Tetraspanins Regulate ADAM10-Mediated Cleavage of TNF- Α and Epidermal Growth Factor

Tetraspanins Regulate ADAM10-Mediated Cleavage of TNF- α and Epidermal Growth Factor

This information is current as Cécile Arduise, Toufik Abache, Lei Li, Martine Billard, of September 25, 2021. Aurélie Chabanon, Andreas Ludwig, Philippe Mauduit, Claude Boucheix, Eric Rubinstein and François Le Naour J Immunol 2008; 181:7002-7013; ; doi: 10.4049/jimmunol.181.10.7002

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Tetraspanins Regulate ADAM10-Mediated Cleavage of TNF-␣ and Epidermal Growth Factor1

Cećile Arduise,*† Toufik Abache,*† Lei Li,*†‡ Martine Billard,*† Aure´lie Chabanon,*† Andreas Ludwig,§ Philippe Mauduit,*† Claude Boucheix,*† Eric Rubinstein,2,3*† and François Le Naour2*†

Several cytokines and growth factors are released by proteolytic cleavage of a membrane-anchored precursor, through the action of ADAM (a disintegrin and metalloprotease) metalloproteases. The activity of these proteases is regulated through largely unknown mechanisms. In this study we show that Ab engagement of several tetraspanins (CD9, CD81, CD82) increases epidermal growth factor and/or TNF-␣ secretion through a mechanism dependent on ADAM10. The effect of anti-tetraspanin mAb on TNF-␣ release is rapid, not relayed by intercellular signaling, and depends on an intact MEK/Erk1/2 pathway. It is also associated with a concentration of ADAM10 in tetraspanin-containing patches. We also show that a large fraction of ADAM10 associates with Downloaded from several tetraspanins, indicating that ADAM10 is a component of the “tetraspanin web.” These data show that tetraspanins regulate the activity of ADAM10 toward several substrates, and illustrate how membrane compartmentalization by tetraspanins can control the function of cell surface proteins such as ectoproteases. The Journal of Immunology, 2008, 181: 7002–7013.

embers of the ADAM (a disintegrin and metallopro- EGF cleavage site leads to a similar defect in heart valve devel- tease domain)4 family of membrane-anchored zinc-de- opment (7). http://www.jimmunol.org/ M pendent metalloproteases play a key role in the con- A central question raised by the essential role of proteolysis as version of membrane-anchored precursors of growth factors or regulator of ectodomain shedding is how this process is regulated. cytokines into soluble mediators and in the ectodomain shedding The fact that several G protein-coupled receptor (GPCR) ligands of various transmembrane proteins (1–4). The importance of are able to transactivate the EGF receptor by a mechanism involv- ADAM-mediated ectodomain shedding is highlighted by the fact ing ADAM metalloproteases (including ADAM10 and ADAM17) that ADAM10 knockout mice die during embryonic development and EGFR ligands strongly suggests that GPCR activation up-reg- (5), whereas ADAM17 inactivation leads to perinatal lethality, ulates ADAM metalloprotease activity (8). In contrast, the activity

probably as a consequence of defects in heart development (6). of ADAM10 and ADAM17 toward certain substrates can be rap- by guest on September 25, 2021 Interestingly heparin-binding epidermal growth factor (HB-EGF) idly up-regulated by ionomycin or 12-O-tetradecanoyl-phorbol 13- is a substrate for ADAM17, and a knock-in deletion of the HB- acetate (TPA), respectively, but the underlying mechanisms remain largely unknown (2, 9–11). Notably, the ability of TPA to up-regulate ADAM17-mediated TNF-␣ cleavage does not require *INSERM U602, Villejuif, France; †Universite´ Paris-Sud, Institut Andre´ Lwoff, the ADAM17 cytoplasmic domain (12), suggesting a possible reg- Villejuif, France; ‡Institute of Urology, The First Affiliated Hospital, Xi’an Jiaotong ulation of its activity at the level of the plasma membrane. University, Xi’an, Shaanxi, China; and §Institute for Pharmacology and Toxicology, RWTH Aachen University, Aachen, Germany The analysis of the sheddases responsible for soluble TNF-␣ Received for publication September 20, 2007. Accepted for publication September generation provides a striking example of a cellular regulation of 19, 2008. an ADAM protease. TNF-␣ is a multifunctional cytokine that The costs of publication of this article were defrayed in part by the payment of page plays a key role in inflammation, autoimmunity, and antitumor charges. This article must therefore be hereby marked advertisement in accordance reaction, and mediates the response to infection (13–15). with 18 U.S.C. Section 1734 solely to indicate this fact. 1 ADAM17/TACE is the primary sheddase responsible for pro- This work was supported by Grants from the Agence Nationale pour la Recherche, ␣ from the Groupement des Entreprises Françaises dans la Lutte contre le Cancer Paris TNF- cleavage in T cells or myeloid cells. Evidence for the Ile de France, the Association pour la Recherche contre le Cancer, and Nouvelles prominent role of ADAM17 in the shedding of TNF-␣ is provided Recherches Biome´dicales-Vaincre le Cancer. T.A. and C.A. were supported by fel- by the finding that mouse T cells or mouse embryonic fibroblasts lowships awarded by the French government. T.A. and L.L. were recipients of a fellowship awarded by the Association Nouvelles Recherches Biome´dicales. L.L. was (MEF) homozygous for a targeted mutation in ADAM17 that in- also the recipient of a fellowship from the Fondation Franco-Chinoise pour la Science activates metalloproteinase activity are strongly deficient in their et ses Applications. A.L. is supported in part by the Interdisziplina¨res Zentrum fu¨r ␣ Klinische Forschung Biomat, RWTH Aachen University, and by Sonderforschungs- ability to shed TNF- (12, 16, 17). More recently it was reported bereich 451, project A12 of the Deutsche Forschungsgemeinschaft. that ADAM17 inactivation in myeloid cells reduced TNF-␣ secre- 2 E.R. and F.L.N. contributed equally to this work. tion in vivo (18). Among ADAM proteases ADAM10 is the most 3 Address correspondence and reprint requests to Dr. Eric Rubinstein, INSERM, closely related to ADAM17 (4) and was initially purified in two U602, 14 Avenue Paul Vaillant Couturier, F-94807 Villejuif Cedex, France. E-mail studies based on its ability to cleave purified pro-TNF-␣ or a address: [email protected] TNF-␣ peptide encompassing pro-TNF-␣ cleavage site (19, 20). 4 Abbreviations used in this paper: ADAM, a disintegrin and metalloprotease; HB- EGF, heparin-binding epidermal growth factor; TPA, 12-O-tetradecanoyl-phorbol 13- Transfection of ADAM10 was shown to induce the release acetate; MEF, mouse embryonic fibroblast; HEK, human embryonic kidney; siRNA, of TNF-␣ from 293EBNA cells (19). However, in subsequent small interfering RNA; PKC, protein kinase C; GPCR, G protein-coupled receptor; studies the transfection of ADAM10 in ADAM17Ϫ/Ϫ MEF did not HA, hemagglutinin. increase the shedding of TNF-␣, and ADAM10Ϫ/Ϫ MEF were Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 shown to process TNF-␣ normally (12, 17). Altogether, these data www.jimmunol.org The Journal of Immunology 7003 suggest that the ability of ADAM10 to cleave TNF-␣ can be lyzed 24 h after the second electroporation. For HEK-293, cells were al- tightly controlled at the cellular level by an unknown mechanism. lowed to recover in complete DMEM for 24 h following electroporation, Tetraspanins compose a family of 33 integral membrane pro- then were trypsinized and submitted to a second ADAM10 siRNA transfection using the INTERFERin reverse protocol as described by the man- teins with four transmembrane domains delimiting three short ufacturer (Polyplus Transfection). Cells were then analyzed for EGF se- intracellular domains and two extracellular regions of unequal cretion 36 h later as described below. In most experiments the siRNA size. They exhibit significant sequence identity as well as spe- oligonucleotide targeting the ADAM10 sequence 5Ј-GGA TTA TCT TAC cific structural features in the large extracellular domain. They are AAT GTG G-3Ј was used. A nonactive siRNA targeting the CD82 sequence 5Ј-ACC TCC TCC AGC TCG CTT A-3Јwas used as a control. In widely distributed and have been implicated in a large variety of some experiments a stealth siRNA (catalog no. HSS100165; Invitrogen) physiological processes such as cell migration, cell fusion (includ- targeting the ADAM10 sequence 5Ј-TAC ACC AGT CAT CTG GTA TTT ing macrophage fusion and sperm-egg fusion), and activation of CCT C-3Ј was also used. lymphoid cells (21–24). Tetraspanins play a role in infection by Recombinant proteins, mAbs, and flow cytometry several viruses including HIV and human T cell leukemia virus (25, 26). Additionally, the tetraspanin CD81 is required for the A recombinant human ADAM10 ectodomain protein was purchased from infection of hepatocytic cells by two major pathogens, the hepatitis Chemicon International. Anti-tetraspanin mAbs used in this study were SYB-1, ALB-6, TS9 (CD9), TS53 (CD53), TS63 (CD63), TS81 (CD81), C virus and the malaria parasite (27–29). TS82 (CD82), TS82b (CD82), TS151 (CD151; Refs. 30, 32, 50), and 5A6 At the molecular level, tetraspanins are believed to be organizers (CD81; Refs. 51). The CD46 mAb (11C5) and the CD55 mAb (12A12) ␣ ␤ of particular microdomains on the plasma membrane that are dif- have been previously described (52). The anti-integrin 4 1 mAb (HP2/1), ferent from the so-called lipid rafts and are collectively referred to the CD19 mAb (B4), and the CD28 mAb (CD28.2) were obtained from Beckman Coulter, the ␤-actin mAb (AC-74) was obtained from Sigma- Downloaded from as the “tetraspanin web” (21–24, 30). These microdomains criti- Aldrich, the CD3 mAb (OKT3) was purchased from American Type Cul- cally depend on the interaction of tetraspanins with one another ture Collection, and the anti-HA mAb (HA-11) was purchased from Covance through a mechanism involving protein palmitoylation and inter- Research Products. The anti TNF-␣ mAbs B-C7 and B-D9 (PE-labeled) were action with lipids (31–36). Tetraspanins also associate with a num- obtained from Diaclone. An anti-phospho Erk1/2 mAb (E10) and a polyclonal ber of nontetraspanin proteins. Although the association with in- Ab to Erk1/2 were obtained from Cell Signaling Technology. Flow cytometry analysis was performed as previously described (53). tegrins and proteins with Ig domains is well characterized (21–23), To generate mAbs, BALB/c mice were injected i.p. three times with 107 recent proteomic analysis demonstrated the presence of several Jurkat cells. Spleen cells were fused with P3X63AG8 mouse myeloma http://www.jimmunol.org/ ectoenzymes in the tetraspanin web, including ADAM10 (37, 38). cells (5 ϫ 107 and 3 ϫ 107 cells respectively) according to standard tech- Tetraspanins may influence the proteins with which they associate niques and distributed into a 96-well tissue culture plate. After 2 wk hybridoma culture supernatants were harvested and tested for Jurkat cell in several ways. For example, CD81 participates in the traffic of staining by indirect immunofluorescence and analyzed using a microplate CD19 to the cell surface in lymphoid B cells and modulates its fluorescence reader (CytoFluor II, Applied Biosystems) and a FACSCali- cosignaling activity (36, 39). CD151 is not essential for the ex- bur flow cytometer (BD Biosciences). Positive supernatants were then fur- pression of the integrins with which it associates (40), but regulates ther characterized by immunoprecipitation. Beside 11G2, the mAbs TS53, post-ligand binding events such as adhesion strengthening and sig- TS81, and TS82 were produced this way. naling (41–43). For most other proteins, including ectoenzymes, Ag purification, in-gel enzymatic digestion, and mass the functional consequences of the association with tetraspanins is spectrometry analysis by guest on September 25, 2021 unknown. We now provide evidence that tetraspanins regulate For purification of 11G2 target Ag 5 ϫ 108 HEL cells were lysed in 12 ml ADAM10 proteolytic activity. of lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and protease inhibitors. Insoluble material was removed by Materials and Methods centrifugation at 12,000 ϫ g for 30 min, and the lysate was successively precleared five times with Sepharose 4B beads (Amersham Biosciences) Cells, cell culture, and generation of transfected cell lines coupled to BSA. After another centrifugation, the lysate was incubated for The lymphoid T cell line Jurkat, the erythromegakaryocytic cell line HEL, 3 h with Sepharose 4B beads coupled to mAb 11G2. The beads were and the B lymphoid cell lines Raji, Raji/CD9, Daudi, and Daudi/CD9 have washed five times with lysis buffer, and the immunoprecipitated proteins been previously described with respect to tetraspanin expression (30, 44, were separated by 5–15% SDS-PAGE under nonreducing conditions. Sil- 45). All hematopoietic cell lines (see Table II) were cultured in RPMI 1640 ver staining, gel piece preparation, and mass spectrometry analysis were medium supplemented with 10% FCS, GlutaMAX, and antibiotics (all performed as previously described (53). from Invitrogen). Human embryonic kidney 293 (HEK-293) cells and the prostate cell line PC3 were cultured in DMEM similarly supplemented. Immunoprecipitation and Western blotting Transfection of TNF-␣ in Raji cells was performed as previously described Labeling of cell surface proteins with EZ-Link-Sulfo-NHS-LC-Biotin (30), and positive cells were sorted using a FACSVantage cell sorter after (Pierce) and immunoprecipitations in the presence of Brij97 were per- fluorescent labeling. HEK-293 cells stably expressing GFP-tagged rat pro- formed as described previously (50). For two-step immunoprecipitations EGF were selected under G-418 following transfection with ExGen (Eu- tetraspanins were immunoprecipitated from Brij 97 lysates, and coimmu- romedex) as described previously (46), and positive fluorescent cells were noprecipitated proteins were eluted in the presence of 1% Triton X-100 and sorted as above. For the construction of a C-terminal GFP-tagged pro-EGF 0.2% SDS before the second immunoprecipitation. Biotin-labeled cell sur- expression plasmid, the pro-EGF cDNA in pcD12-HA plasmid (where face proteins were visualized using labeled streptavidin. Western blotting “HA” is hemagglutinin; Ref. 46) was excised following HindIII/ApaI re- on immunoprecipitates was performed using biotinylated mAbs followed striction and subcloned in-frame into the pEGFP-N2 expression vector (BD by labeled streptavidin. We used a streptavidin-biotinylated HRP complex Clontech). The human TNF-␣ plasmid was provided by Dr. F. Peiretti for ECL (PerkinElmer Life Sciences) revelation or Alexa Fluor 680-la- (INSERM U626, Marseille, France) (47). PBMC were isolated as previ- beled streptavidin (Invitrogen) for the use of the Odyssey Infrared Imaging ously described (48). The cells were used after removal of adherent cells System (LI-COR Biosciences). and CD34-positive cells. They were activated with either PHA-P (1/2000; Difco), a combination of CD3 and CD28 mAb (0.25 and 1 ␮g/ml, respec- Quantification of TNF-␣ release by ELISA and inhibitors tively), or TPA (10 ng/ml). Cells were washed three times in PBS and cultured in complete RPMI 1640 RNA silencing medium with or without 10 ␮g/ml mAb or 10 ng/ml TPA. All mAb used in this assay are of the IgG1 subclass. After incubation at 37°C, the cells were Cells (5–10 ϫ 106 cells in 400 ␮l RPMI 1640 medium) were transfected removed by centrifugation at 200 ϫ g for 15 min. The concentration of with synthetic small interfering RNA (siRNA) oligonucleotides (200 pmol) TNF-␣ in the supernatant was determined using a TNF-␣ ELISA kit ac- by electroporation at room temperature (49) using the Gene Pulser appa- cording to the manufacturer’s instructions (Diaclone). In some experiments ratus (Bio-Rad). The settings were 300 V and 500 microfarads. For Raji the cells were preincubated 30 min before addition of mAb with different cells, electroporation was performed twice 24 h apart and cells were ana- inhibitors: GM6001 (40 ␮M), TAPI-2 (5 ␮M), bisindolylmaleimide I 7004 TETRASPANINS REGULATE ADAM10

(GF109203X, 2 ␮M), Herbimycin A (2 ␮M), U0126 (50 ␮M), and SB203580 (20 ␮M) were obtained from Calbiochem. GI254023X (3 ␮M) and GW280264X (3 ␮M) have been previously described (54). Statistical analysis was performed using the one-way ANOVA followed by the Tukey multiple comparison test. Quantiﬁcation of EGF ectodomain release by Western blotting HEK-293 cells were serum starved overnight before incubation in DMEM supplemented with or without 10 ␮g/ml mAb or 1 ␮M TPA for the indicated time. Cells and conditioned medium were collected and prepared as previously described (46) and analyzed by Western bloting for the presence of EGF species using a combination of HA-11 mAb and Alexa Fluor 680- labeled anti-mouse Ab (Invitrogen). Data acquisition was performed using the Odyssey Infrared Imaging System (LI-COR Biosciences). Confocal microscopy Raji or Raji/TNF-␣ cells were treated or not with Alexa Fluor 568-labeled C CD82 or CD53 mAb (TS82b and TS53) for 2 h, resuspended in PBS supplemented with 0.1% BSA, and chilled at 4°C for 30 min. A second Ag staining was then performed for 45 min at 4°C using mAb (12A12 to CD55; 11G2 to ADAM10; B-C7 to TNF-␣) labeled using the Alexa Fluor

488 Zenon Mouse IgG1 Labeling kit (Invitrogen). Nonstimulated cells Downloaded from were also stained at the same time using labeled CD82 or CD53 mAbs. After three washes in PBS/BSA, the cells were deposited on poly-lysine coated coverslips, fixed 4 min at room temperature with 4% paraformal- dehyde in PBS and then 15 min in Ϫ20°C methanol, and finally mounted in Mowiol. Analysis was performed with a TCS SP1 confocal microscope (Leica Microsystems). http://www.jimmunol.org/ Results D E Production of an anti-ADAM10 mAb selected on its ability to coimmunoprecipitate CD81 To identify new molecules associating with tetraspanins, mice were immunized with the lymphoid T cell line Jurkat, and hybridoma supernatants were selected for their ability to coimmunoprecipitate the ϳ24-kDa tetraspanin CD81. This selection step was performed after biotin-labeling of cell surface proteins and cell lysis in the presence of Brij 97, a detergent preserving any number by guest on September 25, 2021 of interactions within the tetraspanin web, including tetraspanin/ tetraspanin interactions. Except for several anti-tetraspanin mAb, only 1 of 200 hybridoma supernatants tested coimmunoprecipitated a molecule comigrating with CD81, showing the specificity of the interaction. The major band immunoprecipitated by this ϳ mAb (11G2) has a Mr of 67,000 under nonreducing conditions, FIGURE 1. Generation and characterization of an anti-ADAM10 mAb compatible with that of ADAM10, and comigrated with a major screened for its ability to coimmunoprecipitate CD81. A, During the initial band present in the CD81 immunoprecipitate (Fig. 1A). screening, hybridoma supernatants were tested for their ability to coimmu- The protein recognized by mAb 11G2 was purified on a 11G2 noprecipitate CD81 after cell labeling with biotin and lysis in the presence affinity column from ϳ 5 ϫ 108 HEL cells lysed in 1% Triton of Brij 97. The mAb 11G2 immunoprecipitated (IP) a major 67-kDa mol- X-100 (to disrupt the tetraspanin web), yielding two major bands ecule (arrow) and was selected for further analysis because it precipitated (Fig. 1B). After digestion with trypsin and MALDI-TOF mass a band comigrating with CD81. B, The 11G2 target Ag was purified from spectrometry analysis, 36 peptides from the higher band and 24 HEL cells lysed in Triton X-100 on an 11G2 affinity column. Two major from the lower band matched peptides expected for the metallo- bands were visualized by silver staining after SDS-PAGE. C and D, PC3 cells were transfected with a control siRNA (si ctrl) or an siRNA-targeting protease ADAM10 (National Center for Biotechnology Informa- ADAM10 (si ADAM10) and analyzed after 48 h. C, Binding of mAb 11G2 tion accession number 1616601). The peptides covered 47 and was determined by indirect immunofluorescence and flow cytometry. D, 36% of the protein, respectively. The lower band lacked peptides Cells were lysed and the level of 11G2 target Ag was determined by West- covering the N-terminal part of the protein that corresponds to ern blot (WB) analysis. The mAb 11G2 recognizes under nonreducing ADAM10 prodomain, which is removed upon maturation of the conditions two bands of 67 and 80 kDa (arrows), which are consistent with protein. Thus the upper band corresponds to the unprocessed pre- the mature and immature forms of ADAM10, respectively, and these two cursor protein and the lower band to the mature ADAM10 (Table bands were strongly decreased after ADAM10 silencing. E, The ability of I). It should be noted that the lower band is diffuse and is bordered mAb 11G2 to recognize recombinant ADAM10 ectodomain (100 ng) was examined by Western blotting after SDS-PAGE under nonreducing by a more intense thin band with a Mr lower than expected for mature ADAM10. This band probably corresponds to a postlysis conditions. cleavage fragment of ADAM10 generated in Triton X-100 (our unpublished data). No peptide covering the intracellular region was recovered from this band (Table I). blotting two bands of 67 and 80 kDa (under nonreducing condi- In addition, silencing ADAM10 in PC3 cells reduced by ϳ80% tions), which are consistent with the mature and immature forms of the labeling of cells by mAb 11G2, as determined by flow cytom- ADAM10, and these bands were reduced after silencing ADAM10 etry analysis (Fig. 1C). The mAb 11G2 recognizes in Western (Fig. 1D). Finally, 11G2 recognized recombinant ADAM10 The Journal of Immunology 7005

Table I. Assignment of peptide masses to the human ADAM10 sequencea

m/z Submitted MHϩ Ion Matched Delta ppm Peptide Sequence Start End Upper Band Lower Band

1790.0396 1789.8504 106 HYEGLSYNVDSLHQK 31 45 ϩϪ 1201.7545 1201.5960 132 AVSHEDQFLR 52 61 ϩϪ 1847.0216 1846.8705 82 DTSLFSDEFKVETSNK 78 93 ϩϪ 997.6545 997.5107 144 FEGFIQTR 124 131 ϩϪ 1225.7438 1225.5853 129 GGTFYVEPAER 132 142 ϩϪ 1629.8802 1629.8271 32 GGTFYVEPAERYIK 132 145 ϩϪ 2325.3776 2325.1299 107 TLPFHSVIYHEDDINYPHK 148 166 ϩϪ 1679.9353 1679.7236 126 YGPQGGCADHSVFER 167 181 ϩϪ 3067.7865 3067.5159 88 YQMTGVEEVTQIPQEEHAANGPELLRK 185 211 ϩϪ 1928.1618 1927.9376 116 NTCQLYIQTDHLFFK 220 234 ϩϩ 659.3891 659.3153 112 YYGTR 235 239 ϩϩ 1281.7701 1281.7167 42 EAVIAQISSHVK 240 251 ϩϩ 1701.0722 1700.8495 131 AIDTIYQTTDFSGIR 252 266 ϩϩ 838.5107 838.4497 73 NISFMVK 267 273 ϩϩ 994.6239 994.5508 73 NISFMVKR 267 274 ϩϩ ϩϩ 276 267 ءNISFMVKRIR 127 1279.7309 1279.8931 846.5223 846.4110 131 DPTNPFR 285 291 ϩϩ 1731.1110 1730.8860 130 DPTNPFRFPNIGVEK 285 299 ϩϪ 903.5526 903.4934 65 FPNIGVEK 292 299 ϩϩ Downloaded from 2749.5558 2749.2204 122 FLELNSEQNHDDYCLAYVFTDR 300 321 ϩϩ 2494.2010 2494.1560 18 DFDDGVLGLAWVGAPSGSSGGICEK 322 346 Ϫϩ 1025.5584 1025.5626 4 SKLYSDGKK 347 355 ϩϪ 2025.2160 2025.0769 69 SLNTGIITVQNYGSHVPPK 357 375 ϩϩ 1771.0045 1770.8592 82 NLGQKENGNYIMYAR 406 420 ϩϪ 1230.7277 1230.5577 138 ENGNYIMYAR 411 420 ϩϩ

1790.0561 1789.8179 133 ENGNYIMYARATSGDK 411 426 ϩϪ http://www.jimmunol.org/ 2025.2160 2025.0187 97 ATSGDKLNNNKFSLCSIR 421 438 ϩϩ 882.5823 882.4507 149 FSLCSIR 432 438 ϩϩ 1597.6845 1597.6011 52 DECCFDANQPEGR 481 493 Ϫϩ 1986.8036 1986.7930 5 QCSPSQGPCCTAQCAFK 502 518 Ϫϩ 1961.8779 1961.8632 7 HTQVCINGQCAGSICEK 558 574 Ϫϩ 1610.7692 1610.6435 78 DDKELCHVCCMK 589 600 Ϫϩ 1898.0837 1897.8537 121 KMDPSTCASTGSVQWSR 601 617 ϩϩ 1770.0028 1769.7587 138 MDPSTCASTGSVQWSR 602 617 ϩϩ 1605.9787 1605.7695 130 TITLQPGSPCNDFR 623 636 ϩϩ 1047.5803 1047.4392 135 GYCDVFMR 637 644 ϩϩ 1026.7038 1026.5584 142 LVDADGPLAR 647 656 ϩϩ by guest on September 25, 2021 1257.9379 1257.7935 115 LPPPKPLPGTLK 710 721 ϩϪ 1414.0879 1413.8946 137 LPPPKPLPGTLKR 710 722 ϩϪ 1501.0351 1500.8512 123 RRPPQPIQQPQR 723 734 ϩϪ 1344.9346 1344.7500 137 RPPQPIQQPQR 724 734 ϩϪ

a The protein recognized by mAb 11G2 was purified on a 11G2 affinity column, yielding two major bands (Fig. 1B). After digestion with trypsin, the peptide masses of the resulting peptides were measured by MALDI-TOF mass spectrometry and analyzed by searching in the National Center for Biotechnology Information (NCBI) protein database. were compatible with a Met-ox modification. The table is classified according to the position of the peptides on the sequence (ء) The matching of peptides masses labeled with of the protein. Thirty-six peptides from the upper band and 24 from the lower band matched peptides expected for the metalloprotease ADAM10 (NCBI accession number 4557251). The peptides covered 47 and 36% of the protein, respectively. The lower band lacked peptides covering the N-terminal part of the protein that corresponds to the ADAM10 prodomain. Thus the upper band corresponds to the unprocessed precursor protein and the lower band to the mature ADAM10. m/z, mass-to-charge ratio; ppm, parts per million. ectodomain in Western blotting experiments (Fig. 1E). Altogether, cally ADAM10 coimmunoprecipitated several tetraspanins. The these results demonstrate that the mAb 11G2 is directed against the strongest association was observed in HEL cells. Importantly, metalloprotease ADAM10. when CD9 was expressed in Daudi or Raji cells (Fig. 2, A and C), it associated not only with the other tetraspanins (30, 44) but also ADAM10 associates with multiple tetraspanins in lymphoid cell with ADAM10. As a control, the raft resident protein CD55 did lines and PBL not coimmunoprecipitate ADAM10 or tetraspanins (Fig. 2A). Previously published data indicated that ADAM10 associated with Thus, ADAM10 is a component of the tetraspanin web. In all CD9 in colon cancer cells or in transfected COS7 cells (37, 38, 55), experiments, only the lower molecular weight form of ADAM10, and the above data suggested an association of ADAM10 with corresponding to mature ADAM10 after removal of the prodo- CD81 in a cell line (Jurkat) expressing low levels of CD9. main, was associated with tetraspanins. Because removal of ADAM10 could form separate complexes with these two tet- ADAM10 prodomain is mediated by furin and PC7 (56), which are raspanins, or alternatively be part of the tetraspanin web and as- localized in the TGN and endosomal compartments (57), this result sociate with multiple tetraspanins. To investigate this possibility, indicates that the interaction of ADAM10 with the tetraspanins several cell lines (HEL, Raji, Jurkat, Daudi/CD9, Raji/CD9) were studied in this article occurs in a post-Golgi compartment. lysed in the presence of Brij97 and immunoprecipitations were The distribution of ADAM10 in peripheral blood cells has not performed with anti-tetraspanin mAb or the anti-ADAM10 mAb been reported yet. The anti-ADAM10 mAb 11G2 stained polynu- (Fig. 2A). The composition of the immunoprecipitates was ana- clear cells, monocytes, and platelets (Table II). It also stained PBL lyzed by Western blotting. All tetraspanins studied were able to including T cells, B cells, and NK cells as shown by the labeling coimmunoprecipitate a large fraction of ADAM10, and recipro- of CD3ϩ, CD19ϩ, and CD56ϩ cells (Table II), as well as all 7006 TETRASPANINS REGULATE ADAM10 Downloaded from http://www.jimmunol.org/

FIGURE 2. ADAM10 associates with multiple tetraspanins. A, The indicated cells were lysed in the presence of Brij 97, and immunoprecipitations (IP) were performed as indicated on the top of each lane, with anti-tetraspanin mAb (CD9, CD53, CD81, CD82, CD151) or Abs to other surface molecules. The composition of the immunoprecipitates was analyzed by Western blotting using a combination of biotin-labeled mAb and peroxidase-labeled strepta-

␣ ␤ by guest on September 25, 2021 vidin. ADAM10 associates with all tetraspanins studied. Note that although CD19 and the integrin (Int.) 4 1 have been demonstrated to interact with tetraspanins, Brij 97 is not a detergent suitable to visualize these interactions. B, PBL were biotin labeled, lysed in the presence of Brij 97, and immunoprecipitations (IP 1) were performed with anti-tetraspanin (CD81 and CD53) mAb, the anti-ADAM10 mAb, or a CD55 mAb as a control. In the lower panel (IP 2), the coimmunoprecipitated proteins were eluted with 1% Triton X-100 and 0.2% SDS and subjected to a second immunoprecipitation with the anti-ADAM10 mAb. The proteins were visualized using labeled streptavidin. C, Raji/CD9 cells were lysed in the presence of Brij 97 and subjected to two rounds of preclearing with protein G-Sepharose beads precoated with several anti-tetraspanin mAbs (CD9, CD53, CD81, CD82, CD151) and two additional preclearings with protein G-Sepharose beads alone. As a control, the same volume of lysate was treated with an equivalent amount of CD55-precoated protein G beads. Immunoprecipitations were performed as indicated on the top of each lane, with anti-tetraspanin mAbs (CD9, CD53, CD81), the anti-ADAM10 mAb, or the CD55 mAb as a control. The composition of the immunoprecipitates was analyzed by Western blotting using biotin-labeled mAb and streptavidin. The analysis of the material collected during the first preclearing step is also shown at the same exposure. hematopoietic cell lines tested. The level of expression of ADAM10 a mixture of five anti-tetraspanins mAbs (CD9, CD53, CD81, in PBL was similar to that of Raji cells (see below, Fig. 6A). CD82, CD151). There was similar amount of ADAM10 in this To determine whether ADAM10 interacted with tetraspanins in immunoprecipitate as in the anti-ADAM10 immunoprecipitate. PBL, the cells were labeled with biotin and lysed in Brij 97 buffer Additionally, depletion of these five tetraspanins yielded a strong before immunoprecipitation of ADAM10, CD53, CD81, and reduction in the amount of ADAM10 that was immunoprecipitated CD55 as a control (Fig. 2B). As expected, many proteins were by the 11G2 mAb, indicating that a major fraction of ADAM10 coimmunoprecipitated with the tetraspanins CD53 and CD81. The associates with tetraspanins (Fig. 2C). same bands were also present in the ADAM10 immunoprecipitate, ␣ although at a lower level. The major protein coimmunoprecipitated Engagement of tetraspanins stimulates TNF- release by ϳ B lymphoid cells with CD81 has a Mr of 63,000 and most likely corresponds to EWI-2 (53). The identity of the major ϳ180-kDa molecule present It has been reported that CD81 mAbs stimulate the release of in the CD53 immunoprecipitate is unknown, and so is that of the TNF-␣ by murine T lymphocytes and some human B lymphoid ϳ24 and ϳ30 kDa molecules present in the ADAM10 immuno- cell lines (58, 59). Fig. 3 shows that among several anti-tetraspanin precipitate. To prove that ADAM10 was associated with CD53 and (CD9, CD81, CD82, CD53) mAbs tested, only the two CD82 CD81 in PBL, the proteins coimmunoprecipitated with CD53 or mAbs stimulated TNF-␣ release by Raji cells (a B lymphoid cell CD81 were eluted and the presence of ADAM10 in these immu- line), inducing after 24 h incubation a 2- to 3-fold increase of noprecipitations was confirmed by a second immunoprecipitation TNF-␣ concentration in the supernatant (Fig. 3A). The lack of using the mAb 11G2 (Fig. 2B, bottom). effect of CD9 mAbs was expected because Raji cells do not ex- To estimate the fraction of ADAM10 associated with tetraspan- press this tetraspanin (30). The anti-ADAM10 mAb 11G2 did not ins, Raji/CD9 lysates were subjected to immunoprecipitation using stimulate TNF-␣ secretion. The Journal of Immunology 7007

Table II. Example of cells stained by the anti-ADAM10 mAb 11G2a A

Cell Classiﬁcation Cell Name

Peripheral blood Neutrophils, monocytes, platelets, cells lymphocytes (B and T cells), NK cells Myeloid cell lines U937, KG1, K562, HEL, DAMI, MEG01, HL60 Pre-B cell lines NALM-6, KM3, Reh-6, RS4;11 B cell lines DAUDI, Raji, BJAB, JY T cell lines Jurkat, CEM B a PBL were analyzed by multicolor ﬂow cytometry using appropriate lymphocyte markers and a biotin-labeled anti ADAM10 11G2 mAb. The cell lines were tested by indirect immunoﬂuorescence and so far all were positive for 11G2 staining.

The ability of CD82 mAbs to stimulate TNF-␣ release by Raji cells is not due to unique features of CD82. Indeed, several different CD9 mAbs (SYB-1, ALB-6, TS9) were able to stimulate a ␣ 2- to 3-fold increase in release of TNF- by Raji cells stably ex- FIGURE 4. Metalloprotease inhibitors prevent stimulation of TNF-␣ Downloaded from pressing CD9 (Raji/CD9; Fig. 3, B and C and data not shown). All release by CD82 mAbs. The cells were incubated with TPA, anti- CD9-positive clones tested were stimulated by CD9 mAb (data not tetraspanin (CD82 (TS82) and CD53) mAbs, or the CD55 mAb in the Ј ␣ shown). F(ab )2 of the CD9 mAb SYB-1 stimulated TNF- release presence or absence of the ADAM10/ADAM17 inhibitors GW280264X or (Fig. 3C), indicating that this effect is not mediated through a Fc GI254023X (used at 3 ␮M). The production of TNF-␣ was analyzed using receptor-dependent mechanism. The increased release of TNF-␣ an ELISA. A, Raji cells treated for 24 h with mAb or 1 h with TPA. B, ␣ into the supernatant upon incubation with CD9 mAbs is not a Raji/TNF- cells treated for 2 h with mAb or with TPA. http://www.jimmunol.org/ consequence of increased synthesis, as determined by flow cytometry analysis of permeabilized cells stained with an anti-TNF-␣ mAb (data not shown). Additionally, palmitoylation has been shown to contribute to some extent to the interaction of tetraspanins with one another and to some functions of these molecules (31, 34–36). However, CD9 mAb were still able to stimulate the re- C ␣ A lease of TNF- by Raji cells expressing a nonpalmitoylatable CD9, indicating that this posttranslational modification is dispens- able (Fig. 3D). Finally, both basal secretion and CD9-stimulated TNF-␣ secretion were completely inhibited by the broad-spectrum by guest on September 25, 2021 metalloprotease inhibitor GM6001 (data not shown), indicating that the fraction of TNF-␣ released upon addition of this mAb is normally processed through a metalloprotease. To exclude the possibility that changes in TNF-␣ concentrations D induced by anti-tetraspanin mAbs could be the consequence of B modifications of biological phenomena such as proliferation or survival, the concentrations of TNF-␣ were measured at earlier time points. The stimulatory effect of CD9 mAbs on TNF-␣ release by Raji/CD9 cells was already detectable after a 4-h incubation and lasted over a period of 24 h (data not shown). Because of the sensitivity limit of the ELISA, it was not possible to determine whether the mAb could have any stimulatory effect at earlier time points. To overcome this problem, we overexpressed TNF-␣ E in Raji cells that have been shown to be insensitive to the effects of TNF-␣ (60). These cells produced ϳ200 times more TNF-␣ than nontransfected Raji cells after a 4-h incubation (Fig. 3E). The CD82 mAb TS82 stimulated TNF-␣ release 2-fold, and its effect could be detected as early as 30 min after mAb addition. As in wild-type Raji cells, the CD53 and the CD55 mAbs did not stimulate TNF-␣ secretion (data not shown). Thus the effect of anti- tetraspanin mAbs is rapid and most likely independent of new FIGURE 3. Engagement of tetraspanins stimulates TNF-␣ release by protein synthesis. Raji cells. The cells were incubated with the indicated anti tetraspanin mAbs (CD9, CD53, CD81, CD82) or mAbs to other cell surface molecules, TNF-␣ shedding upon CD82 and CD9 engagement is mediated and the release of TNF-␣ in the medium was analyzed using an ELISA by ADAM10 after 24-h incubation except when otherwise indicated. A, Raji cells. B and ␣ C, Raji/CD9 cells. D, Sorted population of Raji cells expressing either a We then tested whether the increased TNF- secretion observed wild-type CD9 or a nonpalmitoylatable CD9 mutant. E, Raji cells stably upon addition of CD9 or CD82 mAbs required an active ADAM10 transfected with TNF-␣ (Raji/TNF-␣) analyzed after various time of incu- (Fig. 4A). We first used two recently described hydroxamate-based bation with the CD82 mAb. Except when otherwise indicated, the CD9 compounds that differ in their capacity to inhibit the activities of mAb was SYB-1. ADAM17 and ADAM10. Whereas GW280264X potently inhibits 7008 TETRASPANINS REGULATE ADAM10

B B Downloaded from

C http://www.jimmunol.org/

FIGURE 5. ␣ ADAM10 silencing prevents stimulation of TNF- release FIGURE 6. ADAM10 in PBL. A, PBL and Raji cells were labeled by by CD82 and CD9 mAbs. Raji or Raji/CD9 cells were transfected with a indirect immunoﬂuorescence using the anti-ADAM10 mAb 11G2 and an- control siRNA (si Control) or a siRNA targeting ADAM10 (si ADAM10) alyzed by ﬂow cytometry. The dotted line corresponds to the control stain- A and analyzed 48 h later. , Flow cytometry analysis of ADAM10, CD9, ing. The same acquisition parameters were used for both cell types. B, PBL and CD82 expression. B, The cells were incubated with the indicated mAb

were stimulated or unstimulated for 24 h with CD3 and CD28 mAbs or by guest on September 25, 2021 left right for 24 h ( ) or with DMSO or TPA for2h( ), and the release of PHA in the presence or absence of the ADAM10/ADAM17 inhibitors ␣ TNF- in the medium was analyzed using an ELISA. The mAbs used were GW280264X or GI254023X (6 ␮M). TNF-␣ secretion was analyzed using SYB-1 (CD9), TS82b (CD82), and TS53 (CD53). an ELISA. ND: not detected. C, PBL were treated with TPA for 24 h in the presence or absence of anti-tetraspanins mAbs (CD81 (5A6), CD82 (TS82b), CD53) or mAbs to nontetraspanin proteins (CD46, CD55). The effect of the ADAM10/ADAM17 inhibitor GW280264X or GI254023X (6 p Ͻ ,ء .both enzymes, GI254023X inhibits ADAM10 in the same concen- ␮M) was tested. TNF-␣ secretion was analyzed using an ELISA tration range of GW280264X but blocks ADAM17 with Ͼ100- 0.05, as compared with TPA-treated cells. fold reduced potency (54). GW280264X but not GI254023X par- tially blocked the basal TNF-␣ secretion, suggesting that it is in ␣ part an ADAM17-dependent mechanism. Both inhibitors com- Anti-tetraspanin mAbs stimulate TNF- release by an pletely abolished the stimulation of TNF-␣ release induced by ADAM10-dependent mechanism in TPA-treated lymphocytes CD9 and CD82 mAbs (Fig. 4A and data not shown), including in The availability of inhibitors discriminating between ADAM10 Raji/TNF-␣ cells (Fig. 4B), strongly implicating ADAM10 in this and ADAM17 activities made it possible to test whether process. As a control, only GW280264X blocked TPA-induced ADAM10, which is expressed by lymphocytes (Fig. 6A, Table II), TNF-␣ release, indicating that only ADAM17 contributes to this contributes to the secretion of TNF-␣ by in vitro activated human cleavage. It should be noted that the strong increase of TNF-␣ lymphocytes. As expected (62), activation of PBL by a combina- secretion observed after TPA treatment is not primarily the con- tion of CD3 and CD28 mAbs or PHA strongly increased the syn- sequence of activation of a protease activity, but the result of a thesis and the secretion of TNF-␣ into the medium. GW280264X strong stimulation of TNF-␣ synthesis (61). but not GI254023X blocked TNF-␣ secretion, indicating that To confirm that the increased TNF-␣ release induced by CD9 or ADAM10 does not participate in the release of TNF-␣ by CD3/ CD82 mAbs was dependent on ADAM10 proteolytic activity, the CD28 or PHA-activated PBL (Fig. 6B). Additionally, TPA treat- expression of ADAM10 in Raji or Raji/CD9 cells was knocked- ment induced the synthesis and the release of TNF-␣ as previously down by RNA interference. The expression of ADAM10 was re- described (62). CD81 and CD82 mAbs, but not mAbs to CD53 or duced by ϳ70–80%, as determined by flow cytometry, and the nontetraspanin proteins (CD46 and CD55), significantly increased expression levels of CD9 and CD82 were not changed (Fig. 5A). the release of TNF-␣ by TPA-treated PBL (Fig. 6C). ADAM10 silencing by two different siRNA (Fig. 5B and data not GW280264X, which potently blocks both ADAM10 and shown) completely abolished the stimulation of TNF-␣ release by ADAM17 activity, prevented the secretion of TNF-␣ by cells stim- CD9 or CD82 mAbs, confirming the prominent role of ADAM10 ulated by TPA alone or TPA with anti-tetraspanin mAb. In contrast in this cleavage. ADAM10 silencing did not reduce TPA-stimu- GI254023X, that inhibits ADAM10 activity but has no effect on lated TNF-␣ release. ADAM17, blocked the stimulating effect of anti-tetraspanin mAb, The Journal of Immunology 7009

C Downloaded from FIGURE 7. Signaling pathways involved in the stimulation of TNF-␣ release by anti-tetraspanin mAb. A, Raji/CD9 cells were mixed with an equal number (5 ϫ 105 cells/ml) of either Raji or Raji/TNF-␣ in the presence or not of CD9, CD82, CD53, or CD55 mAbs for 2 h. TNF-␣ secretion was measured using an ELISA. B, Raji/TNF-␣ cells were incubated for 2 h with mAbs to CD82, CD55, or TPA in the presence or absence of the indicated inhibitors: bisindolylmaleimide I (BIM I, 2 ␮M), herbimycin A (2 ␮M), U0126 (50 ␮M), SB203580 (20 ␮M). The production of TNF-␣ was determined using an ELISA. C, Raji/CD9 cells were stimulated with TPA or CD9 or CD82 mAbs for 5, 15, or 30 min. The phosphorylation of Erk was examined using an anti-phosphoErk Ab (p-Erk; top panel). The level of Erk1/2 in the extract was checked by immunoblotting using an anti-Erk1/2 Ab (bottom panel). C., untreated cells. http://www.jimmunol.org/ but not the secretion induced by TPA stimulation alone. Altogether inhibits p38 MAPK only affected TPA-stimulated release. Impor- these data strongly suggest that ADAM17 is the main TNF-␣ shed- tantly, CD9 and CD82 mAbs did not induce detectable increases in dase for in vitro-activated human PBL, and indicate that engage- Erk1/2 phosphorylation in contrast to TPA (Fig. 7C). We therefore ment of certain tetraspanins on PBL increases TNF-␣ release conclude that, as for other stimuli, the cleavage of TNF-␣ induced through ADAM10 “activation.” by tetraspanins mAb requires an intact MEK/Erk1/2 pathway, but this increased cleavage is not the consequence of a detectable in- ␣ CD9 mAb do not stimulate TNF- release when CD9 crease in Erk1/2 activation. ␣ and TNF- are present in different cells by guest on September 25, 2021 The anti-tetraspanin mAbs that increase ectodomain shedding could increase shedding on the same cell, or alternatively could CD82 mAbs induce a major redistribution of tetraspanins and deliver an extracellular signal that stimulates neighboring cells. To ADAM10 but do not detectably change the level of ADAM10 discriminate between these two hypotheses, experiments where interaction with tetraspanins Raji/CD9 cells were mixed with either Raji or Raji/TNF-␣ cells We then examined whether the engagement of tetraspanins with were performed. Only Raji/CD9 cells can be stimulated by the specific mAb modified the distribution of ADAM10 and TNF-␣ by CD9 mAb, but after a 2-h incubation the amount of TNF-␣ re- using confocal microscopy. In nonstimulated cells, the tetraspanins leased by these cells is negligible, as determined by the analysis of CD82 and CD53, as well as ADAM10, were regularly distributed Raji/CD9 and Raji cocultures. In cocultures of Raji/CD9 and Raji at the surface of Raji cells (Fig. 8 first row, and data not shown). TNF-␣ cells, TNF-␣ is essentially released only by Raji/TNF-␣ Upon incubation with the CD82 mAb TS82b for2hat37°C, cells. As shown in Fig. 7A, under these conditions the CD9 mAb CD82 molecules gathered into one or several patches of various did not stimulate TNF-␣ release in contrast with the CD82 mAb. sizes, to which ADAM10 was for the most part redistributed (Fig. This experiment argues against the requirement for an extracellular 8, second row). The other tetraspanins CD81 and CD53 also signal and strongly suggests that the protease stimulated by the strongly redistributed to these CD82 patches (data not shown), CD9 mAb needs to be in the same cell as the target TNF-␣. whereas the GPI-anchored protein CD55, which does not associate with tetraspanins, was only slightly enriched in these patches (Fig. ␣ CD82-stimulated release of TNF- depends on active 8, third row). The CD9 mAb TS9 also induced the formation of Erk1/2 but does not depend on tyrosine kinase or CD9 patches to which ADAM10 redistributed in Raji/CD9 cells protein kinase C (PKC) activities (data not shown), whereas the mAb TS81 and TS53, which do not Tetraspanins have been shown to be coupled to diverse signal stimulate TNF-␣ secretion, induced only a limited redistribution of transduction pathways, including PKC, tyrosine kinases, and the their target Ag and ADAM10 (Fig. 8, fourth row and data not MAPK pathways (44, 63–67). As expected, the PKC inhibitor shown). The CD82 mAb also induced patches to which ADAM10 bisindolylmaleimide I blocked the release of TNF-␣ induced by was redistributed in Raji/TNF-␣ cells (data not shown). As ex- the PKC activator TPA. However, it did not affect the stimulation pected, pro-TNF-␣ could not be detected by immunofluorescence of TNF-␣ release induced by the CD82 mAb (Fig. 7B). The broad in most of these cells. However, a few expressed pro-TNF-␣ at a tyrosine kinase inhibitor herbimycin A did not reduce the effect of detectable level. The major fraction of pro-TNF-␣ did not redis- the CD82 mAb, but had a partial inhibitory effect on TPA-induced tribute into the CD82 patches (Fig. 8, last row). release. The stimulation by both the CD82 mAb and TPA was These changes in tetraspanin and ADAM10 membrane distri- blocked by U0126, a specific MEK1/2 inhibitor that consequently bution were not accompanied by a change in the level of associ- prevents activation of Erk1/2 MAPK. In contrast, SB203580 that ation. Indeed, the level of ADAM10 coimmunoprecipitated with a 7010 TETRASPANINS REGULATE ADAM10

FIGURE 9. CD82 mAbs do not detectably change the association of ADAM10 with tetraspanins. Raji cells were stimulated or not with CD82 or CD53 mAb for 2 h before lysis in the presence of Brij 97 and immunoprecipitation (IP) using the anti-ADAM10 mAb 11G2 or a mixture of anti-tetraspanin mAbs (Tspan x4: CD81, CD53, CD82, CD151). The composition of the immunoprecipitates was analyzed by Western blotting using biotin or Alexa Fluor 680-labeled mAb to ADAM10, CD81, and CD82. Note that the increased amount of CD82 and CD81 in the ADAM10 immunoprecipitate after stimulation is due to the immunoprecipitation of these tetraspanins by the stimulatory mAb. Cont, Control. Downloaded from

protease mediating the ectodomain cleavage of pro-EGF upon treatment with CD9 and CD81 mAb, GFP-tagged pro-EGF was stably expressed in HEK cells. Two hours of incubation with CD9 or CD81 mAb stimulated respectively an ϳ2- and ϳ3-fold increase of EGF secretion, and this stimulation was nearly completely abolished after ADAM10 silencing (Fig. 10, C and D). http://www.jimmunol.org/ Additionally the level of EGF in the supernatant of nonstimulated cells was decreased by ϳ50% after ADAM10 silencing, whereas the stimulation produced by TPA was for the most part not dependent on ADAM10 expression. In control experiments, we found that the expression of ADAM10 was reduced by ϳ70–80%

FIGURE 8. ADAM10 redistributes into CD82-containing patches upon stimulation with CD82 mAbs. Raji or Raji/TNF-␣ cells were treated or not by guest on September 25, 2021 with Alexa Fluor 568-labeled CD82 or CD53 mAb (red) for2hat37°C. They were then incubated at 4°C with Alexa Fluor 488-labeled mAb directed to ADAM10, CD55, or TNF-␣ (green). Alexa Fluor 568-labeled CD82 was also added at this stage in nonstimulated cells (first row, 4°C). The distribution of the different molecules was analyzed by confocal microscopy. Right panels show a merge image of the green and red fluorescence. Raji cells were used for all experiments except for the labeling of TNF-␣ for which we analyzed Raji/TNF-␣ cells. Bar, 5 ␮m. mixture of anti-tetraspanin mAb was not modified after incubation for 2 h with CD82 or CD53 mAbs (Fig. 9).

Anti-tetraspanin mAbs stimulate EGF release by an ADAM10-dependent mechanism Do anti-tetraspanin mAb specifically stimulate the activity of ␣ FIGURE 10. Engagement of tetraspanins stimulate pro-EGF shedding ADAM10 toward TNF- , or do they also stimulate the cleavage of through ADAM10 activation. HEK cells expressing rat pro-EGF were other substrates? This question was addressed using pro-EGF as a starved for 24 h and incubated in medium containing 1 ␮M TPA, the model of EGFR ligand, the ectodomain of which was predomi- indicated anti-tetraspanin mAbs (CD9, CD81, CD82), or mAbs to other nantly cleaved by ADAM10 (68). HEK cells were transiently cell surface molecules (ADAM10, CD55). Full-length pro-EGF in the cell transfected with pro-EGF and after 48 h of incubation, stimulated lysate (Cell) and ectodomain in the medium (SN) were detected by West- or not with anti-tetraspanin mAb or TPA. The fraction of ectodo- ern blotting using an anti-HA mAb. A, HEK-293 cells transiently trans- main released in the medium as well as cell-associated pro-EGF fected with rat pro-EGF and stimulated for 24 h. B, Quantification of the was quantified by Western blotting. CD9 and CD81 mAbs, respec- experiment shown in A. C, HEK-293 cells stably expressing pro-EGF fused tively, stimulated an ϳ3- and ϳ10-fold increase in the EGF to EGFP and transfected with a control siRNA (si control) or a siRNA targeting ADAM10 (si ADAM10). The right and left panels are from the ectodomain shedding after 24-h incubation (Fig. 10, A and B). same gel and are shown after applying the same settings to the initial 16-bit mAbs directed to CD82, ADAM10, and CD55 did not have any image produced by the Odyssey Imaging System. Stimulation was for 2 h. detectable stimulatory effect. The lack of effect of the CD82 mAb D, Quantification of the experiment shown in C. Data are expressed as may be due to the very low level of expression of CD82 in this cell mean Ϯ SD of three different experiments. In B and D, the shedding of line. As previously described (46), TPA also stimulated pro-EGF pro-EGF in incubation medium is expressed as a percentage of total EGF ectodomain shedding. To determine whether ADAM10 was the in both incubation medium and cell lysates. The Journal of Immunology 7011 after silencing, according to the experiments, and that ADAM10 of MAPK in the cleavage of various cytokines and growth factors, associated with tetraspanins in HEK cells (data not shown). We including TNF-␣ (70, 71). It is therefore possible that the inhibi- conclude that tetraspanins regulate the activity of ADAM10 to- tory activity of the MEK/Erk inhibitor reflects the necessity for a ward several substrates including TNF-␣ and EGF. locally active MEK/Erk pathway for ADAM-mediated cleavage. The fact that ADAM10 associates with tetraspanins makes pos- Discussion sible the hypothesis that tetraspanins directly regulate ADAM10 In this study we have shown that several anti-tetraspanin mAbs can ability to cleave some of its substrates. The inability of ADAM10 rapidly stimulate the release of TNF-␣ or EGF in the medium to cleave TNF-␣ in nonstimulated cells while strongly interacting through an ADAM10-dependent mechanism. Additionally, we with tetraspanins suggests that tetraspanins negatively regulate the have demonstrated a robust interaction of ADAM10 with several activity of ADAM10. The stimulatory effect of CD82 or CD9 mAb tetraspanins in various cell types. Our data suggest that tetraspan- on TNF-␣ release is, however, not a consequence of a disruption ins could be important regulators of ADAM10 activity. of ADAM10/tetraspanin interaction (Figs. 8 and 9). Instead, it is The activity of ADAM metalloproteases is thought to be tightly correlated with the ability of these anti-tetraspanin mAb to redis- regulated, but the underlying mechanisms remain largely unknown tribute ADAM10 into tetraspanin-containing patches (Fig. 8). We (2). A remarkable example of such regulation is that of TNF-␣. propose that this increase in ADAM10 local concentration confers Indeed, although ADAM10 can cleave TNF-␣ or a TNF-␣ peptide on ADAM10 the ability to cleave TNF-␣ (and increase the activity encompassing the cleavage site in vitro (19, 69), it was shown not toward other substrates such as EGF) despite the association with to contribute to TNF-␣ shedding in several studies using MEF (12, tetraspanins.

17), suggesting a cellular inhibition of ADAM10 activity toward Among other ADAM proteases, ADAM10 is most closely re- Downloaded from TNF-␣. We have extended this result by showing that in Raji cells lated to ADAM17 (4). In several experiments using different cell or in vitro activated lymphocytes, ADAM10 does not contribute to lines, we did not observe an interaction between ADAM17 and the cleavage of TNF-␣, similar to what is observed in MEF (12, tetraspanins (data not shown). Additionally, analysis of CD9-as- 17). The demonstration that anti-tetraspanin mAbs can induce sociated molecules in colon cancer cells using proteomic ap- ADAM10-mediated TNF-␣ cleavage indicates that ADAM10 can proaches did not reveal an interaction with ADAM17 (38). How-

cleave TNF-␣ in a cellular context provided it is properly stimu- ever, the detection of ADAM17 in the cell lines tested was always http://www.jimmunol.org/ lated and points to a role for tetraspanins in the regulation of its lower than that of ADAM10 (by flow cytometry, Western blotting, activity. The fact that anti-tetraspanin mAbs also stimulate and immunoprecipitation of biotin-labeled surface proteins, data ADAM10-mediated shedding of EGF suggests that tetraspanins not shown), which may reflect different efficiency of Abs or a regulate the activity of ADAM10 toward several substrates, al- different level of expression. We therefore cannot exclude the pos- though in a less dramatic manner than toward TNF-␣. sibility that ADAM17 is associated with tetraspanins but could not How could tetraspanins regulate ADAM10 activity? We have be identified in tetraspanin immunoprecipitates because of a low tested whether the effect of anti-tetraspanin mAb on ADAM10 level of expression. activity could be the result of stimulating certain signaling path- The list of substrates for ADAM10 has grown quickly in recent ways. The effect of anti-tetraspanin mAbs on TNF-␣ release is years (for review see Ref. 3), and it will have to be determined by guest on September 25, 2021 rapid and does not depend on extracellular signaling. Anti-tet- whether tetraspanins regulate the cleavage of substrates other than raspanins mAbs induce in minutes the phosphorylation of proteins TNF-␣ and EGF. Because TNF-␣ is mainly an ADAM17 sub- on tyrosine residues in certain B or T lymphoid cell lines (44, 63, strate, it will have also to be determined whether tetraspanins pre- 64). However, we did not detect any induction of tyrosine phos- vent the cleavage by ADAM10 of other known ADAM17 sub- phorylation in the Raji cell line incubated with anti-tetraspanin strates. A number of ADAM10/ADAM17 substrates have been mAbs (data not shown), and the tyrosine kinase inhibitor herbi- shown to associate with tetraspanins (mostly CD9) including the mycin A did not inhibit the increased release of TNF-␣ produced EGF receptor ligands HB-EGF and TGF␣, the complement regu- by anti-tetraspanin mAbs, suggesting that the effect does not rely latory molecule CD46, or the hyaluronic acid receptor CD44 (2, on protein tyrosine phosphorylation. Tetraspanins have been 38, 52, 72–75). CD9 expression was shown to inhibit the release of shown to associate with activated PKC (65), and treatment of cells TGF-␣ into the medium (73), a process that like TNF-␣ release is with the PKC activator TPA promotes the release of TNF-␣ critically dependent on ADAM17 activity under the conditions through an ADAM17-dependent mechanism (Refs. 12, 17 and tested so far (2). This raises the hypothesis that tetraspanins control Figs. 4 and 6). The effect of anti-tetraspanin mAbs on TNF-␣ re- the substrate specificity of ADAM10 by interacting with both lease is independent of PKC, because a broad spectrum PKC in- ADAM10 and some of its substrates. In this context, the proteins hibitor did not change the release of TNF-␣ induced by anti- may be in different tetraspanin microdomains and the mAb could tetraspanin mAb while completely preventing that induced by TPA bring different microdomains together, thus promoting the cleav- (Fig. 7). We have shown here that the increased TNF-␣ release age of the substrate. However, not all ADAM10/ADAM17 sub- induced by the CD82 mAb is dependent on an intact MEK/Erk strates associate with tetraspanins. Among the proteins identified pathway, but does not depend on the p38 MAPK pathway. Al- as associating with tetraspanins by mass spectrometry analysis (37, though the engagement of tetraspanins has been shown to activate 38), only CD44 and CD46 are known substrates of metallopro- these pathways in nonhematopoietic cell lines (66, 67), the stim- teases (74, 75). More specifically, we could not coimmunoprecipi- ulation of ADAM10-mediated TNF-␣ shedding by anti-tetraspanin tate the ADAM10 substrates L1, PrP, or E-cadherin (76–78) with mAbs is not a mere consequence of the activation of the Erk path- tetraspanins (our unpublished data). So far we have been unable to way. Indeed, we did not observe Erk activation upon addition of demonstrate an interaction of tetraspanins with pro-TNF-␣ in Raji/ CD82 mAb to Raji cells although TPA caused strong Erk phos- TNF-␣ cells by coimmunoprecipitation. However, our experi- phorylation without inducing ADAM10-mediated TNF-␣ shed- ments with TNF-␣ may lack sensitivity because the expression of ding. ADAM17-dependent ectodomain shedding also requires an pro-TNF-␣ remains very low at the surface of the majority of the intact MEK/Erk pathway because TPA-stimulated TNF-␣ release cells. In addition, the lack of TNF-␣ redistribution into CD82 is blocked to a large extent by the MEK inhibitor (Fig. 7). This patches argues against a strong association of TNF-␣ with result is in agreement with previous studies showing the key role tetraspanins. 7012 TETRASPANINS REGULATE ADAM10

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Evaluation of the con- tribution of different ADAMs to tumor necrosis factor ␣ (TNF␣) shedding and of would be regulated through tetraspanin-enriched microdomains. the function of the TNF␣ ectodomain in ensuring selective stimulated shedding The organization of the tetraspanin web is dynamic because it can by the TNF␣ convertase (TACE/ADAM17). J. Biol. Chem. 279: 42898–42906. Downloaded from be modified by anti-tetraspanin mAbs (this study), by manipulating 18. Horiuchi, K., T. Kimura, T. Miyamoto, H. Takaishi, Y. Okada, Y. Toyama, and C. P. Blobel. 2007. Cutting edge: TNF-␣-converting enzyme (TACE/ADAM17) cholesterol levels (29), or by modifications in the pattern of asso- inactivation in mouse myeloid cells prevents lethality from endotoxin shock. ciated proteins (84), and is therefore compatible with a regulatory J. Immunol. 179: 2686–2689. role. Physiological modifiers of the “web” may have consequences 19. Lunn, C. A., X. Fan, B. Dalie, K. Miller, P. J. Zavodny, S. K. 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