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J. Biol. Chem., Vol. 277, Issue 9, 7118-7126, March 1, 2002

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Interferon--induced Epithelial ICAM-1 Expression and Monocyte Adhesion

INVOLVEMENT OF PROTEIN KINASE C-DEPENDENT c-Src TYROSINE KINASE ACTIVATION PATHWAY^*

Ya-Jen Chang, Michael J. Holtzman, and Ching-Chow Chen^§

From the Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan and the  Department of Medicine and Cell Biology, Washington University, School of Medicine, St. Louis, Missouri 63110

Received for publication, October 13, 2001, and in revised form, December 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interferon- (IFN-) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN--induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN--induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN- and TPA in cells transfected with pIC339-Luc, containing the downstream NF-B and -activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (AP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN--stimulated cells contained STAT1. The IFN--induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-2 mutant inhibited IFN-- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKC or c-Src inhibited both IFN-- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-2, PKC, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN- and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN- activates PLC-2 via an upstream tyrosine kinase to induce activation of PKC- and c-Src or Lyn, resulting in activation of STAT1, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell adhesion mediated by specific cell-surface molecules is important in establishing and maintaining inflammation, bronchial asthma, rheumatoid arthritis, atopic dermatitis, tumor metastasis, and allograft rejection (1-3). It elicits the recruitment of leukocytes from the circulation into the extravascular space, a process involving several steps (4, 5). The initial interaction between leukocytes and the endothelium appears to be transient, resulting in the leukocytes rolling along the vessel wall. These rolling leukocytes then become activated by local factors generated by the endothelium, resulting in their arrest and firm adhesion to the vessel wall. Finally, the leukocytes migrate across the endothelium. These complex processes are regulated, in part, by specific endothelial-leukocyte adhesion molecules. The intercellular adhesion molecule-1 (ICAM-1¹; CD54), an 80-114-kDa inducible surface glycoprotein belonging to the immunoglobulin superfamily, is involved in a wide range of inflammatory and immune responses (6). During inflammation, ICAM-1 binds to two integrins belonging to the ₂ subfamily, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), both expressed by leukocytes and that promote the adhesion and transendothelial migration of leukocytes (7, 8). Similar processes govern leukocyte adhesion to lung airway epithelial cells and may contribute to the damage to these cells seen in asthma (9). ICAM-1 can be up-regulated by bacterial lipopolysaccharide, phorbol esters, platelet-derived growth factor, and inflammatory cytokines, such as tumor necrosis factor  (TNF-), interleukin-1 (IL-1), and interferon- (IFN-) (10-13). This regulation occurs at the transcriptional level and involves the binding of specific homo- or heterodimeric complexes to target DNA sequences located along the ICAM-1 promoter (14-16). The ICAM-1 promoter has been identified and shown to contain two TATA boxes, two NF-B sites, two AP-1 sites, two AP-2 sites, two glucocorticoid receptor element sites, and one IFN--activated (GAS) site (17-19).

IFN-, a lymphocyte effector molecule produced by T cells and natural killer cells, plays an important role in macrophage activation and is implicated in the pathogenesis of a number of inflammatory diseases of infectious or presumed autoimmune origin (20). The intracellular signaling of IFN- has been shown to act through the JAK/STAT pathway in several different tissues (21, 22), and the mechanism of IFN--mediated gene induction has been elucidated (21-23). Following IFN- binding, the IFN- receptor oligomerizes and brings the Janus kinases (JAKs) into juxtaposition, leading to their cross-phosphorylation and activation. The JAKs in turn phosphorylate tyrosine residues on receptors that lack intrinsic kinase activity, thereby providing the docking site for downstream signaling proteins. The signal transducers and activators of transcription (STATs), which are recruited to the JAK-receptor complex via their Src homology 2 (SH2) domain, are phosphorylated on a conserved tyrosine residue in the C-terminal region. This phosphorylation results in STAT dimerization and forms a protein complex first identified as -activated factor (GAF). The GAF complex then translocates to the nucleus, where it binds to the specific promoter DNA sequence, GAS, thereby affecting the expression of multiple target genes, such as ICAM-1 (24, 25). In addition to the JAK-STAT pathway, other signaling components are involved; these include phospholipase D (PLD)-dependent arachidonic acid release to activate protein kinase C (PKC) in endothelial cells (26, 27), PC-PLC and PKC activation to induce inducible nitric-oxide synthase expression in J774 macrophages (28), or PKC activation to induce ICAM-1 in endothelial cells (29). The intracellular signaling pathways by which IFN- causes ICAM-1 expression are not well understood but have been suggested to be involved; these include tyrosine kinase activation (e.g. JAKs and their downstream transcriptional factors, the STATs) (25), PKC, and intracellular Ca²⁺ concentration (29, 32). However, the relationship between these pathways is unknown. In the present study, we explored the intracellular signaling pathway involved in IFN--induced ICAM-1 expression in a human alveolar epithelial cell line, NCI-H292. The results show that IFN- activates phosphatidylinositol-phospholipase C-2 (PI-PLC-2), resulting in the activation of PKC, c-Src or Lyn, STAT1, and GAS in the ICAM-1 promoter, followed by ICAM-1 expression and monocyte adhesion.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Mouse monoclonal anti-human ICAM-1 antibody (11C81) and recombinant human IFN- were purchased from R & D Systems (Minneapolis, MN). Rabbit polyclonal antibodies specific for p65, p91 (STAT-1), c-Src, Lck, Lyn, or Fyn were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). RPMI, fetal calf serum (FCS), penicillin, and streptomycin were obtained from Invitrogen. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was purchased from LC Services (Woburn, MA). Staurosporine, pyrrolidinedithiocarbamate (PDTC), O-phenylenediamine dihydrochloride, and rabbit muscle enolase were obtained from Sigma. D609, U73122, U73343, propranolol, Ro 31-8220, PP2, genistein, and herbimycin were obtained from Calbiochem. 2',7'-Bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was obtained from Molecule Probes; reagents for SDS-PAGE were from Bio-Rad; T4 polynucleotide kinase was from New England Biolabs (Beverly, MA); poly(dI-dC) was from Amersham Biosciences; [-³²P]ATP (3,000 Ci/mmol) was from PerkinElmer Life Sciences; fluorescein isothiocyanate-conjugated goat anti-mouse IgG was from Cappel (Aurora, OH), and Tfx^TM-50 and the luciferase assay kit were from Promega (Madison, WI).

Plasmids-- The four ICAM-1 promoter constructs, pIC1352, pIC339, pIC135, and pIC135(AP2) (pIC135 with the 105/38 region deleted), were the generous gifts from Dr. P. T. Van der Saag (Hubrecht Laboratory, Utrecht, Netherlands). The PLC-2 wild type and mutant, SH₂(N), in which Arg-564 is replaced by Ala, and the PKC wild type and dominant negative mutant (K/R) were gifts from Drs. T. Kurosaki (Kansai Medical University, Japan) and A. Altman (La Jolla Institute for Allergy and Immunology, San Diego, CA), respectively. The JAK1 wild type and dominant negative JAK1 and JAK2 mutants were gifts from Dr. Rothman (Department of Microbiology, College of Physicians and Surgeons of Columbia University) and Dr. Levy (Department of Pathology, New York University, New York), respectively. The STAT3(DN) was gift from Dr. Nakajima (Department of Molecular Oncology, Osaka university, Japan).

Cell Culture-- The human alveolar epithelial cell carcinoma cell line, NCI-H292, was obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 supplemented with 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin in 6-well plates for ICAM-1 promoter transfection, on 24-mm glass coverslips in 35-mm dishes for ICAM-1 immunofluorescence studies, in 6-cm dishes for kinase activity measurements, or in 10-cm dishes for the gel-shift assay.

The human monocytic leukemia cell line, U937, was obtained from the Department of Microbiology, College of Medicine, National Taiwan University, Taiwan, and cultured in RPMI 1640 medium supplemented with 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were split and fed every 3-4 days.

Quantification of ICAM-1 Expression-- The level of cell-surface ICAM-1 expression was determined using an enzyme-linked immunosorbent assay (ELISA) as described previously (33, 34). Each assay was performed in triplicate, and the basal absorbance (about 0.2 units) was subtracted. In pretreatment experiments, cells were incubated with the tyrosine kinase inhibitors, genistein and herbimycin, the PC-PLC inhibitor, D609, the PI-PLC inhibitor, U73122, the phosphatidate phosphohydrolase inhibitor, propranolol, or the PKC inhibitors, staurosporine and Ro 31-8220, and Src inhibitor, PP2 for 30 min before addition of IFN- or TPA. None of these inhibitors affected the basal ICAM-1 expression.

Immunofluorescence Staining-- NCI-H292 cells, grown on coverslips, were treated for 18 h with IFN- or TPA in growth medium. Immunofluorescence staining was performed as described previously (33).

Cell Adhesion Assay-- NCI cells, grown in 96-well plates, were treated for 18 h at 37 °C with IFN- or TPA and then washed twice with PBS. U937 cells were labeled for 30 min at 37 °C with 10 ng/ml BCECF and washed twice with growth medium, and then 2.5 × 10⁵ (100 µl) of the labeled cells were added to the NCI monolayer and the cultures incubated in a CO₂ incubator for 1 h. Non-adherent cells were removed from the plate by gentle washing with PBS and the number of adherent cells determined by measuring the fluorescence intensity using a CytoFluor 2300 (Millipore, Bedford, MA).

To determine the contribution of ICAM-1 to IFN--induced monocytes adherence, NCI cells were treated with anti-ICAM-1 antibody at a concentration of 10 µg/ml for 30 min at 37 °C before the BCECF-labeled U937 cells were added.

Transient Transfection and Luciferase Activity Assay-- NCI-H292 cells, grown in 6-well plates, were transfected with the human ICAM-1 promoter-firefly luciferase constructs, pIC1352, pIC339, pIC135, or pIC135(AP2), using Tfx^TM-50, as described previously (35). The following day, cells were exposed to 10 ng/ml IFN- or 1 µM TPA for 5 h; cell extracts were then prepared and the luciferase and -galactosidase activities measured, and the luciferase activity of each well was normalized to the -galactosidase activity. In dominant negative mutant experiments, cells were cotransfected with reporter/-galactosidase and the mutant PLC-2 SH₂(N), the dominant negative PKC-(K/R), c-Src (K295M),² JAK1, JAK2, STAT1 (Y701M) or STAT3 mutant, or the empty vector.

In wild type experiments, cells were cotransfected with reporter/-galactosidase and the PLC-2, PKC, c-Src, JAK1, or STAT1 wild type plasmids, or the empty vector using SuperFect Transfection Reagent (Qiagen). Briefly, wild type plasmid or empty vector (1.5 µg), pIC135 (0.5 µg), and -galactosidase (0.25 µg) were mixed with 1.87 µl (1:0.5) of SuperFect in 600 µl of serum-free RPMI 1640 medium. After 10 min of incubation at room temperature, 300 µl of serum-free RPMI 1640 medium was then applied to the cells. Eight hours later, 100 µl of FCS was added, and the cells were grown in medium containing 10% FCS. On the following day, the cell extracts were prepared. The luciferase (Promega) and -galactosidase activities were measured, and the luciferase activity of each well was normalized to -galactosidase activity. In PLC-2 (wt), PKC (wt), or c-Src(wt) and dominant negative c-Src(KM) or STAT1(Y701M) mutant experiments, the wild types (1.5 µg) and dominant negative mutants (2.0 µg) or the empty vector were cotransfected.

Preparation of Nuclear Extracts and the Electrophoretic Mobility Shift Assay (EMSA)-- Cells were incubated for 10 min, 1 h, or 24 h with IFN- and then nuclear extracts were prepared as described previously (35). Oligonucleotides corresponding to the GAS consensus sequence in the human ICAM-1 promoter (5'-CGAGGTTTCCGGGAAAGCAGC-3') were synthesized, annealed, and end-labeled with [-³²P]ATP using T4 polynucleotide kinase, and EMSA was performed as described previously (35). When supershift assays were performed, polyclonal antibodies specific for p91 (STAT1-) or p65 were added to the nuclear extracts 30 min before the binding reaction, and the DNA-nuclear protein complexes were separated on a 4.5% polyacrylamide gel.

In Vitro c-Src and Lyn Activity Assay-- After treatment with IFN- or TPA for 10, 30, or 60 min, with or without pretreatment with various inhibitors for 30 min at 37 °C, the cells were rapidly washed with PBS and then lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM NaF, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml of leupeptin, 20 µg/ml of aprotinin, 1 mM Na₃VO₄, 1% Triton X-100). A sample of total cell extract containing 50 µg of protein was incubated for 1 h at 4 °C with 0.5 µg of anti-c-Src or anti-Lyn antibody, and the antibody-bound protein was collected using protein A-Sepharose CL-4B beads (Sigma). The beads were then washed three times with lysis buffer without Triton X-100 and incubated for 30 min at 30 °C in 20 µl of kinase reaction mixture (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 5 mM MnCl₂, 0.1 mM Na₃VO₄, 1 mM dithiothreitol, 5 µg of enolase, and 10 µM [-³²P]ATP). The reaction was then stopped by addition of 20 µl of Laemmli buffer and the proteins subjected to 10% SDS-PAGE; the, phosphorylated enolase was visualized by autoradiography. Quantitative data were obtained using a densitometer with ImageQuant software.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IFN- Induces Cell Surface ICAM-1 Expression in, and U937 Adhesion to, NCI-H292 Cells-- When NCI-H292 cells were treated with 100 ng/ml IL-1, TNF-, or IFN-, with 1 µg/ml lipopolysaccharide or with 1 µM TPA, only IFN- and TPA stimulated ICAM-1 expression, as measured by ELISA (data not shown). IFN- induced ICAM-1 expression in a concentration- and time-dependent manner (Fig. 1). With an exposure period of 18 h, maximal ICAM-1 expression was seen using 10 ng/ml IFN- (Fig. 1A), and when cells were treated with 10 ng/ml IFN- for various times, ICAM-1 expression was significantly increased after 5 h and was maximal at 18 h, remaining at this level for at least 40 h (Fig. 1B). Induction of ICAM-1 by IFN- was also demonstrated by immunofluorescence staining. As shown in Fig. 2, ICAM-1 was not seen in the basal state (Fig. 2B) but appeared on the cell surface following treatment with IFN- or TPA (Fig. 2, D and F). In the following ICAM-1 expression experiments, the cells were treated with 10 ng/ml IFN- for 18 h. Under these conditions, both the transcriptional and translational inhibitors, actinomycin and cycloheximide, inhibited the IFN--induced ICAM-1 expression (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Concentration- and time-dependent IFN--induced ICAM-1 expression in NCI-H292 epithelial cells. Cells were incubated at 37 °C with various concentrations of IFN- for 18 h (A) or with 10 ng/ml IFN- for various time intervals (B). Surface expression of ICAM-1 was measured by ELISA using anti-ICAM-1 antibody, as described under "Experimental Procedures." Results are expressed as the mean ± S.E. of three independent experiments performed in triplicate.

View larger version (99K): [in this window] [in a new window]   Fig. 2.   Localization of ICAM-1 on the cell surface. Immunofluorescent staining of NCI-H292 epithelial cells with affinity-purified anti-ICAM-1 antibody (1:100). Control cells (A and B), cells after 18 h treatment with 10 ng/ml IFN- (C and D), and cells after 18 h treatment with 1 µM TPA (E and F) were fixed and stained as described under "Experimental Procedures." Bar, 200 µm.

To determine whether IFN-- or TPA-induced monocytes adherence to NCI-H292 cells correlated with cell-surface ICAM-1 expression, we analyzed U937 cell adhesion to NCI-H292 cells (Fig. 3). After 18 h of treatment with IFN-, adherence was increased by ~11-fold, and anti-ICAM-1 antibody reduced adherence to below the basal level, showing that IFN--induced U937 cell adhesion to NCI-H292 cells was because of ICAM-1 expression. Similarly, 18 h of treatment with TPA resulted in a 10-fold increase in adherence, which was inhibited by 70% by anti-ICAM-1 antibody, indicating a role of ICAM-1 in TPA-induced U937 cell adhesion.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Adhesion of U937 cells to IFN-- or TPA-activated NCI-H292 epithelial cells. U937 cells, labeled with BCECF, were added to NCI-H292 cells pretreated with IFN- (10 ng/ml) or TPA (1 µM) for 18 h, and culture was continued at 37 °C for 1 h, and then adhesion was measured as described under "Experimental Procedures." Where indicated, anti-ICAM-1 antibody (10 µg/ml) was added to the NCI-H292 cells 30 min before addition of BCECF-labeled U937 cells. Results are expressed as the mean ± S.E. of three independent experiments, each value being the mean of six determinations. *, p < 0.05 as compared with the basal value.

Inhibitory Effect of Tyrosine Kinase, PI-PLC, or PKC Inhibitors on IFN--induced ICAM-1 Expression-- To study the intracellular signaling pathway involved in IFN--induced ICAM-1 expression, NCI-H292 cells were pretreated for 30 min with the tyrosine kinase inhibitors, genistein and herbimycin. Under these conditions, IFN--induced ICAM-1 expression was inhibited 34, 45, or 47%, respectively, by 30 or 100 µM genistein or 1 µM herbimycin (Fig. 4A). When cells were pretreated with the PI-PLC inhibitor, U73122, at 10 or 30 µM, IFN--induced ICAM-1 expression was inhibited by 31 or 50%, respectively, whereas 30 µM U73343 (an inactive analogue of U73122), 100 µM D609 (a PC-PLC inhibitor), or propranolol (a phosphatidate phosphohydrolase inhibitor) had no effect (Fig. 4B).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of various inhibitors on IFN--induced ICAM-1 expression in NCI-H292 epithelial cells. NCI-H292 cells were pretreated for 30 min with 30 or 100 µM genistein or 1 µM herbimycin (A), or 10 or 30 µM U73122, 30 µM U73343, 100 µM propranolol, or 100 µM D609 (B), or with the indicated concentration of staurosporine or Ro 31-8220 (C) before incubation with 10 ng/ml IFN- for 18 h. Surface expression of ICAM-1 was measured by ELISA using anti-ICAM-1 antibody, as described under "Experimental Procedures." Results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05 as compared with IFN- alone.

Because IFN--induced ICAM-1 expression was inhibited by U73122, indicating involvement of the PI-PLC pathway, which increases diacylglycerol levels and then activates PKC, the PKC inhibitors, staurosporine and Ro 31-8220, were used to determine whether PKC was involved in IFN--induced ICAM-1 expression. Following pretreatment of cells with 10, 30, or 100 nM staurosporine or with 0.1, 0.3, or 1 µM Ro 31-8220, IFN--induced ICAM-1 expression was inhibited in a dose-dependent manner (Fig. 4C).

Because PKC had been shown to be involved, the effect of direct TPA-mediated PKC activation on ICAM-1 expression was examined. TPA (1 µM) also induced a time-dependent increase in ICAM-1 expression, which was significant at 4.5 h and maximal at 16 h and then declined after 20 h (Fig. 5A). Induction of ICAM-1 expression by TPA was also demonstrated by immunofluorescence staining (Fig. 2F) and U937 cell adhesion (Fig. 3). When cells were pretreated with 100 nM staurosporine or 10, 30, and 100 µM genistein, or 1 µM herbimycin, TPA-induced ICAM-1 expression was inhibited by 89, or 40, 62, 80, or 52%, respectively (Fig. 5B). The specific Src inhibitor, PP2 (56), inhibited IFN-- or TPA-induced ICAM-1 expression dose-dependently (Fig. 5C).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Time-dependent TPA-induced ICAM-1 expression in NCI-H292 epithelial cells and the inhibitory effect of genistein, herbimycin, or PP2. A, cells were incubated at 37 °C with 1 µM TPA for various time intervals. B, cells were pretreated for 30 min with 100 nM staurosporine or the indicated concentrations of genistein or 1 µM herbimycin before incubation with 1 µM TPA for 18 h. C, cells were pretreated for 30 min with the indicated concentrations of PP2 before incubation with 10 ng/ml IFN- or 1 µM TPA for 18 h. Surface expression of ICAM-1 was measured by ELISA using anti-ICAM-1 antibody, as described under "Experimental Procedures." Results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05 as compared with TPA alone.

Induction of ICAM-1 Promoter Activity by IFN- and the Inhibitory Effect of Genistein, Herbimycin, U73122, Staurosporine, Ro 31-8220, PLC-2 Mutant, or Dominant Negative Mutants of PKC- or c-Src-- To study further the involvement of the PI-PLC-dependent PKC pathway in IFN--induced ICAM-1 expression, transient transfection was performed using the human ICAM-1 promoter-luciferase constructs, pIC1352 (1352/+1), which contains full-length human ICAM-1 promoter; pIC339 (339/+1), which contains the downstream NF-B and GAS sites in the ICAM-1 promoter; pIC135 (135/+1), which contains the GAS site but not the NF-B site; and pIC135(AP2), which does not contain either the NF-B or the GAS site but contains the proximal TATA box site. Treatment with 10 ng/ml IFN- or 1 µM TPA led to a 4.1- or 5.7-fold increase, respectively, using pIC1352, and a 3.7- or 5.3-fold increase, respectively, using pIC339, and a 3.5- or 3-fold increase, respectively, using pIC135. However, using pIC135(AP2), IFN-, or TPA treatment only resulted in a 1.4- or 1.2-fold increase, respectively, in ICAM-1 promoter activity (Fig. 6). These results indicate that the GAS cis-acting element is responsible for mediating both IFN-- and TPA-induced ICAM-1 expression in NCI-H292 cells and that NF-B may be involved in TPA- but not IFN--induced ICAM-1 expression.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   GAS-dependent activation of the ICAM-1 promoter by IFN- and TPA. Upper diagram, schematic diagram of the 5' regulatory region of the human ICAM-1 gene. Rectangles indicate the location of potential binding sites for the transcription factors AP-1, AP-2, Ets, NF-B, and Sp-1 and the binding sites TRE and GAS. The arrow above the initiation codon ATG indicates the translation start site. The cells were transfected with the pIC1352, pIC339, pIC135, or pIC135(AP2) luciferase expression vector, as under "Experimental Procedures," then incubated for 5 h with 10 ng/ml IFN- or 1 µM TPA. Cell extracts were prepared and assayed for luciferase and -galactosidase activity, and then the luciferase activity was normalized using the -galactosidase activity and expressed as the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05 as compared with pIC339.

By using pIC339, the induction of ICAM-1 promoter activity mediated by IFN- was attenuated by genistein, herbimycin, U73122, staurosporine, or Ro 31-8220, but not by PDTC, whereas that induced by TPA was inhibited by genistein, herbimycin, staurosporine, or PDTC (Fig. 7A). In cotransfection experiments, the induction of ICAM-1 promoter activity by IFN- was inhibited by the mutant PLC-2 SH₂(N) or by the dominant negative PKC-/KR or c-Src(KM) mutants, whereas that induced by TPA was inhibited by the dominant negative PKC-/KR or c-Src(KM) mutants but not by the mutant PLC-2 SH₂(N) (Fig. 7B). By using pIC135, the IFN--induced ICAM-1 promoter activity was also inhibited by cotransfection with the above three mutants. Furthermore, induction of ICAM-1 promoter activity by IFN- was inhibited by cotransfection with the dominant negative JAK1, JAK2, or STAT1 (Y701M) but not by STAT3 (Fig. 8A). The ICAM-1 promoter activity was enhanced by cotransfection with wild type PLC-2(wt), PKC(wt), c-Src(wt), JAK1(wt) or STAT1(wt), which led to 4.8-, 4.4-, 4.9-, 4.1-, or 6.4-fold increase, respectively (Fig. 8B). The induction of ICAM-1 promoter activity by wild type PLC-2 (wt) or PKC (wt) was blocked by cotransfection with dominant negative c-Src(KM) or STAT1 (Y701M) mutant and that by wild type c-Src (wt) was blocked by cotransfection with dominant negative STAT1 (Y701M) mutant (Fig. 8C).

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effect of inhibitors, mutants, or dominant negative mutants on IFN-- or TPA-induced ICAM-1 promoter activity. A, NCI-H292 cells were transfected with the pIC339 luciferase expression vector and then pretreated for 30 min with 100 µM genistein, 1 µM herbimycin, 30 µM U73122, 100 nM staurosporine, 1 µM Ro 31-8220, or 100 µM PDTC before incubation with 10 ng/ml IFN- or 1 µM TPA for 5 h. B, NCI-H292 cells were cotransfected with pIC339 and the PLC-2 mutant, the dominant negative PKC- or c-Src mutant, or empty vector. Luciferase activity was assayed as under "Experimental Procedures." The results were normalized using the -galactosidase activity and expressed as the mean ± S.E. of three independent experiments performed in triplicate; *, p < 0.05 as compared with IFN- or TPA alone.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Effect of mutants or dominant negative mutants on IFN- or wild type plasmid-induced ICAM-1 promoter activity. A, NCI-H292 cells were cotransfected with pIC135 (0.5 µg) and the PLC-2 (SH2(N)), PKC(K/R), c-Src(K/M), JAK1(DN), JAK2(DN), or STAT1(Y701M) (1.5 µg) or the empty vector before incubation with 10 ng/ml IFN- for 5 h. B, NCI-H292 cells were cotransfected with pIC135 (0.5 µg) and the wild type PLC-2(wt), PKC(wt), c-Src(wt), JAK1(wt), or STAT1(wt) (1.5 µg) or the empty vector. C, NCI-H292 cells were cotransfected with wild type PLC-2(wt), PKC(wt), or c-Src(wt) (1.5 µg) and the dominant negative c-Src(KM) or STAT1(Y701M) (2.0 µg) mutant or the empty vector. Luciferase activity was assayed as under "Experimental Procedures." The results were normalized using the -galactosidase activity and expressed as the mean ± S.E. of three independent experiments performed in triplicate. A, *, p < 0.05 as compared with IFN- alone. B, *, p < 0.05 as compared with control vector. D, *, p < 0.05 as compared with PLC-2(wt). **, p < 0.05 as compared with PKC(wt). ***, p < 0.05 as compared with c-Src(wt).

IFN- and TPA Induce STAT1- Binding to the GAS Site of the ICAM-1 Promoter-- The ICAM-1 promoter contains a complex array of transactivating binding sites. To determine whether the GAS element was involved in ICAM-1 gene transcription following IFN- stimulation, GAF complex formation was examined by EMSA. Although no GAF-GAS DNA-protein binding was seen in nonstimulated cells, IFN- rapidly (10 min) stimulated GAF-GAS DNA-protein binding, with similar activation being seen after 1 and 24 h (Fig. 9A). TPA resulted in a similar pattern of GAF-GAS DNA-protein binding (data not shown). In subsequent EMSA experiments, cells were treated with IFN- for 1 h. To identify the specific subunits involved in the formation of the GAF-GAS complex after IFN- stimulation, supershift assays were performed in the presence of antibodies specific for STAT-1 (p91) or p65 (NF-B). As shown in Fig. 9, B and C, incubation of nuclear extracts with anti-STAT-1 antibody induced attenuation of GAF-GAS DNA-protein binding (Fig. 9, B and C, lane 3), but no shift or attenuation occurred in the presence of anti-p65 antibody (Fig. 9B, lane 4), indicating that the GAF complex induced by IFN- contained STAT1. Excess cold GAS probe blocked the GAF-GAS DNA-protein binding (Fig. 9C, lane 4).

View larger version (34K): [in this window] [in a new window]   Fig. 9.   Kinetics of IFN--induced GAF-GAS DNA-protein binding in NCI-H292 epithelial cells. A, cells were treated for 10 min, 1 h, or 24 h with 10 ng/ml IFN-, and then nuclear extracts were prepared and tested using the GAS oligonucleotide probe to measure the DNA-protein binding activity by EMSA as under "Experimental Procedures." B, supershift assays were performed using 2 µg of the indicated antibodies as described under "Experimental Procedures." C, excess cold GAS probe was used as competitor as described under "Experimental Procedures." NS, nonspecific binding.

Induction of c-Src and Lyn Activation by IFN- or TPA and the Inhibitory Effect of U73122, Staurosporine, or Herbimycin-- Because IFN-- or TPA-induced ICAM-1 expression was inhibited by genistein, herbimycin, and PP2 (Fig. 4A and Fig. 5, B and C), and the induced ICAM-1 promoter activity was inhibited by a dominant negative c-Src(KM) mutant (Fig. 7B), these results indicated that c-Src was involved downstream of PKC in the induction of ICAM-1 expression. Western blot analysis using antibodies against the Src family members, c-Src, Lck, Lyn, or Fyn, showed that c-Src and Lyn were expressed substantially in NCI-H292 cells (data not shown). To determine whether IFN- or TPA induced activation of these two tyrosine kinases, c-Src and Lyn were isolated by immunoprecipitation using anti-c-Src or anti-Lyn antibody and tested for in vitro kinase activity, using enolase as substrate. When cells were treated for 10, 30, or 60 min with 10 ng/ml IFN- or 1 µM TPA, IFN- induced c-Src and Lyn activation that was significant at 10 min and maximal at 60 min (Fig. 10A), although TPA also induced c-Src and Lyn activation, the kinetics were different, with activation being significant at 10 min, maximal at 30 min, and declining after 60 min (Fig. 10B). The activation of c-Src and Lyn induced by IFN- was inhibited by U73122, staurosporine, and herbimycin and that induced by TPA was inhibited by staurosporine and herbimycin (Fig. 11). These inhibitors alone had no effect on basal c-Src or Lyn activity (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 10.   Time-dependent activation of c-Src or Lyn tyrosine kinase activity by IFN- or TPA in NCI-H292 cells. Cells were treated for 10, 30, or 60 min with 10 ng/ml IFN- (A) or with 1 µM TPA (B), and then whole cell lysates were immunoprecipitated (IP) with anti-c-Src or anti-Lyn antibody, followed by autoradiography of phosphorylated enolase as described under "Experimental Procedures." The amount of c-Src or Lyn in immunoprecipitates was determined by Western blot (WB) using anti-c-Src or anti-Lyn antibody.

View larger version (30K): [in this window] [in a new window]   Fig. 11.   Effect of various inhibitors on IFN-- or TPA-induced c-Src or Lyn activation in NCI-H292 epithelial cells. Cells were pretreated for 30 min with 30 µM U73122, 100 nM staurosporine, or 1 µM herbimycin before incubation with 10 ng/ml IFN- for 60 min or with 1 µM TPA for 30 min. Whole cell lysates were immunoprecipitated (IP) with anti-c-Src (A) or anti-Lyn (B) antibody, followed by autoradiography for phosphorylated enolase as described under "Experimental Procedures." The amount of c-Src or Lyn in immunoprecipitates was determined by Western blot (WB) using anti-c-Src or anti-Lyn antibody.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we have shown that IFN- induced ICAM-1 expression in the plasma membrane of NCI-H292 epithelial cells, and this resulted in increased adhesion of U937 cells. The transcriptional factor binding site, GAS, appears to be essential for the enhanced ICAM-1 expression seen after exposure to IFN- in human monocytes (36). To test whether the NF-B or GAS site was involved in IFN--induced ICAM-1 promoter activity in NCI-H292 cells, we used different deletion mutants of the ICAM-1 promoter-Luc construct, pIC1352, pIC339, pIC135, or pIC135 (AP2). The results showed that the GAS site was essential for both IFN-- and TPA-induced ICAM-1 promoter activity and that the downstream NF-B site reported to be critical for TNF- and IL-1 to induce ICAM-1 expression (33, 34) was only involved in TPA-induced ICAM-1 promoter activity (Fig. 6). Experiments using PDTC, an NF-B inhibitor, further supported this notion, because PDTC inhibited TPA-, but not IFN--, induced ICAM-1 promoter activity. In the EMSA, IFN- increased GAF-GAS DNA-protein binding, indicating that the GAS site in the ICAM-1 promoter was involved in IFN--mediated ICAM-1 induction. GAF, the protein complex binding to GAS sequences in IFN--treated cells, is a dimer of STAT1, which is the prototype of a family of cytokine-responsive transcriptional factors. The component of the GAF transcriptional complex in NCI-H292 cells was identified as STAT1. This result is consistent with previous findings that IFN- mediates ICAM-1 induction via translocation of activated STAT1 (30, 31).

We demonstrated that the PKC inhibitors, staurosporine and Ro 31-8220, inhibited the IFN--mediated induction of ICAM-1 expression in a dose-dependent manner, indicating that PKC activation is an obligatory event in IFN--induced ICAM-1 expression in these cells. This was further confirmed by the result that the dominant negative PKC- mutant, PKC- (K/R), inhibited IFN--induced ICAM-1 promoter activity (Fig. 7B), and overexpression of wild type PKC- (wt) enhanced ICAM-1 promoter activity (Fig. 8B). PKC is activated by the physiological activator, diacylglycerol, which can be generated either directly, by the action of PLC, or indirectly, by a pathway involving the production of phosphatidic acid by PLD, followed by a dephosphorylation reaction catalyzed by phosphatidate phosphohydrolase. Normally, the PLC involved in the production of diacylglycerol is PI-PLC, but PC-PLC can also be involved (37, 38). The PI-PLC inhibitor, U73122, inhibited IFN--induced ICAM-1 expression, whereas the PC-PLC inhibitor, D609, the phosphatidate phosphohydrolase inhibitor, propranolol, and the inactive U73122 analogue, U73343, did not. Tyrosine kinase inhibitors blocked IFN--induced ICAM-1 expression, indicating that the PI-PLC involved might be PLC-, because PLC- contains an SH2 domain used to link phosphotyrosine-containing sequences in a receptor protein or in cytoplasmic protein tyrosine kinase to PI hydrolysis (39). In the present study, we used the PLC-2 N-terminal SH2 (SH₂(N)) mutant (40) to further determine the role of PLC-, and we found that it inhibited IFN-- but not TPA-induced ICAM-1 promoter activity, indicating the possible involvement of PLC-2 in IFN--induced ICAM-1 expression in NCI-H292 cells. This was further confirmed by the result that cotransfection with wild type PLC-2 (wt) increased ICAM-1 promoter activity (Fig. 8B). Thus, IFN- acts through the PI-PLC-2 pathway, but not through the PC-PLC or PC-PLD pathway, to induce PKC activation in NCI-H292 cells. Although IFN- has been reported to act mainly via JAK-STAT pathway to regulate most gene expression, in some cases IFN- acts via PI-PLC to induce Ca²⁺ signals in neutrophils (41), and it acts via PC-PLC to induce PKC activation in macrophages (28) or via PC-PLD to activate PKC in endothelial cells (26, 27).

Because PKC had been shown to be involved in the IFN- signaling, direct activation of PKC by TPA was tested and was found to induce ICAM-1 expression, as shown both by ELISA and immunofluorescence staining. TPA-induced ICAM-1 expression was inhibited in a dose-dependent manner by genistein, herbimycin, or PP2, as was IFN--induced ICAM-1 expression. TPA also stimulated GAF-GAS DNA-protein binding (data not show). The induction of ICAM-1 promoter activity by TPA was inhibited by genistein or herbimycin and the dominant negative c-Src(KM) mutant, as was that induced by IFN-. Furthermore, the induction of ICAM-1 promoter activity by PKC(wt) was also inhibited by cotransfection with dominant negative c-Src(KM) mutant (Fig. 8C). These results suggest that the protein tyrosine kinase, c-Src, acts downstream of PKC in the induction of STAT1 activation and ICAM-1 expression. Two cytoplasmic protein tyrosine kinases, c-Src and Lyn, were found to be activated by IFN- and TPA in NCI-H292 cells. These effects were inhibited by staurosporine and herbimycin, indicating the involvement of PKC-dependent c-Src or Lyn activation in IFN--mediated ICAM-1 induction. This might be a common signal pathway for inducible gene expression, because TNF-- or IL-1-induced ICAM-1 or cyclooxygenase-2 expression in human alveolar epithelial cells has also been demonstrated to be involved in PKC-dependent activation of c-Src or Lyn tyrosine kinase (33-35). In addition to gene expression, a similar signal pathway has also been reported in the development of ischemic preconditioning in the conscious rabbit, which involved PKC-dependent Src and Lck activation (42), in the G protein-coupled receptors regulating N-methyl-D-aspartic acid receptor in CA1 pyramidal neurons, which involved PKC-dependent c-Src activation (43), and in the cellular response to oxidative stress, which involved PKC-dependent c-Abl activation (44).

STATs are latent cytoplasmic transcription factors that transduce signals from the cell membrane to the nucleus upon activation by tyrosine phosphorylation. Several protein tyrosine kinases can induce phosphorylation of STATs in cells, including JAK and Src family kinases. Previous studies (45-47) have shown that IFN--induced tyrosine 701 phosphorylation of STAT-1 is mediated by JAK tyrosine kinases. However, many cytokines and growth factors are reported to recruit STATs via the cytoplasmic Src tyrosine kinase family (48). A role for Src kinase in STAT activation was first suggested by studies aimed at investigating the molecular mechanisms associated with Src-mediated transformation of fibroblasts and hematopoietic cell lines. STAT3 activation is required for v-Src-mediated transformation of NIH3T3 cells (49, 50), and a direct association of v-Src and STAT3 has been found in 32Dcl3 cells (51). In addition to v-Src, c-Src also plays a role in IL-3-, epidermal growth factor-, and platelet-derived growth factor-induced STAT3 or STAT1 activation (52-54). In A431 cells, epidermal growth factor-induced activation of STAT and c-Src kinase and direct association of these two components were demonstrated (53). In NIH3T3 cells, both tyrosine phosphorylation and DNA binding activity of STAT1 and STAT3 were up-regulated in c-Src-overexpressing cells, and coimmunoprecipitation of STAT1 and c-Src was also seen (54). In Sf9 insect cells, overexpression of c-Src kinase without expression of JAK family members can directly activate functional STAT1 and STAT3, indicating that c-Src can phosphorylate STATs independent of JAKs (55). In the present NCI-H292 cells, STAT1 but not STAT3 is shown to be involved in IFN--induced ICAM-1 expression (Fig. 8, A and B), and STAT1 acts downstream of c-Src (Fig. 8C). Phosphorylation of STAT1 at Tyr-701 by IFN- and TPA is seen, and this effect is inhibited by inhibitors of PKC and c-Src,³ indicating that the phosphorylation of STAT1 at Tyr-701 is possibly due to c-Src via IFN--induced PKC activation. Furthermore, direct association of c-Src and STAT1 is seen.³ Thus, c-Src can directly phosphorylate STAT1 in NCI-H292 cells. JAK1 is also involved in IFN--induced ICAM-1 expression (Fig. 8, A and B). However, its role in c-Src-induced STAT1 phosphorylation is unknown and is currently under investigation. The IFN--induced ICAM-1 expression in NCI-H292 cells was not affected by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, PD98059, or the p38 inhibitor, SB203580 (data not shown), excluding the involvement of p44/42 mitogen-activated protein kinase and p38 in IFN--induced effect in this type of cells. However, p44/42 mitogen-activated protein kinase was reported to be activated by IFN- to stimulate c/EBP--dependent gene expression in RAW 264.7 cells (57), and p38 activation by IFN- was required for STAT1 serine phosphorylation in HeLa S3 cells (58).

In summary, the signaling pathway involved in IFN--induced ICAM-1 expression in human NCI-H292 epithelial cells has been explored. IFN- activates phospholipase C-2 via an upstream tyrosine kinase to induce activation of PKC- and c-Src or Lyn, resulting in STAT1 activation, and activation of GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.

	FOOTNOTES

^* This work was supported by Research Grant NSC-90-2315-B002-004 from the National Science Council.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.

^§ To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, National Taiwan University, No.1, Jen-Ai Rd., 1st Section, Taipei 10018, Taiwan. Tel.: 886-2-23123456 (Ext. 8321); Fax: 886-2-23947833; E-mail: ccchen@ha.mc.ntu.edu.tw.

Published, JBC Papers in Press, December 20, 2001, DOI 10.1074/jbc.M109924200

² W. C. Huang, J. J. Chen, and C.-C. Chen, manuscript in preparation.

³ Y.-J. Chang, M. J. Holtzman, and C.-C. Chen, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; EMSA, electrophoretic mobility shift assay; GAS, -activated site; IFN, interferon; JAK, Janus family kinase; STAT, signal transducers and activators of transcription; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; ELISA, enzyme-linked immunosorbent assay; PI-PLC, phosphatidylinositol-phospholipase C; IL-1, interleukin-1; PKC, protein kinase C; BCECF, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein; PDTC, pyrrolidinedithiocarbamate; FCS, fetal calf serum; wt, wild type; PBS, phosphate-buffered saline; SH2, Src homology 2; GAF, -activated factor; PLD, include phospholipase D.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES