Tumor Necrosis Factor-α Converting Enzyme (TACE/ADAM-17) Mediates the Ectodomain Cleavage of Intercellular Adhesion Molecule-1 (ICAM-1)

Nina L. Tsakadze *, Srinivas D. Sithu *, Utpal Sen, William R. English1, Gillian Murphy1

and Stanley E. D’Souza Department of Physiology and Biophysics, University of Louisville, Louisville, KY 40292, USA; 1Department of Oncology, Cambridge Institute for Medical Research, Addenbrooke’s Hospital,

Cambridge, CB2 2XY, UK * These authors contributed equally to this paper.

Running Title: TACE (ADAM-17) induces ICAM-1 shedding Address correspondence to: Stanley E. D’Souza, Department of Physiology and Biophysics, Health Sciences Center A-1115, University of Louisville, Louisville, KY 40292, USA. Tel. 502-852-3194, E-mail: sedsou01@gwise.louisville.edu Ectodomain shedding has emerged as

an important regulatory step in the function of transmembrane proteins. ICAM-1, an adhesion receptor that mediates inflammatory and immune response, undergoes shedding in the presence of inflammatory mediators and PMA. The shedding of ICAM-1 in ICAM-1 transfected 293 cells upon PMA stimulation, and in endothelial cells (EC) upon stimulation with TNFα was blocked by metalloproteinase inhibitors, while serine protease inhibitors were ineffective. APMA, a mercuric compound, which is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3, but not TIMP-1 or -2, effectively blocked cleavage. This profile suggests the involvement of the ADAM family of protease in the cleavage of ICAM-1. The introduction of enzymatically active TACE in ICAM-1 expressing cells up-regulated cleavage. siRNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE (-/-) fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1 transfected TACE (+/+) cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cellular adhesive function, as ICAM-1 mediated cell adhesion was up-regulated in the presence of TACE siRNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage indicating that TACE mediated

cleavage of ICAM-1 may not be sequence specific.

ICAM-1, a cell adhesion molecule expressed on various cell types, plays a key role in mediating the inflammatory and immune responses (1). It functions as a costimulatory molecule during antigen presentation to T cells. ICAM-1 interaction with leukocyte integrins LFA-1 and Mac-1 promotes firm adhesion of leukocytes and their transmigration to the sites of inflammation (1-3). ICAM-1 consists of five Ig-like domains, a single transmembrane domain and a short cytoplasmic tail. ICAM-1 is shed as soluble ICAM-1 (sICAM-1) in blood and other biological fluids. Elevated plasma levels of sICAM-1 have been reported in inflammatory, neoplastic, autoimmune and vascular diseases (4), and is utilized as a marker of inflammation and a predictor of prognosis (5, 6). Multiple signaling pathways regulate the shedding of ICAM-1 in 293 cells transfected with ICAM-1 (293ICAM-1), including MAPK and PI-3 kinases (7). How these signaling cascades mediate ICAM-1 release is currently unknown. Previous reports indicate that ICAM-1 is a substrate for the matrix metalloprotease (MMP)-9 (8), as well as human leukocyte elastase and cathepsin G (9, 10).

Ectodomain shedding is an important regulatory mechanism in the function of membrane-bound cell surface molecules (reviewed in 11, 12). Cytokines and their receptors, growth factors, ectoenzymes, adhesion molecules and proteoglycans undergo shedding (13-16). The most widely studied inducer of shedding is phorbol 12-myristate-13 acetate (PMA), which activates protein kinase C (PKC) (16, 18-20).

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Calcium ionophores, cytokines, growth factors and chemotactic peptides also induce shedding (22). The majority of the shedding events are mediated by the zinc-dependent metalloproteinases of the metzincin family, which includes MMPs and proteases containing a disintegrin and a metalloproteinase domain (ADAMs). Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of MMPs and some ADAMs. There are four distinct TIMPs. TIMP-1, -2, -3 and -4 inhibit a wide range of MMPs, whilst TIMP-3 is also an inhibitor of ADAM-17. ADAM-10 is inhibited by TIMP-1 and TIMP-3 whereas ADAM-8 and -9 are very poorly inhibited by, and are unlikely to be physiologically regulated by, TIMPs. To date no ADAM has been shown to be inhibited by TIMP-2. ADAMs are type I transmembrane glycoproteins and in addition to their proteolytic activity, they also mediate cell adhesion. TACE plays a central role in ectodomain shedding (16). Cell lines derived from the TACE deficient mice (TACE-/-) verify that TACE is involved in the PMA-induced shedding of a number of structurally and functionally diverse proteins including pro-TNFα, L-selectin, β amyloid precursor protein (βAPP), TGFα, heparin binding-epidermal growth factor (HB-EGF) and vascular cell adhesion molecule-1 (VCAM-1) (14, 17-21), suggesting a common shedding mechanism regulated by a PKC-TACE axis. Mice lacking TACE have a severe phenotypic defect that resembles EGF receptor and TGFα deficient mice (23) suggesting an essential role for TACE in development and emphasizes the importance of ectodomain shedding in vivo (14). Our results now provide evidence that the TACE (ADAM-17) mediates ICAM-1 shedding. Shedding of ICAM-1 reduced the adhesive capacity of the cells. TACE cleavage site in ICAM-1 is not sequence specific but appears to be non-selective as has been reported for other TACE substrates.

EXPERIMENTAL PROCEDURES Reagents - Ethylenediamine tetraacetic acid (EDTA) and ethyleneglycol tetraacetic acid (EGTA) were purchased from J.T.Baker (Phillipsburg, NJ). Phosphoramidon and MMP inhibitor III (MMPI-III) were from Calbiochem (La Jolla, CA), soybean trypsin inhibitor (SBTI)

was from Gibco BRL (Paisley, UK), and BB-94 was from British Biotech (Oxford, UK). TNFα protease inhibitor-1 (TAPI-1) from Peptide International (Louisville, KY) was a kind gift from Drs. R. Grey and A. Spatola. TIMP-1, -2 and -3 were prepared as described previously (24-26). PMA, p-aminophenylmercuric acetate (APMA), phenylmethylsulfonyl fluoride (PMSF) and trichloroacetic acid (TCA) were obtained from Sigma (St. Louis, MO). The BCA protein assay kit was from Pierce (Rockford, IL) and electrochemiluminescence (ECL) detection reagents were from Amersham (Piscataway, NJ). R98 a polyclonal antibody directed against cytoplasmic sequence of ICAM-1 (AQKGTPMKPNTQATPP) was developed in our laboratory (7) as were antibodies against ICAM-1 ectodomain (R686 and R803). LB-2, a monoclonal antibody against ICAM-1 ectodomain, was from BD Bioscience (Franklin Lakes, NJ). Monoclonal antibodies against integrin subunits αL, αM and β2 were purchased from BioLegend (San Diego, CA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from BioRad (Richmond, CA). The antibody to TACE was obtained from QED Bioscience Inc. The ELISA kit for the detection of human sICAM-1 was purchased from Diaclone (Immuno-Diagnostic Systems, UK). Cell culture - 293 cells of human kidney fibroblast origin, obtained from the ATCC (Rockville, MD), were transfected with ICAM-1 as previously described (27, 28). 293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) F-12 (Biowhittaker, Walkersville, MD) complemented with 5 % fetal calf serum (FCS) and 1.0 mM L-glutamine at 37°C, 5% CO2, and 100 % humidity. Cells were plated on 24-well tissue culture plates (50,000/well) and grown to 70-80 % confluency. Human umbilical vein endothelial cells (EC) purchased from Clonetics (San Diego, CA) were grown in DMEM F-12 supplemented with 20 % FCS and EGM-2 (27). EC from passages 2-6 were used for this study. EC, removed by brief trypsin treatment, were coated on 12-well plates at a density of 25,000 cells/well and grown to confluence. TACE (-/-) and (+/+) fibroblasts (EC2 and EC4 cell lines) (14) were provided by Dr. R.A. Black (Immunex Corporation, Seattle, WA). EC2 and EC4 mouse fibroblasts were cultured in DMEM, 10 % FCS, 1

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mM L-glutamine, penicillin/streptomycin at 37°C, 5% CO2, and 100 % humidity. Monocytic cells, THP-1 were obtained from the ATCC (Rockville, MD) and cultured in DMEM with 5 % FCS. Transfection - Wild type Human TIMP3 cDNA (WT-hTIMP3) was a gift from Dr. D. Edwards (UEA, Norwich, U.K). Mutant C1S-TIMP3 cDNA (m-hTIMP3) a gift from Dr. M. Bond (29) (Bristol Heart Institute, Bristol, UK) was subcloned into pEGFP-N1 (Clontech, Palo Alto, CA) replacing the EGFP cDNA (30). The mouse TACE cDNA in pMOS (mTACE) was a gift from Celltech Chiroscience (Bothell, WA) (28). The TACE cDNA was subcloned into pCDNA3.1 zeo+ (Invitrogen, San Diego, CA) and a FLAG peptide inserted immediately preceding the proposed pro-protein convertase cleavage site using standard PCR technology and sequenced. In order to transiently express WT-hTIMP3, m-hTIMP3 or mTACE in 293ICAM-1 cells, cells were grown to 60-80% confluency in 12-well plates and transfected with 0.5-2.0 µg of the appropriate DNA using Lipofectamine PlusTM (Invitrogen). Briefly, for each well in transfection, DNA was diluted in 25 µl of DMEM without serum, containing 5 µl of Plus reagent. This mixture was incubated for 15 minutes at room temperature. 2 µl of Lipofectamine reagent were mixed into 25 µl of DMEM without serum. The diluted DNA and Lipofectamine reagent were mixed and incubated for 15 minutes at room temperature to allow DNA-liposome complexes to form. While complexes were being formed, the media on the cells was replaced with 0.4 ml of DMEM without serum. For each transfection, 50 µl of the DNA-liposome mixture was overlaid and mixed gently. The plate was incubated at 37˚C in a CO2 incubator. After 5 h of incubation, the media was replaced with media containing serum and incubated further. Cell lysates were assayed for transient gene expression 48 h after transfection. siRNA Transfection - TACE siRNA was purchased from Dharmacon (Lafayette, CO). Transfection of siRNA was performed using GenePORTER Transfection Reagent (Gene Therapy Systems, San Diego, CA). Briefly, 293ICAM-1 cells / EC were grown to 60-80% confluence in 24 well plates. 5 µl of the transfection reagent was diluted in 0.125 ml of DMEM. 2 µg of siRNA was also separately diluted in 0.125 ml of DMEM. They were mixed

together and incubated at room temperature for 45 min. The mixture was overlaid on the cells and incubated at 37°C in a CO2 incubator. After 2-5 hours, the overlaid siRNA-reagent mixture was diluted by adding 0.25 ml of complete medium containing 5% FCS and further incubated for 24-48 hours. The transfected 293 cells were stimulated with PMA (3µM) for 3 h. Transfected EC were stimulated with TNF-α (10ng/mL) for 16h. Both the non-stimulated and stimulated lysates were collected and stored at 4°C for analysis. Transfection of immortalized mouse fibroblasts EC2 and EC4 with human ICAM-1 cDNA and analysis of ICAM shedding - EC2 and EC4 fibroblasts were seeded into 6-well dishes in DMEM and transfected with either pcDNA3.1 alone or with human ICAM-1 cDNA using Fugene 6 (Roche Diagnosistcs, Indianapolis, IN) according to the manufacturer’s instructions for 16 h. The medium was then replaced with 1 ml pre-warmed fresh medium prior to incubation with or without BB-94 for 30 min at final concentration of 10 µM. Cells were then incubated with or without 3 µM PMA for 6 h. Medium was then harvested and subjected to centrifugation in a microfuge for 5 min at 16,000 x g before analysis by ELISA. EC2 and EC4 cell lines were found to transfect at different efficiencies. To derive a correction factor, separate wells of each cell line were transfected with 0.5 µg pEGFP-N1 (Clontech, Palo Alto, CA) in duplicate and EGFP fluorescence of the cells quantitated on a TECAN spectrafluor plus plate reader using the appropriate filters. Analysis of ICAM-1 shedding and inhibitor assays - Treatment of 293ICAM-1 cells was conducted under serum deprivation conditions. One hour before the experiment, media was replaced with DMEM containing 1% FCS. Media substitution caused little cell death, as cells remained firmly attached to the plate surface. Inhibitors were added at indicated concentrations and incubated for 3 h at 37°C. After stimulation with PMA (3 µM) cells were incubated for an additional 3 h at 37°C. EC were complemented with fresh medium supplemented with 10% FCS 1 h before the experiment. Cells were stimulated with TNFα and incubated for 6 h, after which time inhibitors were added as indicated and cells incubated for an

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additional 18 h at 37°C. After treatment, cell morphology and degree of adhesion were assessed and cell viability was estimated using trypan blue exclusion assay. Drug concentrations were maintained in a range that did not affect cell viability. After treatment, cells were rinsed with DMEM, and then lysed with ice-cold lysis buffer (Tris 10 mM, pH 7.5, EDTA 5 mM, NaCl 50 mM, Triton X-100 0.5%, SDS 0.1% Nonidet-40 1%). Protein concentration was measured using a BCA kit. Aliquots containing equal amount of total protein were analyzed on 16.5% Tricine SDS-PAGE (32). Proteins were transferred to PVDF membranes and immunoblotted with a primary antibody followed by an HRP-conjugated secondary antibody. Blots were developed using ECL detection system. Intensity of signals on autoradiograms was quantitated using UNSCAN-IT software. The signal intensity in the absence of inhibitors was taken as 100 % and the degree of inhibition was expressed accordingly as a percentage. Each data point represents results of at least three separate experiments and is presented as means ± SE. Adhesion assay - Adhesion of THP-1 cells to EC was performed as previously described (7). Briefly, EC grown on 24-well culture wells were stimulated with TNFα and treated with inhibitors. Monocytic THP-1 cells were gently added to the EC monolayer and incubated for 1 h at 37°C. Non-adherent cells were removed. The adherent cells were stained with 0.25% Rose Bengal. The excess stain was removed and cells were washed. The stain was extracted with ethanol-DPBS and optical density (OD) was measured at wavelength of 570 nm. The OD value from the non-stimulated EC in the absence of THP-1 cells was taken as background and subtracted from the other values. Each data point represents results of three separate experiments. Statistical analysis was performed with the Student’s t-test. Differences were considered significant when p ≤ 0.05. Cleavage site analysis - The cleavage site was assessed by molecular mass analysis using the Micromass Instrumentation that utilizes electrospray ionization (ESI) time-of-flight Mass Spectrometry (ESI/TOF-MS). The ~7kDa fragment of ICAM-1 was eluted out of gel, desalted and then were subjected to ESI/MS. Several point mutations at the “stalk” region of

WT-ICAM-1 on pcDNA3.1 (7, 27) namely R433A, R433K, R431A, E438A, R451A, N446A, RK441/442-AA were generated using Quick Change

TM site-directed mutagenesis kit

(Stratagene, La Jolla, CA). 293 cells were transiently transfected with each of the above mutations and PMA induced cleavage was examined and compared with WT-ICAM-1 as described above.

RESULTS MMPs regulate ICAM-1 shedding - Cleavage was monitored utilizing an antibody directed against the cytoplasmic sequence of ICAM-1, which recognizes both the full-length ICAM-1 (MW~93 kDa) and a cytoplasmic fragment (MW~7 kDa) remaining after ectodomain cleavage. In 293ICAM-1 cells, there is a certain level of constitutive shedding, which is increased 2-3 fold upon stimulation with PMA (7). The detection of the membrane-bound 7 kDa fragment in cell lysates corresponded to the appearance of sICAM-1 in the culture media (7). Protease inhibitors of different classes were utilized to identify enzyme(s) responsible for the shedding of ICAM-1. EDTA and EGTA – metal chelators of divalent cations dose-dependently inhibited cleavage of ICAM-1 in PMA-stimulated 293 cells and in TNFα-stimulated EC, indicating that a metal-dependent or metal-containing protease(s) was involved in the release of ICAM-1. EDTA and EGTA at 5 mM inhibited cleavage by over 80 % in 293ICAM-1 and by over 90 % in EC (Table 1). We observed no significant effect with SBTI and PMSF, thus indicating that serine proteases were unlikely to be involved in ICAM-1 shedding. However, three different inhibitors of MMPs were effective in blocking cleavage. Phosphoramidon, the least effective of the three reagents, displayed inhibitory activity at higher concentrations and at 80 µM caused 72 % inhibition in 293ICAM-1, while in EC the degree of inhibition was about 55 % (Table 1). Phosphoramidon is not a potent inhibitor of the metzincin class of metalloproteinases and its ineffectiveness is to be expected if an ADAM or MT-MMP is involved in shedding of ICAM-1. TAPI-1, known to inhibit conventional MMPs and ADAMs, at a concentration of 75 µM caused over 90 %

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inhibition in 293ICAM-1 cells. At even 10 µM, TAPI-1 was very effective. In EC the degree of inhibition was 65 % at 75 µM. MMPI-III (effective against MMP-1, -2, -3, -7 and -13) at 100 µM blocked cleavage by over 80 % in both cell types (Table 1). As the S2’ position is not known to contribute significantly to an inhibitor’s interaction with TACE, MMPI-III can be considered similar in properties to Ro-31-9790. MMPI-III has the same specificity for MMP-1, 2 and -3 as Ro-31-9790 and it is likely that its IC50 for TACE will be approximately similar, i.e. 30-50 µM (Calbiochem) (31), in agreement with its potency in inhibiting ICAM-1 shedding. These inhibitors yielded very similar effect on the constitutive shedding of ICAM-1 (results not shown).

The mercuric compound APMA is known to activate in vitro MMPs, as well as ADAMs (20). As shown in Fig. 1A, APMA up-regulated cleavage of ICAM-1 by over two-fold with the corresponding production of sICAM-1 in culture media in a dose- and time-dependent manner. Interestingly, the cytoplasmic fragment generated upon stimulation with APMA migrates at the same ~7 kDa level, as the one induced by PMA stimulation. Soluble ICAM-1 detected in culture media after APMA and PMA treatment is also indistinguishable (data not shown). Therefore, a protease(s) induced by these two compounds causes cleavage at a common site or at sites in close proximity. When APMA and PMA were applied simultaneously, no further increase in the amount of ICAM-1 shedding was observed (data not shown). These results indicate that ICAM-1 shedding is mediated by a member of the MMP-like family of proteases. The differing proteinase specificities of TIMPs permit the identification of whether MMPs or ADAMs are implicated in the ectodomain processing (33, 34). The effects of purified TIMP-1, -2 and -3 application at 500 nM concentration to stimulated EC and 293ICAM-1 was assessed. TIMP-1 had no effect on ICAM-1 shedding. TIMP-2 caused minimal inhibition, while TIMP-3 caused about 50 % inhibition in both cell types (Fig. 1B). At the concentrations used in our study, TIMP-1 and -2 inhibit almost all known MMPs and TIMP-1 would be expected to inhibit ADAM-10 (35). Transfection of TIMP-3 into 293ICAM-1 resulted in

the inhibition of ICAM-1 shedding by over 50% (Fig. 1C). TIMP-3 is active against TACE (31), while TIMP-1 and to a lesser extent TIMP-3 are effective against ADAM-10. Both ADAM-10 and -17 are present in EC, but only inhibition by TIMP-3 is observed. This strongly indicates that TACE is involved in the shedding of ICAM-1. The effectiveness of TAPI-1 in blocking ICAM-1 cleavage (Table 1) also indicates a possible role for TACE in ICAM-1 shedding. TACE (ADAM17) mediates ICAM-1 shedding - To determine if TACE mediates ICAM-1 shedding, TACE was transfected into ICAM-1 expressing 293 cells, and cells were stimulated with PMA. TACE increased the amount of ICAM-1 cleavage by over 100 % (Fig. 2A). These results indicate that TACE is a potential sheddase mediating ICAM-1 release. This was further verified by the application of siRNA directed against TACE. siRNA was transfected into 293 cells, cells were stimulated with PMA, and ICAM-1 cleavage was assessed using R98 antibody. We found that siRNA blocked ICAM-1 release in 293 cells. As shown on Fig. 2B (upper panel) PMA-stimulated shedding of ICAM-1 was inhibited by 57 %. In EC, TACE siRNA inhibited ICAM-1 shedding by 74 % (Fig. 2C, upper panel). Moreover, when we analyzed for the effectiveness of the siRNA, we found that siRNA completely blocked TACE expression in both cell types (Fig. 2B and 2C, lower panels). Interestingly, although siRNA was very effective in blocking the expression of TACE (over 95% inhibition in both cell types), cleavage of ICAM-1 was not completely inhibited, indicating a possible role for other proteases in the shedding process.

To confirm the involvement of TACE in shedding, fibroblasts derived from the TACE knock-out mice were transfected with ICAM-1 and cleavage was assessed by measuring the concentration of sICAM-1 in the culture media by an ELISA specific for human ICAM-1. As shown on Fig. 3, both constitutive and PMA-stimulated shedding was deficient in TACE-/- cells. Constitutive shedding of ICAM-1 was inhibited by 52 %, while PMA-stimulated release by 73 %. Both constitutive and stimulated shedding was sensitive to BB-94 - a broad spectrum and potent inhibitor of MMPs and ADAMs (33 % and 71 % of inhibition, respectively). These results confirm

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that TACE is required for both constitutive and PMA-induced ICAM-1 release. Functional implication of ICAM-1 shedding -We studied the effect of TACE inhibition on cell adhesion mediated through ICAM-1 (7). In the assay performed, THP-1 adhesion to TNF-α stimulated EC is a result of ICAM-1 ligation on EC with β2 integrins on THP-1. Anti-ICAM-1 antibody (LB2) when preincubated with EC at 20 µg/mL for 1h blocked THP-1 adhesion by 80% while antibodies against integrin subunits αL /αM (20 µg/mL), when preincubated with THP-1 cells blocked adhesion by 80%. Antibody against β2 integrin abolished 95% of THP-1 adhesion, thus indicating the specificity of the adhesive interaction.

EC were treated with TIMP and TACE siRNA as shown in Fig 1B and 2C, respectively. Monocytic THP-1 cells were allowed to adhere to EC to assess whether inhibition of cleavage affected ICAM-1 adhesive function. As shown on Fig. 4A, TIMP-3 dramatically (by 91%) enhanced adhesion of THP-1 cells to EC. Similarly, TACE siRNA up-regulated adhesion by 89 % (Fig. 4B). These results indicate that TACE-mediated ICAM-1 shedding reduced functionally intact ICAM-1 and consequently reduced EC adhesive function with leukocytes. Identification of ICAM-1 cleavage site -Analysis of previous reports, regarding TACE cleavage site in several different substrates demonstrated preference for the site with a hydrophobic residue at the membrane-proximal site (36-39). Mass spectrometric analyses of the 7 kDa PMA–induced fragment of ICAM-1 from 293 cells indicated heterogeneous fragmentation following residues R431 and R433 and K442, thus, suggesting non-selective cleavage as has been previously noted for other proteins (37, 38). For further verification on the cleavage specificity of ICAM-1, we introduced several point mutations in the membrane-proximal region of ICAM-1, including those identified by mass spectrometry to examine if cleavage would be hampered through mutations (Fig. 5). However, none of the mutations prevented the shedding of ICAM-1. In R431A we noted appearance of doublet, indicating that cleavage also occurs at the alternative site. These results indicate that the ectodomain cleavage site of

ICAM-1 is not sequence specific, and occurs in a non-selective manner.

DISCUSSION

This study provides evidence indicating that ICAM-1 shedding is mediated by TACE (ADAM-17). This evidence was developed initially by using protease inhibitors which suggested that the candidate sheddase was a metal-dependant protease belonging to the large family of MMPs or ADAMs (Table 1). Since TIMP-1 and -2 were ineffective in blocking ICAM-1 cleavage in both 293 cells and EC (Fig. 1B), the role for classical MMPs in ICAM-1 shedding was eliminated. The indication that TIMP-3 specifically blocked cleavage suggested the role for ADAMs in the shedding process (Fig. 1B and 1C). TIMP-3 is an effective inhibitor of TACE (31). We focused on TACE as this protease is present in 293 cells and EC and has been involved in PMA-induced cleavage of other proteins (16-21). Transfection of TACE into ICAM-1 expressing cells augmented ICAM-1 cleavage (Fig. 2A). The introduction of siRNA, to specifically inactivate TACE, in ICAM-1 expressing cells resulted in reduction of ICAM-1 cleavage (Fig. 2B and 2C). Further evidence was obtained using fibroblasts from TACE deficient mice. ICAM-1 transfected into TACE (-/-) fibroblast were resistant to cleavage upon stimulation with PMA, whereas TACE (+/+) cells processed ICAM-1 in a normal manner (Fig. 3). Therefore, ICAM-1 belongs to the growing number of transmembrane proteins representing substrates of TACE.

TACE has been recognized as an important mediator of the regulated ectodomain shedding. Previous studies have identified the preferential site for TACE cleavage as Ala-Val. However, more recent studies demonstrated efficient shedding by TACE at divergent cleavage sites (40). There is one Ala-Val pair located within the transmembrane domain of ICAM-1, and none within the juxtamembrane “stalk” domain, where the majority of the shedding events are known to occur (11). However, apart from the Ala-Val site in TNFα (36) and TGF-α (37), TACE cleaves at Lys-Ser in L-selectin (38) and amphiregulin (AR) (37), at Arg-Ser in ACE (39), and at Pro-Val in HB-EGF (37). Therefore, TACE seems to have

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preference for the site with a hydrophobic residue at the membrane-proximal side. We initially attempted to identify the site of ICAM-1 cleavage by mass spectrometry analysis. However, this indicated multiple cleavage sites. We, therefore, resorted to introducing point mutations in the membrane-proximal region of ICAM-1. None of the mutations prevented the shedding of ICAM-1 (Figure 5). Similar results have been shown in several earlier reports that mutations close to the cleavage site have had very little effect on processing of different TACE substrates (22, 41, 42).

There appears to be several structural characteristics of substrate that are important for its recognition by an enzyme, including sequence, length of stalk, distance from membrane, and features of distal ectodomain, as well as tertiary structure of enzyme itself (16). There is considerable variability between different TACE substrates as to which features are functional in individual molecules. Stalk sequence appears to be important in many instances, as substitution of stalk sequences from shed proteins to those of unshed conferred shedding in the latter group (41, 43). In the case of TNFα, cleavage site is sufficient for its cleavage by TACE, as cleavage still occurs even in the absence of ectodomain (36). With ACE, the conservation of the amino acid sequence is not essential for shedding. The critical parameter is conformation of stalk rather than sequence (44, 45). Length of stalk or distance from membrane appears to be important in the cleavage of TNFα, growth hormone receptor, L-selectin and ACE, presumably reflecting an essential role of the accessibility of cleavage site by the enzyme. Deletion of several residues in the membrane-proximal domain, even with preservation of the cleavage site prevented cleavage (36, 42). In the case of Kit ligand, chimeras containing repeated stalk sequences, the cleavage occurred only in the membrane-proximal cleavage site, emphasizing the importance of the distance from membrane (46). The shedding of the EGF receptor family members (TGFβ, AR, HB-EGF) is determined by stalk length and target bond sequence (37). Several studies using constructs combining shed and unshed molecules underline the importance of structural features of distal ectodomain for efficient cleavage (42, 45). Even with the same stalk sequence testis ACE is

cleaved more efficiently, probably due to a distal structural motif, which is recognized by the enzyme (47). Interestingly, the cleavage of ACE is dramatically increased with mutation in the close proximity to the cleavage site. This could perhaps be due to the optimal positioning of the ectodomain loop for the cleavage to occur (39). The catalytic domain of TACE shows distinctive surface projections, which may play a role in protease-substrate recognition (48), and may in fact contact site(s) remote from the actual cleavage site. While these factors may be functional in ICAM-1 shedding site recognition by TACE, cleavage itself appears to occur in a non-selective manner.

Recently, MMP-9 was shown to be implicated in ICAM-1 shedding in human promyelocytic leukemia cell line HL-60, and this process modified tumor cell resistance to natural killer cell-mediated cytotoxicity (8). In this study MMP-9 was shown to co-localize with ICAM-1 and induced its cleavage in vitro, which was blocked by the MMP-specific inhibitors. ICAM-1 is also susceptible to cleavage by elastase and cathepsin G (9). Alternative splicing of ICAM-1 modifies its susceptibility to cleavage by these enzymes with the common form being most resistant to cleavage, and may thus, modify immune interactions mediated through ICAM-1 (10). It is conceivable that different proteases may become activated under different conditions, with ICAM-1 being a substrate for more than one enzyme in different cell lines.

Several shed molecules can be cleaved by more than one enzyme in different cell lines and under different physiological conditions: APP shedding is mediated by ADAM-10 / -9 in 293 cells (49) and by TACE in CHO cells (50); similarly, HB-EGF cleavage is mediated ADAM-9 in Vero cells (51), and by the TACE in CHO cells (20); TNFα can be cleaved by ADAM-10 (52) and MMP-7 (53) in addition to TACE. The reverse is also true, some enzymes can cleave more than one substrate, and a good example is TACE. Up-regulation of the shedding rate by PMA is considered to be a common feature of the shedding events mediated by TACE. In our cell system cleavage was up-regulated upon stimulation with PMA (7). Similarly, in other reports ICAM-1 shedding was enhanced by the PMA (8). In contrast, ICAM-1 release was not found to be

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affected by the PMA in NIH 3T3 cells, while VCAM-1 shedding was significantly up-regulated (21). Thus, different cell types may vary considerably in their shedding characteristics. TACE was found to be involved in both constitutive and PMA-induced shedding of ICAM-1, yet it mediated only PMA-stimulated VCAM-1 shedding (21). We have previously shown that signaling mechanisms mediating ICAM-1 shedding in different cell types activated with different agonists have common as well as some unique features (7). Thus, the involvement of different proteases is quite possible. As ICAM-1 is expressed on diverse cell types, the differential regulation may be designed to target only selected cell populations under different conditions.

Diversity of ICAM-1 architecture and presentation on cell surface, including the ability to dimerize may affect its susceptibility to proteolysis by the different agents. Recently, ICAM-1 was shown to shed as a dimer in the pleural spaces during inflammation (54). It is not yet known how the protease and its substrate are brought together. It is believed that shedding of transmembrane proteins by ADAMs requires the presence of the membrane-bound enzyme and its substrate in cis orientation in the membrane (13).

The physiological role of ICAM-1 shedding is currently unknown. The processing of cell surface molecules by the ectodomain shedding may represent an important regulatory mechanism for cellular function (11). Mechanistically, removal of cell surface ICAM-1 prevents ICAM-1-mediated events (e.g. leukocyte adhesion/transmigration, fibrinogen deposition) and limits the extent of local inflammation. Since sICAM-1 is capable of ligand interaction, it may decrease ICAM-1-mediated interactions on the vasculature (55). Our results indicate that shedding interferes with ICAM-1 function and affects intercellular adhesion mediated through ICAM-1 (Figure 4A and 4B). Soluble ICAM-1 induces angiogenesis and is involved in the production of cytokines (56). Other studies demonstrate a unique role played by the membrane-anchored fragments, generated by the ectodomain cleavage. A membrane-anchored cytoplasmic fragment of CD44 is further cleaved, resulting in the release of a smaller cytoplasmic fragment, which translocates to the nucleus and functions as a transcriptional activator (57). Thus, ectodomain shedding may affect cellular functions in diverse ways.

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ACKNOWLEDGEMENTS

This work was supported by grants from the National Institutes of Health (HL43721), an Established Investigator Award from the American Heart Association (AHA) and Jewish Hospital Foundation Grant (999497) (to S.E.D.), the AHA Ohio Valley Affiliate Postdoctoral Fellowship Award (to N.L.T. and S.D.S.), The Wellcome Trust (G.M.) and The Medical Research Council (W.R.E. and G.M.). We would like to thank Drs. R.A. Black, M. Bond, D. Edwards, R. Grey, and A. Spatola for their generous gifts of reagents.

ABBREVIATIONS ADAM- a disintegrin and metalloproteinase domain containing protease; APMA – aminophenylmercuric acetate; ICAM-1 – intercellular adhesion molecule-1; MMP – matrix metalloproteinase; PMA – phorbol myristate acetate; TACE – TNF-alpha converting enzyme; TAPI-1 – TNF-alpha protease inhibitor-1; TIMP – tissue inhibitor of metalloproteinase, siRNA - small interference RNA.

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FIGURE LEGENDS Table 1. Effect of metalloprotease and serine protease inhibitors on ICAM-1 shedding in EC and 293ICAM-

1. Serum-depleted EC were stimulated with TNFα (10 ng/mL) for 6 h. Inhibitors were added and incubated for additional 18 h at 37°C. 293ICAM-1 cells were stimulated with PMA (3 µM) for 3 h at 37°C. Inhibitors were added at indicated concentrations and incubated for an additional 3 h. Cells were lysed, subjected to 16.5% Tricine SDS-PAGE and Western blotting with R98 (which recognizes the cytoplasmic tail of ICAM-1). Intensity of signals on autoradiogram was quantitated using Unscan-It software. Intensity of signals obtained from PMA-stimulated cells was taken as 100 % and degree of inhibition expressed accordingly as percentage. The data shown are representative of at least three independent experiments. Fig. 1. A, Effect of APMA. Serum-depleted 293ICAM-1 were incubated with APMA at indicated concentrations for 1 h. Cells were lysed and subjected to 16.5% Tricine SDS PAGE and Western blotting with R98 (left panel). Cell culture media was TCA-precipitated and subjected to 10 % SDS PAGE and Western blotting using R686 directed against ectodomain of ICAM-1 (right panel). B, Effect of TIMP-1, TIMP-2 and TIMP-3. Serum-depleted EC were stimulated with TNFα (10 ng/mL) for 6 h, TIMP-1, -2 and -3 were added at 500 nM and incubated for an additional 18 h at 37°C. Serum-depleted 293ICAM-1 cells were stimulated with PMA (3 µM) for 3 h at 37°C. TIMP-1, -2 and -3 were added at 500 nM and incubated for an additional 3 h. Cells were lysed and subjected to 16.5 % Tricine-SDS PAGE and Western blot with R98. C, Effect of TIMP-3 transfection. 293ICAM- 1 cell were transiently transfected with TIMP-3 cDNA. Following transfection cells were stimulated with PMA (3 µM) for 3 h. Cells were lysed at indicated time points and lysates were subjected to SDS PAGE, immunoblotting, and the results analyzed as described in legend for Table 1. The data shown are representative of at least three independent experiments. Fig. 2. A, Effect of TACE . ICAM-1 expressing 293 cells were transiently transfected with TACE cDNA. Following transfection, cells were stimulated with PMA for 3 h. Lysates were subjected to SDS PAGE and immunoblotting with R98. B, Effect of siRNA. ICAM-1 expressing 293 cells were transfected with TACE siRNA. Transfected cells were stimulated with PMA (3 µM) for 3 h at 37°C. Lysates were subjected to SDS PAGE and immunoblotting with R98 (upper panel) or anti-TACE antibody (lower panel). C, EC transfected with TACE siRNA were stimulated with TNF-α (10 ng/mL) for 16 h at 37°C. The stimulated cell lysates were subjected to SDS-PAGE and immunoblotting with R98 (upper panel) or anti-TACE antibody (lower panel). Fig. 3. ICAM-1 shedding in TACE WT and KO fibroblasts. Murine immortalized fibroblasts either -/- or +/+ for TACE were transfected with either pcDNA3.1 (vector control) or pCDNA3.1 expressing human ICAM-1 in the presence or absence of 3 µM PMA or 10 µM BB-94. Soluble ICAM-1 released into the medium was quantitated by ELISA. Fig. 4. The effect of ICAM-1 shedding on the adhesion of monocytic THP-1 cells to EC. TNF-α stimulated EC were treated with TIMP-1 or -3 (500 nM) for 6 h as in Fig 1B (A). EC were transfected with TACE siRNA and stimulated with TNF-α (10 ng/mL) for 16 h (B). THP-1 cells were allowed to adhere to EC. Adherent cells were stained with Rose Bengal and the OD of supernatant was measured at wavelength 570 nm. Each data point represents results of triplicate determinations from three separate experiments. Data are presented as means ± SE. * P ≤ 0.001 vs. control. Fig. 5. Identification of ICAM-1 cleavage site. Several point mutations were introduced at the “stalk” domain of WT-ICAM-1 of 293 cells. Serum-depleted cells were stimulated with PMA (3 µM) for 3 h at 37°C. Cells were lysed, subjected to 16.5 % Tricine-SDS PAGE and Western blotting with R98.

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Table 1

EC 293 WT Inhibitors

% Inhibition

EDTA (5mM) 95% 77%

EGTA (5mM) 95% 84%

Phosphoramidon (80µM) 55% 72%

TAPI-1 (75µM) 65% 95%

MMP Inhibitor-3 (100µM) 82% 88%

SBTI (200µg/mL) 0% 17%

PMSF (2mM) 7% 0%

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Figure 5.

Stanley E. D'SouzaNina L. Tsakadze, Srinivas D. Sithu, Utpal Sen, William R. English, Gillian Murphy and

ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1) converting enzyme (TACE/ADAM-17) mediates theαTumor necrosis factor-

published online December 6, 2005J. Biol. Chem. 

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