Article |
Address correspondence to Cécile Gauthier-Rouvière, Centre de Recherche de Biochimie Macromoléculaire (CRBM), CNRS UPR 1086, 1919 Route de Mende, 34293 Montpellier Cedex, France. Tel.: 33-4-6761-3355. Fax: 33-4-6752-1559. E-mail: gauthier{at}crbm.cnrs-mop.fr
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Abstract |
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Key Words: N-cadherin; Rho GTPases; JNK; ß-catenin, myogenesis; PL, polylysine
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Introduction |
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In the present study, we have investigated the influence of N-cadherindependent cellcell contacts on mouse C2C12 myoblasts myogenesis and on Rho GTPases and MAPK activity. Using antibodies that specifically recognize the extracellular domain of N-cadherin or N-cadherin ligand, allowing us to mimic N-cadherinmediated adhesion, we demonstrate that N-cadherindependent cellcell adhesion negatively regulates the activity of Rac1, Cdc42Hs, and JNK but increases RhoA activity and induces three skeletal muscle-specific promoters. We also observed RhoA-dependent ß-catenin accumulation at the cellcell contact sites. Together, these results suggest a crucial role for N-cadherindependent cellcell contacts in the regulation of RhoA, Rac1, Cdc42Hs, and JNK activity during myogenesis.
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Results |
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N-cadherindependent cellcell contact decreases Rac1 and Cdc42Hs activities
Since active Rac1 and Cdc42Hs inhibit myogenesis (Meriane et al., 2000), we examined whether N-cadherindependent cellcell contacts control Rac1 and Cdc42Hs activity. Cadherins require Ca2+ to form homophilic cellcell adhesions; thus Ca2+ chelation with EGTA, followed by Ca2+ readdition, offers a simple method to study the adhesive properties of these surface molecules (Volberg et al., 1986). Whereas control cells present a typical pattern of N-cadherin and ß-catenin immunostaining at the level of cellcell contacts (Fig. 1, a and f), in EGTA-treated cells, N-cadherin and ß-catenin are lost from the cellcell contact sites (Fig. 1, b and g). Upon restoration of extracellular Ca2+, cellcell contacts were rapidly reformed as shown by both N-cadherin and ß-catenin staining (Fig. 1, c and h). In contrast, the presence of antiN-cadherin antibodies blocked calcium-restored N-cadherin or ß-catenin accumulation at the cellcell contact sites (Fig. 1, d and i) as did addition of antiN-cadherin antibodies during contact establishment (Fig. 1, e and j). Higher magnification and deconvolved views of N-cadherin and ß-catenin at the contact sites are shown in Fig. 1 (k to y).
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To address the question of whether cadherins themselves signal directly to Rac1 and Cdc42Hs, we examined N-cadherin engagement in the absence of cellcell adhesion. Isolated C2C12 were plated on dishes coated with either Fc fragment (Fc), polylysine (PL), or Ncad-Fc ligand, which allowed us to mimic N-cadherinmediated adhesion. We verifited that adhesion to Ncad-Fccoated surfaces is dependent on calcium (unpublished data). 2 h after plating, we observed a marked Rac1 activation on Fc and PL and Cdc42Hs activity on PL (Fig. 2 E). No such Rac1 and Cdc42Hs activity was detected when C2C12 myoblasts were plated on surface coated with Ncad-Fc. These data demonstrate that N-cadherin engagement strongly inhibited Rac1 and Cdc42Hs activity.
N-cadherindependent cellcell contact decreases JNK activity without modification of p38 activity
Activation of the JNK pathway by Rac1 and Cdc42Hs GTPases inhibits myogenesis (Meriane et al., 2000). Therefore we examined whether N-cadherindependent cellcell contact regulates JNK activity (Fig. 3 A). In cells treated with EGTA, JNK activity was similar to that in isolated cells. Addition of antiN-cadherin antibody blocked the calcium-dependent decrease in JNK activity. To assess whether the control of JNK activity by cellcell contacts depends on Rac1 and Cdc42Hs activity, we transfected C2C12 myoblasts with constructs expressing the dominant negative T17N mutants of Rac1 and Cdc42Hs (Fig. 3 B). The level of JNK activity in isolated cells is decreased by expression of Rac1N17 or Cdc42HsN17. In addition, Rac1N17 or Cdc42HsN17 expression impaired JNK activation by EGTA treatment of cells in contact.
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N-cadherindependent cellcell contact activates muscle-specific promoters
We then determined whether the effect of N-cadherindependent cellcell contact extends to muscle-specific promoters shown previously to be controlled by RhoA, the skeletal -actin (SkA), and the myosin light chain (MLC)1A promoters (Fig. 6, A and B) (Carnac et al., 1998; Wei et al., 1998). Both reporter constructs are significantly more active in cells in contact, whereas the effect is weak with the SkA promoter. We also manipulated cadherin-mediated junction formation using the calcium switch procedure. EGTA addition reduced the promoter activity of both constructs to that measured in isolated cells. After Ca2+ addition, SkA and MLC1A promoter activities increased, although to different extents. This increase was partially (MLC1A) or fully (SkA) sensitive to the addition of antiN-cadherin antibodies. Controls were performed with serum addition and expression of constitutively active RhoA. Serum addition, which as contact formation, activates endogenous pathway led to a twofold increased in the SkA promoter, whereas expression of constitutively active RhoA strongly activates the MLC1A promoter.
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RhoA activity is required for ß-catenin recruitment to intercellular adhesions sites
Recently, ß-catenin was shown to be translocated and accumulated to adherens junctions in differentiating myoblasts (Goichberg et al., 2001). Moreover, Rho GTPases have been shown to regulate adherens junction assembly. We wondered whether the N-cadherindependent RhoA increase we observed might regulate ß-catenin translocation to cellcell contact sites. We first analyzed RhoA and ß-catenin localization (Fig. 7 A). Endogenous RhoA (Fig. 7 A, a) and ß-catenin (Fig. 7 A, b) were closely associated at cellcell junctions (Fig. 7 A, c, merge). We next examined RhoA localization during the calcium switch procedure by time-lapse microscopy. C2C12 cells were transfected with GFPRhoA. Fig. 7 B (videos 15 available at http://www.jcb.org/cgi/content/full/jcb.200202034/DC1) shows that under normal calcium conditions, GFPRhoAWT, is both cytoplasmic and found at cellcell contact sites (Fig. 7 B, a and d; videos 1 and 4). In cells treated with EGTA, GFPRhoAWT is cytoplasmic but barely detectable at the cellcell contact sites (Fig. 7 B, b; video 2). Upon addition of extracellular Ca2+, GFPRhoAWT was rapidly revisualized at cellcell contact sites (Fig. 7 B, c; video 3), except when antiN-cadherin antibodies were added (Fig. 7 B, e; video 5).
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Discussion |
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To address these signaling pathways, we investigated whether Rho GTPase activities might be affected by N-cadherinmediated cellcell adhesion. We found that N-cadherindependent adhesion decreases the activity of Rac1 and Cdc42Hs, two GTPases shown previously to have an inhibitory role in myogenesis (Gallo et al., 1999; Meriane et al., 2000). Two mechanisms have been proposed to explain their inhibitory effect. The first one involves JNK activation, which inhibits Myf5 nuclear localization (Meriane et al., 2000). We found here that JNK activity is diminished after N-cadherindependent cellcell adhesion. This is in agreement with previous results showing that in fibroblasts cell density decreases JNK activity (Lallemand et al., 1998). The second one consists of active Rac1 and Cdc42Hs inhibiting cell cycle withdrawal in myoblasts (Heller et al., 2001; Meriane et al., 2002), a prerequisite for differentiation, and activation of muscle-specific gene expression (Lassar et al., 1994). Interestingly, cadherin-dependent adhesion is important for contact-dependent growth inhibition in CHO cells and regulates the level of the cyclin-dependent kinase inhibitor p27 (St Croix et al., 1998; Levenberg et al., 1999). We demonstrate here that the inhibition of N-cadherindependent adhesion impairs the expression of the two cyclin-dependent kinase inhibitors p21 and p27, which are normally upregulated during myogenesis (Guo et al., 1995; Ragolia et al., 2000). The N-cadherindependent decrease of Rac1 and Cdc42Hs activity that we observed might thus contribute to both cell cycle withdrawal and extinction of a myogenic inhibitory signal, such as JNK activity (Fig. 8). Notably, the decreased level of Rac1 and Cdc42Hs activity upon N-cadherindependent cellcell contact formation remains sufficient to allow p38 activity (Cuenda and Cohen, 1999; Zetser et al., 1999; Meriane et al., 2000).
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Not only was RhoA activated in our system, but it was also recruited to sites of N-cadherinmediated cellcell adhesion. The localization and activity of Rho GTPases is controlled by three classes of regulators: Rho GDP dissociation inhibitors, guanine nucleotide exchange factors (GEFs), and GTPase-activating proteins. All of these regulators are potential interesting candidates to link N-cadherindependent adhesion to Rho GTPase activity. Regulators of Rho GDP dissociation inhibitor activity (ezrin/radixin/moesin proteins) and GEF family members were shown to localize or interact with the cadherincatenins complexes (Takahashi et al., 1998; Hiscox and Jiang, 1999; Sander et al., 1999). Recently, it has been shown that free p120 catenin, which normally binds to the cytoplasmic domain of the cadherins in the juxtamembrane region interacts with the GEF Vav2 and modulates the activity of Rho GTPases, suggesting that regulating p120 localization might also control Rho GTPases activity (Noren et al., 2000). Whether the activity of these Rho GTPases regulators is directly controlled by cadherin activation or results from a functional interaction between activated cadherin and classical growth receptors remains to be determined. Recently, N-cadherin has been shown to interact with the FGF receptor in neuronal cells (Williams et al., 2001). A balance between Rac1-Cdc42Hs and Rho activities determines the cellular phenotype and biological behavior in various cell systems: actin cytoskeleton organization (Sander et al., 1999; Vignal et al., 2001), formation of focal adhesions (Rottner et al., 1999), neurite extension (Kozma et al., 1997; Leeuwen et al., 1997), and myogenesis (Meriane et al., 2000). This was clearly demonstrated in NIH3T3, where Rac1 signaling is able to antagonize Rho activity directly at the GTPase level (Sander et al., 1999). Various other studies reported a similar antagonism, but the precise molecular mechanism remains a debated issue. Nevertheless, expression of activated form of RhoA in C2C12 myoblasts decreases Rac1 and Cdc42Hs activities, suggesting that N-cadherin signaling leading to RhoA activation might in turn downregulate Rac1 and Cdc42Hs activities (unpublished data). The identification of the missing links between N-cadherin and Rho GTPases will shed light on this process.
RhoA activity is clearly required for ß-catenin localization at the contact sites, an event involved in myogenic induction (Goichberg et al., 2001). RhoA GTPase activity is mediated by downstream effectors, such as Rho kinase family of serine/threonine kinases and the proline-rich formin homology domaincontaining proteins mDia1 and 2, which regulate actin stress fiber organization and alignment of actin bundles and microtubules (Bishop and Hall, 2000). Whether these RhoA effectors participate in the regulation of ß-catenin localization through signaling toward actin filaments remains to be determined.
Previous studies examining the regulation of the Rho family GTPases by the formation of adherens junctions have concentrated on E-cadherindependent events. Interestingly, the results are the inverse of those obtained in myoblasts, since they found a stimulation of Rac1 and Cdc42Hs and a decrease of RhoA activities (Fukata and Kaibuchi, 2001). The differences between E- and N-cadherindependent intracellular signaling might be due to the cadherin molecule itself and/or to the different cell types in which these cadherins are expressed. Further studies are required to address this issue.
In conclusion, our data show that N-cadherindependent adhesion controls different crucial steps of the myogenic differentiation program. In the developing embryo, N-cadherinmediated RhoA activation might play an important role during somitogenesis, myotome formation, and also terminal skeletal myogenesis, which lead to formation of the different body muscles. This would thus constitute an integral part of the community effect process which governs myogenesis. Further studies will be necessary to further correlate the activity of Rho GTPases with the skeletal myogenesis during development.
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Materials and methods |
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For density experiments, C2C12 cells were trypsinized and extensively homogenized to generate a dispersed cell suspension. Isolated cells were plated at a density of 1,250 cells/cm2, and confluent cells were plated at a density of 6,250 cells/cm2. Cells were allowed to recover for 20 h before measurement of Rac1, Cdc42Hs, RhoA, JNK, and p38 activities as described later. Quantification was performed by densitometric analysis of Western blots using Aida/2D densitometry software. The relative amount of active protein was determined by measuring the amount of protein sedimented or immunoprecipitated relative to the amount in whole cell lysates.
Cellcell contact manipulation
Isolated C2C12 myoblasts were either left untreated, treated with EGTA for 60 min, or treated with EGTA for 60 min and allowed to recover in fresh medium. Confluent C2C12 myoblasts were either left untreated, treated with EGTA for 60 min, treated with EGTA for 60 min, and allowed to recover in fresh medium in the presence or not of antiN-cadherin antibodies for 30 min. Confluent C2C12 myoblasts were allowed to establish cellcell contacts with preimmune serum or antiN-cadherin antibody. Alternatively, isolated cells were settle onto Ncad-Fccoated Petri dishes.
Polyclonal antiN-cadherin antibody production
The EcoRV fragment of N-cadherin (NM 007664) corresponding to amino acids 75706 was cloned in the pGEX5X vector. GSTN-cadherin fragment was produced as described (Mary et al., 2002).
Three rabbits were injected with 80100 µg of GSTN-cadherin protein. Antisera were tested by immunoblotting and immunofluorescence. Affinity purification was performed by incubation with the GSTN-cadherin fragment spotted onto nitrocellulose.
Differentiation inhibition assays
C2C12 cells were plated in 35-mm dish in DME/HAM F-12 supplemented with 10% FCS (HyClone). 2 h after plating, antiN-cadherin antibody was added. To induce differentiation, growth medium was replaced with differentiation medium (DME/HAM F-12 supplemented with 2% FCS) containing antiN-cadherin antibody. The medium was replaced every 24 h for 7 d. The differentiation of control or antiN-cadherintreated cells was followed by time-lapse imaging, analyzing the expression of three markers for myoblast differentiation (myogenin, Troponin T and MHC) on fixed cells, or measuring the levels of cyclin-dependent kinase inhibitors p21 and p27 by immunoblotting.
Transfection and kinase assay
C2C12 myoblasts cultured in 60-mm dishes were transfected or cotransfected with 2 µg HA-JNK, HA-p38 (provided by B. Dérijard, Centre National de la Recherche Scientifique, UMR 6548, Nice, France), GFPRac1N17, GFPCdc42HsN17 plasmids as described by the supplier (Life Technologies). 4 h after the transfection, the medium was replaced with DME/HAM F12 supplemented with 10% FCS. 24 h after transfection, cellcell contacts were manipulated, and cells were lysed and processed as described (Meriane et al., 2000). Scanned autoradiographs were quantified using Aida/2D densitometry software and normalized as a function of the expression of the various proteins.
Preparation of Ncad-Fccoated dishes
The Ncad-Fc chimera (chicken N-cadherin ectodomain fused to the Fc fragment of mouse IgG2b) was produced in eukaryotic cells as described (Lambert et al., 2000). The production media was collected every 23 d and analyzed for the presence of the fusion protein by Western blot analysis. 100-mm Petri dishes were first coated with goat antimouse Fc antibody (Jackson ImmunoResearch Laboratories) at 1 µg/cm2 in 0.1 borate buffer (pH 8.5) for 18 h at 4°C. Dishes were washed with borate buffer and incubated with Ncad-Fc medium (375 µg to obtain a theoretical coating value of 7 µg/cm2). Dishes were washed in PBS and used immediately. Coating efficiency was controlled by Western blot analysis of the coated material in a dish. Petri dishes were also coated with poly-L-lysine (0.001% in PBS; Sigma-Aldrich) for 10 min. RhoA, Rac1, and Cdc42Hs activity was measured 2 h after plating.
GTPase activity assays
After cellcell contact manipulation, cells were processed as described to measure Rac1 and Cdc42Hs activity (Meriane et al., 2002). For RhoA activity assay, C2C12 cells were lysed in 50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 500 mM NaCl, 10 mM MgCl2, 1 mM PMSF, and cocktail protease inhibitors (Sigma-Aldrich). Cleared lysate was incubated with 25 µg of GST fusion protein containing the RhoA-binding domain of Rhotekin (GST-TRBD) beads for 40 min at 4°C, and then the beads were washed four times in Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 0.1 mM PMSF and cocktail protease inhibitors before addition of SDS sample buffer containing DTT. Rho A fractions were analyzed by Western blotting with RhoA antibody (Santa Cruz Biotechnology, Inc.). Scanned autoradiographs were quantified using Aida/2D densitometry software and normalized as a function of the expression of the various proteins.
Luciferase assay
C2C12 cells plated in 35-mm dishes (20,000 cells for subconfluent condition and 50,000 cells for confluent condition) were cotransfected using the lipofectamine method with 0.7 µg of chimeric construct containing the -394/+24 skeletal actin (SkA) promoter fused to the luciferase gene (MacLellan et al., 1994), 0.7 µg of pEGFP empty vector (CLONETECH Laboratories, Inc.), and 40 ng of pRL cytomegalovirus (CMV) vector (renilla luciferase CMV). 48 h after transfection, cellcell junctions were manipulated and luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega).
Chloramphenicol acetyltransferase assay
C2C12 cells plated in 60-mm dishes (40,000 cells for subconfluent condition and 100,000 cells for confluent condition) were cotransfected using Lipofectamine with 0.8 µg of chimeric construct containing the first 630 bp of the mouse MLC1A promoter driving chloramphenicol acetyltransferase (CAT) gene expression (Catala et al., 1995) (provided by M. Buckingham, Institut Pasteur, Paris, France) with either 0.8 µg of pEGFP empty vector or GFP-tagged RhoA V14 and 0.4 µg of CMV driving ß-galactosidase expression vector (CMVß-gal). 48 h after transfection, cells were submitted to different treatments, and CAT (CAT Elisa; Roche Molecular Biology) and ß-gal (ß-gal reporter gene assay, chemiluminescent; Roche Molecular Biology) activities were determined as described by the supplier. CAT activity was corrected with respect to ß-gal activity.
ß-Gal assay
C27 stably expressing MyoD promoter fused to ß-gal reporter gene (Carnac et al., 1998) were analyzed for ß-gal activity in isolated or contacting culture conditions as described by the supplier. Alternatively, cells were allowed to form contacts in the presence of antiN-cadherin antibodies or C3 transferase for 24 h. Protein determination (BCA assay) was determined to normalize results.
Immunofluorescence
Cells growing onto 35-mm dishes were fixed in 3.7% formaldehyde in PBS followed by a 5-min permeabilization in 0.1% Triton X-100 in PBS and incubated in PBS containing 0.1% BSA. Myogenin and Troponin T expression was detected as described (Meriane et al., 2000). Antiß-catenin and antiN-cadherin antibodies are from Transduction Laboratories (Interchim). All of these mouse antibodies were revealed with either an Alexa Fluor 546 or an Alexa Fluor 488conjugated goat antimouse antibody (Molecular Probes and Interchim). Anti-Rho antibody is from Upstate Biotechnology (Euromedex), revealed by a either an Alexa Fluor 546 or an Alexa Fluor 488conjugated goat antirabbit antibody (Molecular Probes and Interchim). Cells were stained for F-actin using TRITC-conjugated phalloidin (Sigma-Aldrich). Cells were prepared and observed as described (Mary et al., 2002). Fluorescent images were deconvolved using the maximum likelihood estimation algorithm (Huygens, Scientific Volume Imaging). The restored images were saved as Tif files that were mounted using Adobe Photoshop® and Adobe Illustrator®.
Gel electrophoresis and immunoblotting
Cells cultured in 60-mm dishes were rinsed in cold PBS and lysed in 50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM NaPPi, 15 mMpNPP, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM vanadate, and 1% Nonidet P-40. 15 µg of proteins were loaded on a 12.5% polyacrylamide gel and then transferred onto nitrocellulose. After saturation in 5% milk in PBS, membranes were incubated with rabbit anti-p21 and anti-p27 antibodies (Santa Cruz Biotechnology, Inc.). After washing, membranes were processed as described (Mary et al., 2002).
The relative amounts of p21 and p27 in each sample were calculated by densitometric analysis of Western blots using Aida/2D densitometry software. A 100% value was arbitrary affected to the control condition.
Time-lapse imaging
Time-lapse epifluorescence microscopy was performed as described previously (Mary et al., 2002). Fluorescent images were deconvolved using the maximum likelihood estimation algorithm (Huygens, Scientific Volume Imaging). The restored images were saved as Tif files that were edited with NIH Image and compiled into QuickTime movies or directly mounted using Adobe Photoshop® and Illustrator®.
Online supplemental material
Videos 15, available at http://www.jcb.org/cgi/content/full/jcb.200202034/ DC1, correspond to Fig. 7. C2C12 cells were transfected with GFPRhoAWT cDNA, and images of GFP fluorescence were recorded 18 h after transfection. Note the accumulation of GFPRhoA at the cellcell contact sites in control cells (videos 1 and 2), whereas no such GFPRhoA accumulation is detectable in EGTA- (video 3) or antiN-cadherintreated cells (video 4). GFPRhoA is again found at the cellcell contact sites upon restoration of extracellular Ca2+ (video 5).
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Footnotes |
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* Abbreviations used in this paper: ß-gal, ß-galactosidase; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; GEF, guanine nucleotide exchange factor; JNK, c-jun NH2-terminal kinase; MLC, myosin light chain; SkA, skeletal
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Acknowledgments |
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This work was supported by contracts for the Ligue Nationale contre le Cancer ("Equipe labelisee"), the Association pour la Recherche contre le Cancer (contract no. 5668), the Association Francaise contre les myopathies, and institutional grants from Centre National de la Resherche. During this work S. Charrasse was supported by a fellowship from the Association pour la Recherche sur le Cancer.
Submitted: 8 February 2002
Revised: 11 July 2002
Accepted: 12 July 2002
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