Research Article 543 Integrin-mediated functional polarization of Caco-2 cells through E-cadherin–actin complexes Cyrille Schreider, Gregory Peignon, Sophie Thenet, Jean Chambaz and Martine Pinçon-Raymond* INSERM U505, Université Pierre et Marie Curie, EPHE, 15 rue de l’Ecole de Médecine, 75006 Paris, France Author for correspondence (e-mail: [email protected]) Accepted 25 October 2001 Journal of Cell Science 115, 543-552 (2002) © The Company of Biologists Ltd Summary Enterocyte differentiation is a dynamic process during type IV collagen or laminin 2, which suggests a common which reinforcement of cell-cell adhesion favours migration pathway of induction through integrin receptors. Indeed, along the crypt-to-villus axis. Functional polarization of these effects were antagonized by blocking anti-β1- and Caco-2 cells, the most commonly used model to study anti-α6-integrin antibodies and directly induced by a intestinal differentiation, is assessed by dome formation stimulating anti-β1-integrin antibody. These results and tightness of the monolayer and is under the control demonstrate that integrin-dependent cell to ECM adhesion of the extracellular matrix (ECM). Furthermore, our reinforces E-cadherin-dependent cell-cell adhesion in biochemical and confocal microscopy data demonstrate Caco-2 cells and further support the notion that enterocyte that the ECM dramatically reinforces E-cadherin targeting differentiation is supported by a molecular crosstalk to the upper lateral membrane, formation of the apical between the two adhesion systems of the cell. actin cytoskeleton and its colocalization with E-cadherin in functional complexes. In our model, these effects were Key words: Caco-2 cells, β1 integrin, E-cadherin, Extracellular produced by native laminin-5-enriched ECM as well as by matrix, Actin cytoskeleton Introduction developing kidney (Klein et al., 1990). Similarly, the addition The epithelium forms a barrier made of polarized cells joined of laminin boosts the formation of polarized alveoles in various by a complex set of cell-cell junctions. The assembly of types of epithelial cells, including mouse mammary (Li et al., adherens junctions through the interaction of E-cadherin of 1987), human salivary (Hoffman et al., 1996) and rat lung adjacent cells initiates this process (Gumbiner, 1996; Kemler, (Matter and Laurie, 1994) cells in culture. ECM-integrin 1992). The importance of cell-cell adhesion in differentiation interactions have either been demonstrated to be directly and in the maintenance of the differentiated phenotype is well involved in ECM control of cell functions or found to be established in epithelial cells (Braga et al., 1999). In addition, aberrant in embryos or animals carrying mutations in integrin epithelial cells are separated from the underlying connective genes (Wang et al., 1999). tissue by a basement membrane that is composed of a variety Both cell-ECM and cell-cell adhesion systems are connected of extracellular matrix (ECM) molecules that control cell to the cytoskeleton, which controls cell polarization. Numerous differentiation in many tissues through interactions with their studies have established that the interaction between ECM and cellular receptors, for example, with integrins (Boudreau and integrin results in cytoskeletal rearrangements (Larjava et Bissell, 1998). al., 1990; Wang et al., 1999). Integrins are heterodimeric The basement membrane is mostly composed of type IV transmembrane receptors composed of α and β subunits collagen, different types of laminins, entactin and heparan associated in a noncovalent manner (Hynes, 1987; Yamada and sulfate proteoglycan (Beaulieu, 1997). ECM molecules, Miyamoto, 1995). Integrin initiates, through its β1 cytoplasmic originating from both epithelial and underlying mesenchymal domain, the assembly of specialized cytoskeletal and signaling cells, create a framework that is essential for maintaining tissue protein complexes at the contacting membrane (Gimond et integrity (Simon-Assmann and Kedinger, 1993). Besides this al., 1999). In the same way, epithelial cells forming strong cell- structural role, ECM proteins are involved in the control of cell junctions assemble a subcortical actin skeleton instead of adhesion, migration, proliferation, differentiation and gene focal adhesion and actin stress fibers (Larjava et al., 1990). expression of adjacent cells, which emphasizes the dynamic Cadherins are also dependent on cytoskeletal organization reciprocity between epithelial and mesenchymal cells (Bissell (Tsukita et al., 1992); correct function of the E-cadherin– et al., 1982). Additionally, ECM is able to control the effects catenin complex requires association with the cytoskeleton of trophic factors by sequestration outside of the cell (Simon- (Skoudy et al., 1996). In epithelial cells, about one half of Assmann et al., 1998) and by crosstalk between their signaling plasma membrane E-cadherin is connected to the actin pathways (Yamada and Geiger, 1997). It is admitted that cell cytokeleton: the rest is free within the membrane (Sako et al., adhesion to the ECM contributes to the apical-to-basal axis of 1998). The linkage between E-cadherin and the F-actin polarity, in vivo as well as in vitro. Appearance of polarized cytoskeleton is mediated through direct binding of the cells coincides with the expression of laminin 1 (LN1) in the cytoplasmic domain of E-cadherin to β-catenin, which binds 544 Journal of Cell Science 115 (3) to α-catenin (Aberle et al., 1994; Jou et al., 1995) in a 1:1:1 of methotrexate (Lesuffleur et al., 1990) and cultured without the drug stochiometry. Crosstalk between the two adhesion systems has and named HT29-MTX (9th passage) were cultured at 37°C with 10% also been demonstrated in mammary epithelial cells through CO2 in Dulbecco’s minimal essential medium (DMEM), 25 mM the integrin signaling pathway. In these cells, integrins promote glucose (Gibco), pen/strept (50 µg/ml) and non-essential amino acid the formation of morphologically differentiated acini-like (1%) (Gibco) supplemented with 5% foetal calf serum (Boehringer). structures, which involves the assembly of adherens junctions Mesenchymal intestinal cells C9, C11, C20 obtained from M. Kedinger (Fritsch et al., 1999) (28th, 29th and 14th passages, through the relocalization of E-cadherin at the lateral side of respectively) were cultured at 37°C with 7.5% CO2 in RPMI 1640 the cells (Weaver et al., 1997). medium, pen/strept (50 µg/ml) (Gibco), supplemented with 10% The mammalian intestinal epithelium is peculiar in that it is foetal calf serum (Boehringer). Muscle 129CB3 cells were cultured a constantly renewing monocellular epithelium, which as described (Pinçon-Raymond et al., 1991) to form contracting migrates ‘en cohorte’ along the basement membrane from the myotubes and secrete a large amount of ECM. proliferative undifferentiated compartment in the crypts to the tips of the villi. Enterocytes can probably glide over the basement membrane through loose adhesion, through them Extracellular matrix preparation and coating being tied to each other by strong cell-cell junctions. Whereas Native ECM was prepared from 129CB3 myotubes, mesenchymal type IV collagen is constantly present in the basement C9, C11, C20 cells (at confluence), HT29-MTX cells (3 days post- confluence) or Caco-2 cells (12d post-confluence) as described membrane, LN2 is preferentially found in the proliferative previously (Le Beyec et al., 1997). Coating of plastic petri dishes compartment, LN5 in the villus and LN1 at the junction of the was performed by overnight incubation with poly-D-lysine, 5 two compartments (Vachon et al., 1993; Lorentz et al., 1997). µg/cm2, collagen type IV, 10 µg/cm2 and merosin LN2, 8.4 µg/cm2 Similarly, villus and crypt epithelial cells display a different at 4°C. pattern of integrins, β1-containing integrins being more abundant in the villi than in the crypts. Furthermore, β1 is mainly associated with α2 in the crypt and with α3 integrins Perturbation experiments in the villus (Beaulieu, 1992). Whereas α2β1 integrin Caco-2 cells were seeded at 125,000 cells/cm2 (pre-confluence) in preferentially binds to collagen IV but also to LN1 and LN2, 24-well plates coated or not with native ECM or ECM components. α β At the time of plating, cells were mixed with control mouse IgG or 3 1 integrin binds to both collagen IV and LN5 (Beaulieu, β α α β anti- 1-integrin monoclonal blocking antibody (6S6) or anti- 6- 1999; Rousselle and Garrone, 1998). Integrin 6 4 binds to integrin used to block β4 integrin (CD49F) or anti-E-cadherin both LN1 and LN5 (Fleischmajer et al., 1998). This differential monoclonal blocking antibody (HECD-1) at the indicated dilutions. pattern of expression of ECM proteins and their receptors Under these conditions, control cells were confluent within 24 hours. along the crypt-to-villus axis parallels the differentiation For each kinetics experiment, triplicate wells were observed using process of epithelial cells. One can wonder whether changes in a phase contrast microscope. Confluence was evaluated, and ECM-integrin interactions at the crypt to villus junction are counting triplicate wells on a phase contrast microscope numerated accompanied by changes in cell-cell adhesion, which allow cell the domes. migration to the tip of the villus. The colon cancer Caco-2 cell line in culture mimics Ribonuclease protection assay enterocyte differentiation. We previously showed that ECM was A specific 400 bp cDNA encoding the human apoA-IV gene was required for