Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2006) 30, 784–792 (Printed in Great Britain)
Contribution of α2β1, α3β1, α6β4 integrins and 67kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4
Olga A. Cherepanova*1, Natalia Kalmykova, Yury P. Petrov, Miralda Blinova and George Pinaev
Department of Cell Culture, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia


Abstract

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of α2β1, α3β1, α6β4 integrins and 67kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins α2β1, α3β1, α6β4 and 67kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. α2β1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between α2β1 and α3β1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.


Keywords: Laminin-2/4, Integrins, 67kDa Laminin receptor, A-431 cells.

1Contract grant sponsor: Russian Foundation for Basic Research; Contract grant number: 03-04-48251.

*Corresponding author. Cardiovascular Research Center, 415 Lane Road, MR5 RM 1328, Charlottesville, VA 22908, USA. Tel.: +1 434 924 5993; fax: +1 434 982 0055.


1 Introduction

The mechanisms of cell regulation are the main problems of cell biology. In tissues of living organisms, this regulation is realized by interaction of ambient bioactive molecules with cell receptors. The extracellular matrix (ECM) glycoproteins, which interact with cell adhesion receptor integrins and regulate proliferation, differentiation and motility of cells, are types of bioactive molecules (Adams and Watt, 1993; Roskelly et al., 1995).

The interaction of cells with ECM is a complicated process, consisting of cell attachment, spreading, focal contacts and stress fiber formation followed by cell migration or cell stabilization. ECM glycoproteins are large multidomain molecules and they interact with cell surface receptors by means of short amino acid sequences. The fibronectin sequence RGD mediates cell adhesion and cell spreading; collagen I and IV have both RGD and other sequences (Yamada and Kennedy, 1985; Raghow, 1994). The formation of complexes of ECM glycoproteins with cell receptors is accompanied by activation of signaling pathways and as a result of this the expression of specific genes occurs (Van der Flier and Sonnenberg, 2001; Hynes, 2002; DeMali et al., 2003). Little is known about the contribution of the sequences of ECM molecules and cell surface receptors at different stages of cell–matrix interactions.

The ECM glycoprotein laminin has the largest number of sequences for cell surface receptors (Nomizu et al., 1995; Yoshida et al., 1999). Laminins along with collagen IV and proteoglycans form the structure of basement membrane (BM), a network of specific ECM proximately touching cells (Engbring and Kleinman, 2003; Sasaki et al., 2004). Currently, at least 12 different isoforms of laminin in various tissues are known (Patarroyo et al., 2002). The most characterized isoform is laminin-1 from Engelberth-Holm-Swarm mouse sarcoma. It is composed of α1, β1 and γ1 chains bound by S-S links (Beck et al., 1990). Laminin-2/4 (α2β1/2γ1) from human placenta has 50% homology to laminin-1 amino acid sequence and equal domain composition (Vuolteenaho et al., 1994). The laminin α2 chain (laminin-2/4) was found in BM of skeletal muscle, heart muscle and Schwann cells (Leivo and Engvall, 1988; Ehrig et al., 1990). It has been shown that mutations in the laminin α2 chain result in muscular dystrophy (Wewer and Engvall, 1996; Cohn et al., 1998; Guo et al., 2003).

Although laminin-2/4 has been found in the skin (Sollberg et al., 1992; Sewry et al., 1996; Squarzoni et al., 1997), its specific interactions with skin cells are not as well characterized as those of laminin-1 and laminin-5. In addition, it has been reported that the expression of the α2 subunit of laminin correlates with increased cell adhesion and metastatic propensity (Jenq et al., 1994). Thus, laminin-2/4 can play an essential role in both normal skin morphogenesis and skin carcinogenesis. We have shown that laminin-2/4 is a better adhesion agent for human keratinocytes and epidermoid carcinoma A-431 cells than laminin-1 (Gorelik et al., 2001; Voronkina et al., 1999).

In this study, we applied a number of methods to compare the contribution of laminin receptors to different stages of the interaction of cells with laminin-2/4 (from attachment to motility). We used human epidermoid carcinoma cells, A-431, as a model, since a wealth of information about the receptors to laminin-1 is available.

2 Materials and methods

2.1 Cells and reagents

The transformed human epidermoid carcinoma cell line A-431 was obtained from the Russian Cell Culture Collection (Institute of Cytology RAS, St. Petersburg) and maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum.

Laminin isoforms were isolated from murine EHS-sarcoma (laminin-1) and human placenta (laminin-2/4) as previously described (Palm and Furcht, 1983, Gorelik et al., 2001). Proteins were stored at −70°C in the presence of protease inhibitors. To exclude the possibility of the presence of laminin-10/11 in the protein samples a Western blotting assay was performed using 4C7 antibodies to α5 laminin chain (laminin-10/11). The 4C7 monoclonal antibodies were kindly provided by Dr. E. Engvall (The La Jolla Cancer Research Foundation, USA).

To determine the contribution of α2β1, α3β1, α6β4 integrins and 67kDa LR to cell attachment and spreading, the A-431 cells were incubated with monoclonal antibodies to β1- (W1B1O, Sigma, 25μg/ml), α2- (α2\VLA-2α, BD Biosciences, 25μg/ml), α3- (clon P1B5, DAKO), β4-integrin chains (25μg/ml) and YIGSR peptide (Sigma, 40μg/ml) for 30min at 0°C (on ice). The monoclonal antibodies to β4-integrin chains were kindly provided by Dr. E. Engvall (The La Jolla Cancer Research Foundation, USA). For the cell adhesion inhibition assay and the cell migration assay, the same monoclonal antibodies were individually incubated with cells in serum-free medium at room temperature for 30min: anti-α2 (25μg/ml), anti-α3 (10μg/ml), β4 (25μg/ml).

2.2 Protein-coated latex bead assay

Latex beads D=1.09μ (Sigma) were incubated with laminin-1 or laminin-2/4 in 100μg/ml solutions on a shaker for 45min. A bovine serum albumin (BSA, Sigma) solution of the same concentration was used as a negative control. Protein-covered beads were precipitated by centrifuging at 12 000rpm for 15min. The pellet was suspended in 0.5ml HEPES buffer (10mM HEPES, 150mM NaCl, 10mg/ml BSA, pH 7.2) and sonicated for 30s. The A-431 cell suspension was brought to a concentration of 106 cells/ml in HEPES buffer with 0.1mM CaCl2, 0.2mM MgCl2 and 25μl protein-coated beads were added to 0.5ml of the suspension. Incubation was conducted on a shaker for 1min at 0°C (on ice). Then the cells were suspended with the addition of 0.4ml 3.7% paraformaldehyde (Merk) in PBS (137mM; NaCl, 7mM Na2HPO4, pH 7.4, 1.5M KH2PO4, 267mM KCl). Fixed cells were observed in phase contrast optics and the amount of cell-attached beads was counted. For each individual experiment 100 cells were analyzed. Student's t-test was used to determine if differences existed among the obtained means (P<0.05).

2.3 Cell adhesion assay

Multiwell tissue culture plates (96-wells, Nunc) were coated with laminin-1 or laminin-2/4 (20μg/ml) at 4°C overnight and then blocked with PBS containing 1% bovine serum albumin at 37°C for 1h. Cells in the serum-free medium were added to wells (4x104 /well) followed by 30min incubation at 37°C. After fixation of the adherent cells with 3.7% formaldehyde in PBS, the extent of adhesion was determined by staining with 0.1% crystal violet and color reading at 570nm with LKB Multiskan or by counting cells in each well under an inverted light microscope. Seven fields were counted in each well and the average number of cells per field was used. All experiments were performed at least three times with three replicates within each study. The statistical significance of differences between obtained means was assessed by a paired Student's t-test (P<0.05).

2.4 Cell motility assay

The cell motility assay was performed as described in Albrecht-Buehler (1977) with some modifications. Cover slips were treated with extracellular matrix proteins laminin-1 or laminin-2/4 (20μg/ml) at 4°C overnight. After rinsing three times in PBS, the cover slips were coated with latex beads D=0.8μ (Sigma). Cells were plated in the serum-free medium in the absence or presence of EGF (10 ng/ml). After 24h incubation, cells were fixed in 3.7% formaldehyde in PBS and photographed under dark-field optics. Moving cells formed motility tracks (area free of latex beads). The area of the motility tracks was measured and presented as a percentage of the total cover slip area. Typically 10 fields were counted on each cover slip. All experiments were performed at least three times with three replicates within each study. The ArcSin transformation was used for normalization of obtained means. After determination of differences by a paired Student's t-test (P<0.05) the means were retransformed to percentages.

2.5 Cytoskeleton staining assay

Cytoskeleton staining assay was performed as described previously (Are et al., 2001). Glass cover slips, treated with Repell-Silane (Pharmacia Biotech, Sweden) were coated with laminin-1 or laminin-2/4 at 20μg/ml at 37°C for 1h or at 4°C by overnight. Then 1% heat denatured BSA was added for 1h at 37°C to block nonspecific interactions with the solid substrata.

For visualization of the actin filament system, detached cells were plated on ligand-coated glass cover slips in CO2-incubator at 37°C for 1h. Adherent intact A-431 cells were fixed with 3% paraformaldehyde prepared in PBS (10min at room temperature), permeabilized with 0.1% Triton X-100 in PBS (10min at room temperature). Actin distribution was visualized by staining the cells with rhodamine-phalloidin (10min at 37°C) followed by washing of the samples with PBS. The preparations were analyzed using Opton ICM microscope (Zeiss). The polarization of cells was quantified by digital analysis and the criterion of polarization was defined as described below. For each individual experiment 3x100 cells were analyzed. Student's t-test was used to determine if differences existed among the obtained means (P<0.05).

3 Results

3.1 Integrins α2β1, α3β1, α6β4 participate in early stage of interaction of A-431 cells with laminin-2/4

A latex bead assay was proposed for the study of the early adhesion stage (attachment) (Gorelik et al., 2001). The incubation of human epidermoid carcinoma A-431 cells with protein-coated latex beads was performed on ice for 1min. The phagocytosis of laminin-coated beads but not BSA-coated beads by A-431 cells occurred after 5min incubation. Fig. 1 shows the fractions of cells that attached latex beads covered by laminin-1, laminin-2/4 and BSA (as the control) (Fig. 1a) and the comparative numbers of beads per cell (Fig. 1b). It was found that laminin-2/4-beads attached to the A-431 cells twice as effectively as laminin-1 beads.


Fig. 1

The comparison of laminin-1 and laminin-2/4-covered latex beads binding to human epidermoid carcinoma A-431 cells after 1min incubation on ice. The percentage of cells with beads (a), the average number of beads per cell (b). Bars=SEM. Asterisks represent statistically significant difference (P<0.05) as determined by Student's t-test.


Previously it has been shown that the A-431 cell surface has α2β1, α3β1, α6β4 integrins and 67kDa receptor (67 LR) for laminin-1 (Ardini et al., 1997). However, little information is available for laminin-2/4. To test whether these receptors might also interact with laminin-2/4, we treated the cells with antibodies to β4-, β1-, α2- and α3-integrin subunits and YIGSR peptide, which is a specific site for 67 LR (Graf et al., 1987; Massia et al., 1993). The results are shown in Fig. 2. After treatment of the cells with antibodies to β1-, β4, α2 and α3-integrin subunits the attachment of the cells to latex beads covered with laminin-2/4 was reduced by up to 40–60% (in comparison with the untreated A-431 cells), indicating that α2β1, α3β1, α6β4 integrins participate in the attachment of A-431 cells to laminin-2/4. Moreover, it was shown that the simultaneous treatment of A-431 cells with antibodies to α2- and α3-integrin subunits mediated a decrease in cell attachment of laminin-2/4-beads. This decrease is similar to the decrease that takes place after treatment with antibodies to the β1-integrin subunit. Treatment of the cells with YIGSR peptide did not change the efficiency of the attachment of cells to latex beads, suggesting no 67kDa laminin receptor participation in early adhesion stage to laminin-2/4.


Fig. 2

The differences in the relative number of latex beads covered by laminin-2/4 attached to A-431 cells after incubation of cells with Abs to α2-integrins, α3-integrins, α2- and α3-integrins simultaneously, β1-integrins, β4-integrins and YIGSR peptide. The certain changes of the laminin-2/4-bead number were observed after treatment of cells with Abs to α2-integrins, α3-integrins, α2- and α3-integrins simultaneously, β1-integrins, β4-integrins, but not YIGSR peptide. The data represent the percentage of latex beads covered with laminin-2/4 relative to the control (untreated cells). Bars=SEM. Asterisks represent statistically significant difference (P<0.05) as determined by Student's t-test.


3.2 Contribution of receptors to spreading of A-431 cells on laminin-2/4 differs from that to the attachment of the cells

The latex bead assay describes early interaction between receptors and laminin only. The classical quantitative adhesion assay for cell spreading on substrate was applied as the model characterizing cell receptor–laminin interactions in vivo, when the cells are polarized and attached to BM by their basal surface.

Previously, it has been shown that the adhesion of A-431 cells to laminin-2/4 substratum is more effective than the adhesion to laminin-1 (Voronkina et al., 1999). The results of the influence of the cell pretreatments with antibodies and YIGSR peptide on A-431 cells adhesion to laminin-2/4 substratum are summarized in Fig. 3. Although antibodies to α2- and α3-integrin subunits alone could not inhibit cell adhesion to laminin-2/4, the simultaneous treatment of the cells with these antibodies decreased adhesion approximately by a factor of two, indicating cooperative action of α2β1 and α3β1 integrins. Preincubation with antibodies to β4-integrin subunit alone resulted in a small increase in adhesion of A-431 cells to laminin-2/4, supporting the fact that α6β4 integrins are not essential for adhesion at this stage. In contrast, treatment of the cells with YIGSR peptide resulted in a significant decrease in adhesion of the cells to laminin-2/4. From these observations we concluded that the contribution of receptors is different at different stages of interaction of laminins with cells.


Fig. 3

The alteration of A-431 cell adhesion to laminin-2/4-substratum after treatment of cells with Abs to α2-integrins, α3-integrins, α2- and α3-integrins simultaneously (a), β4-integrins and YIGSR peptide (b). Cell adhesion was measured as crystal violet staining intensity at 570nm (a) and as the number of adhered cells (b). A significantly lower number of cells adhered to laminin-2/4 after treatment of the cells with Abs to α2- and α3-integrins simultaneously and YIGSR peptide alone when compared with non-treatment cells. Bar=SEM. Asterisks represent statistically significant difference (P<0.05) as determined by Student's t-test.


3.3 Effect of antibody treatment on the polarization of cells spread on the laminin-covered substrata

A cytoskeleton assay was suggested for the study of cell phenotypes in the late adhesion stage (spreading). A-431 cells did not demonstrate visible alterations in cytoskeleton patterns on substrata coated by laminin-1 or laminin-2/4 (Fig. 4) either before or after treatments with antibodies to β4-, β1-, α2- and α3-integrin subunits. In order to quantify these data, the notion of the criterion of polarization was introduced as follows. We separated five general groups of the cells after 1h spreading on laminin-coated substratum (Fig. 5). Group 1 consisted of unpolarized cells. Group 2 – evenly spreading round cells. Group 3 – ellipse-like polarized cells with lamellae on leading edge. Group 4 – polarized cells forming rays in different directions. Group 5 – cells with lamellae on the leading edge stretched along one axis. To quantity the spreading degree for different cells each cell was presented as an ellipse (Fig. 5f). The ratio of the ellipse major and minor axises (h) was given as the characters of groups 2–5. The mean value of h was defined as the criterion of polarization. The criteria of polarization for cells on laminin-1 and laminin-2/4 substrata after 1h were 2.60±0.04 and 3.27±0.05, respectively, indicating that the cells on laminin-2/4 substratum are more polarized than on laminin-1 substratum.


Fig. 4

Organization of actin cytoskeleton patterns in the A-431 cells spread on laminin-1 (a) and laminin-2/4 (b) substrata X 100. The cells were plated on cover slips (100μl, 105 cells/ml) pretreated with Repell-Silane and covered with the laminins. The spread cells were fixed with 3% paraformaldehyde after 1h incubation, treated with 0.1% Triton X-100 in PBS, and stained with rhodamin-phalloidin.


Fig. 5

Five groups of A-431 cells after spreading on laminin-coated substrata. After incubation as described in the legend to Fig. 4, the spread cells were fixed and stained with rhodamin-phalloidin X 100. To quantify the spreading degree for different cells, each cell was represented as an ellipse (f). Five general groups of the cells were separated. Group 1 consists of unpolarized cells. The ratios of the lengths of the major and minor axes of an ellipse (h) were given as the characters of groups 2–5.



The criterion of polarization has allowed an estimate of the variations in the degree of polarization of the cells after treatment with different antibodies. Fig. 6 demonstrates the relative change of the criterion of polarization of A-431 cells after different treatments (ΔK). The criterion of polarization of untreated cells was taken as zero (the X-axis). The negative values of ΔK correspond to reduction of cell polarization after pretreatment with an antibody; the positive values correspond to increase of cell polarization. Since information about laminin-1 receptors on A-431 cells surface was available, laminin-1 action was used for comparison with laminin-2/4 action. After treatment of cells with antibodies to β1-integrin subunit alone and simultaneously with antibodies to α2- and α3-integrin subunits, the polarization of the cells on the laminin-1-coated substratum was almost the same. After treatment of cells with antibodies to β1-integrin the criterion of polarization of A-431 cells on laminin-2/4-coated substratum decreased, and it was constant after simultaneous treatment with antibodies to α2- and α3-integrin subunits, confirming the observation about cooperative action of α2β1 and α3β1 integrins in the adhesion assay. The antibodies to β4-integrin subunit had no effect on the criterion of polarization of A-431 cells on laminin-1-coated substratum (Klam-1), while the criterion of polarization of A-431 cells on laminin-2/4-coated substratum (Klam-2/4) was decreased significantly (Fig. 6). Unexpectedly, preincubation with YIGSR peptide increased Klam-1 and decreased Klam-2/4.


Fig. 6

The differences in the criterion of polarization (ΔK) for A-431 cells on laminin-1 and laminin-2/4 substrata after cell treatment with Abs to α2-integrins, α3-integrins, α2- and α3-integrins simultaneously, β1-integrins, β4-integrins and YIGSR peptide. The ΔK of non-treated cells is taken as zero (the X-axis). Bars=SEM. Asterisks represent statistically significant difference (P<0.05) as determined by Student's t-test.


Previously, co-regulation and physical association of the 67 LR and α6β4 integrin during A-431 cells interaction with laminin-1 were reported (Ardini et al., 1997). In order to check this possibility for laminin-2/4, preincubation with a combination of antibodies to β4-integrin subunit and YIGSR peptide was performed. There was an increase of Klam-1 different from the average effect of antibodies and YIGSR peptide alone, indicating a cooperative action between α6β4 integrin and 67 LR with laminin-1. In contrast, the alteration of Klam-2/4 after simultaneous treatment of the cells with antibodies to β4-integrin subunit and YIGSR peptide led to the average value of these agents' action, indicating that α6β4 integrin and 67 LR interact with laminin-2/4 independently of one another. We suggest that these data may be one of the reasons why the response of the same cells to laminin-1 and laminin-2/4 is different.

3.4 Contribution of α2β1, α3β1, α6β4 integrins to the EGF-induced A-431 cell motility

To determine which integrin(s) are involved in the cell motility on laminin-2/4, we employed the modified phagokinetic assay as previously described (Albrecht-Buehler, 1977). We used latex beads (D=0.8μ) instead of colloid gold applied in the original method (Gorelik et al., 1998). Fig. 7 demonstrates the typical survey of areas cleaned of latex beads by cells during motility. It was found that A-431 cell motility on laminin-2/4-coated substratum occurred in the presence of EGF; no cell motility on laminin-2/4-coated substratum was observed without the promoting EGF effects.


Fig. 7

The micrograph of A-431 cells on laminin-2/4-covered substratum without motility (a) and the cells migrated on laminin-2/4-covered substratum in the presence of EGF (b) after 24h incubation. ×20.


The preincubation of A-431 cells with antibodies to β4-integrin subunit caused a fivefold increase in motility in the absence of EGF. After simultaneous exposure of antibodies to β4-integrin subunit and EGF a significant additional increase in motility was observed. Antibodies to β1-integrin subunit alone had no effect on motility. After treatment of the cells with antibodies to β1-integrin subunit, EGF-induced motility was less than EGF-induced migration of untreated cells. These results suggest that β1-integrins and α6β4 integrins play opposite roles in A-431 cell motility on laminin-2/4.

Since β1-integrins are represented by both α2β1 and α3β1, we investigated the effect of antibodies to α2- and α3-integrin subunits on the A-431 cell motility on laminin-2/4 (Fig. 8). Antibodies to α2- and α3-integrin subunits had no influence on the motility of A-431 cells in the absence of EGF stimulation. Similarly, removing α3β1 from interaction had no effect on the A-431 cell EGF-induced motility. However, antibodies to the α2-integrin subunit almost completely blocked EGF-induced motility. These findings indicate that α2β1 integrin plays a determinative role in EGF-induced motility of A-431 cells on laminin-2/4-coated substratum.


Fig. 8

The alteration of A-431 cell motility on laminin-2/4-coated substratum after cell treatment with Abs to β1-integrins, β4-integrins (a), α2-integrins and α3-integrins (b). The area of the motility tracks was measured and presented as the percentage of total cover slip area. Bar=confidence intervals. Asterisks represent statistically significant difference (P<0.05) as determined by paired Student's t-test (a). The ArcSin transformation was used for the normalization of the obtained means.


4 Discussion

Our attention was focused on the laminin-2/4 interaction with human epidermoid carcinoma A-431 cells (transformed keratinocytes). In this study, we compared the contribution of A-431 cell receptors, earlier described as receptors for laminin-1, to different stages of the interaction of A-431 cells with laminin-2/4. We proposed a number of methods for the investigation of different stages of cell–matrix interaction.

The α6β4 integrin possesses unique dual properties. On one hand, α6β4 is a fundamental component of stable adhesion complexes in the normal epithelium termed hemidesmosomes and links the EM with intermediate filaments (Borradori and Sonnenberg, 1999; Burgeson and Christiano, 1994). On the other hand, α6β4 induces migration of carcinoma cells through its ability to interact with the actin cytoskeleton (Rabinovitz and Mercurio, 1997; Rabinovitz et al., 1999). Thus, α6β4 integrin is an important receptor for malignant cell transformation. Our study demonstrates that the α6β4 integrins are involved in the primary interaction of A-431 cells with laminin-2/4, but their contribution to adhesion cell spreading on substratum is ambiguous, while the elimination of α6β4 from the interaction with laminin-2/4 resulted in only a small influence on the number of adhered cells. It has been previously established that α6β4 integrin does not participate in early steps of keratinocytes adhesion. Instead, it stabilizes the stable adhesion complexes that are formed in later stages of the adhesion process (Carter et al., 1990). However, α6β4 integrin may participate in early steps of adhesion when cells directly interact with laminin without needing to synthesize their own matrix (Karecla et al., 1994).

It should be noted that the hydrophobic surface of laminin-covered latex beads and the culture plates hydrophilic surface covered with laminins may differ. It was shown that the polarization of cells on laminin-2/4-coated substratum, but not on laminin-1-covered substratum, depends strongly on α6β4. Furthermore, the anti-α6β4 antibodies increase both the EGF-induced motility of A-431 cells and motility without EGF stimulation. Taken together, these data suggest that a determinate stage of α6β4-linked activation of signal pathways leading to A-431 cell motility is the earlier stage (attachment) of the interaction of cells with laminin-2/4.

In contrast, our data on the preincubation of A-431 cells with YIGSR peptide indicate that the 67 LR does not participate at an earlier interaction stage of A-431 cells with laminin-2/4. Recent studies described 67 LRs as the most numerous receptors on the A-431 cell surface (Ardini et al., 1997). Moreover, expression of 67 LR has been found to correlate with the metastatic potential of tumors (Sobel, 1993). Our experiments demonstrate that the elimination of 67 LR from the interaction of A-431 cells with laminin-2/4 leads to a decrease in cell polarization on laminin-2/4 and to an increased one on laminin-1. A number of studies have demonstrated the co-expression and correlation between the level of 67 LR and integrins with α6-subunits (α6β1 and α6β4) and their adhesion to laminin-1 (Ardini et al., 1997; Pellegrini et al., 1994; Romanov et al., 1994). Thus, the fact that YIGSR peptide did not have an influence on the attachment of A-431 cells to laminin-covered latex beads may be explained in the following way: 67 LR does not participate in the primary interaction of A-431 cells with laminins but it can participate in cell spreading on laminin-coated substrata which is compatible with our data obtained in the adhesion assay and cytoskeleton assay. Since both laminin isoforms have the YIGSR sequence, we speculated that the opposing action of the YIGSR peptide treatment on cell spreading (ΔK) for laminin-1 and laminin-2/4 could be due to the interference of α6β4 integrin and 67 LR. To check this possibility, the simultaneous action of anti-β4 antibodies and YIGSR peptide was studied. The average value (as the result of two individual treatments) must be obtained if the cooperation between α6β4 integrin and 67 LR does not occur. Thus, the mutual interaction between α6β4 and 67 LR was found for A-431 cell spreading on laminin-1, but not on laminin-2/4. It is possible that cooperative action for α6β4 and 67 LR is one of the causes of the different effects of laminin-1 and laminin-2/4 on the cell response.

We next examined the contribution of α2β1 and α3β1 integrins to different stages of the interaction of A-431 cells with laminin-2/4. We showed that both α2β1 and α3β1 integrins participate in the attachment of A-431 cells to laminin-2/4-coated latex beads. In contrast, we found no effect of the anti-α2- or anti-α3-antibody treatment if either of them is used alone. It was demonstrated that laminin-2/4 is not a ligand for α3β1 integrin (Eble et al., 1998; Nishiuchi et al., 2003). However, since a combination of α2β1 and α3β1 integrin-blocking antibodies has significantly decreased the cell adhesion, we suggested that the interplay between these receptors is probably involved at the A-431 cell interaction with laminin-2/4. In support of this suggestion, we found that α3β1 integrin alone did not affect cell polarization on laminin-2/4-coated substratum, but it mediated α2β1 integrin-dependent cell spreading.

The consistency of our observations with earlier findings about a role of α3β1 as a trans-dominant inhibitor of fibronectin and collagen IV integrin receptors in keratinocytes (Hodivala-Dilke et al., 1998) further strengthens the view that the interplay between α2β1 and α3β1 integrins takes place during A-431 cell interaction with laminin-2/4.

It was shown that α3β1 and α6β4 integrins are the receptors for keratinocyte adhesion and migration on laminin-5 (Shang et al., 2001; Gagnoux-Palacios et al., 2001). The α2β1 integrin was shown to be the basic collagen receptor as well as a receptor for laminin (Pfaff et al., 1994; Colognato et al., 1997). Furthermore, studies on the integrin expression revealed the presence of α2β1 integrin correlated with expression of the laminin-2 isoform in metastatic melanoma cells (Han et al., 1999). Our cell motility assay results showed that α2β1 integrin took direct place in the EGF-induced A-431 cell motility on laminin-2/4, while α6β4 integrin participates in the formation of stable cell–laminin-2/4 contacts, preventing cell migration. We also demonstrated that integrin α3β1 did not influence the A-431 cell motility on laminin-2/4-coated substrate. It is noteworthy that α2β1 integrin is colocolized with EGF receptors in A-431 cells (Yu et al., 2000). It is well known that the crosstalk between integrins and growth factor receptors is an important factor in the development of biochemical responses in different cells (Eliceiri, 2001). This evidence may explain the fact that certain integrins have definitive properties permanent for different cell types.

Overall, our conclusions can be summarized as follows: (1) the interaction of epidermoid carcinoma A-431 cells with laminin-2/4 is mediated by α2β1, α3β1, α6β4 integrins and 67 LR; (2) the interaction of α6β4 integrins with laminin-2/4 mediates a stable adhesion and prevents cell motility; (3) α2β1 participates in the EGF-induced A-431 cell motility on laminin-2/4. From the aforementioned it seems reasonable to suggest that (1) the α6β4 integrin ligation on the earlier stage of interaction determines the different character of interactions of 67 LR with laminin-1 and laminin-2/4 on spreading stage; (2) the cooperative action of α2β1 and α3β1 integrins affects the adhesion of A-431 cells on laminin-2/4. Furthermore, our results demonstrate that the contribution of the same type of receptors to various stages of interaction of cells with laminin can be different.

Acknowledgments

We thank Dr. E. Engvall for kindly providing antibodies to β4-integrin subunit and 4C7. We are grateful to Drs. Irina Kropacheva, Elena Kuzminykch, Olga Petoukhova and Lidia Turoverova, Irina Voronkina and Svyatoslav Tkachenko for helpful ideas and contributions to this work. This work was supported by a grant from the Russian Foundation for Basic Research No. 03-04-48251.

References

Adams JC, Watt, FM. Regulation of development and differentiation by the extracellular matrix. Development 1993:117:1183-98
Medline   1st Citation  

Albrecht-Buehler G. The phagokinetic traks of 3T3 cells. Cell 1977:11:395-404
Crossref   Medline   1st Citation   2nd  

Ardini E, Tagliabue, E, Magnifico, A, Buto, S, Castronovo, V, Colnaghi, MI. Co-regulation and physical association of the 67-kDa monomeric laminin receptor and the α6β4 integrin. J Biol Chem 1997:272:4:2342-5
Crossref   Medline   1st Citation   2nd   3rd   4th  

Are A, Pinaev, G, Burova, E, Lindberg, U. Attachment of A-431 cells on immobilized antibodies to the EGF receptor promotes cell spreading and reorganization of the microfilament system. Cell Motil Cytoskeleton 2001:48:24-36
Crossref   Medline   1st Citation  

Beck K, Hunter, I, Engel, J. Structure and function of laminin: anatomy of multidomain glycoprotein. FASEB J 1990:4:148-60
Medline   1st Citation  

Borradori L, Sonnenberg, A. Structure and function of hemidesmosomes: more than simple adhesion complexes. J Invest Dermatol 1999:112:411-8
Crossref   Medline   1st Citation  

Burgeson RE, Christiano, AM. The dermal–epidermal junction. Curr Opin Cell Biol 1994:9:651-8
Crossref   1st Citation  

Carter WG, Kaur, P, Gil, SG, Gahr, PJ, Wayner, EA. Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J Cell Biol 1990:111:6:3141-54
Crossref   Medline   1st Citation  

Cohn RD, Herrmann, R, Sorokin, L, Wewer, UM, Voit, T. Laminin α2 chain-deficient congenital muscular dystrophy: variable epitope expression in severe and mild cases. Neurology 1998:51:94-100
Medline   1st Citation  

Colognato H, MacCarrick, M, O'Rear, JJ, Yurchenco, PD. The laminin α2-chain short arm mediates cell adhesion through both the α1β1 and α2β1 integrins. J Biol Chem 1997:272:46:29330-6
Crossref   Medline   1st Citation  

DeMali KA, Wennerberg, K, Burridge, K. Integrin signaling to the actin cytoskeleton. Curr Opin Cell Biol 2003:15:572-82
Crossref   Medline   1st Citation  

Eble JA, Wucherpfennig, KW, Gauthier, L, Dersch, P, Krukonis, E, Isberg, RR. Recombinant soluble human α3β1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner. J Biochem 1998:37:10945-55
Crossref   1st Citation  

Ehrig K, Leivo, I, Argraves, WS, Ruoslahti, E, Engvall, E. Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc Natl Acad Sci U S A 1990:87:3264-8
Crossref   Medline   1st Citation  

Eliceiri BP. Integrin and growth factor receptor crosstalk. Circ Res 2001:89:1104-10
Crossref   Medline   1st Citation  

Engbring JA, Kleinman, HK. The basement membrane matrix in malignancy. J Pathol 2003:200:4:465-70
Crossref   Medline   1st Citation  

Gagnoux-Palacios L, Allegra, M, Spirito, F, Pommeret, O, Romero, C, Ortonne, J-P. The short arm of the laminin γ2 chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion. J Cell Biol 2001:153:4:835-49
Crossref   Medline   1st Citation  

Gorelik Yu, Diakonov, IA, Kuhoreva, LV, Blinova, MI, Pinaev, GP. Effects of extracellular matrix elements on pseudopodial activity of rat keratinocytes. Tsitologiia 1998:40:12:1037-44
Medline   1st Citation  

Gorelik JV, Cherepanova, OA, Voronkina, IV, Diakonov, IA, Blinova, MI, Pinaev, GP. Laminin-2/4 from human placenta is a better adhesion agent for primary keratinocytes than laminin-1 from EHS sarcoma. Cell Biol Int 2001:25:5:395-402
Crossref   Medline   1st Citation   2nd   3rd  

Graf J, Ogle, RC, Robey, FA, Sasaki, M, Martin, GR, Yamada, Y. A pentapeptide from the laminin B1 chain mediates cell adhesion and binds the 67 000 laminin receptor. Biochemistry 1987:26:6896-900
Crossref   Medline   1st Citation  

Guo LT, Zhang, XU, Kuang, W, Xu, H, Liu, LA, Vilquin, JT. . Neuromuscul Disord 2003:13:3:207-15
Crossref   Medline   1st Citation  

Han J, Jenq, W, Kefalides, NA. Integrin alpha2beta1 recognizes laminin-2 and induces C-erb B2 tyrosine phosphorylation in metastatic human melanoma cells. Connect Tissue Res 1999:40:4:283-93
Crossref   Medline   1st Citation  

Hodivala-Dilke KM, DiPersio, CM, Kreidberg, JA, Hynes, RO. Novel roles for α3β1 integrin as a regulator of cytoskeletal assembly and as a trans-dominant inhibitor of integrin receptor function in mouse keratinocytes. J Cell Biol 1998:142:1357-69
Crossref   Medline   1st Citation  

Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002:110:673-87
Crossref   Medline   1st Citation  

Jenq W, Wu, SJ, Kefalides, NA. Expression of the alpha 2-subunit of laminin correlates with increased cell adhesion and metastatic propensity. Differentiation 1994:58:1:29-36
Crossref   Medline   1st Citation  

Karecla PL, Timpl, R, Watt, F. Adhesion of human epidermal keratinocytes to laminin. Cell Adhes Cummun 1994:2:309-18
Crossref   1st Citation  

Leivo I, Engvall, E. Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development. Proc Natl Acad Sci U S A 1988:85:5:1544-8
Crossref   Medline   1st Citation  

Massia SP, Rao, SS, Hubbell, JA. Covalently immobillized laminin peptide tyr-ile-gly-ser-arg (YIGSR) supports cell spreading and co-localization of the 67-kilodalton laminin receptor with α-actinin and vinculin. J Biol Chem 1993:268:11:8053-9
Medline   1st Citation  

Nishiuchi R, Murayama, O, Fujiwara, H, Gu, J, Kawakami, T, Aimoto, S. Characterization of the ligand-binding specificities of integrin α3β1 and α6β1 using a panel of purified laminin isoforms containing distinct α chains. J Biochem 2003:134:497-504
Crossref   Medline   1st Citation  

Nomizu M, Woo, Hoo Kim, Yamamura, K, Utani, A, Sang-Yong, Song, Otaka, A. Identification of cell binding sites in the laminin α1 chain carboxyl-terminal globular domain by systematic screening of synthetic peptides. J Biol Chem 1995:270:35:20583-90
Crossref   Medline   1st Citation  

Palm SL, Furcht, LT. Production of laminin and fibronectin by Schwannoma cells: cell–protein interactions in vitro and protein localization in peripheral nerve in vivo. J Cell Biol 1983:96:1218-26
Crossref   Medline   1st Citation  

Patarroyo M, Tryggvason, K, Virtanen, I. Laminin isoforms in tumor invasion, angiogenesis and metastasis. Semin Cancer Biol 2002:12:197-207
Crossref   Medline   1st Citation  

Pellegrini R, Martignone, S, Menard, S, Colnaghi, MI. Laminin receptor expression and function in small-cell lung carcinoma. Int J Cancer Suppl 1994:8:116-20
Medline   1st Citation  

Pfaff M, Gohring, W, Brown, JC, Timpl, R. Binding of purified collagen receptors (alpha 1 beta 1, alpha 2 beta 1) and RGD-dependent integrins to laminins and laminin fragments. Eur J Biochem 1994:225:975-84
Crossref   Medline   1st Citation  

Rabinovitz I, Mercurio, AM. The integrin α6β4 function in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures. J Cell Biol 1997:139:7:1873-84
Crossref   Medline   1st Citation  

Rabinovitz I, Toker, A, Mercurio, AM. Protein kinase C-dependent mobilization of the α6β4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells. J Cell Biol 1999:146:1147-60
Crossref   Medline   1st Citation  

Raghow R. The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB J 1994:8:823-31
Medline   1st Citation  

Romanov V, Sobel, ME, pinto da Silva, P, Menard, S, Castronovo, V. Cell localization and redistribution of the 67kDa laminin receptor and alpha 6 beta 1 integrin subunits in response to laminin stimulation: an immunogold electron microscopy study. Cell Adhes Commun 1994:2:3:201-9
Crossref   Medline   1st Citation  

Roskelly CD, Srebow, A, Bissell, MJ. A hierarchy of ECM-mediated signaling regulates tissue-specific gene expression. Curr Opin Cell Biol 1995:7:736-47
Crossref   Medline   1st Citation  

Sasaki T, Fassler, R, Hohenester, E. Laminin: the crux of basement membrane assembly. J Cell Biol 2004:164:7:959-63
Crossref   Medline   1st Citation  

Sewry CA, Dalessandro, M, Wilson, LA, Sorokin, LM, Naom, I, Bruno, S. Expression of laminin chains in skin in merosin deficient congenital muscular dystrophy. Neuropediatrics 1996:28:217-22
Crossref   1st Citation  

Shang M, Koshikawa, N, Schenk, S, Quaranta, V. The LG3 module of laminin-5 harbors a binding site for integrin α3β1 that promotes cell adhesion, spreading, and migration. J Biol Chem 2001:276:35:39045-52
1st Citation  

Sobel ME. Differential expression of the 67kDa laminin receptor in cancer. Semin Cancer Biol 1993:4:5:311-7
Medline   1st Citation  

Sollberg S, Peltonen, J, Uitto, J. Differential expression of laminin isoforms and β4 integrin epitopes in the basement membrane zone of normal human skin and basal cell carcinomas. J Invest Dermatol 1992:98:864-70
Crossref   Medline   1st Citation  

Squarzoni S, Villanova, M, Sabatelli, P, Malandrini, A, Toti, P, Pini, A. Intracellular detection of laminin alpha 2 chain in skin by electron microscopy immunocytochemistry: comparison between normal and laminin alpha 2 chain deficient subjects. Neuromuscul Disord 1997:7:2:91-8
Crossref   Medline   1st Citation  

Van der Flier A, Sonnenberg, A. Function and interactions of integrins. Cell Tissue Res 2001:305:285-98
Crossref   Medline   1st Citation  

Voronkina IV, Gorelik, YV, Are, AF, Kalmykova, NV, Potokin, IL, Pinaev, GP. The peculiarities of attachment and spreading of normal and transformed epithelial human cells on different extracellular matrix proteins. NATO Science Series A: life science 1999:311:171-81
1st Citation   2nd  

Vuolteenaho R, Nissinen, M, Sainio, K, Byers, M, Eddy, R, Hirvonen, H. Human laminin M chain (Merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues. J Cell Biol 1994:124:381-9
Crossref   Medline   1st Citation  

Wewer UM, Engvall, E. Merosin/laminin-2 and muscular dystrophy. Neuromuscul Disord 1996:6:6:409-18
Crossref   Medline   1st Citation  

Yamada KM, Kennedy, DW. Amino acid sequence specificities of an adhesive recognition signal. J Cell Biol 1985:28:99-104
1st Citation  

Yoshida I, Tashiro, K-I, Monji, A, Nagata, I, Hayashi, Y, Mitsuyama, Y. Identification of heparin binding site and the biological activities of the laminin α1 chain carboxy-terminal globular domain. J Cell Phys 1999:179:18-28
Crossref   1st Citation  

Yu X, Miyamoto, S, Mekada, E. Integrin α2β1-dependent EGF receptor activation at cell-cell contact sites. J Cell Sci 2000:113:2139-47
Medline   1st Citation  


Received 4 January 2006/4 May 2006; accepted 23 June 2006

doi:10.1016/j.cellbi.2006.06.012


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)