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First published online 8 December 2005
doi: 10.1242/jcs.02698

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Laminin-5-integrin interaction signals through PI 3-kinase and Rac1b to promote assembly of adherens junctions in HT-29 cells

Nicolas T. Chartier^1,*, Michèle Lainé^1,*, Stéphanie Gout¹, Géraldine Pawlak¹, Christiane A. Marie¹, Paulo Matos², Marc R. Block¹ and Muriel R. Jacquier-Sarlin^1,

¹ Laboratoire d'Etude de la Différenciation et de l'Adhérence Cellulaires, UMR UJF/CNRS 5538, Institut Albert Bonniot, Faculté de Médecine de Grenoble, Domaine de la Merci, 38706 La Tronche Cedex, France
² Centro de genética Humana, Instituto Nacional de Saùde `Dr Ricardo Jorge' Avenida Padre Cruz, 1649-016 Lisboa, Portugal

View larger version (129K): [in a new window]   Fig. 1. Laminin 5 induces E-cadherin-mediated intercellular adhesion in HT-29 cells. (A) Morphological changes: phase-contrast images of HT-29 cells grown in standard medium (HT-29), in differentiating medium for 10 days (HT-29 Gal), or on laminin 5 matrix in standard medium for 24 hours (HT-29 LN 5). Bars, 100 µm. (B) Cell aggregation assays. Upper panel: HT-29 cells grown on plastic (PL) or laminin 5 matrix (LN 5) for 48 hours were resuspended in PBS containing 10 mM Hepes + 2 mM CaCl2. Suspensions were incubated for 60 minutes at 37°C in a gyratory shaker at 75 rpm. Cell aggregation was observed under phase contrast with a Zeiss Axiovert 135 inverted microscope. Lower panel: HT-29 cells grown for 48 hours on laminin 5 matrix were resuspended in PBS containing 10 mM Hepes + 2 mM CaCl2 in the presence of 4 mM EDTA or 10 µg/ml blocking anti-human E-cadherin (HECD1) antibody and then assayed for cell aggregation. Aggregation of HT-29 cells cultured on laminin 5 matrix was mediated by E-cadherin. Bars, 100 µm.

View larger version (33K): [in a new window]   Fig. 2. Laminin 5 increases the expression of E-cadherin in HT-29 cells. (A) Upper panel: western blot analysis of the total amount of E-cadherin during the time course of HT-29 cell culture onto laminin 5 matrix. Expression of actin was used as a loading control. Lower panel: the band densitometry was normalized with actin to allow quantification. Data indicate a significant increase in E-cadherin expression during the first 24 hours of culture. Data represent mean ± s.e.m. of three independent experiments. *P<0.05; **P<0.01, as determined by Student's t-test. (B) Upper panel: western blot analysis of the total amount of E-cadherin after 24 hours of culture onto various concentrations of purified laminin 5. Expression of actin was used as a loading control. Lower panel: the band densitometry was normalized with actin to allow quantification. Data indicate a dose-dependent induction of E-cadherin expression. Data represent mean ± s.e.m. of three independent experiments. **P<0.01, as determined by Student's t-test. (C) ECM proteins differentially increase expression of E-cadherin. HT-29 cells were grown for various times (0-24 hours) on plastic (PL) or different ECM proteins used at 10 µg/ml (FN, fibronectin; CO IV, collagen type IV; LN 5, laminin 5). Cell lysates were assayed for E-cadherin expression, using actin as an internal control. The density ratio of E-cadherin:actin is expressed as a relative factor of the value obtained from HT-29 cells in suspension (mean ± s.e.m. of three independent experiments). (D) Increase in E-cadherin expression is mediated by laminin 5-integrin interactions. Western blot analysis of the total amount of E-cadherin in HT-29 cells cultured for 24 hours on plastic (PL) or 5 µg/ml purified laminin 5 (LN5) in the presence of mAbs directed against integrin subunits at a concentration of 10 µg/ml: P4C10 (anti-ß1), ASC8 (anti-ß4), BHA2.1 (anti-2), P1B5 (anti-3) and GoH3 (anti-6) or laminin 5 (3 chain), with a 1/10 diluted BM165 mAb (from cell culture supernatant). Expression of actin was used as a control. Band densitometry was normalized with actin to allow quantification and the % of control (i.e. laminin 5-induced E-cadherin expression without mAbs) was reported on the histogram. Data represent mean ± s.e.m. of three independent experiments. *P<0.05; **P<0.01, as determined by Student's t-test. Laminin 5-mediated increase in E-cadherin expression was dependent on 6ß4 and 3ß1 integrins.

View larger version (21K): [in a new window]   Fig. 3. Laminin 5 does not modulate expression of ß-catenin and p120ctn in HT-29 cells. Western blot analysis of the total amount of ß-catenin (A) and p120ctn (B) during the time course of HT-29 cell culture onto laminin 5 matrix. Expression of actin was used as a loading control. Lower panel: the band densitometry was normalized with actin to allow quantification. Data indicate no significant variation in ß-catenin or p120ctn expression. Data represent mean ± s.e.m. of three independent experiments. P values determined by Student's t-test were not significant.

View larger version (42K): [in a new window]   Fig. 4. Culture of HT-29 cells on laminin 5 triggers E-cadherin accumulation at cell-cell contacts and colocalization with actin. (A) Western blot analysis of E-cadherin, p120ctn and ß-catenin distribution in Triton-soluble fractions (TS) and Triton-insoluble fractions (TI) obtained from lysates of HT-29 cells cultured in standard medium (HT-29) or on laminin 5 matrix for 24 hours (HT-29 LN 5). Histograms showing the percentage of molecules associated with the TI fraction were determined by densitometry of the blots visualized by chemiluminescence (Amersham ECL reagents) using the Image Master VDS-CL (Amersham-Pharmacia Biotech) imaging device. The data were compiled from five independent experiments; error bars represent mean ± s.e.m. The proportion of E-cadherin and ß-catenin associated with the cytoskeleton is increased in HT-29 cells cultured on laminin 5 matrix. (B) Reconstituted images along the Z axis from a series of optical sections. HT-29 cells grown in standard medium (left panels, HT-29) or on laminin 5 matrix for 24 hours (right panels, HT-29 LN 5) were immunostained with anti-E-cadherin (green) and TRITC-conjugated phalloidin (red), and analyzed by confocal microscopy as described under the Materials and Methods. In HT-29 LN 5 cells, E-cadherin was localized to the apicolateral membrane in association with the actin cytoskeleton. Bars, 16 µm.

View larger version (28K): [in a new window]   Fig. 5. Laminin 5-induced activation of PI 3-kinase in HT-29 cells. (A) Effect of different ECM proteins on PI 3-kinase activity. HT-29 cells were allowed to adhere for 24 hours to dishes uncoated (PL) or coated with purified laminin 5 (LN 5), collagen IV (CO IV), or fibronectin (FN) at 10 µg/ml. To test the specificity of the interaction with laminin 5, HT-29 cells were preincubated with the anti-3 chain of laminin 5 BM165 at a concentration of 20 µg/ml and then allowed to adhere to laminin 5. (B) Time course of PI 3-kinase activation by laminin 5 matrix. HT-29 cells were allowed to adhere for 1, 4, 24 or 48 hours to dishes coated with laminin 5 matrix. In (A) and (B), total cell lysates were prepared and equivalent amounts of proteins were used in immunoprecipitation and kinase assays. Western blots performed on immunoprecipitated p85 subunit were carried out as controls. Phosphorylated lipids were resolved by thin layer chromatography. The amount of radiolabeled PtdIns(3,4,5)P3 (PIP3) was determined for each condition by densitometry with ImageQuant software. Data represent the average of percentage of control activity (mean ± s.e.m. of three to four independent experiments) and one representative chromatogram is shown. *P<0.05; **P<0.01, as determined by Student's t-test. (C) HT-29 cells were incubated with 2 mM EGTA or 10 µg/ml of integrin or E-cadherin-specific antibodies and allowed to adhere to plates coated with laminin 5 matrix for 24 hours. mAbs specific for 3 (P1B5), 6 (GoH3) or ß1 (P4C10) integrin subunits and E-cadherin (HECD1) were used at 10 µg/ml. Total cell lysates were prepared and equivalent amounts of proteins were subjected to immunoprecipitation and kinase assays as described previously. **P<0.01, as determined by Student's t-test.

View larger version (38K): [in a new window]   Fig. 6. PI 3-kinase inhibition interferes with E-cadherin expression and its association with the cytoskeleton. (A) HT-29 cells grown for 24 hours on laminin 5 matrix (LN 5) were treated with increasing concentrations of LY294002 as indicated and then lyzed. Whole-cell lysates were separated by SDS-PAGE and subjected to immunoblotting for E-cadherin. Expression of actin was used for normalization. *P<0.05; **P<0.01, as determined by Student's t-test. (B) Upper panel: whole lysates of HT-29 cells expressing the vector alone (pBabe) or p85 were subjected to immunoblotting for p85. Expression of tubulin was used as a loading control. Lower panel: HT-29 cells expressing the vector alone (pBabe) or p85 grown on plastic or laminin 5 matrix for 24 hours were lyzed, then analyzed for E-cadherin expression by western blotting. The band densitometry was normalized with tubulin to allow quantification. The results are representative of three independent experiments. (C) Upper panel: HT-29 cells grown for 24 hours on plastic (PL) or laminin 5 matrix (LN 5) treated with (+) or without (-) 50 µM LY294002 were lyzed and then the Triton-soluble (TS) fraction and the Triton-insoluble (TI) fraction (i.e. cytoskeleton-associated proteins) were separated by SDS-PAGE and immunoblotted for E-cadherin. The results are representative of at least three independent experiments. Lower panel: HT-29 cells expressing vector alone (pBabe) or p85 were grown on plastic or laminin 5 matrix for 24 hours. E-cadherin and F-actin were detected by immunofluorescence using HECD1 antibody and TRITC-conjugated phalloidin, respectively. Optical sections were performed using a confocal microscope. Bars, 50 µm.

View larger version (55K): [in a new window]   Fig. 7. PI 3-kinase associates with E-cadherin complexes upon cell adhesion on laminin 5. Upper panel: western blot analysis of the distribution of the p85 regulatory subunit of PI 3-kinase. HT-29 cells grown for 24 hours on plastic (PL) or laminin 5 matrix (LN 5) were fractionated as described under the Materials and Methods, to separate nuclear-, cytosolic- and membrane-enriched fractions, then analyzed by western blot using an anti-p85 antibody. The blot shown is representative of four independent experiments. Middle panel: HT-29 cells grown for 24 hours on plastic (PL) or laminin 5 matrix (LN 5) were immunostained with anti-E-cadherin (green) and anti-p85 (red) antibodies and analyzed by confocal microscopy on optical sections. Bar, 30 µm. Lower panel: the p85 subunit of PI 3-kinase was immunoprecipitated (IP) from 500 µg of proteins from whole-cell lysates of HT-29 cells cultured for 24 hours on plastic or laminin 5 matrix. Proteins of the immunoprecipitates were solubilized into Laemmli's buffer and resolved by SDS-PAGE before western blotting (WB) to determine the amount of PI 3-kinase and E-cadherin in the immunoprecipitates. The blot shown is representative of three independent experiments.

View larger version (63K): [in a new window]   Fig. 8. PI 3-kinase signaling is necessary for adherens junction biogenesis. HT-29 cells were cultured for 24 hours on laminin 5 matrix and subsequently treated with 4 mM EGTA for 30 minutes in the absence or presence of 50 µM LY294002. The EGTA-containing medium was then replaced with Ca2+-containing medium for 60 minutes. Cells were fixed and stained with anti-E-cadherin (green) and TRITC-conjugated phalloidin (red) before confocal microscopy analysis on optical sections. Bars, 25 µm.

View larger version (41K): [in a new window]   Fig. 9. Rac1b activation is dependent on PI 3-kinase. Time course of Rac1 and Rac1b activation by laminin 5 matrix. GST-PAK-CRIB specifically pulls down GTP-loaded Rac. Lysates were prepared from HT-29 cells cultured on laminin 5 matrix (LN5) for various time periods (0-72 hours) or for 72 hours on plastic (PL). The amount of Rac1-GTP (upper panel) and Rac1b-GTP (upper middle panel) were estimated by pulling down the GTP-loaded proteins with GST-PAK-CRIB, followed by a western blot analysis with antibodies specific for Rac1 and Rac1b. Samples of the whole-cell lysates were also fractionated to determine the total levels of Rac1 and Rac1b (lower middle panel). Expression of actin was used as a loading control (lower panel). (B) Analysis of Rac1b activation upon inhibition of PI 3-kinase by LY294002: HT-29 cells were grown for 24 hours on laminin 5 matrix in the presence of increasing concentrations of LY294002 (0-100 µM). Then, the level of Rac1b-GTP was estimated as described above. Lower panel: the band densitometry was determined and the ratio of Rac1b-GTP to total Rac1b was used to allow quantification on the histogram. Results are representative of four experiments. **P<0.01, as determined by Student's t-test. (C) Subcellular distribution of Rac1b: HT-29 cells grown for 24 hours on plastic or laminin 5 matrix were fixed and stained with anti-Rac1b antibodies before confocal microscopy analysis on optical sections. Bars, 16 µm.

View larger version (79K): [in a new window]   Fig. 10. Rac1 localization at nascent cell-cell contacts is dependent on PI 3-kinase signaling. HT-29 cells were cultured for 24 hours on laminin 5 matrix and then treated with 4 mM EGTA for 30 minutes in the absence or presence of 50 µM LY294002. The EGTA-containing medium was then replaced by Ca2+-containing medium for 60 minutes. Cells were fixed and stained for Rac1 before confocal microscopy analysis on optical sections. Bars, 25 µm.

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