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The Journal of Immunology, 2008, 181, 7002 -7013
Copyright © 2008 by The American Association of Immunologists, Inc.
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Tetraspanins Regulate ADAM10-Mediated Cleavage of TNF-{alpha} and Epidermal Growth Factor1

Cécile Arduise*,{dagger}, Toufik Abache*,{dagger}, Lei Li*,{dagger},{ddagger}, Martine Billard*,{dagger}, Aurélie Chabanon*,{dagger}, Andreas Ludwig§, Philippe Mauduit*,{dagger}, Claude Boucheix*,{dagger}, Eric Rubinstein2,3,*,{dagger} and François Le Naour2,*,{dagger}

* INSERM U602, Villejuif, France; {dagger} Université Paris-Sud, Institut André Lwoff, Villejuif, France; {ddagger} Institute of Urology, The First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi, China; and § Institute for Pharmacology and Toxicology, RWTH Aachen University, Aachen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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-{alpha} secretion through a mechanism dependent on ADAM10. The effect of anti-tetraspanin mAb on TNF-{alpha} 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Members of the ADAM (a disintegrin and metalloprotease domain)4 family of membrane-anchored zinc-dependent metalloproteases play a key role in the conversion of membrane-anchored precursors of growth factors or cytokines into soluble mediators and in the ectodomain shedding of various transmembrane proteins (1, 2, 3, 4). The importance of ADAM-mediated ectodomain shedding is highlighted by the fact that ADAM10 knockout mice die during embryonic development (5), whereas ADAM17 inactivation leads to perinatal lethality, probably as a consequence of defects in heart development (6). Interestingly heparin-binding epidermal growth factor (HB-EGF) is a substrate for ADAM17, and a knock-in deletion of the HB-EGF cleavage site leads to a similar defect in heart valve development (7).

A central question raised by the essential role of proteolysis as regulator of ectodomain shedding is how this process is regulated. The fact that several G protein-coupled receptor (GPCR) ligands are able to transactivate the EGF receptor by a mechanism involving ADAM metalloproteases (including ADAM10 and ADAM17) and EGFR ligands strongly suggests that GPCR activation up-regulates ADAM metalloprotease activity (8). In contrast, the activity of ADAM10 and ADAM17 toward certain substrates can be rapidly up-regulated by ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate (TPA), respectively, but the underlying mechanisms remain largely unknown (2, 9, 10, 11). Notably, the ability of TPA to up-regulate ADAM17-mediated TNF-{alpha} cleavage does not require the ADAM17 cytoplasmic domain (12), suggesting a possible regulation of its activity at the level of the plasma membrane.

The analysis of the sheddases responsible for soluble TNF-{alpha} generation provides a striking example of a cellular regulation of an ADAM protease. TNF-{alpha} is a multifunctional cytokine that plays a key role in inflammation, autoimmunity, and antitumor reaction, and mediates the response to infection (13, 14, 15). ADAM17/TACE is the primary sheddase responsible for pro-TNF-{alpha} cleavage in T cells or myeloid cells. Evidence for the prominent role of ADAM17 in the shedding of TNF-{alpha} is provided by the finding that mouse T cells or mouse embryonic fibroblasts (MEF) homozygous for a targeted mutation in ADAM17 that inactivates metalloproteinase activity are strongly deficient in their ability to shed TNF-{alpha} (12, 16, 17). More recently it was reported that ADAM17 inactivation in myeloid cells reduced TNF-{alpha} secretion in vivo (18). Among ADAM proteases ADAM10 is the most closely related to ADAM17 (4) and was initially purified in two studies based on its ability to cleave purified pro-TNF-{alpha} or a TNF-{alpha} peptide encompassing pro-TNF-{alpha} cleavage site (19, 20). Transfection of ADAM10 was shown to induce the release of TNF-{alpha} from 293EBNA cells (19). However, in subsequent studies the transfection of ADAM10 in ADAM17–/– MEF did not increase the shedding of TNF-{alpha}, and ADAM10–/– MEF were shown to process TNF-{alpha} normally (12, 17). Altogether, these data suggest that the ability of ADAM10 to cleave TNF-{alpha} can be tightly controlled at the cellular level by an unknown mechanism.

Tetraspanins compose a family of 33 integral membrane proteins with four transmembrane domains delimiting three short intracellular domains and two extracellular regions of unequal size. They exhibit significant sequence identity as well as specific structural features in the large extracellular domain. They are widely distributed and have been implicated in a large variety of physiological processes such as cell migration, cell fusion (including macrophage fusion and sperm-egg fusion), and activation of lymphoid cells (21, 22, 23, 24). Tetraspanins play a role in infection by several viruses including HIV and human T cell leukemia virus (25, 26). Additionally, the tetraspanin CD81 is required for the infection of hepatocytic cells by two major pathogens, the hepatitis C virus and the malaria parasite (27, 28, 29).

At the molecular level, tetraspanins are believed to be organizers of particular microdomains on the plasma membrane that are different from the so-called lipid rafts and are collectively referred to as the "tetraspanin web" (21, 22, 23, 24, 30). These microdomains critically depend on the interaction of tetraspanins with one another through a mechanism involving protein palmitoylation and interaction with lipids (31, 32, 33, 34, 35, 36). Tetraspanins also associate with a number of nontetraspanin proteins. Although the association with integrins and proteins with Ig domains is well characterized (21, 22, 23), recent proteomic analysis demonstrated the presence of several ectoenzymes in the tetraspanin web, including ADAM10 (37, 38). Tetraspanins may influence the proteins with which they associate in several ways. For example, CD81 participates in the traffic of CD19 to the cell surface in lymphoid B cells and modulates its cosignaling activity (36, 39). CD151 is not essential for the expression of the integrins with which it associates (40), but regulates post-ligand binding events such as adhesion strengthening and signaling (41, 42, 43). For most other proteins, including ectoenzymes, the functional consequences of the association with tetraspanins is unknown. We now provide evidence that tetraspanins regulate ADAM10 proteolytic activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells, cell culture, and generation of transfected cell lines

The lymphoid T cell line Jurkat, the erythromegakaryocytic cell line HEL, and the B lymphoid cell lines Raji, Raji/CD9, Daudi, and Daudi/CD9 have been previously described with respect to tetraspanin expression (30, 44, 45). All hematopoietic cell lines (see Table II) were cultured in RPMI 1640 medium supplemented with 10% FCS, GlutaMAX, and antibiotics (all from Invitrogen). Human embryonic kidney 293 (HEK-293) cells and the prostate cell line PC3 were cultured in DMEM similarly supplemented. Transfection of TNF-{alpha} in Raji cells was performed as previously described (30), and positive cells were sorted using a FACSVantage cell sorter after fluorescent labeling. HEK-293 cells stably expressing GFP-tagged rat pro-EGF were selected under G-418 following transfection with ExGen (Euromedex) as described previously (46), and positive fluorescent cells were sorted as above. For the construction of a C-terminal GFP-tagged pro-EGF expression plasmid, the pro-EGF cDNA in pcD12-HA plasmid (where "HA" is hemagglutinin; Ref. 46) was excised following HindIII/ApaI restriction and subcloned in-frame into the pEGFP-N2 expression vector (BD Clontech). The human TNF-{alpha} plasmid was provided by Dr. F. Peiretti (INSERM U626, Marseille, France) (47). PBMC were isolated as previously described (48). The cells were used after removal of adherent cells 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 µg/ml, respectively), or TPA (10 ng/ml).


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

 
RNA silencing

Cells (5–10 x 106 cells in 400 µl RPMI 1640 medium) were transfected with synthetic small interfering RNA (siRNA) oligonucleotides (200 pmol) by electroporation at room temperature (49) using the Gene Pulser apparatus (Bio-Rad). The settings were 300 V and 500 microfarads. For Raji cells, electroporation was performed twice 24 h apart and cells were analyzed 24 h after the second electroporation. For HEK-293, cells were allowed to recover in complete DMEM for 24 h following electroporation, then were trypsinized and submitted to a second ADAM10 siRNA transfection using the INTERFERin reverse protocol as described by the manufacturer (Polyplus Transfection). Cells were then analyzed for EGF secretion 36 h later as described below. In most experiments the siRNA oligonucleotide targeting the ADAM10 sequence 5'-GGA TTA TCT TAC AAT GTG G-3' was used. A nonactive siRNA targeting the CD82 sequence 5'-ACC TCC TCC AGC TCG CTT A-3'was used as a control. In some experiments a stealth siRNA (catalog no. HSS100165; Invitrogen) targeting the ADAM10 sequence 5'-TAC ACC AGT CAT CTG GTA TTT CCT C-3' was also used.

Recombinant proteins, mAbs, and flow cytometry

A recombinant human ADAM10 ectodomain protein was purchased from Chemicon International. Anti-tetraspanin mAbs used in this study were SYB-1, ALB-6, TS9 (CD9), TS53 (CD53), TS63 (CD63), TS81 (CD81), TS82 (CD82), TS82b (CD82), TS151 (CD151; Refs. 30, 32, 50), and 5A6 (CD81; Refs. 51). The CD46 mAb (11C5) and the CD55 mAb (12A12) have been previously described (52). The anti-integrin {alpha}4β1 mAb (HP2/1), the CD19 mAb (B4), and the CD28 mAb (CD28.2) were obtained from Beckman Coulter, the β-actin mAb (AC-74) was obtained from Sigma-Aldrich, the CD3 mAb (OKT3) was purchased from American Type Culture Collection, and the anti-HA mAb (HA-11) was purchased from Covance Research Products. The anti TNF-{alpha} mAbs B-C7 and B-D9 (PE-labeled) were obtained from Diaclone. An anti-phospho Erk1/2 mAb (E10) and a polyclonal Ab to Erk1/2 were obtained from Cell Signaling Technology. Flow cytometry analysis was performed as previously described (53).

To generate mAbs, BALB/c mice were injected i.p. three times with 107 Jurkat cells. Spleen cells were fused with P3X63AG8 mouse myeloma cells (5 x 107 and 3 x 107 cells respectively) according to standard techniques and distributed into a 96-well tissue culture plate. After 2 wk hybridoma culture supernatants were harvested and tested for Jurkat cell staining by indirect immunofluorescence and analyzed using a microplate fluorescence reader (CytoFluor II, Applied Biosystems) and a FACSCalibur flow cytometer (BD Biosciences). Positive supernatants were then further characterized by immunoprecipitation. Beside 11G2, the mAbs TS53, TS81, and TS82 were produced this way.

Ag purification, in-gel enzymatic digestion, and mass spectrometry analysis

For purification of 11G2 target Ag 5 x 108 HEL cells were lysed in 12 ml 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 centrifugation at 12,000 x g for 30 min, and the lysate was successively precleared five times with Sepharose 4B beads (Amersham Biosciences) coupled to BSA. After another centrifugation, the lysate was incubated for 3 h with Sepharose 4B beads coupled to mAb 11G2. The beads were washed five times with lysis buffer, and the immunoprecipitated proteins were separated by 5–15% SDS-PAGE under nonreducing conditions. Silver staining, gel piece preparation, and mass spectrometry analysis were performed as previously described (53).

Immunoprecipitation and Western blotting

Labeling of cell surface proteins with EZ-Link-Sulfo-NHS-LC-Biotin (Pierce) and immunoprecipitations in the presence of Brij97 were performed as described previously (50). For two-step immunoprecipitations tetraspanins were immunoprecipitated from Brij 97 lysates, and coimmunoprecipitated proteins were eluted in the presence of 1% Triton X-100 and 0.2% SDS before the second immunoprecipitation. Biotin-labeled cell surface proteins were visualized using labeled streptavidin. Western blotting on immunoprecipitates was performed using biotinylated mAbs followed by labeled streptavidin. We used a streptavidin-biotinylated HRP complex for ECL (PerkinElmer Life Sciences) revelation or Alexa Fluor 680-labeled streptavidin (Invitrogen) for the use of the Odyssey Infrared Imaging System (LI-COR Biosciences).

Quantification of TNF-{alpha} release by ELISA and inhibitors

Cells were washed three times in PBS and cultured in complete RPMI 1640 medium with or without 10 µg/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 removed by centrifugation at 200 x g for 15 min. The concentration of TNF-{alpha} in the supernatant was determined using a TNF-{alpha} ELISA kit according to the manufacturer’s instructions (Diaclone). In some experiments the cells were preincubated 30 min before addition of mAb with different inhibitors: GM6001 (40 µM), TAPI-2 (5 µM), bisindolylmaleimide I (GF109203X, 2 µM), Herbimycin A (2 µM), U0126 (50 µM), and SB203580 (20 µM) were obtained from Calbiochem. GI254023X (3 µM) and GW280264X (3 µM) 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 µg/ml mAb or 1 µM 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-{alpha} cells were treated or not with Alexa Fluor 568-labeled 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-{alpha}) labeled using the Alexa Fluor 488 Zenon Mouse IgG1 Labeling kit (Invitrogen). Nonstimulated cells 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% paraformaldehyde in PBS and then 15 min in –20°C methanol, and finally mounted in Mowiol. Analysis was performed with a TCS SP1 confocal microscope (Leica Microsystems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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 ~24-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 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 mAb (11G2) has a Mr of ~67,000 under nonreducing conditions, compatible with that of ADAM10, and comigrated with a major band present in the CD81 immunoprecipitate (Fig. 1A).


Figure 1
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  		FIGURE 1. Generation and characterization of an anti-ADAM10 mAb screened for its ability to coimmunoprecipitate CD81. A, During the initial screening, hybridoma supernatants were tested for their ability to coimmunoprecipitate CD81 after cell labeling with biotin and lysis in the presence of Brij 97. The mAb 11G2 immunoprecipitated (IP) a major 67-kDa molecule (arrow) and was selected for further analysis because it precipitated a band comigrating with CD81. B, The 11G2 target Ag was purified from HEL cells lysed in Triton X-100 on an 11G2 affinity column. Two major 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 ADAM10 (si ADAM10) and analyzed after 48 h. C, Binding of mAb 11G2 was determined by indirect immunofluorescence and flow cytometry. D, Cells were lysed and the level of 11G2 target Ag was determined by Western blot (WB) analysis. The mAb 11G2 recognizes under nonreducing conditions two bands of 67 and 80 kDa (arrows), which are consistent with the mature and immature forms of ADAM10, respectively, and these two bands were strongly decreased after ADAM10 silencing. E, The ability of mAb 11G2 to recognize recombinant ADAM10 ectodomain (100 ng) was examined by Western blotting after SDS-PAGE under nonreducing conditions.

 
The protein recognized by mAb 11G2 was purified on a 11G2 affinity column from ~ 5 x 108 HEL cells lysed in 1% Triton X-100 (to disrupt the tetraspanin web), yielding two major bands (Fig. 1B). After digestion with trypsin and MALDI-TOF mass spectrometry analysis, 36 peptides from the higher band and 24 from the lower band matched peptides expected for the metalloprotease ADAM10 (National Center for Biotechnology Information accession number 1616601). 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 ADAM10 prodomain, which is removed upon maturation of the protein. Thus the upper band corresponds to the unprocessed precursor protein and the lower band to the mature ADAM10 (Table I). It should be noted that the lower band is diffuse and is bordered by a more intense thin band with a Mr lower than expected for mature ADAM10. This band probably corresponds to a postlysis 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).


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

 
In addition, silencing ADAM10 in PC3 cells reduced by ~80% the labeling of cells by mAb 11G2, as determined by flow cytometry analysis (Fig. 1C). The mAb 11G2 recognizes in Western blotting two bands of 67 and 80 kDa (under nonreducing conditions), which are consistent with the mature and immature forms of ADAM10, and these bands were reduced after silencing ADAM10 (Fig. 1D). Finally, 11G2 recognized recombinant ADAM10 ectodomain in Western blotting experiments (Fig. 1E). Altogether, these results demonstrate that the mAb 11G2 is directed against the metalloprotease ADAM10.

ADAM10 associates with multiple tetraspanins in lymphoid cell lines and PBL

Previously published data indicated that ADAM10 associated with CD9 in colon cancer cells or in transfected COS7 cells (37, 38, 55), and the above data suggested an association of ADAM10 with CD81 in a cell line (Jurkat) expressing low levels of CD9. ADAM10 could form separate complexes with these two tetraspanins, or alternatively be part of the tetraspanin web and associate with multiple tetraspanins. To investigate this possibility, several cell lines (HEL, Raji, Jurkat, Daudi/CD9, Raji/CD9) were lysed in the presence of Brij97 and immunoprecipitations were performed with anti-tetraspanin mAb or the anti-ADAM10 mAb (Fig. 2A). The composition of the immunoprecipitates was analyzed by Western blotting. All tetraspanins studied were able to coimmunoprecipitate a large fraction of ADAM10, and reciprocally ADAM10 coimmunoprecipitated several tetraspanins. The strongest association was observed in HEL cells. Importantly, 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 with ADAM10. As a control, the raft resident protein CD55 did not coimmunoprecipitate ADAM10 or tetraspanins (Fig. 2A). Thus, ADAM10 is a component of the tetraspanin web. In all experiments, only the lower molecular weight form of ADAM10, corresponding to mature ADAM10 after removal of the prodomain, was associated with tetraspanins. Because removal of ADAM10 prodomain is mediated by furin and PC7 (56), which are localized in the TGN and endosomal compartments (57), this result indicates that the interaction of ADAM10 with the tetraspanins studied in this article occurs in a post-Golgi compartment.


Figure 2
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  		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 streptavidin. ADAM10 associates with all tetraspanins studied. Note that although CD19 and the integrin (Int.) {alpha}4β1 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.

 
The distribution of ADAM10 in peripheral blood cells has not been reported yet. The anti-ADAM10 mAb 11G2 stained polynuclear cells, monocytes, and platelets (Table II). It also stained PBL including T cells, B cells, and NK cells as shown by the labeling of CD3+, CD19+, and CD56+ cells (Table II), as well as all hematopoietic cell lines tested. The level of expression of ADAM10 in PBL was similar to that of Raji cells (see below, Fig. 6A).


Figure 6
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  		FIGURE 6. ADAM10 in PBL. A, PBL and Raji cells were labeled by indirect immunofluorescence using the anti-ADAM10 mAb 11G2 and analyzed by flow cytometry. The dotted line corresponds to the control staining. The same acquisition parameters were used for both cell types. B, PBL were stimulated or unstimulated for 24 h with CD3 and CD28 mAbs or PHA in the presence or absence of the ADAM10/ADAM17 inhibitors GW280264X or GI254023X (6 µM). TNF-{alpha} secretion was analyzed using 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 µM) was tested. TNF-{alpha} secretion was analyzed using an ELISA. *, p < 0.05, as compared with TPA-treated cells.

 
To determine whether ADAM10 interacted with tetraspanins in PBL, the cells were labeled with biotin and lysed in Brij 97 buffer before immunoprecipitation of ADAM10, CD53, CD81, and CD55 as a control (Fig. 2B). As expected, many proteins were coimmunoprecipitated with the tetraspanins CD53 and CD81. The same bands were also present in the ADAM10 immunoprecipitate, although at a lower level. The major protein coimmunoprecipitated with CD81 has a Mr of ~63,000 and most likely corresponds to EWI-2 (53). The identity of the major ~180-kDa molecule present in the CD53 immunoprecipitate is unknown, and so is that of the ~24 and ~30 kDa molecules present in the ADAM10 immunoprecipitate. To prove that ADAM10 was associated with CD53 and CD81 in PBL, the proteins coimmunoprecipitated with CD53 or CD81 were eluted and the presence of ADAM10 in these immunoprecipitations was confirmed by a second immunoprecipitation using the mAb 11G2 (Fig. 2B, bottom).

To estimate the fraction of ADAM10 associated with tetraspanins, Raji/CD9 lysates were subjected to immunoprecipitation using a mixture of five anti-tetraspanins mAbs (CD9, CD53, CD81, CD82, CD151). There was similar amount of ADAM10 in this immunoprecipitate as in the anti-ADAM10 immunoprecipitate. Additionally, depletion of these five tetraspanins yielded a strong reduction in the amount of ADAM10 that was immunoprecipitated by the 11G2 mAb, indicating that a major fraction of ADAM10 associates with tetraspanins (Fig. 2C).

Engagement of tetraspanins stimulates TNF-{alpha} release by B lymphoid cells

It has been reported that CD81 mAbs stimulate the release of TNF-{alpha} by murine T lymphocytes and some human B lymphoid cell lines (58, 59). Fig. 3 shows that among several anti-tetraspanin (CD9, CD81, CD82, CD53) mAbs tested, only the two CD82 mAbs stimulated TNF-{alpha} release by Raji cells (a B lymphoid cell line), inducing after 24 h incubation a 2- to 3-fold increase of TNF-{alpha} concentration in the supernatant (Fig. 3A). The lack of effect of CD9 mAbs was expected because Raji cells do not express this tetraspanin (30). The anti-ADAM10 mAb 11G2 did not stimulate TNF-{alpha} secretion.


Figure 3
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  		FIGURE 3. Engagement of tetraspanins stimulates TNF-{alpha} release by Raji cells. The cells were incubated with the indicated anti tetraspanin mAbs (CD9, CD53, CD81, CD82) or mAbs to other cell surface molecules, and the release of TNF-{alpha} in the medium was analyzed using an ELISA 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 wild-type CD9 or a nonpalmitoylatable CD9 mutant. E, Raji cells stably transfected with TNF-{alpha} (Raji/TNF-{alpha}) analyzed after various time of incubation with the CD82 mAb. Except when otherwise indicated, the CD9 mAb was SYB-1.

 
The ability of CD82 mAbs to stimulate TNF-{alpha} 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-{alpha} by Raji cells stably expressing CD9 (Raji/CD9; Fig. 3, B and C and data not shown). All CD9-positive clones tested were stimulated by CD9 mAb (data not shown). F(ab')2 of the CD9 mAb SYB-1 stimulated TNF-{alpha} release (Fig. 3C), indicating that this effect is not mediated through a Fc receptor-dependent mechanism. The increased release of TNF-{alpha} into the supernatant upon incubation with CD9 mAbs is not a consequence of increased synthesis, as determined by flow cytometry analysis of permeabilized cells stained with an anti-TNF-{alpha} 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, 35, 36). However, CD9 mAb were still able to stimulate the release of TNF-{alpha} by Raji cells expressing a nonpalmitoylatable CD9, indicating that this posttranslational modification is dispensable (Fig. 3D). Finally, both basal secretion and CD9-stimulated TNF-{alpha} secretion were completely inhibited by the broad-spectrum metalloprotease inhibitor GM6001 (data not shown), indicating that the fraction of TNF-{alpha} released upon addition of this mAb is normally processed through a metalloprotease.

To exclude the possibility that changes in TNF-{alpha} concentrations induced by anti-tetraspanin mAbs could be the consequence of modifications of biological phenomena such as proliferation or survival, the concentrations of TNF-{alpha} were measured at earlier time points. The stimulatory effect of CD9 mAbs on TNF-{alpha} 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-{alpha} in Raji cells that have been shown to be insensitive to the effects of TNF-{alpha} (60). These cells produced ~200 times more TNF-{alpha} than nontransfected Raji cells after a 4-h incubation (Fig. 3E). The CD82 mAb TS82 stimulated TNF-{alpha} 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-{alpha} secretion (data not shown). Thus the effect of anti-tetraspanin mAbs is rapid and most likely independent of new protein synthesis.

TNF-{alpha} shedding upon CD82 and CD9 engagement is mediated by ADAM10

We then tested whether the increased TNF-{alpha} secretion observed upon addition of CD9 or CD82 mAbs required an active ADAM10 (Fig. 4A). We first used two recently described hydroxamate-based compounds that differ in their capacity to inhibit the activities of ADAM17 and ADAM10. Whereas GW280264X potently inhibits both enzymes, GI254023X inhibits ADAM10 in the same concentration range of GW280264X but blocks ADAM17 with >100-fold reduced potency (54). GW280264X but not GI254023X partially blocked the basal TNF-{alpha} secretion, suggesting that it is in part an ADAM17-dependent mechanism. Both inhibitors completely abolished the stimulation of TNF-{alpha} release induced by CD9 and CD82 mAbs (Fig. 4A and data not shown), including in Raji/TNF-{alpha} cells (Fig. 4B), strongly implicating ADAM10 in this process. As a control, only GW280264X blocked TPA-induced TNF-{alpha} release, indicating that only ADAM17 contributes to this cleavage. It should be noted that the strong increase of TNF-{alpha} secretion observed after TPA treatment is not primarily the consequence of activation of a protease activity, but the result of a strong stimulation of TNF-{alpha} synthesis (61).


Figure 4
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  		FIGURE 4. Metalloprotease inhibitors prevent stimulation of TNF-{alpha} release by CD82 mAbs. The cells were incubated with TPA, anti-tetraspanin (CD82 (TS82) and CD53) mAbs, or the CD55 mAb in the presence or absence of the ADAM10/ADAM17 inhibitors GW280264X or GI254023X (used at 3 µM). The production of TNF-{alpha} was analyzed using an ELISA. A, Raji cells treated for 24 h with mAb or 1 h with TPA. B, Raji/TNF-{alpha} cells treated for 2 h with mAb or with TPA.

 
To confirm that the increased TNF-{alpha} release induced by CD9 or CD82 mAbs was dependent on ADAM10 proteolytic activity, the expression of ADAM10 in Raji or Raji/CD9 cells was knocked-down by RNA interference. The expression of ADAM10 was reduced by ~70–80%, as determined by flow cytometry, and the expression levels of CD9 and CD82 were not changed (Fig. 5A). ADAM10 silencing by two different siRNA (Fig. 5B and data not shown) completely abolished the stimulation of TNF-{alpha} release by CD9 or CD82 mAbs, confirming the prominent role of ADAM10 in this cleavage. ADAM10 silencing did not reduce TPA-stimulated TNF-{alpha} release.


Figure 5
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  		FIGURE 5. ADAM10 silencing prevents stimulation of TNF-{alpha} release by CD82 and CD9 mAbs. Raji or Raji/CD9 cells were transfected with a control siRNA (si Control) or a siRNA targeting ADAM10 (si ADAM10) and analyzed 48 h later. A, Flow cytometry analysis of ADAM10, CD9, and CD82 expression. B, The cells were incubated with the indicated mAb for 24 h (left) or with DMSO or TPA for 2 h (right), and the release of TNF-{alpha} in the medium was analyzed using an ELISA. The mAbs used were SYB-1 (CD9), TS82b (CD82), and TS53 (CD53).

 
Anti-tetraspanin mAbs stimulate TNF-{alpha} release by an ADAM10-dependent mechanism in TPA-treated lymphocytes

The availability of inhibitors discriminating between ADAM10 and ADAM17 activities made it possible to test whether ADAM10, which is expressed by lymphocytes (Fig. 6A, Table II), contributes to the secretion of TNF-{alpha} by in vitro activated human lymphocytes. As expected (62), activation of PBL by a combination of CD3 and CD28 mAbs or PHA strongly increased the synthesis and the secretion of TNF-{alpha} into the medium. GW280264X but not GI254023X blocked TNF-{alpha} secretion, indicating that ADAM10 does not participate in the release of TNF-{alpha} by CD3/CD28 or PHA-activated PBL (Fig. 6B). Additionally, TPA treatment induced the synthesis and the release of TNF-{alpha} as previously described (62). CD81 and CD82 mAbs, but not mAbs to CD53 or nontetraspanin proteins (CD46 and CD55), significantly increased the release of TNF-{alpha} by TPA-treated PBL (Fig. 6C). GW280264X, which potently blocks both ADAM10 and ADAM17 activity, prevented the secretion of TNF-{alpha} by cells stimulated by TPA alone or TPA with anti-tetraspanin mAb. In contrast GI254023X, that inhibits ADAM10 activity but has no effect on ADAM17, blocked the stimulating effect of anti-tetraspanin mAb, but not the secretion induced by TPA stimulation alone. Altogether these data strongly suggest that ADAM17 is the main TNF-{alpha} sheddase for in vitro-activated human PBL, and indicate that engagement of certain tetraspanins on PBL increases TNF-{alpha} release through ADAM10 "activation."

CD9 mAb do not stimulate TNF-{alpha} release when CD9 and TNF-{alpha} are present in different cells

The anti-tetraspanin mAbs that increase ectodomain shedding could increase shedding on the same cell, or alternatively could deliver an extracellular signal that stimulates neighboring cells. To discriminate between these two hypotheses, experiments where Raji/CD9 cells were mixed with either Raji or Raji/TNF-{alpha} cells were performed. Only Raji/CD9 cells can be stimulated by the CD9 mAb, but after a 2-h incubation the amount of TNF-{alpha} released by these cells is negligible, as determined by the analysis of Raji/CD9 and Raji cocultures. In cocultures of Raji/CD9 and Raji TNF-{alpha} cells, TNF-{alpha} is essentially released only by Raji/TNF-{alpha} cells. As shown in Fig. 7A, under these conditions the CD9 mAb did not stimulate TNF-{alpha} release in contrast with the CD82 mAb. This experiment argues against the requirement for an extracellular signal and strongly suggests that the protease stimulated by the CD9 mAb needs to be in the same cell as the target TNF-{alpha}.


Figure 7
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  		FIGURE 7. Signaling pathways involved in the stimulation of TNF-{alpha} release by anti-tetraspanin mAb. A, Raji/CD9 cells were mixed with an equal number (5 x 105 cells/ml) of either Raji or Raji/TNF-{alpha} in the presence or not of CD9, CD82, CD53, or CD55 mAbs for 2 h. TNF-{alpha} secretion was measured using an ELISA. B, Raji/TNF-{alpha} 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 µM), herbimycin A (2 µM), U0126 (50 µM), SB203580 (20 µM). The production of TNF-{alpha} 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.

 
CD82-stimulated release of TNF-{alpha} depends on active Erk1/2 but does not depend on tyrosine kinase or protein kinase C (PKC) activities

Tetraspanins have been shown to be coupled to diverse signal transduction pathways, including PKC, tyrosine kinases, and the MAPK pathways (44, 63, 64, 65, 66, 67). As expected, the PKC inhibitor bisindolylmaleimide I blocked the release of TNF-{alpha} induced by the PKC activator TPA. However, it did not affect the stimulation of TNF-{alpha} release induced by the CD82 mAb (Fig. 7B). The broad tyrosine kinase inhibitor herbimycin A did not reduce the effect of the CD82 mAb, but had a partial inhibitory effect on TPA-induced release. The stimulation by both the CD82 mAb and TPA was blocked by U0126, a specific MEK1/2 inhibitor that consequently prevents activation of Erk1/2 MAPK. In contrast, SB203580 that inhibits p38 MAPK only affected TPA-stimulated release. Importantly, CD9 and CD82 mAbs did not induce detectable increases in Erk1/2 phosphorylation in contrast to TPA (Fig. 7C). We therefore conclude that, as for other stimuli, the cleavage of TNF-{alpha} induced by tetraspanins mAb requires an intact MEK/Erk1/2 pathway, but this increased cleavage is not the consequence of a detectable increase in Erk1/2 activation.

CD82 mAbs induce a major redistribution of tetraspanins and ADAM10 but do not detectably change the level of ADAM10 interaction with tetraspanins

We then examined whether the engagement of tetraspanins with specific mAb modified the distribution of ADAM10 and TNF-{alpha} by using confocal microscopy. In nonstimulated cells, the tetraspanins CD82 and CD53, as well as ADAM10, were regularly distributed at the surface of Raji cells (Fig. 8 first row, and data not shown). Upon incubation with the CD82 mAb TS82b for 2 h at 37°C, CD82 molecules gathered into one or several patches of various sizes, to which ADAM10 was for the most part redistributed (Fig. 8, second row). The other tetraspanins CD81 and CD53 also strongly redistributed to these CD82 patches (data not shown), whereas the GPI-anchored protein CD55, which does not associate with tetraspanins, was only slightly enriched in these patches (Fig. 8, third row). The CD9 mAb TS9 also induced the formation of CD9 patches to which ADAM10 redistributed in Raji/CD9 cells (data not shown), whereas the mAb TS81 and TS53, which do not stimulate TNF-{alpha} secretion, induced only a limited redistribution of their target Ag and ADAM10 (Fig. 8, fourth row and data not shown). The CD82 mAb also induced patches to which ADAM10 was redistributed in Raji/TNF-{alpha} cells (data not shown). As expected, pro-TNF-{alpha} could not be detected by immunofluorescence in most of these cells. However, a few expressed pro-TNF-{alpha} at a detectable level. The major fraction of pro-TNF-{alpha} did not redistribute into the CD82 patches (Fig. 8, last row).


Figure 8
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  		FIGURE 8. ADAM10 redistributes into CD82-containing patches upon stimulation with CD82 mAbs. Raji or Raji/TNF-{alpha} cells were treated or not with Alexa Fluor 568-labeled CD82 or CD53 mAb (red) for 2 h at 37°C. They were then incubated at 4°C with Alexa Fluor 488-labeled mAb directed to ADAM10, CD55, or TNF-{alpha} (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-{alpha} for which we analyzed Raji/TNF-{alpha} cells. Bar, 5 µm.

 
These changes in tetraspanin and ADAM10 membrane distribution were not accompanied by a change in the level of association. Indeed, the level of ADAM10 coimmunoprecipitated with a mixture of anti-tetraspanin mAb was not modified after incubation for 2 h with CD82 or CD53 mAbs (Fig. 9).


Figure 9
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  		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.

 
Anti-tetraspanin mAbs stimulate EGF release by an ADAM10-dependent mechanism

Do anti-tetraspanin mAb specifically stimulate the activity of ADAM10 toward TNF-{alpha}, or do they also stimulate the cleavage of other substrates? This question was addressed using pro-EGF as a model of EGFR ligand, the ectodomain of which was predominantly cleaved by ADAM10 (68). HEK cells were transiently transfected with pro-EGF and after 48 h of incubation, stimulated or not with anti-tetraspanin mAb or TPA. The fraction of ectodomain released in the medium as well as cell-associated pro-EGF was quantified by Western blotting. CD9 and CD81 mAbs, respectively, stimulated an ~3- and ~10-fold increase in the EGF ectodomain shedding after 24-h incubation (Fig. 10, A and B). mAbs directed to CD82, ADAM10, and CD55 did not have any detectable stimulatory effect. The lack of effect of the CD82 mAb may be due to the very low level of expression of CD82 in this cell line. As previously described (46), TPA also stimulated pro-EGF ectodomain shedding. To determine whether ADAM10 was the 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 ~2- and ~3-fold increase of EGF secretion, and this stimulation was nearly completely abolished after ADAM10 silencing (Fig. 10, C and D). Additionally the level of EGF in the supernatant of nonstimulated cells was decreased by ~50% 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 ~70–80% after silencing, according to the experiments, and that ADAM10 associated with tetraspanins in HEK cells (data not shown). We conclude that tetraspanins regulate the activity of ADAM10 toward several substrates including TNF-{alpha} and EGF.


Figure 10
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  		FIGURE 10. Engagement of tetraspanins stimulate pro-EGF shedding through ADAM10 activation. HEK cells expressing rat pro-EGF were starved for 24 h and incubated in medium containing 1 µM TPA, the indicated anti-tetraspanin mAbs (CD9, CD81, CD82), or mAbs to other cell surface molecules (ADAM10, CD55). Full-length pro-EGF in the cell lysate (Cell) and ectodomain in the medium (SN) were detected by Western blotting using an anti-HA mAb. A, HEK-293 cells transiently transfected with rat pro-EGF and stimulated for 24 h. B, Quantification of the experiment shown in A. C, HEK-293 cells stably expressing pro-EGF fused 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 same gel and are shown after applying the same settings to the initial 16-bit image produced by the Odyssey Imaging System. Stimulation was for 2 h. D, Quantification of the experiment shown in C. Data are expressed as mean ± SD of three different experiments. In B and D, the shedding of pro-EGF in incubation medium is expressed as a percentage of total EGF in both incubation medium and cell lysates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we have shown that several anti-tetraspanin mAbs can rapidly stimulate the release of TNF-{alpha} or EGF in the medium through an ADAM10-dependent mechanism. Additionally, we have demonstrated a robust interaction of ADAM10 with several tetraspanins in various cell types. Our data suggest that tetraspanins could be important regulators of ADAM10 activity.

The activity of ADAM metalloproteases is thought to be tightly regulated, but the underlying mechanisms remain largely unknown (2). A remarkable example of such regulation is that of TNF-{alpha}. Indeed, although ADAM10 can cleave TNF-{alpha} or a TNF-{alpha} peptide encompassing the cleavage site in vitro (19, 69), it was shown not to contribute to TNF-{alpha} shedding in several studies using MEF (12, 17), suggesting a cellular inhibition of ADAM10 activity toward TNF-{alpha}. We have extended this result by showing that in Raji cells or in vitro activated lymphocytes, ADAM10 does not contribute to the cleavage of TNF-{alpha}, similar to what is observed in MEF (12, 17). The demonstration that anti-tetraspanin mAbs can induce ADAM10-mediated TNF-{alpha} cleavage indicates that ADAM10 can cleave TNF-{alpha} in a cellular context provided it is properly stimulated and points to a role for tetraspanins in the regulation of its activity. The fact that anti-tetraspanin mAbs also stimulate ADAM10-mediated shedding of EGF suggests that tetraspanins regulate the activity of ADAM10 toward several substrates, although in a less dramatic manner than toward TNF-{alpha}.

How could tetraspanins regulate ADAM10 activity? We have tested whether the effect of anti-tetraspanin mAb on ADAM10 activity could be the result of stimulating certain signaling pathways. The effect of anti-tetraspanin mAbs on TNF-{alpha} release is rapid and does not depend on extracellular signaling. Anti-tetraspanins mAbs induce in minutes the phosphorylation of proteins on tyrosine residues in certain B or T lymphoid cell lines (44, 63, 64). However, we did not detect any induction of tyrosine phosphorylation in the Raji cell line incubated with anti-tetraspanin mAbs (data not shown), and the tyrosine kinase inhibitor herbimycin A did not inhibit the increased release of TNF-{alpha} produced by anti-tetraspanin mAbs, suggesting that the effect does not rely on protein tyrosine phosphorylation. Tetraspanins have been shown to associate with activated PKC (65), and treatment of cells with the PKC activator TPA promotes the release of TNF-{alpha} through an ADAM17-dependent mechanism (Refs. 12, 17 and Figs. 4 and 6). The effect of anti-tetraspanin mAbs on TNF-{alpha} release is independent of PKC, because a broad spectrum PKC inhibitor did not change the release of TNF-{alpha} induced by anti-tetraspanin mAb while completely preventing that induced by TPA (Fig. 7). We have shown here that the increased TNF-{alpha} release induced by the CD82 mAb is dependent on an intact MEK/Erk pathway, but does not depend on the p38 MAPK pathway. Although the engagement of tetraspanins has been shown to activate these pathways in nonhematopoietic cell lines (66, 67), the stimulation of ADAM10-mediated TNF-{alpha} shedding by anti-tetraspanin mAbs is not a mere consequence of the activation of the Erk pathway. Indeed, we did not observe Erk activation upon addition of CD82 mAb to Raji cells although TPA caused strong Erk phosphorylation without inducing ADAM10-mediated TNF-{alpha} shedding. ADAM17-dependent ectodomain shedding also requires an intact MEK/Erk pathway because TPA-stimulated TNF-{alpha} release is blocked to a large extent by the MEK inhibitor (Fig. 7). This result is in agreement with previous studies showing the key role of MAPK in the cleavage of various cytokines and growth factors, including TNF-{alpha} (70, 71). It is therefore possible that the inhibitory activity of the MEK/Erk inhibitor reflects the necessity for a locally active MEK/Erk pathway for ADAM-mediated cleavage.

The fact that ADAM10 associates with tetraspanins makes possible the hypothesis that tetraspanins directly regulate ADAM10 ability to cleave some of its substrates. The inability of ADAM10 to cleave TNF-{alpha} in nonstimulated cells while strongly interacting with tetraspanins suggests that tetraspanins negatively regulate the activity of ADAM10. The stimulatory effect of CD82 or CD9 mAb on TNF-{alpha} release is, however, not a consequence of a disruption of ADAM10/tetraspanin interaction (Figs. 8 and 9). Instead, it is correlated with the ability of these anti-tetraspanin mAb to redistribute ADAM10 into tetraspanin-containing patches (Fig. 8). We propose that this increase in ADAM10 local concentration confers on ADAM10 the ability to cleave TNF-{alpha} (and increase the activity toward other substrates such as EGF) despite the association with tetraspanins.

Among other ADAM proteases, ADAM10 is most closely related to ADAM17 (4). In several experiments using different cell lines, we did not observe an interaction between ADAM17 and tetraspanins (data not shown). Additionally, analysis of CD9-associated molecules in colon cancer cells using proteomic approaches did not reveal an interaction with ADAM17 (38). However, the detection of ADAM17 in the cell lines tested was always lower than that of ADAM10 (by flow cytometry, Western blotting, and immunoprecipitation of biotin-labeled surface proteins, data not shown), which may reflect different efficiency of Abs or a different level of expression. We therefore cannot exclude the possibility that ADAM17 is associated with tetraspanins but could not be identified in tetraspanin immunoprecipitates because of a low level of expression.

The list of substrates for ADAM10 has grown quickly in recent years (for review see Ref. 3), and it will have to be determined whether tetraspanins regulate the cleavage of substrates other than TNF-{alpha} and EGF. Because TNF-{alpha} is mainly an ADAM17 substrate, it will have also to be determined whether tetraspanins prevent the cleavage by ADAM10 of other known ADAM17 substrates. A number of ADAM10/ADAM17 substrates have been shown to associate with tetraspanins (mostly CD9) including the EGF receptor ligands HB-EGF and TGF{alpha}, the complement regulatory molecule CD46, or the hyaluronic acid receptor CD44 (2, 38, 52, 72, 73, 74, 75). CD9 expression was shown to inhibit the release of TGF-{alpha} into the medium (73), a process that like TNF-{alpha} release is critically dependent on ADAM17 activity under the conditions tested so far (2). This raises the hypothesis that tetraspanins control the substrate specificity of ADAM10 by interacting with both ADAM10 and some of its substrates. In this context, the proteins may be in different tetraspanin microdomains and the mAb could bring different microdomains together, thus promoting the cleavage of the substrate. However, not all ADAM10/ADAM17 substrates associate with tetraspanins. Among the proteins identified as associating with tetraspanins by mass spectrometry analysis (37, 38), only CD44 and CD46 are known substrates of metalloproteases (74, 75). More specifically, we could not coimmunoprecipitate the ADAM10 substrates L1, PrP, or E-cadherin (76, 77, 78) with tetraspanins (our unpublished data). So far we have been unable to demonstrate an interaction of tetraspanins with pro-TNF-{alpha} in Raji/TNF-{alpha} cells by coimmunoprecipitation. However, our experiments with TNF-{alpha} may lack sensitivity because the expression of pro-TNF-{alpha} remains very low at the surface of the majority of the cells. In addition, the lack of TNF-{alpha} redistribution into CD82 patches argues against a strong association of TNF-{alpha} with tetraspanins.

The physiological activators of ADAM metalloproteases are poorly characterized (3). The activity of several ADAM proteases can be up-regulated through GPCR activation, at least in certain circumstances (8). In addition, apoptosis, pore-forming toxins, and cholesterol depletion have been shown to increase the shedding of ADAM protease substrates (79, 80, 81, 82). The most frequently used activators of ADAM-mediated shedding events are ionomycin and TPA, which have been shown to up-regulate the activity of ADAM10 and ADAM17, respectively (2, 10, 11, 83), through largely unknown mechanisms. Notably, the ability of TPA to up-regulate ADAM17-mediated TNF-{alpha} cleavage does not require the ADAM17 cytoplasmic domain (12), suggesting a regulatory mechanism at the level of the plasma membrane. ADAM17 was recently shown to partition into low-density fractions of sucrose gradients after lysis in Triton X-100, suggesting an interaction with classical lipid rafts (47). This raises the hypothesis that ADAM17 could be regulated through classical raft microdomains, whereas ADAM10 would be regulated through tetraspanin-enriched microdomains. The organization of the tetraspanin web is dynamic because it can be modified by anti-tetraspanin mAbs (this study), by manipulating cholesterol levels (29), or by modifications in the pattern of associated proteins (84), and is therefore compatible with a regulatory role. Physiological modifiers of the "web" may have consequences for the activity of ADAM10 toward TNF-{alpha} and other substrates. In this regard, the GPCR ligand bombesin increased the interaction of ADAM10 with CD9 in ADAM10-transfected COS7 cells (55).

In conclusion, we have demonstrated that ADAM10 strongly associates with several tetraspanins, and that engagement of tetraspanins through mAbs induces ADAM10-mediated shedding of EGF and TNF-{alpha}. These data implicate tetraspanins in the control of ADAM10 activity and raise the possibility that under particular physiological or pathological conditions, the activity of ADAM10 is modulated through a modification of its interaction with the tetraspanin web.


   Acknowledgments
 
We are grateful to Dr. Levy for the generous gift of CD81 mAb and Dr. Peiretti for the TNF-{alpha} plasmid. We thank Dr. M. Tomlinson for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 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 Ile de France, the Association pour la Recherche contre le Cancer, and Nouvelles Recherches Biomédicales-Vaincre le Cancer. T.A. and C.A. were supported by fellowships awarded by the French government. T.A. and L.L. were recipients of a fellowship awarded by the Association Nouvelles Recherches Biomédicales. L.L. was also the recipient of a fellowship from the Fondation Franco-Chinoise pour la Science et ses Applications. A.L. is supported in part by the Interdisziplinäres Zentrum für Klinische Forschung Biomat, RWTH Aachen University, and by Sonderforschungsbereich 451, project A12 of the Deutsche Forschungsgemeinschaft. Back

2 E.R. and F.L.N. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Eric Rubinstein, INSERM, U602, 14 Avenue Paul Vaillant Couturier, F-94807 Villejuif Cedex, France. E-mail address: eric.rubinstein{at}inserm.fr Back

4 Abbreviations used in this paper: ADAM, a disintegrin and metalloprotease; HB-EGF, heparin-binding epidermal growth factor; TPA, 12-O-tetradecanoyl-phorbol 13-acetate; MEF, mouse embryonic fibroblast; HEK, human embryonic kidney; siRNA, small interfering RNA; PKC, protein kinase C; GPCR, G protein-coupled receptor; HA, hemagglutinin. Back

Received for publication September 20, 2007. Accepted for publication September 19, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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