Chem.

10.1074/jbc.M005067200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

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Intercellular Adhesion Molecule-1 (ICAM-1) gene expression in