Signals mediating cleavage of intercellular adhesion molecule-1
Nina L. Tsakadze,1
Utpal Sen,1
Zhendong Zhao,1
Srinivas D. Sithu,1
William R. English,2 and
Stanley E. D'Souza1
1Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky 40292; and 2Department of Oncology, Cambridge Institute of Medical Research, Cambridge CB2 2XY, United Kingdom
Submitted 29 December 2003
; accepted in final form 13 February 2004
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ABSTRACT
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ICAM-1, a membrane-bound receptor, is released as soluble ICAM-1 in inflammatory diseases. To delineate mechanisms regulating ICAM-1 cleavage, studies were performed in endothelial cells (EC), human embryonic kidney (HEK)-293 cells transfected with wild-type (WT) ICAM-1, and ICAM-1 containing single tyrosine-to-alanine substitutions (Y474A, Y476A, and Y485A) in the cytoplasmic region. Tyrosine residues at 474 and 485 become phosphorylated upon ICAM-1 ligation and associate with signaling modules. Cleavage was assessed by using an antibody against the cytoplasmic tail of ICAM-1, which recognizes intact ICAM-1 and the 7-kDa membrane-bound fragment remaining after cleavage. Cleavage in HEK-293 WT cells was accelerated by phorbol ester PMA, whereas in EC it was induced by tumor necrosis factor-
. In both cell types, a 7-kDa ICAM-1 remnant was detected. Tyrosine phosphatase inhibitors dephostatin and sodium orthovanadate augmented cleavage. PD-98059 (MEK kinase inhibitor), geldanamycin and PP2 (Src kinase inhibitors), and wortmannin (phosphatidylinositol 3-kinase inhibitor) dose-dependently inhibited cleavage in both cell types. SB-203580 (p38 inhibitor) was more effective in EC, and D609 (PLC inhibitor) mostly affected cleavage in HEK-293 cells. Cleavage was drastically decreased in Y474A and Y485A, whereas it was marginally reduced in Y476A. Surprisingly, phosphorylation was not detectable on the 7-kDa fragment of ICAM-1. These results implicate distinct pathways in the cleavage process and suggest a preferred signal transmission route for ICAM-1 shedding in the two cell systems tested. Tyrosine residues Y474 and Y485 within the cytoplasmic sequence of ICAM-1 regulate the cleavage process.
ectodomain shedding; signaling; tyrosine phosphorylation
INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1; 93 kDa) is a highly glycosylated adhesion receptor expressed on different cell types, including endothelial cells (EC). ICAM-1 is a member of the immunoglobulin (Ig) gene superfamily and contains five Ig-like domains on the extracellular surface, a transmembrane region, and a short cytoplasmic tail of 28 amino acids. The interactions of leukocytic integrins LFA-1 (
L
2) and Mac-1 (
M
2) with ICAM-1 permit the adhesion and transmigration of leukocytes through the endothelium. ICAM-1 also functions as a costimulatory molecule on antigen-presenting cells to activate major histocompatibility complex class II restricted T cells (9, 50, 53). ICAM-1 levels on EC are upregulated by cytokines such as tumor necrosis factor-
(TNF-
), interleukin (IL)-1, and bacterial lipopolysaccharide, as well as by oxygen radicals and hypoxia. Elevated levels of ICAM-1 have been detected in affected tissues with malignancies, inflammatory diseases, atherosclerosis, ischemia, and allogenic organ transplant (11, 53).
ICAM-1 undergoes proteolytic cleavage, which releases soluble ectodomain from the cell surface. A soluble form of ICAM-1 (sICAM-1), detectable in the blood and other body fluids, contains most of the extracellular and perhaps all five Ig-like domains. sICAM-1 is produced by diverse cell types, including EC, carcinoma cells, keratinocytes, and astrocytes (16, 18, 32, 34). Cytokines induce rapid shedding of ICAM-1 in EC (16, 32). Elevated levels of sICAM-1 predict a risk of cardiovascular disease (31, 43). Recently, matrix metalloproteinase-9 and human leukocyte elastase have been implicated in ICAM-1 cleavage (18, 45). sICAM-1 is functionally active and may modulate inflammation by binding to the leukocyte integrins (30, 44).
A wide group of transmembrane proteins, including adhesion molecules, TNF-
receptor, transforming growth factor-
(TGF-
), angiotensin-converting enzyme,
-amyloid precursor protein, and syndecan, undergo ectodomain cleavage (for review, see Refs. 2, 25, 46, and 48). Cleavage in numerous cases is activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA), indicating the involvement of protein kinase C (PKC) in cleavage regulation. The extracellular signal-regulated kinase (ERK1/2) has been implicated in the cleavage of some of these proteins (19, 22, 52, 54). However, the mechanisms regulating the shedding of ICAM-1 are largely unknown. To evaluate ICAM-1 cleavage, an antibody with recognition specificity against the cytoplasmic tail of ICAM-1 was developed. This antibody recognized a 7-kDa fragment of ICAM-1 in EC and ICAM-1-transfected human embryonic kidney 293 (HEK-293) cells. Our results indicate that ICAM-1 cleavage is regulated by multiple kinases. Specific tyrosine residues within the cytoplasmic region of ICAM-1 appear to be necessary for the cleavage process to occur.
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METHODS
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Biological reagents and synthetic peptides.
TNF-
and IL-1 were obtained from Genzyme (Boston, MA). PD-98059, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2), geldanamycin, wortmannin, SB-203580, dephostatin, and D609 were purchased from Calbiochem (La Jolla, CA). PMA, sodium orthovanadate trichloroacetic acid (TCA), Rose Bengal, and ProteoSilver silver stain kit were obtained from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM)-F-12 medium, fetal calf serum (FCS), Hanks' balanced salt solution, and Dulbecco's phosphate-buffered saline (DPBS) were purchased from BioWhittaker (Walkersville, MD). EC growth supplements (EGM-2 SingleQuots) were purchased from Clonetics (San Diego, CA). The bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL), and electrochemiluminescence (ECL) detection reagents were purchased from Amersham (Piscataway, NJ). Anti-phosphotyrosine antibody 4G10 was obtained from Upstate Biotechnology (Lake Placid, NY). LB-2 (anti-CD54), a monoclonal antibody (MAb) directed against the first two domains of ICAM-1, was purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA). R803, a polyclonal antibody against the first three domains of ICAM-1, was developed in our laboratory. Anti-SHP-2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated secondary antibodies and protein G-Sepharose 4B were obtained from Zymed (San Francisco, CA). The ICAM-1 cytoplasmic peptide AKQGTPMKPNTQATPP and peptides ICAM-1(821), ICAM-1(4052), and ICAM-1(130145) matching the ectodomain of sequences in ICAM-1 were synthesized by F-moc chemistry on an Applied Biosystems instrument (Foster City, CA) (14, 20).
Cell culture.
Human umbilical vein EC purchased from Clonetics (San Diego, CA) were grown in combined DMEM-F-12 medium supplemented with 20% FCS and EGM-2 (14, 39). EC from passages 26 were used. HEK-293 fibroblast cells and U-937 (human promonocytic leukemia) cells obtained from the American Type Culture Collection (Manassas, VA) were maintained in combined DMEM-F-12 medium supplemented with 5% FCS and 1.0 mM L-glutamine (39).
ICAM-1 cDNA constructs for transfection of HEK-293 cells were prepared as described previously (39). To generate tyrosine substitution within the cytoplasmic tail of ICAM-1 Y485A, Y474A, and Y476A, single-point mutations were introduced using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The DNA constructs were verified by sequence analysis. HEK-293 cells were stably transfected in the absence of serum using Lipofectamine Plus reagent (Life Technologies, Gaithersburg, MD) and 15 µg of pcDNA3.1 containing wild-type (WT) ICAM-1 cDNA (designated HEK-293 WT), or mutant cDNA (Y485A, Y474A, Y476A, P495A, and P498A), or pcDNA3.1 vector alone as control. Transfected cells were selected using G418 (Invitrogen, Carlsbad, CA). Cells expressing ICAM-1 were detected with a fluorescence-activated cell sorter by using anti-ICAM-1 MAb (LB-2) and fluorescein isothiocyanate-conjugated anti-mouse IgG (19, 39). The cell surface expression of ICAM-1 on each of these cell lines appeared to be equivalent to levels on HEK-293 WT cells.
Sequence-specific antibody R98.
New Zealand albino rabbits were immunized with a peptide corresponding to the cytoplasmic sequence of ICAM-1 (AQKGTPMKPNTQATPP) that was coupled to keyhole limpet hemocyanin. The IgG fraction from serum was prepared by 45% ammonium sulfate precipitation and by protein A-Sepharose affinity chromatography. The bound IgG antibody was eluted at pH 3.2 by using glycine HCl buffer. The IgG fraction was isotyped, and specific activity was assessed by ELISA. In this assay, the cytoplasmic peptide and control peptides were coated on plastic dishes at 2.5 µg/ml for 16 h at 4°C and then postcoated with 3% gelatin. Serial dilutions (100 µl) of the rabbit antiserum were added and incubated for 2 h at 22°C, after which 125I-labeled anti-rabbit IgG was applied for 1 h. Wells were washed and counted on a gamma counter (14).
Immunoprecipitation.
Cell lysates were precleared by incubation with 20 µl of protein G-Sepharose at 22°C for 1 h. Aliquots containing equivalent amounts (400 µg) of protein were mixed with 5 µg of primary antibody and incubated at 4°C overnight, in some instances in the presence of ICAM-1(130145) peptide or ICAM-1 cytoplasmic tail peptide (100 µg). The immune complexes were recovered after 20 µl of protein G-Sepharose were added for 1 h at 22°C. Precipitated complexes were extracted by boiling in nonreducing SDS gel-loading buffer and subjected to immunoblotting.
Cleavage assays and Western blot analysis.
EC, removed by brief trypsin treatment, were coated on 12-well plates at a density of 25,000 cells/well and grown to confluence. EC were complemented with fresh medium supplemented with 10% FCS before the experiment. Inhibitors were added as indicated, and cells were stimulated with TNF-
and incubated at 37°C for an additional 1824 h. HEK-293 cells were plated on the 24-well plates at a density of 50,000 cells/well and grown to 7080% confluence. HEK-293 cells were treated under serum deprivation conditions because growth factors in culture medium were shown to affect the cleavage process (22). Cells were maintained in combined DMEM-F-12 medium supplemented with 1% FCS 1824 h before the experiment. Inhibitors were added for 3 h at 37°C. After stimulation with 3 µM PMA, cells were incubated for an additional 3 h. After treatment, cell morphology and the degree of adhesion were assessed, and cell viability was estimated by performing trypan blue exclusion assay. Drug concentrations were maintained in a range not affecting cell viability. IC50 values for each inhibitor provided by the manufacturer are as follows: dephostatin, 7.718 µM; PP2, 4600 nM; geldanamycin, 75 nM; PD-98059, 2 µM; wortmannin, 5200 nM; SB-203580, 34600 nM; and D609, 8 µg/ml (
30 µM). These concentrations apply to the purified enzyme systems. These reagents were used at slightly higher concentrations that were in the range applied by other investigators (15, 19, 22, 52, 54). After treatment, cells were lysed in ice-cold lysis buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 50 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and 1% Nonidet-40). Lysates were clarified by centrifugation, and protein concentration was measured with the use of the BCA kit. Lysates containing equal amounts of total soluble protein were loaded on gel and analyzed on 16.5% tricine SDS-PAGE (47). In some instances, we applied the alternative sequential cell lysis protocol described by Gilbert et al. (24), using nondetergent lysis buffers of different tonicity (hypo-, iso-, and hypertonic), which allows analysis of proteins in different cellular compartments. After electrophoresis, proteins were transferred to the polyvinylidene difluoride membrane Immobilon-PSQ (Millipore, Bedford, MA). Membranes were blocked with 5% nonfat milk solution in Tween-containing Tris-buffered saline. Membranes were immunoblotted with primary antibody R98 for 1 h at 22°C followed by HRP-linked secondary antibody and then developed using the ECL detection system. In some instances, membranes were stripped with a stripping buffer (0.1 M glycine, pH 2.8, 3 M NaCl, and 0.1% Tween 20) and reprobed with other antibodies, such as the anti-phosphotyrosine MAb 4G10. The intensity of signals on autoradiograms was quantitated with UnScan-It software. The intensity of signals obtained from stimulated cells (EC with TNF-
or HEK-293 cells with PMA) in the absence of inhibitors was interpreted as 100%, and the degree of inhibition or activation was expressed accordingly as a percentage. Data represent the results of at least triplicate determinations.
TCA precipitation.
Cell culture medium was clarified by centrifugation at 9,000 g for 20 min. Ice-cold 40% TCA was added to a final concentration of 10%, and samples were incubated on ice for 1 h. Samples were centrifuged for 10 min at 9,000 g and washed three times with 70% ethanol. Samples were subjected to 10% SDS-PAGE and Western blotting with the use of LB-2, which recognizes the ectodomain of ICAM-1, for the detection of sICAM-1.
Adhesion assay.
Adhesion of monocytic cells (U-937) to EC was performed as described previously (3). EC were grown on 12-well plates to confluence in combined DMEM-F-12 medium with 20% FCS and growth supplements. Twenty-four hours before assay, the medium was changed to DMEM with 5% FCS. Cells were stimulated with TNF-
, treated with inhibitors, and incubated overnight at 37°C. U-937 cells were washed with HBSS containing 0.1% BSA, resuspended in HBSS, 0.1% BSA, 1 mM CaCl2, and 1 mM MgCl2, and stimulated with PMA (3 µM) for 1 h. U-937 cells (1 x 105) were added and incubated for 1 h at 37°C. Nonadherent cells were removed, and plates were washed with DMEM. Rose Bengal (250 µl of 0.25% in DPBS) was added for 5 min at room temperature. The stain was removed, and cells were washed with DPBS. Ethanol-DPBS (1:1) was added for 30 min at 22°C. Supernatant was collected and clarified with centrifugation, and optical density (OD) was measured at a wavelength of 570 nm. The OD value from monocyte adhesion to stimulated EC in the absence of inhibitors was interpreted as 100%, and the relative degree of adhesion was expressed accordingly as a percentage. Each data point represents the results of triplicate determinations. Statistical analysis was performed with the Student's t-test. Differences were considered significant when P
0.05.
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RESULTS
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Specificity of the anti-ICAM-1 cytoplasmic sequence antibody R98.
Antiserum from rabbits immunized with the peptide corresponding to sequence 488505 of human ICAM-1 reacted specifically against the immunized peptide, but not with ICAM-1(821) and ICAM-1(4052) sequences from the ectodomain (Fig. 1A). On Western blots, the antiserum reacted against intact ICAM-1 (93 kDa) and also against a 7-kDa cytoplasmic fragment from lysates of TNF-
-treated EC. The reactivity of LB-2, a MAb that interacts with the first two extracellular Ig domains, was solely with intact ICAM-1. The 7-kDa fragment of ICAM-1 appeared only after treatment of EC with TNF-
(Fig. 1B). Thus TNF-
not only caused the upregulation of ICAM-1 but also stimulated the release of sICAM-1, leaving a 7-kDa remnant. Cell lysates mixed with ICAM-1 cytoplasmic peptide or control peptide ICAM-1(130145) were immunoprecipitated with R98 antibody. In the presence of the cytoplasmic peptide, the 7-kDa fragment was detectable in low amounts, indicating immunodepletion of R98, whereas in the presence of a control peptide ICAM-1(130145), the level remained unchanged (Fig. 1C). These results establish the specificity of R98. To verify that the 7-kDa fragment is the result of proteolytic cleavage of ICAM-1, we used several different approaches. Culture supernatant from HEK-293 WT cells was immunoprecipitated with ectodomain-specific antibody (R803) or R98, followed by Western blotting with LB-2. The results, shown in Fig. 1D, demonstrate an 86-kDa band corresponding to sICAM-1, which is increased after stimulation, whereas in the same assay, R98 failed to detect sICAM-1. Furthermore, TCA-precipitated culture medium of HEK-293 WT and EC, immunoblotted with LB-2, shows that the 86-kDa band (sICAM-1) increased after stimulation (Fig. 1E). After performing ELISA, we detected sICAM-1 with antibody recognizing the ectodomain of ICAM-1 (R803) but not with R98 (results not shown). These results indicate that the appearance of a 7-kDa fragment corresponds to the release of sICAM-1 in culture medium.

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Fig. 1. A: reactivity of anti-ICAM-1 cytoplasmic tail antibody (R98). Peptide (100 µl) of ICAM-1(821), ICAM-1(4052), and ICAM-1 cytoplasmic tail at 2.5 µg/ml was coated for 16 h at 4°C and then postcoated with 3% gelatin in PBS. Antibody (100 µl) against ICAM-1 cytoplasmic tail (R98) was incubated at different dilutions, and 125I-labeled anti-rabbit IgG was used as the secondary antibody. After washing, plates were counted. B: analysis of ICAM-1 shedding in endothelial cells (EC) after stimulation with tumor necrosis factor- (TNF- ; 10 ng/ml) for 24 h. Cell lysates from resting EC and TNF- -stimulated EC were analyzed by 16.5% tricine-SDS-PAGE. Anti-human ICAM-1 MAb (LB-2) directed against the first domain of ICAM-1 and polyclonal antibody against the cytoplasmic tail of ICAM-1 (R98) were used for detection by Western blotting. C: cell lysates were immunoprecipitated and immunoblotted with R98. Lane 1, R98; lane 2, R98 plus peptide 130145 (100 µg/ml); lane 3, R98 plus cytoplasmic tail peptide (100 µg/ml); lane 4, R98 plus cytoplasmic tail peptide (50 µg/ml). D: culture supernatants of human embryonic kidney (HEK)-293 wild-type (WT) cells immunoprecipitated with antibody recognizing ICAM-1 ectodomain (R803) and immunoblotted with LB-2. Cell lysate of PMA-stimulated HEK-293 WT cells served as a positive control. IP, immunoprecipitate; WB, Western blot. E: culture supernatant of HEK-293 WT cells in the presence or absence of PMA and EC in the presence or absence of TNF- clarified by centrifugation was precipitated with TCA and then subjected to 10% SDS-PAGE and Western blotting with LB-2.
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Cleavage in TNF-
-stimulated EC and PMA-treated HEK-293 cells expressing ICAM-1.
ICAM-1 cleavage was followed in EC treated with TNF-
(10 ng/ml) for varying time periods (Fig. 2A). Cell lysates analyzed on 16.5% tricine gels to detect low-molecular-weight proteins were immunoblotted with R98. The 7-kDa ICAM-1 fragment was detected on TNF-
-treated EC, but the fragment was not detectable in the absence of stimuli. Intact ICAM-1 expression was detectable at 2 h, whereas the fragment was generated at 6 h after stimulation. The expression of the 7-kDa fragment peaked at 3256 h after TNF-
stimulation, and the levels of the fragment exceeded those of intact ICAM-1 at the later periods. ICAM-1 cleavage also occurred after IL-1 stimulation (results not shown). Longer incubation with TNF-
did not result in the appearance of smaller ICAM-1 fragments with the intact cytoplasmic sequence. To establish the fraction of ICAM-1 that was cleaved, we estimated the percentage of cleaved ICAM-1 (7-kDa fragment) compared with that of intact ICAM-1 (93 kDa) on the basis of densitometric data for each time point (Fig. 2A). At 6 h, when cleavage becomes detectable,
40% of the expressed ICAM-1 was cleaved. Shedding gradually increased to
85% at 810 h. At 3256 h, the amount of cleaved ICAM-1 exceeded the level of expressed ICAM-1 by >60%, presumably because of the accumulation of cytoplasmic fragments inside the cell, whereas ICAM-1 was continuously expressed and shed. The cleavage of ICAM-1 and the appearance of the 7-kDa fragment in cell lysate directly corresponded to the appearance of sICAM-1 in culture medium (Fig. 2A, bottom blot).

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Fig. 2. Time course of ICAM-1 ectodomain shedding in EC and HEK-293 WT cells. A: EC maintained in DMEM with 20% FCS and EGM-2 were stimulated with TNF- (10 ng/ml) and incubated at 37°C. B: serum-depleted HEK-293 WT cells were stimulated with PMA (3 µM). At indicated time points, cells were lysed and precleared by centrifugation. Cell-free samples containing the equivalent amount of protein were subjected to 16.5% tricine-SDS-PAGE and Western blotting with R98. EC culture medium clarified by centrifugation was precipitated with TCA and subjected to 10% SDS-PAGE and Western blotting with LB-2 for the detection of soluble ICAM-1 (sICAM-1). C: serum-depleted HEK-293 WT cells were stimulated with 3 µM PMA for 3 h and subjected to nondetergent sequential cell lysis and subcellular fractionation (23). Aliquots of membrane (M) or cytosolic (C) fractions containing an equivalent amount of protein were subjected to 16.5% SDS-PAGE and immunoblotting with R98 (right) or protein silver staining (left).
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Although the expression and cleavage of ICAM-1 occurred upon stimulation in EC, there was a certain level of constitutive shedding of ICAM-1 in transfected HEK-293 WT cells. TNF-
and IL-1 were weak agonists in HEK-293 WT cells (data not shown). However, constitutive shedding was increased more than twofold upon PMA stimulation (Fig. 2B). Cleavage reached its maximum level at 3 µM PMA, and further increase of PMA concentration did not augment cleavage. Upon stimulation with 3 µM PMA, cleavage was upregulated at 1 h, reaching maximum levels between 3 and 12 h, and by 1824 h, the levels had returned to baseline (Fig. 2B). To establish the fraction of expressed ICAM-1 that is cleaved, we estimated the percentage of cleaved ICAM-1 vs. intact ICAM-1 for each time point and corrected for baseline constitutive shedding (by taking the nonstimulated background signal value as 0). Upon PMA stimulation (36 h) in HEK-293 WT cells, cleavage was increased by
92% above the constitutive level. In subsequent experiments, 3 µM PMA was applied for 3 h to assess cleavage in HEK-293 WT cells. It is interesting to note that ICAM-1 cleavage, mediated through diverse stimuli such as PMA and TNF-
and in different cell types, results in generation of the 7-kDa fragment. To establish whether the 7-kDa fragment is membrane bound, we performed nondetergent cell lysis and subcellular fractionation (24) followed by SDS-PAGE and immunoblotting with R98 and by protein silver staining of the membrane and cytosolic fractions. As shown on Fig. 2C, the 7-kDa fragment was detected only in the membrane fraction. This result indicates that the fragment is membrane bound. Interestingly, a small amount of full-length ICAM-1 was detectable in the cytosolic fraction.
Activation of signaling pathways upregulates cleavage.
To learn whether intracellular kinases are involved in the cleavage process, we used inhibitors of protein phosphatases (41). Dephostatin enhanced shedding in HEK-293 WT cells in both the absence and the presence of PMA (Fig. 3, A and B). At 10 µM dephostatin, cleavage was increased more than twofold. Dephostatin also appeared to increase the expression of intact ICAM-1 by
45%. At 0.1 mM, sodium orthovanadate (inhibitor of protein tyrosine phosphatase) potentiated PMA-induced shedding by
40% (Fig. 3C). These results suggest that reagents that maintain persistent activation of the signaling pathways induce ICAM-1 cleavage and therefore implicate protein phosphorylation in cleavage regulation.

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Fig. 3. Effect of protein phosphatase inhibitors dephostatin and sodium orthovanadate on ICAM-1 shedding in HEK-293 WT. Serum-depleted HEK-293 WT cells were preincubated with dephostatin or sodium orthovanadate for 3 h at 37°C. After 3 h, some cells were stimulated with 3 µM PMA and incubated for an additional 3 h. Cells were lysed and then subjected to SDS-PAGE and Western blotting as described in Fig. 2. Intensity of signals on autoradiograms was quantitated using UnScan-It software. Intensity of signals obtained from nonstimulated HEK-293 cells (A) or stimulated cells (3 µM PMA) (B and C) was interpreted as 100%, and degree of activation or inhibition was expressed accordingly as a percentage. Data shown represent at least 3 independent experiments.
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Signaling pathways involved in ICAM-1 shedding.
Src and MAP kinases (ERK1/2) become activated upon the binding of fibrinogen to ICAM-1 and are involved in the proliferation and survival process mediated through ICAM-1 ligation (20, 40). To investigate the involvement of Src kinases in ICAM-1 shedding, we examined the effect of the inhibitor of the Src family of kinases, PP2, and the inhibitor of pp60Src, geldanamycin (Fig. 4, A and B). At the concentrations applied in this study, these inhibitors blocked the ICAM-1-dependent cell proliferation of transfected HEK-293 cells and the survival of EC, as reported previously (21, 40). These reagents caused dose-dependent inhibition of the amount of cleavage in EC and HEK-293 WT cells. However, PP2 was effective in HEK-293 WT cells, whereas it was much less active in EC. At 20 µM, PP2 blocked ICAM-1 cleavage in EC by 55%, whereas the blockage appeared to be >90% in HEK-293 WT cells. The constitutive shedding of ICAM-1 in HEK-293 WT cells (in the absence of PMA) was also examined. PP2 at 20 µM inhibited constitutive shedding of ICAM-1 by 35%, whereas geldanamycin at 5 µM caused >60% inhibition. These results implicate Src kinase(s), specifically pp60Src, in ICAM-1 shedding in the two cell types studied and suggest a common signal driving the cleavage process through two different stimuli. Herbimycin and lavendustin, Src kinase inhibitors, also blocked PMA-induced cleavage of ICAM-1 (data not shown). To examine whether the MAPK pathway is involved in cleavage regulation, we applied MEK inhibitor PD-98059 (19, 22). Inhibition of MAPK dose-dependently inhibited cleavage of ICAM-1 in both EC and HEK-293 WT cells (Fig. 5A), indicating the involvement of the Ras-Raf-MEK cascade in the shedding process. PD-98059 (20 µM) also inhibited constitutive shedding of ICAM-1 in HEK-293 WT cells by >30%. Because PKC is known to induce MAPK activation, inhibition of PMA-induced shedding by the MEK inhibitor suggests that it may be a result of PKC-induced induction of MAPK. Inhibition was more prominent when HEK-293 WT cells were deprived of serum before the experiment. These results are consistent with the induction of MAPK signaling and ectodomain shedding by the growth factors (15, 22). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin also blocked shedding in activated EC and HEK-293 WT cells (Fig. 5B). It also inhibited the constitutive shedding of ICAM-1 in HEK-293 WT cells by 38% at 30 µM. The presence of the p38 inhibitor SB-203580 inhibited cleavage in EC, whereas in HEK-293 WT cells it was much less effective (Fig. 6A). The intensity of inhibition was 82% in EC, compared with 36% in HEK-293 WT cells. This reagent had virtually no effect on constitutive shedding of ICAM-1 in HEK-293 WT cells. To the contrary, phospholipase C inhibitor D609 was effective in HEK-293 WT cells (
60% inhibition), whereas in EC, it slightly augmented cleavage (Fig. 6B). Constitutive shedding was inhibited by 22% at 100 µM D609. It is important to note that in each of these inhibitor studies, the concentrations applied were in a range that did not significantly affect cell viability. These results demonstrate the involvement of the distinct cascades in the cleavage process and also suggest a preferred pathway for signal transduction mediating cleavage induced by different agonists in the cell systems tested. Constitutive shedding of ICAM-1 in HEK-293 WT cells appears to follow a pattern similar to that of PMA-stimulated shedding. However, we observed a lower degree of inhibition with constitutive shedding.

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Fig. 4. Involvement of Src kinase in ICAM-1 ectodomain shedding in EC and HEK-293 WT cells. EC were stimulated with TNF- (10 ng/ml) and incubated at 37°C. After 4 h, Src inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) (A, left) or geldanamycin (B, left) was added at indicated concentration and incubated for an additional 20 h. Serum-depleted HEK-293 WT cells were preincubated with PP2 (A, right) and geldanamycin (B, right) for 3 h at 37°C. After 3 h, cells were stimulated with PMA (3 µM) and incubated for an additional 3 h. Cells were processed, and results were analyzed as in Fig. 2. Data shown represent at least 3 independent experiments.
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Fig. 5. Role of MAPK and phosphatidylinositol 3-kinase (PI3K) in ICAM-1 ectodomain shedding in EC and HEK-293 WT cells. EC were stimulated with TNF- (10 ng/ml) and incubated at 37°C. After 4 h, MEK inhibitor PD-98059 (A, left) or PI3K inhibitor wortmannin (B, left) was added at indicated concentrations and incubated for an additional 20 h. Serum-depleted HEK-293 WT cells were preincubated with PD-98059 (A, right) or wortmannin (B, right) for 3 h at 37°C, stimulated with PMA (3 µM), and incubated for an additional 3 h. Cells were processed, and the results were analyzed as described in Fig. 2. Data shown represent at least 3 independent experiments.
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Fig. 6. Involvement of p38 kinase and phospholipase C (PLC) in ICAM-1 shedding in EC and HEK-293 WT cells. EC were stimulated with TNF- (10 ng/ml) and incubated at 37°C. After 4 h, p38 kinase inhibitor SB-203580 (A, left) or PLC inhibitor D609 (B, left) was added at indicated concentration and incubated for an additional 20 h. Serum-depleted HEK-293 WT cells were preincubated with SB-203580 (A, right) or D609 (B, right) for 3 h at 37°C. After 3 h, cells were stimulated with PMA (3 µM) and incubated for an additional 3 h. Cells were processed, and results were analyzed as described in Fig. 2. Data shown represent at least 3 independent experiments.
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Role of tyrosine phosphorylation in ICAM-1 cleavage.
We previously reported that ICAM-1 becomes phosphorylated at tyrosine residues 474 and 485 upon ligation with fibrinogen (39, 40). To assess the role of tyrosine phosphorylation sites in ICAM-1 shedding, we used HEK-293 cells transfected with ICAM-1 with single amino acid tyrosine-to-alanine substitution at positions 474, 476, and 485 and proline-to-alanine substitutions at 495 and 498. As shown in Fig. 7, PMA-stimulated cleavage was dramatically decreased in Y474A and Y485A, whereas it was marginally reduced in Y476A. This finding was consistent in 10 independent experiments. Moreover, in control mutations P495A and P498A, cleavage occurred under baseline conditions and was increased upon stimulation with PMA. These results demonstrate that tyrosine residues at 474 and 485 within the cytoplasmic domain of ICAM-1 appear critical for the cleavage process. To determine whether the 7-kDa fragment becomes phosphorylated, we examined tyrosine phosphorylation in HEK-293 WT cells at different time points upon treatment with PMA (Fig. 8A) or with pervanadate (Fig. 8B). Although a large number of intracellular proteins become phosphorylated at various time points upon stimulation, no detectable phosphotyrosine signal was observed in the region to which the 7-kDa fragment migrates. Moreover, by using immunoprecipitation with R98 and an alternative cell lysis protocol, allowing for better preservation of the tyrosine phosphorylation pattern (24), we still were not able to detect phosphorylation of the cytoplasmic fragment (data not shown). Our laboratory has reported that phosphorylated ICAM-1 becomes associated with SH2-containing phosphatase (SHP-2) (39). To verify whether the 7-kDa fragment becomes associated with SHP-2, we performed immunoprecipitation. The 7-kDa fragment was not associated with SHP-2 (Fig. 8C), which is consistent with the data shown in Fig. 8, A and B.

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Fig. 8. Tyrosine phosphorylation of ICAM-1 induced by PMA and pervanadate. HEK-293 WT cells were stimulated with PMA (3 µM) (A) or with pervanadate (0.5 mM sodium orthovanadate and 0.25 mM H2O2) (B) and incubated at 37°C. At indicated time points, cells were lysed and then subjected to SDS-PAGE and Western blotting. Blots were immunostained with R98 (A and B, left) and anti-phosphotyrosine antibody 4G10 (A and B, right). HEK-293 WT cells were stimulated with PMA (3 µM) for 3 h. Cell lysates were immunoprecipitated with R98 and anti-SHP-2 (Src homology-2 domain-containing phosphatase) antibody and then subjected to SDS-PAGE and Western blotting with R98 (C). Data shown represent at least 3 independent experiments.
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Functional implication of ICAM-1 shedding.
To establish the functional role of ICAM-1 shedding in intercellular interactions, we performed EC-monocyte adhesion experiments. Adhesion of monocytic cells (U-937) to EC was studied in the absence and presence of signaling inhibitors, which were found to interfere with the cleavage of ICAM-1. As shown in Fig. 9, the adhesion of monocytes to EC was significantly increased upon treatment with PP2 (
45% increase), PD-98059 (
48% increase), and SB-203580 (
72% increase), whereas D609, which served as a negative control, had virtually no effect on the degree of adhesion. Wortmannin affected the adherence of EC under culture conditions and showed variable results. These results indicate that shedding interferes with ICAM-1 function and affects intercellular adhesion. Downregulation of ICAM-1 cleavage upon treatment with inhibitors of signaling pathways augmented intercellular adhesion, probably increasing cell surface ICAM-1 availability for ligand interaction.
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DISCUSSION
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This study brings to light important features of ICAM-1 cleavage. 1) A 7-kDa ICAM-1 remnant is retained on HEK-293 WT cells and EC after stimulation with PMA and TNF-
, respectively. This fragment is not further degraded with longer or persistent stimulation. 2) ICAM-1 cleavage is regulated through MAPK, Src kinase, and PI3K pathways. 3) The tyrosine residues within the cytoplasmic sequence at 474 and 485 are critical for the cleavage process. 4) Although tyrosine phosphorylation sites are critical for cleavage, phosphorylation of the 7-kDa fragment was not detectable.
The present results demonstrate the role of the Ras-Raf-MEK cascade in the ICAM-1 cleavage regulation and extend the role for MAPK in ICAM-1 function (39, 40). The kinase-to-phosphatase ratio is essential for cell activity regulation. Phosphatase inhibitors dramatically accelerated cleavage, presumably causing persistent hyperactivation of kinases (Fig. 3). MAPK has been implicated in the cleavage of several cellular receptors, including TGF-
(15), syndecan-3 and -4 (19), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (52). p38 is involved in cleavage of several proteins, including TGF-
, TNF-
, L-selectin (15), TrkA (13), and HB-EGF (51). Interestingly, inhibition of p38 prevents the appearance in blood of sICAM-1 and several other adhesion molecules after endotoxin challenge in humans (17). Figure 10 summarizes our findings and presents a simplified scheme of putative signaling mechanisms involved in ICAM-1 shedding.

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Fig. 10. Signaling mechanisms mediating shedding of ICAM-1 in TNF- stimulated EC and PMA-stimulated HEK-293 WT cells. Signaling pathways predominantly mediating cleavage in EC upon stimulation with TNF- are shown in black; pathways inducing cleavage in HEK-293 WT cells activated with PMA are shown in dark gray. Common pathways activated in both cell systems are shown in light gray. Preferred pathways for TNF- -induced shedding of ICAM-1 in EC and PMA-induced shedding in HEK-293 cells are indicated with arrows. Inhibitors are shown in italics. In EC, TNF- binds to TNF- receptor 1 (TNF R1), which recruits TNF R1-associated death domain protein (TRADD). TRADD in turn forms a complex with TNF- receptor-associated factor 2 (TRAF2). TRAF2 activates several MAPK, including PI3K, Raf, and p38 (4). In HEK-293 cells, PMA induces activation of PKC, the best characterized effect of phorbol esters, followed by the activation of Src, Ras, PI3K, and PLC. PKC also can act directly on Raf, circumventing Ras involvement (33). Signaling pathways in both cell systems converge on Ras-Raf-MAPK pathway. Activation of Raf recruits MEK, which in turn activates ERK1/2, which in turn phosphorylates several downstream components, including transcription factors. It also downregulates upstream part of cascade phosphorylating inhibitory sites on MEK, Raf, and other components (26). Tyrosine residues 474 and 485 within cytoplasmic sequence of ICAM-1, which become phosphorylated upon ICAM-1 ligation with fibrinogen and bind SHP-2, are shown within circles. These residues appear to be essential for cleavage of ICAM-1, probably mediating assembly of signaling modules on ICAM-1 and activation of the signaling cascades, resulting in the secretion and/or activation of protease(s) and its translocation to the membrane-proximal region to cleave ICAM-1 in a juxtamembrane site.
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Single amino acid substitution at tyrosine residues 474 and 485 drastically reduced the amount of cleavage, implicating an important regulatory role for these residues in the cleavage process. Surprisingly, we could not detect phosphorylation of the 7-kDa cytoplasmic fragment of ICAM-1 remaining after ectodomain cleavage. This may be due to a transient and/or weak phosphorylation level that was beyond the detection level of our assay system. Although we were not able to provide direct evidence of phosphorylation of the cytoplasmic domain of ICAM-1, we think that dramatic reduction of cleavage upon tyrosine-to-alanine substitution at these critical positions serves as indirect evidence of the important role tyrosine phosphorylation plays in the shedding process. It is quite possible that phosphorylation at these two critical tyrosine residues mediates the assembly of signaling modules essential for cleavage. The cleavage may occur after ICAM-1 has been dephosphorylated. The signaling pathways become activated almost immediately upon ICAM-1:Fg ligation and remain so for 6090 min (40). By 2 h, ERK, SHP-2, and ICAM-1 itself become dephosphorylated. In PMA-stimulated HEK-293 cells, cleavage is upregulated 1 h after stimulation (Fig. 2). It is therefore possible that the protease activation necessary for cleavage takes place by the end of the dephosphorylation phase. Another intriguing possibility may be the transactivation pattern whereby cleavage occurs in nonphosphorylated ICAM-1 induced by a signal from phosphorylated ICAM-1, which remains intact. To our knowledge, this study is the first to demonstrate specific tyrosine residues regulating ectodomain cleavage.
Several other reports also have indicated involvement of a cytoplasmic sequence in ectodomain cleavage. Cleavage of L-selectin is regulated by the interaction of calmodulin with the cytoplasmic domain of L-selectin (28). The presence of COOH-terminal valine in the cytoplasmic domain of pro-TGF-
is required for the ectodomain cleavage (5, 7). On the contrary, the cytoplasmic tail is not required for ectodomain cleavage of p55 TNF receptor (6), TrkA (12), IL-6 (36), and human growth hormone receptor (hGHR) (1). PMA-induced shedding occurred with even higher intensity in truncated hGHR than in the full-length molecule (1). However, cleavage of TrkA resulted in the generation of a tyrosine-phosphorylated cell-bound fragment (8). Interestingly, phosphorylation of the cytoplasmic tail of angiotensin-converting enzyme has recently been shown to abrogate cleavage and promote its retention in the plasma membrane (29).
ICAM-1 appears to exist on the cell surface predominantly as a dimer, as shown by cross-linking studies and ultrastructural analysis (27, 42). Recently, ICAM-1 was reported to be shed as a dimeric molecule in the pleural space under inflammatory conditions (35). One can speculate that mutations within the cytoplasmic tail may affect the biochemical properties of ICAM-1, including its ability to dimerize, as well as cleavage. However, it seems unlikely, because putative dimerization sites are located within domain 1 (10) and within the transmembrane domain (42). Cleavage was normal in Y476A, P495A, and P498A. Thus we think that residues Y474 and Y485 have a more specific role in ICAM-1 cleavage.
Shedding of ICAM-1 may affect the extent of ongoing inflammation by regulating the amount of membrane-bound ICAM-1 available for interaction with ligands. Also, sICAM-1 is functionally active and retains the ability to inhibit leukocyte-EC interaction (30, 44). Signaling inhibitors that blocked ICAM-1 shedding (Figs. 36) also augmented monocytic cell adhesion to stimulated EC (Fig. 9). Therefore, we presume that increased availability of cell surface ICAM-1 in the presence of signaling inhibitors favors adhesive interactions. sICAM-1 has also been reported to promote angiogenesis (23) and induce the production of TNF-
, IFN-
, IL-6, and macrophage inflammatory protein-2 (37, 49). Thus sICAM-1 may also have proinflammatory potential. Therefore, ectodomain shedding affects the intensity of receptor-mediated reactions in at least two different ways: by regulating receptor density on the cell surface on the one hand and the concentration of soluble receptor on the other. Although surface localization restricts activity to microenvironment, shedding may result in more distal and/or more generalized effects (38). The combined effects of ICAM-1 shedding in a physiological setting could be highly complex.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-43721 and an Established Investigator Award from the American Heart Association (AHA) (to S. E. D'Souza) and by the AHA Ohio Valley Affiliate Postdoctoral Fellowship Award (to N. L. Tsakadze).
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FOOTNOTES
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Address for reprint requests and other correspondence: S. E. D'Souza, Dept. of Physiology and Biophysics, Univ. of Louisville, Health Sciences Center A-1115, Louisville, KY 40292.
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.
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