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Integrin-mediated functional polarization of Caco-2 cells through E-cadherin—actin complexes

INSERM U505, Université Pierre et Marie Curie, EPHE, 15 rue de l'Ecole de Médecine, 75006 Paris, France

View larger version (23K): [in a new window]   Fig. 1. Native ECM differentially increases expression of the differentiation marker apoA-IV in Caco-2 cells. The Caco-2 cells were grown until confluence on plastic or on different native ECM previously secreted and deposited by mesenchymal cells from the intestine (C9; C11; C20) or the muscle (129CB3), or by colon tumour epithelial cells (HT29-MTX; Caco-2 at 12 day post confluence). Composition of these ECMs is described in Table 1. Total RNA was isolated, and the abundance of apoA-IV transcripts was assayed by RNAse Protection Assay, using ß actin mRNA as an internal control. The mRNA ratio of apoA-IV:ß actin (mean±s.e.m. of three independent cultures performed in triplicate) is expressed as a percentage of the value obtained from Caco-2 on plastic. * and ** differ from the control at P<0.05 and P<0.01 (t-test), respectively. Note that LN5-rich ECM deposited from HT29 MTX cells is the most effective ECM to induce apoA-IV expression in Caco-2 cells.

View larger version (49K): [in a new window]   Fig. 2. Native LN5-rich ECM reinforces cell-cell interactions between Caco-2 cells. Caco-2 cells were plated at 20,000 cells/cm2 on plastic () or on native LN5-rich ECM () deposited by HT29-MTX cells. (A) Phase contrast micrography (X 25; bar, 200 µm) shows that domes appear on cultures grown on ECM 2 days earlier than on plastic support. (B) shows the confluence rates observed by phase microscopy. (C) At the indicated times, domes were counted. Values represent mean±s.m.d. from triplicate wells from three independent cultures. (D) shows a 3D confocal reconstruction of the diffusion of FITC-conjugated biotin added at the apical side of Caco-2 cultures grown on plastic (a) or on LN5-rich ECM (b). (bar, 10 µm). Note that the presence of ECM restricts biotin diffusion at the apical compartment, which indicates a reinforcement of cell-cell interactions as compared to cultures grown on plastic without ECM. The result is representative of three independent experiments.

View larger version (18K): [in a new window]   Fig. 3. Native LN5-rich ECM induces E-cadherin targeting to the lateral membrane. Caco-2 cells were plated at 20,000 cells/cm2 on glass or on native LN5-rich-ECM deposited by HT29-MTX cells, and RNA and proteins were prepared for further analysis. (A) RNase Protection Assay of apoA-IV and E-cadherin mRNA, using ß actin mRNA as a control. The ratio of apoA-IV or E-cadherin to ß actin mRNA on LN5-rich ECM was compared to that measured in Caco-2 cells grown on glass and expressed as a percentage. Note that ECM increases apoA-IV but not E-cadherin mRNA levels. (B) Western blot analysis of the total amount of E-cadherin and ß-catenin. Note that the total amount of either E-cadherin or ß-catenin proteins does not vary. (C) Western blot analysis of E-cadherin after immunoprecipipation of biotinylated membrane-associated proteins. Total E-cadherin (a,c) and biotinylated E-cadherin (b,d) amounts of E-cadherin in cells grown on plastic without ECM (a,b) or LN5-rich ECM (c,d). Note the increase in E-cadherin localized at the cell surface in cultures grown on ECM. The data are means ±s.e.m. of three independent cultures performed in triplicate. * differs from the control at P<0.05.

View larger version (53K): [in a new window]   Fig. 4. Effect of ECM on cell-cell interactions, E-cadherin and actin subcellular localization. (A) Caco-2 cells were plated at 20,000 cells/cm2 on glass (a) or on native LN5-rich ECM (b), processed for immunofluorescence with anti E-cadherin at confluence and analysed by confocal microscopy. Note that E-cadherin is mostly located at the cell-cell junction membrane on domes formed by cells grown on ECM. 3D confocal reconstruction (XZ) shows an E-cadherin signal at the basal side of the Caco-2 cells grown on glass (c), which disappears in cells grown on ECM (d) where the localization of E-cadherin aggregation (i.e. 25 µm from the basal side out of a 30 µm total height) is compatible with the position adherens junction (bar, 10 µm). (B) Caco-2 cells were plated at 50,000 cells/cm2 on glass (a,a'), polylysine (b,b'), native LN5-rich ECM (c,c') type IV collagen (d,d'), or LN2 (e,e'), processed for immunofluorescence with anti-E-cadherin and FITC (green) and TRITC-conjugated phalloidin (red) after 72 hours of culture and analysed by confocal microscopy. Note that ECM increases E-cadherin-actin colocalization (yellow merge signal) and allows the formation of cortical actin cytoskeleton (bar, 10 µm). (C) Actin labelling in cells grown (72 hours) on glass in presence of the ß1-integrin-activating antibody (bar, 10 µm). The results are representative of three independent experiments.

View larger version (14K): [in a new window]   Fig. 5. Dome formation is under the control of both E-cadherin and ß1 integrin expressions. Caco-2 cells were plated at 125,000 cells/cm2 on plastic ([UNK]) or on native LN5-rich ECM () in the presence of 10 µg/ml mouse non-specific IgG (), 10 µg/ml () or 2.5µg/ml () anti-ß1-integrin blocking antibody. (A) shows the appearance of domes as a function of time. Note that anti-ß1-integrin antibody dose dependently decreases the number of domes formed on ECM substrate as early as 2 days in culture. These perturbations are not correlated with the confluence rate. (B) shows the confluence rate of Caco-2 cells. The presence of ß1-integrin antibody or plastic support delays confluence, which is reached 2 days later than in untreated cells grown on ECM or in the presence of control IgG antibody.

View larger version (22K): [in a new window]   Fig. 6. ß1 integrin mediates the effects of ECM on E-cadherin targeting to the lateral membrane. Caco-2 cells were plated at 125,000 cells/cm2 on plastic, native LN5-rich ECM, type IV collagen or LN2 in the presence or absence of either anti-ß1- or anti-6-integrin blocking antibodies. Cells were then processed for immunofluorescence with anti-E-cadherin and FITC (green) or TRITC-conjugated phalloidin (red) and analysed by confocal microscopy. Colocalization (yellow merge signal) of E-cadherin and actin at the apical-lateral side of cells was estimated by computer analysis of confocal stack series. (A) shows that ECM components increase the colocalization of E-cadherin and actin as compared to plastic without ECM, as expressed in arbitrary units. (B) 3D reconstruction of cells grown on type IV collagen in the absence (a) or in the presence (b) of anti-ß1-integrin blocking antibody (bar, 10µm). The result is representative of three independent experiments. (C) Effect of anti-ß1-integrin blocking antibody on the colocalization of E-cadherin and actin in cells grown on LN2. (D) Effect of anti-6-integrin blocking antibody on the colocalization of E-cadherin and actin in cells grown on native LN5-rich ECM. Note that anti-ß1 antibody treatment results in delocalization of the E-cadherin-actin in cells grown on native ECM, type IV collagen and LN2. Data, expressed as the percentage of control in absence of blocking antibody, are means ±s.e.m. of three independent cultures performed in triplicate. *, **, and *** differ from the control at P<0.05, P<0.01 and P<0.001, respectively.