Article |
Address correspondence to Kiyotoshi Sekiguchi, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8617. Fax.: 81-6-6879-8619. email: sekiguch{at}protein.osaka-u.ac.jp
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Abstract |
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Key Words: cell adhesion; tetraspanin; integrin; actin cytoskeleton; Rho family GTPase
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Introduction |
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Recently, CD151 and some other tetraspanins were shown to associate with conventional PKC (cPKC; Zhang et al., 2001) and modulate integrin-dependent cell morphology (Kazarov et al., 2002). cPKC has been shown to regulate a variety of biological events, including cellcell and cellECM adhesion and the inside-out activation of integrins (Shattil and Brass, 1987; Shimizu et al., 1990). PKC, one of the cPKCs, was shown to be involved in actin reorganization during cellcell adhesion (Cowell and Garrod, 1999). It is generally accepted that cytoplasmic actin dynamics are regulated by the Rho family of small GTPases, often leading to the extension of the cell front as filopodia or lamellipodia, depending on the stimuli (Mackay and Hall, 1998). These dynamic processes are dependent on integrin-mediated cellECM adhesion, and cell adhesion to different ECM ligands leads to differential activation of Rho GTPases. Thus, cell adhesion to fibronectin via integrin
5ß1 selectively activates Rho, whereas cell adhesion to laminin-10/11 via integrin
3ß1 activates Rac, but not Rho (Gu et al., 2001). However, it is not known whether CD151 is involved in the ligand-dependent, differential activation of Rho GTPases through specific association with integrin
3ß1.
The Rho family GTPases have also emerged as crucial regulators of cadherin-dependent cellcell adhesion (Braga, 2000; Fukata and Kaibuchi, 2001). A filopodia-based mechanism of intercellular junction formation has been shown to operate during embryonic development of Caenorhabditis elegans (Raich et al., 1999) and Drosophila melanogaster (Jacinto et al., 2000). In keratinocytes, filopodium formation was reported to be the driving force of cellcell adhesion, leading to the "adhesion zipper" model of epithelial cellcell adhesion (Vasioukhin et al., 2000). In support of this model, E-cadherinmediated cellcell adhesion is associated with activation of Cdc42 (Kim et al., 2000), although the mechanisms regulating small GTPases during cellcell adhesion are only poorly understood. We address the possible involvement of CD151 in cellcell adhesion and its associated actin reorganization. Given the localization of CD151 at sites of epithelial cellcell contacts as a stable complex with integrin 3ß1, and the functional association of CD151 with cPKC, it is tempting to speculate that CD151 may play a role in epithelial cellcell adhesion through cPKC-dependent actin reorganization. Here, we provide evidence that CD151 is a novel regulator of cPKC- and Cdc42-dependent actin reorganization, thereby stimulating E-cadherindependent cellcell adhesion.
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Results |
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Cadherin-mediated cellcell adhesion is central to the maintenance of epithelial integrity. To ascertain whether cadherin-mediated cellcell adhesion was disturbed by pretreatment with anti-CD151 mAb, we examined the distribution of E-cadherin in mAb-pretreated A431 cells overexpressing CD151-GFP. Although a significant fraction of E-cadherin remained localized at cellcell contact sites, it was evident that E-cadherin distribution became irregular and discontinuous compared with untreated cells (Fig. 4). In contrast, integrin 3ß1, known to tightly associate with CD151 and localize at cellcell contacts, was coalesced into aggregates, colocalizing with the aggregates of CD151-GFP. Given that cortical actin filaments were severely disrupted in cells pretreated with mAb (Fig. 3 A), it seems unlikely that E-cadherin at cellcell contact sites was fully functional because strong E-cadherinmediated cellcell adhesion requires
-catenindependent anchorage of E-cadherin to cortical actin filaments.
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Overexpression of CD151 promotes E-cadherin puncta formation through PKC-dependent signaling pathway
Given enhanced cellcell adhesion and cell polarization by CD151 overexpression, the prominent filopodial extension by basal engagement of CD151 raised the possibility that CD151 might be involved in the initial stage of cellcell adhesion through regulating filopodia-based adhesion zipper formation. To explore this possibility, we examined the effect of CD151 overexpression on the formation of E-cadherin puncta, an indication of the initial stage of E-cadherinmediated cellcell adhesion (Adams et al., 1998), by the calcium switch experiment. E-Cadherin puncta were not detectable at 10 min after calcium restoration, but became evident after 60 min (Fig. 7 A). The number and size of the E-cadherin puncta were significantly greater in CD151-overexpressing cells than control GFP-transfected cells, indicating that the initial stage of E-cadherinmediated cellcell adhesion was accelerated upon overexpression of CD151. It was noted that CD151-GFP was also brought into punctate aggregates, partially overlapping the E-cadherin puncta.
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Among known cPKCs, PKC has been shown to be the major cPKC isoform in A431 cells (Szekeres et al., 2000). Because activation of PKC
has been shown to be associated with its translocation to plasma membrane (Vallentin et al., 2001), we examined the localization of PKC
in control and CD151-overexpressing A431 cells. Although PKC
only accumulated to a marginal extent at cellcell contact sites in control cells, a clear accumulation of PKC
was observed with CD151-overexpressing cells (Fig. 7 C). The enhanced accumulation of PKC
at cellcell contacts was not due to the increased expression of PKC
because the level of PKC
expression remained unchanged in CD151-overexpressing cells (unpublished data). A significant fraction of CD151-GFP was found to colocalize with PKC
at cellcell contact sites, consistent with a previous paper that PKC
coprecipitated with CD151 (Zhang et al., 2001).
CD151 overexpression promotes E-cadherin anchorage to cytoskeletal matrix
Throughout E-cadherinmediated cellcell adhesion, anchorage of E-cadherin to actin filaments is a critical, rate-limiting event securing the mechanical strength of intercellular adhesion. Given the possible role of CD151 in regulating actin cytoskeleton, it is conceivable that the anchorage of E-cadherin to actin cytoskeleton is enhanced in cells overexpressing CD151. To explore this possibility, cells were lysed with NP-40 and the amounts of E-cadherin in detergent-soluble (cytoplasmic) and -insoluble (cytoskeleton associated) fractions were determined by immunoblotting. In control cells, the majority of the E-cadherin was recovered in the insoluble fractions, leaving 2025% in the soluble fractions, depending on the detergent concentration used (Fig. 8, A and B). However, in CD151-overexpressing cells, most of the E-cadherin was recovered in the insoluble fractions with <10% remaining in the soluble fractions, supporting the possibility that CD151 overexpression promotes the anchorage of E-cadherin to actin cytoskeletal matrix.
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E-cadherin is not a prerequisite for CD151-mediated actin reorganization
Although E-cadherin serves as one of the regulators of actin dynamics during epithelial cellcell adhesion and polarization, we finally addressed the possibility that homophilic interaction of E-cadherin might be involved in the filopodial extension induced by basal engagement of CD151. Therefore, we used mouse L cells lacking cadherin activity and their E-cadherin transfectants (designated EL cells). When L and EL cells were transfected with CD151-GFP and plated on the substrates coated with anti-CD151 mAb, both cells extended numerous filopodia regardless of whether E-cadherin was present or absent (Fig. 9). These results indicate that basal engagement of CD151 by itself can trigger the Cdc42-dependent filopodial extension without collaboration with E-cadherin.
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Discussion |
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Another line of evidence for the role of CD151 in actin dynamics is the observation that treatment of cells with anti-CD151 mAb impaired not only epithelial polarization but also the assembly of cortical actin belts. Rac has been implicated in the actin reorganization into cortical belts because assembly of cortical actin filaments was potentiated by the dominant-active form of Rac (Takaishi et al., 1997; Jou and Nelson, 1998). Enhanced activation of Rac in CD151-overexpressing cells indicates that CD151 is involved in actin assembly into cortical belts through Rac activation, although the underlying mechanism remains to be defined. Given the accumulating evidence that homophilic interaction of E-cadherin activates Rac at cellcell contact sites (Nakagawa et al., 2001; Noren et al., 2001; Betson et al., 2002), accelerated E-cadherin puncta formation in CD151-overexpressing cells may be responsible for the enhanced Rac activation in these cells. It is generally accepted that strong cadherin-mediated cellcell adhesion requires anchorage of cadherins to cortical actin filaments via catenins. Because E-cadherin anchorage to detergent-insoluble actin cytoskeletal matrix was enhanced in CD151-overexpressing cells, it is conceivable that CD151 overexpression promotes E-cadherinmediated cellcell adhesion by potentiation of not only filopodia-based adhesion zipper formation at an initial stage but also E-cadherin anchorage to cortical actin filaments through enhanced Rac activation at later stages.
Our data show that cPKC is involved in the CD151-mediated actin cytoskeletal dynamics. Thus, calphostin C, a cPKC inhibitor, blocked filopodial extension induced by substrate-adsorbed anti-CD151 mAb as well as enhanced E-cadherin puncta formation in CD151-overexpressing cells. Furthermore, translocation of PKC, the major cPKC isoform in A431 cells (Szekeres et al., 2000), to cellcell contact sites was significantly promoted in CD151-overexpressing cells. These results are consistent with a recent paper that CD151 and some other tetraspanins coprecipitate with cPKC (Zhang et al., 2001). In support of the physical association of CD151 with cPKC, we found that a significant fraction of CD151 colocalized with PKC
during the calcium-stimulated epithelial cellcell adhesion. Together, these results indicate that CD151 directly or indirectly associates with PKC
, thereby regulating actin reorganization during filopodia-based cellcell adhesion and subsequent assembly of cortical actin filaments. Involvement of cPKC in filopodial extension has also been demonstrated with epithelial and neuronal cells (Beckmann et al., 1995; Cheng et al., 2000).
It remains unclear what activates the CD151-dependent signaling pathway that, in turn, leads to the activation of Cdc42 and Rac during epithelial cellcell adhesion. One possible scenario is that an initial E-cadherin homophilic interaction induces local clustering of CD151, which in turn activates Cdc42 via cPKC, thereby promoting filopodia-based E-cadherin puncta formation. Thus, there is a positive feedback loop between the E-cadherin homophilic interaction and the CD151-dependent actin reorganization. In fact, calcium switch experiments showed that E-cadherin puncta formation was associated with the clustering of CD151 into punctate aggregates. Another scenario for the mechanism activating the CD151 signaling pathway could be that there is an unknown counter-receptor for CD151 on the epithelial cell surface, which interacts with CD151 in trans at cellcell contact sites. The interaction of the substrate-adsorbed mAb with CD151 on the cell surface may mimic such physiological transinteraction of CD151 with its counter-receptor, thereby triggering the signaling pathway downstream of CD151. Indeed, there is accumulating evidence that ligand capture by substrate-immobilized antibodies mimics cellular responses induced by physiological ligandreceptor interactions (Miyamoto et al., 1995; Fang et al., 1999). Although no counter-receptor for CD151 has ever been identified, CD9 was recently identified as the receptor for the pregnancy-specific glycoprotein 17 (Waterhouse et al., 2002).
CD151 forms a stable complex with integrin 3ß1 and localizes at cellcell contact sites. Although integrin
3ß1 is the primary adhesion receptor for laminin-5 and laminin-10/11, and it plays a central role in adhesion of epithelial cells to basement membranes, its preferential localization at cellcell contact sites implies that it may also be involved in cellcell adhesion. In support of this possibility, integrin
3ß1 has been shown to interact with integrin
2ß1 at cellcell contact sites (Symington et al., 1993). The role of integrin
3ß1 in epithelial cellcell adhesion was also addressed by Weitzman et al. (1995), who examined the intercellular adhesion of integrin
3transfected cells but obtained no clear evidence supporting the role of integrin
3ß1 in cellcell adhesion. Recently, Wang et al. (1999) reported that the assembly of cortical actin filaments was severely impaired in integrin
3ß1deficient epithelial cells. The anchorage of the E-cadherincatenin complex to the actin cytoskeleton was also significantly impaired in these cells. Given the stable association of integrin
3ß1 with CD151 and the role of CD151 in E-cadherinmediated cellcell adhesion, it is tempting to speculate that integrin
3ß1 also serves as a modulator of E-cadherinmediated cellcell adhesion and actin reorganization through its tight association with CD151. Although the mechanisms operating downstream of CD151 still remain to be defined, studies on the role of the integrin
3ß1CD151 complex in actin cytoskeletal organization in epithelial cells should shed light on the delineation of the mechanisms underlying epithelial cellcell adhesion and polarization.
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Materials and methods |
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Antibodies and reagents
The mAb against E-cadherin (ECCD-2) was obtained from TAKARA Shuzo. The mAb against Rac was obtained from Transduction Laboratories. Polyclonal antibodies against Cdc42, Rho, ZO-1, and PKC were obtained from Santa Cruz Biotechnology, Inc. The mAb against
2 integrin was obtained from Chemicon. Polyclonal antibody against actin was obtained from Sigma-Aldrich. The mAb 3G8 against
3 integrin was prepared as described previously (Kikkawa et al., 2000). The mAb 8C3 against CD151 was isolated along with the mAb 3G8 as another mAb against
3 integrin, but was later found to recognize CD151 tightly associated with integrin
3ß1 (Sterk et al., 2000; Yanez-Mo et al., 2001). The mAb 4F5 against
5 integrin was produced by immunizing mice with purified integrin
5ß1 as described previously (Manabe et al., 1997). The mAb against HA was purchased from BAbCo. TRITC-conjugated anti
3 integrin mAb was prepared as described by Goding (1976). Rhodamine-labeled phalloidin was obtained from Molecular Probes Inc.; calphostin C was obtained from Wako; rottlerin was obtained from Calbiochem; LY294002 was obtained from Sigma-Aldrich. Y27632, a ROCK inhibitor, was a gift from A. Yoshimura (Yoshitomi Pharmaceutical, Saitama, Japan). Laminin-5 was purified from the conditioned medium of MKN45 cells by immunoaffinity chromatography as described previously (Fukushima et al., 1998). Fibronectin was purified from human plasma by gelatin affinity chromatography.
Expression plasmids
A full-length cDNA encoding human CD151 (Hasegawa et al., 1996) was inserted in frame into pEGFP-N3 (CLONTECH Laboratories, Inc.) at the HindIII and ApaI sites to produce an expression vector (pEGFP-CD151) for CD151 fused with EGFP at its COOH terminus. The expression plasmids for GST-RB (a fusion protein of GST to the Rho-binding domain of Rho kinase) and GST-CRIB (a fusion protein of GST to the Cdc42/Rac-interactivebinding domain of PAK1) as well as the dn forms of HA-tagged Cdc42 and Rho were provided by K. Kaibuchi (Nagoya University Medical School, Nagoya, Japan).
DNA transfection and selection of stable transfectants
A431 cells were transfected with pEGFP-CD151 by electroporation using a Gene Pulser (Bio-Rad Laboratories). Cells were passaged at a 1:3 dilution 12 h after transfection and maintained in medium containing 1 mg/ml G418 to select stable transfectant clones. A431 cells were also transfected with pEGFP-N3 as a control. 20 µg of expression plasmids of dn-Cdc42 and dn-Rho were cotransfected with 5 µg of pHA262Puro, a puromycin-resistant marker, into 3 x 106 HeLa S3 cells using LipofectAMINETM Plus (GIBCO BRL). Cells were passaged at a 1:3 dilution 12 h after transfection and maintained for 48 h in medium containing 10% FBS and 1 µg/ml puromycin.
Cell labeling and immunoprecipitation
A431 transfectants were washed twice with biotinylation buffer (0.1 M HEPES-HCl, pH 8.0, 50 mM NaCl, 1 mM PMSF, and 20 µg/ml leupeptin) and surface labeled with 2 mg/ml sulfo-NHS-LC-biotin for 20 min at RT. Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 150 mM NaCl, 1 mM PMSF, and 2 µg/ml leupeptin) and immunoprecipitated with the indicated mAbs. The precipitates were eluted into sample treatment buffer and resolved by 10% SDS-PAGE under nonreducing conditions. Proteins were transferred to PVDF membranes and visualized with HRP-conjugated streptavidin.
Immunofluorescence
Cells were fixed with 3% PFA or in methanol, permeabilized with 0.1% Triton X-100 for 5 min, and incubated with either appropriate primary antibodies or rhodamine-labeled phalloidin for 1 h at RT, followed by incubation with secondary antibodies for 1 h. The secondary antibodies used were Alexa 596labeled goat antirabbit IgG (Molecular Probes) or Cy3-labeled goat antirat IgG (Jackson ImmunoResearch Laboratories). After three washes with PBS, cells were mounted and examined with a confocal microscope (model LSM5 PASCAL; Carl Zeiss MicroImaging, Inc.). All images were imported into Adobe Photoshop as TIFFs for contrast manipulation and figure assembly.
Pretreatment of cells with mAbs
To examine the effects of mAb treatment on epithelial integrity, cells were detached with trypsin/EDTA, washed twice with DME containing 1% BSA, and resuspended in the same medium containing 20 µg/ml of the mAbs to be examined. Cells were kept in suspension for 10 min at RT, and then plated on substrates coated with 10 µg/ml laminin-5. The cells were incubated on the substrates for 6 h at 37°C, fixed, and stained with appropriate antibodies or rhodamine-labeled phalloidin. The stained cells were examined with a confocal microscope.
Pull-down assay of GTP-loaded Cdc42, Rac, and Rho
Pull-down assays of GTP-loaded Cdc42, Rac, and Rho were performed as described previously (Gu et al., 2001), except that cells were grown to confluence in serum-containing medium before the assay. Cells adhering to the substrates as specified were lysed in 50 mM Tris-HCl, pH 7.4, containing 1% NP-40, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF. The lysates were clarified by centrifugation at 20,000 g for 15 min at 4°C, and then incubated with either 30 µg of GST-CRIB or GST-RB for 60 min at 4°C in the presence of 30 µl of glutathione-agarose beads. The precipitates were washed three times with lysis buffer and resolved in 12% SDSpolyacrylamide gels. After electrophoretic transfer to PVDF membranes, the membranes were probed with anti-Rac mAb or polyclonal antibodies against Cdc42 or Rho.
Wound closure assay
A431 transfectants were seeded on 24-well plates and grown to confluence. The medium was replaced with fresh DME containing 1% FBS 8 h before the onset of the assay. The assay was started by scratching the confluent cells with a pipette tip to make a wound 0.3 mm wide. Wounded cells were incubated in the same medium at 37°C for 24 h to heal. Cells were photographed at 12 and 24 h after the onset of the assay using a phase-contrast microscope (model CK40; Olympus).
Cell motility assay
The cell motility of individual cells was quantified by time-lapse videomicroscopy. A431 transfectants were replated on 35-mm culture dishes at 2.5 x 105 cells/dish in DME containing 10% FBS and allowed to adhere to the substrates. 12 h after replating, the medium was replaced with DME containing 1% FBS. Cell migration was monitored using an inverted microscope (model S-25; Carl Zeiss MicroImaging, Inc.) equipped with a built-in CO2 incubator. Video images were collected at 15-min intervals using Image-Pro software (Media Cybernetics). The positions of the nuclei were tracked to quantify the cell motility. Velocities were calculated in micrometers per 8 h using the same software.
Detergent extraction of actin and E-cadherin
Detergent extraction of E-cadherin from A431 transfectants was performed as described previously (Nagafuchi and Takeichi, 1988). In brief, subconfluent cells grown on 6-cm culture dishes were collected into a 1.5-ml centrifuge tube with a rubber policeman, centrifuged at 5,000 g for 1 min, and then lysed in 200 µl HBS/C (HEPES-buffered saline containing 1 mM CaCl2) containing 0.02 or 0.2% NP-40 on ice for 10 min with gentle pipetting. The lysates were centrifuged at 100,000 g for 30 min, and the supernatants were mixed with 100 µl of 3x SDS sample treatment buffer and used as the detergent-soluble fractions. The pellets were dissolved in 300 µl of 1x SDS sample treatment buffer and used as the detergent-insoluble fractions. The detergent-soluble and -insoluble fractions were subjected to SDS-PAGE, followed by immunoblotting with antiactin polyclonal antibody or antiE-cadherin mAb. Densitometric quantification of the relative intensity of the bands visualized with an ECL chemiluminescence detection kit (Amersham Biosciences) was performed using NIH Image software.
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Acknowledgments |
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This work was supported by Special Coordination Funds and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Submitted: 21 January 2003
Accepted: 21 August 2003
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