Isolation and Characterization of Laminin-10/11 Secreted by Human Lung Carcinoma Cells
LAMININ-10/11 MEDIATES CELL ADHESION THROUGH INTEGRIN alpha 3beta 1*

Yamato Kikkawa, Noriko Sanzen, and Kiyotoshi SekiguchiDagger

From the Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo, Izumi, Osaka 594-1101, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

A panel of human tumor cell lines was screened for selective expression of laminin alpha 5 chain, a newly identified laminin subunit comprising laminin-10 (alpha 5beta 1gamma 1) and -11 (alpha 5beta 2gamma 1). The lung adenocarcinoma cell line A549 was found to express the alpha 5 chain at relatively high levels but no detectable amounts of other alpha  chains. The laminin variants containing alpha 5 chain were purified from the conditioned medium of A549 cells by immunoaffinity chromatography using the anti-laminin monoclonal antibody 4C7 which was shown recently to recognize the laminin alpha 5 chain (Tiger, C.-F., Champliaud, M.-F., Pedrosa-Domellof, F., Thornell, L.-E., Ekblom, P., and Gullberg, D. (1997) J. Biol. Chem. 272, 28590-28595). The purified laminin variants consisted of three chains with molecular masses of 350, 220, and 210 kDa. The 350-kDa chain was specifically recognized by another anti-alpha 5 chain monoclonal antibody capable of recognizing denatured alpha 5 chain on immunoblots, whereas the 210-kDa chain was recognized by an anti-gamma 1 chain antibody. The purified alpha 5 chain-containing laminin variants (hereafter referred to as laminin-10/11) were highly active in mediating adhesion of A549 cells to the substratum with potency as high as that of laminin-5 and significantly higher than those of laminin-1, laminin-2/4, or fibronectin. Adhesion to substrata coated with laminin-10/11 was specifically inhibited by anti-integrin antibodies directed against the integrin alpha 3 or beta 1 subunit but not by those against alpha 2 or alpha 6 subunit, indicating that laminin-10/11 is specifically recognized by integrin alpha 3beta 1. Given the wide distribution of laminin-10/11 in the basement membrane of various tissue types and dominant expression of integrin alpha 3beta 1 in most epithelial cells, specific interaction of laminin-10/11 with integrin alpha 3beta 1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the basement membrane.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Laminins are a family of basement membrane proteins implicated in diverse functions of epithelial and neuronal cells including adhesion, migration, proliferation, differentiation, and programmed cell death. Laminins are disulfide-linked heterotrimers of three distinct but distantly related subunit chains termed alpha , beta , and gamma . Nine genetically distinct laminin chains, i.e. alpha 1-5, beta 1-3, and gamma 1-2, have been identified in man and mouse (1). Combinations of these chains generate at least 11 different laminin variants, although the differences in biological functions among these variants are understood only poorly.

Interaction of cells with laminins is mediated by a variety of cell surface receptors including integrins, membrane-bound proteoglycans (e.g. dystroglycan), and other membrane glycoproteins, of which integrins are of crucial importance with respect to the control of growth and differentiation of cells by the basement membrane (2). To date, nine different integrins (alpha 1beta 1, alpha 2beta 1, alpha 3beta 1, alpha 6beta 1, alpha 6beta 4, alpha 7beta 1, alpha 9beta 1, alpha vbeta 3, and alpha ?beta 8) have been suggested to be receptors for laminins (3). Specificities of interactions of various laminin variants with integrins have been investigated extensively with laminin-1 (a prototype laminin purified from the EHS1 tumor), laminin-2/4 (merosin), and laminin-5 (also referred to as kalinin, epiligrin, nicein, or ladsin), but those of other newly identified laminins, particularly those containing alpha 4 and alpha 5 chains, remain to be defined.

Laminin-10/11 is composed of alpha 5, beta 1/2, and gamma 1 chains (4). The laminin alpha 5 chain was cloned initially in mouse and found to be more related to a Drosophila laminin alpha  chain than to other laminin alpha  chains (5). cDNA clones encoding the G-domain of the human alpha 5 chain were isolated, and the gene encoding it has been mapped to chromosome 20q13.2-13.3 (6). In contrast to other laminin alpha  chains, alpha 5 is expressed widely in adult tissues including placenta, heart, lung, skeletal muscle, kidney, and pancreas (4-10), suggesting that laminin-10/11 may be the major laminin isoforms in the adult basal laminae. Despite its wide distribution in the body, however, the biological functions and integrin binding specificity of laminin-10/11 are yet to be defined with purified proteins.

In the present study we screened a panel of human tumor cell lines for those selectively expressing laminin-10/11. One of the human lung adenocarcinoma cell lines, A549, was found to express the alpha 5 chain at high levels but no detectable amounts of other alpha  chains. Purification of laminin-10/11 from the conditioned medium of A549 cells allowed us to characterize the cell adhesive activity and integrin binding specificity of these widely expressed laminin variants.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Laminin-1 was purified from mouse EHS tumor tissues by the method of Paulsson et al. as described previously (11). Laminin-5 was purified from the conditioned medium of the human gastric carcinoma line MKN45 by immunoaffinity chromatography using affinity-purified rabbit polyclonal antibody against human laminin gamma 2 chain (12). Human laminin-2/4 (merosin) was purchased from Chemicon (Temecula, CA). Plasma fibronectin was purified from outdated human plasma by gelatin-affinity chromatography (13).

Cell Lines and Culture Conditions-- The human lung adenocarcinoma cell line A549 and other human tumor cell lines used in this study were obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan), except for the human lung squamous carcinoma cell RERF-LC-AI and the cervix epidermoid carcinoma cell CaSki, which were obtained from RIKEN Gene Bank (Tsukuba, Japan) and American Type Culture Collection (Rockville, MD), respectively. These cells were grown in DMEM supplemented with 15 mM HEPES (pH 7.2), 100 units/ml penicillin G, 0.1 mg/ml streptomycin sulfate, and 10% fetal bovine serum (JRH Bioscience, Lenexa, KS) unless otherwise indicated, at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.

RT-PCR and Isolation of cDNAs Encoding Human Laminin alpha  Chains-- Total RNA was extracted from cultured cells by the acid guanidinium isothiocyanate method (14). cDNA was synthesized using a First Strand Synthesis Kit (Amersham Pharmacia Biotech) according to the manufacturer's protocol. A cDNA fragment encoding domain IIIb of the human laminin alpha 5 chain was amplified by RT-PCR from CaSki cells using the primers 5'-TGTATCTGTCCACCACGCACTG-3' (sense strand) and 5'-ACATCTTGAGCCCTGCACGTTC-3' (antisense strand), which were modeled after the cDNA sequence 4258-4587 of mouse laminin alpha 5 chain (5). The resulting 330-base PCR product was isolated on a low melting point agarose gel and ligated into EcoRV-cleaved pBluescript II KS(+). The nucleotide sequence of the amplified cDNA, deposited into GenBank under accession number AB010099, was 86% homologous to the corresponding sequence of mouse alpha 5 chain cDNA. A pair of nested primers, 5'-GACTGCCTGCTGTGCCAGC-3' (sense strand) and 5'-GGGGTAGCCATGAAAGCCCG-3' (antisense strand), was used for routine amplification of the laminin alpha 5 chain transcript by RT-PCR. The RNA transcripts encoding the domain IIIb of human laminin alpha 1 chain were also amplified by RT-PCR using primers 5'-AAGTGTGAAGAATGTGAGGATGGG-3' (sense strand; nucleotides 3020-3043 (7)) and 5'-CACTGAGGACCAAAGACATTTTCCT-3' (antisense strand; nucleotides 3312-3336). Similarly, the transcripts encoding human laminin beta 1, beta 2, and gamma 1 chains were amplified using the PCR primers 5'-AACTGTGAGCAGTGCAAGCCGTTT-3' (sense strand for beta 1 chain; nucleotides 1054-1077 (15)), 5'-CAACCAAATGGATCTTCACTGCTT-3' (antisense strand for beta 1 chain; nucleotides 1278-1301), 5'-CACTGTGAGCTCTGTCGGCCCTTC-3' (sense strand for beta 2 chain; nucleotides 1153-1176 (16)), 5'-CAAGGAGTGCTCCCAGGCACTGTG-3' (antisense strand for beta 2 chain; nucleotides 1427-1451), 5'-CACTGTGAGAGGTGCCGAGAGAAC-3' (sense strand for gamma 1 chain; nucleotides 1033-1056 (17)), and 5'-CATCCTGCTTCAGTGAGAGAATGG-3' (antisense strand for gamma 1 chain; nucleotides 1203-1226). PCR products were amplified under the following conditions: 30 cycles at 94 °C for 1 min, 61 °C (alpha 5 and beta 2 chains), 57 °C (gamma 1 chain), 55 °C (alpha 1 chain) or 53 °C (beta 1 chain) for 1 min, and 72 °C for 1 min. PCR products were analyzed by electrophoresis using 2% agarose gels.

Monoclonal Antibodies-- Monoclonal antibodies against human laminin alpha 5 and alpha 1 chains were produced by fusion of SP2/0 mouse myeloma cells with splenocytes from mice immunized with GST fusion proteins containing the IIIb domain of each laminin alpha  chain. GST fusion proteins were expressed in Escherichia coli using pGEX4T-1 (Amersham Pharmacia Biotech) and purified on glutathione-Sepharose. Hybridomas were first screened for reactivity with GST fusion proteins used as immunogens and then selected for reactivity on immunoblots with intact human laminin alpha  chains secreted by human lung carcinoma cells. Monoclonal antibodies against human laminin beta 1 chain (4E10) and gamma 1 chain (2E8) were purchased from Chemicon. Monoclonal antibodies against integrin alpha 5 and beta 1 subunits, 8F1 and 4G2, were produced and characterized in our laboratory and were described previously (18). Monoclonal antibodies against laminin-2/4 were also produced by immunizing mice with human laminin-2/4 and screened for positive reactivity with reduced, denatured alpha 2 chain on immunoblots. One of these antibodies, 10G1, specifically stained ~300-kDa alpha 2 chain but not ~200-kDa beta /gamma chains. Monoclonal antibodies against human integrin alpha 2 and alpha 3 subunits, P1E6 and P1B5, respectively, were purchased from Life Technologies, Inc., and the monoclonal antibody against human integrin alpha 6 subunit (GoH3) was from Cosmo Bio (Tokyo).

Screening of Human Tumor Cells for Expression of Laminin Variants-- 31 human tumor-derived cell lines (13 lung carcinomas, 4 gastric carcinomas, 3 cervix carcinomas, 3 gliomas, 2 kidney carcinomas, 2 choriocarcinomas, 1 fibrosarcoma, 1 hepatoma, 1 oral carcinoma, and 1 pancreatic carcinoma) were grown to confluence in 15-cm culture dishes with DMEM containing 10% fetal bovine serum. The conditioned media were harvested and clarified by sequential centrifugation at 1,500 × g for 10 min and 15,000 × g for 30 min followed by precipitation with ammonium sulfate at 40% saturation. The resulting precipitates were collected by centrifugation at 15,000 × g for 40 min and then dissolved in and dialyzed against 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl. The precipitates were screened for the expression of laminin alpha  chains by immunoblotting with antibodies specific to each alpha  chain.

Purification of Laminin 10/11-- The human lung adenocarcinoma cell line A549 was grown to confluence in 1,700-cm2 roller bottles with DMEM containing 10% fetal bovine serum (400 ml/bottle). After the cells reached confluence, the conditioned medium was harvested once every 6 days and clarified by centrifugation. Pooled conditioned medium (3-5 liters) was first precipitated with 40% ammonium sulfate and then dissolved in and dialyzed against phosphate-buffered saline (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4). The precipitated proteins were subjected to immunoaffinity chromatography with the monoclonal anti-human laminin antibody 4C7 which was shown recently to recognize the laminin alpha 5 chain (19). The affinity column was prepared by coupling 1 mg of 4C7 IgG purified from ascites (Life Technologies, Inc.) using protein G-Sepharose 4B (Amersham Pharmacia Biotech) to CNBr-Sepharose 4B (Pharmacia). The bound proteins were eluted from the 4C7 column with 0.1 M triethylamine (pH 11.5), neutralized, and dialyzed against phosphate-buffered saline.

Electrophoretic Analysis and Immunoblotting-- SDS-PAGE was carried out on 4% gels under nonreducing or reducing conditions (20). For immunoblotting, proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Proteins on the membrane were reacted with chain-specific monoclonal antibodies followed by incubation with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (EY Laboratories, San Mateo, CA). Bound antibodies were visualized with ECL Western blotting detection regents (Amersham Pharmacia Biotech).

Cell Adhesion Assay-- Cell adhesion assay was performed as described previously (21). Briefly, 96-well microtiter plates (Nunc, Wiesbaden, Germany) were incubated with different types of laminins or fibronectin at 37 °C for 1 h and then blocked with phosphate-buffered saline containing 1% bovine serum albumin for another h at the same temperature. A549 cells were trypsin treated and suspended in serum-free DMEM at a density of 3 × 105 cells/ml; then 0.1 ml of the cell suspension was added to each well of the plates followed by incubation at 37 °C for 1 h. The attached cells were fixed and stained with a 0.4% crystal violet in methanol (w/v) for 30 min. After washing with distilled water, the stained cells were extracted with 0.1 M citrate in 50% ethanol. The absorbance of each well of the plates was measured at 590 nm with a model 3550 microplate reader (Bio-Rad). Photomicrographs of cells stained with Diff-Quik (International Reagents Corp., Kobe, Japan) were taken on Minicopy films (Fuji Photo Film Co., Ltd., Tokyo) with an Olympus IMT-2 microscope (Olympus Optical Co., Ltd., Tokyo).

To identify the receptor for laminin-10/11, monoclonal antibodies against different types of integrins were preincubated individually with A549 cells in a volume of 0.05 ml of incubation solution (4 × 105 cells/ml) at room temperature for 15 min. The preincubated cells were transferred onto plates precoated with different proteins and then incubated further at 37 °C for 30 min. After staining with crystal violet, the attached cells were quantified as described above.

Determination of Protein Concentration-- Protein concentration was determined by the dye method using a Bio-Rad protein assay kit.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Screening of Human Tumor Cell Lines for Expression of Laminin alpha 5 Chain-- To purify and characterize human laminin-10/11, we screened by RT-PCR a panel of 31 human tumor cell lines for expression of the laminin alpha 5 chain. The PCR primers were designed according to the nucleotide sequence of human alpha 5 chain cDNA encoding the IIIb domain, which had been cloned by RT-PCR from total RNA extracted from human cervix epidermoid carcinoma cells using primers modeled after the mouse alpha 5 cDNA sequence (5). Expression of the laminin alpha 1 chain, the alpha  chain of the classical laminin-1, was also screened by RT-PCR in parallel to select cells expressing alpha 5 but not alpha 1. One of the human lung adenocarcinoma cell lines, A549, was found to express the alpha 5 chain mRNA at relatively high levels but not that of alpha 1 chain, although another lung carcinoma cell line, RERF-LC-AI, expressed alpha 1 but not alpha 5 (Fig. 1A).


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Fig. 1.   Expression of laminin-10/11 in human lung carcinoma cells. Panel A, detection of transcripts encoding laminin alpha 5 and alpha 1 chains by RT-PCR. Transcripts encoding the IIIb domain of the alpha 5 and alpha 1 chains were amplified by RT-PCR from two human lung carcinoma cell lines, A549 and RERF-LC-AI, using the primer sets described under "Experimental Procedures." The expected sizes of the amplified cDNA fragments corresponding to the alpha 5 and alpha 1 chains were 197 and 317 base pairs, respectively. Panel B, detection of laminin (LN) alpha 5, alpha 1, and gamma 1 chains by immunoblotting. The conditioned media of A549 and RERF-LC-AI cell lines were subjected to SDS-PAGE on 4% polyacrylamide gels under nonreducing conditions and transferred onto polyvinylidene difluoride membranes followed by immunostaining with monoclonal antibodies against human laminin alpha 5 chain (15H5), alpha 1 chain (5A3), or gamma 1 chain (2E8). The anti-gamma 1 chain antibody was used as a panspecific antibody detecting all of the known laminin variants except laminin-5 (alpha 3beta 3gamma 2). The position of the nonreduced EHS laminin-1 (~800 kDa) is indicated in the left margin. Panel C, expression of laminin beta 1, beta 2, and gamma 1 chains in A549 and RERF-LC-AI cells. Transcripts encoding beta 1, beta 2, and gamma 1 chains were amplified by RT-PCR by using the primer sets described under "Experimental Procedures." The expected sizes of the amplified cDNA fragments corresponding to the beta 1, beta 2, and gamma 1 chains were 248, 299, and 194 base pairs, respectively.

To confirm the selective expression of the alpha 5 chain by A549 cells, the conditioned medium of A549 cells was analyzed for expression of alpha 5 and alpha 1 chains by immunoblotting with monoclonal antibodies specific to each laminin alpha  chain. The monoclonal antibodies were produced by immunizing mice with recombinant GST fusion proteins containing the IIIb domain of either the alpha 5 or alpha 1 chain. Immunoblotting with the monoclonal antibody against the alpha 5 chain (clone 15H5) specifically detected a protein band migrating at the ~800-kDa region in the conditioned medium of A549 cells but not that of RERF-LC-AI cells (Fig. 1B). In contrast, the monoclonal antibody against the alpha 1 chain (clone 5A3) specifically stained a protein band migrating at the same region in the conditioned medium of RERF-LC-AI cells, but not that of A549 cells. The ~800-kDa protein secreted by A549 cells was strongly reactive with a monoclonal antibody specific to the laminin gamma 1 chain and migrated only slightly above the alpha 1-containing laminin variant(s) secreted by RERF-LC-AI cells. Immunoblotting with antibodies specific for the alpha 2 or alpha 3 showed that no detectable amounts of these laminin alpha  chains were expressed by A549 cells (data not shown). Because the anti-gamma 1 chain antibody did not detect any bands corresponding to the molecular masses of the alpha 4 chain-containing laminin-8 or laminin-9 (i.e. 500-600 kDa) in the conditioned medium of A549 cells, it is likely that A549 cells express only laminin-10 (alpha 5beta 1gamma 1) or laminin-11 (alpha 5beta 2gamma 1) among the 11 laminin variants identified to date. Because laminin-10 and laminin-11 differ in their beta  chain types, we examined the expression of laminin beta 1 and beta 2 chains in A549 and RERF-LC-AI cells by RT-PCR (Fig. 1C). The results showed that both beta 1 and beta 2 chains were expressed in both cell types, indicating that A549 cells expressed both laminin-10 and laminin-11. The relative amounts of the PCR products for beta 1 and beta 2 chains also indicated that A549 cells expressed more beta 1 chain than beta 2 chain, whereas RERF-LC-AI cells expressed more beta 2 than beta 1. These results suggest that laminin-10 is the major laminin variant expressed in A549 cells.

Purification of Laminin-10/11-- Laminin-10/11 in the conditioned medium of A549 cells was purified by fractionation with 40% ammonium sulfate followed by immunoaffinity chromatography using the monoclonal antibody 4C7, which was previously considered to recognize the alpha 1 chain but has recently been shown to recognize the alpha 5 chain (19). The protein eluted from a 4C7-Sepharose column gave a single band with molecular mass of ~800 kDa on SDS-PAGE under nonreducing conditions and three bands with molecular masses of 350, 220, and 210 kDa under reducing conditions (Fig. 2A). The nonreduced ~800-kDa band was recognized by the anti-alpha 5 monoclonal antibody 15H5 and also by monoclonal antibodies specific for the beta 1 or gamma 1 (Fig. 2B), confirming that the purified ~800-kDa protein was either laminin-10 or a mixture of laminin-10 and laminin-11. In support of this conclusion, the 350-kDa and 210-kDa bands on the reducing gel were specifically stained by monoclonal antibodies specific for the alpha 5 chain (350-kDa band) and for the gamma 1 chain (210-kDa band), respectively (Fig. 2B). Weak reactivity of the anti-beta 1 monoclonal antibody with reduced protein prevented identification of the subunit type(s) of the 220-kDa chain (data not shown). However, the apparent size of the chain was consistent with that of the beta 1 or beta 2 chain, supporting the conclusion that the laminin variants purified from the conditioned medium of A549 cells were alpha 5 chain-containing laminin-10/11. The purified laminin-10/11 did not show any detectable bands at ~150 kDa, indicating that nidogen/entactin was not associated with the purified protein.


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Fig. 2.   SDS-PAGE and immunoblotting analyses of purified laminin-10/11. Panel A, laminin-10/11 purified from conditioned medium of A549 cells was subjected to SDS-PAGE using 4% polyacrylamide gels under nonreducing and reducing conditions. Proteins were visualized by silver staining. Panel B, proteins separated by SDS-PAGE under nonreducing (2ME-) and reducing (2ME+) conditions were transferred onto polyvinylidene difluoride membranes followed by immunostaining with monoclonal antibodies specific for alpha 5, beta 1, and gamma 1 chains. Positions of EHS laminin-1 under reducing conditions (400 and 200 kDa) are indicated in the right margin.

Cell Adhesion Activity and Receptor Binding Specificity of Laminin-10/11-- The cell adhesive activity of the purified laminin-10/11 was compared with those of other laminin variants (i.e. laminin-1, laminin-2/4, and laminin-5) and fibronectin using A549 cells. A549 cells readily attached and spread onto surfaces coated with laminin-10/11, as was the case with surfaces coated with laminin-5 (Fig. 3). Cells spread on the laminin-10/11-coated surface assumed an elongated, spindle-shape morphology with thin processes, as opposed to the cells on the laminin-5-coated surface which displayed a well spread cobblestone-like morphology with greater cell-substratum contact area. Cells were less adherent to the surfaces coated with laminin-1, laminin-2/4, or fibronectin with limited cell spreading at the same coating concentration. In support of this conclusion, quantitative analysis of cells adhering to surfaces coated with increasing concentrations of different adhesion proteins showed that laminin-10/11 and laminin-5 were almost equally active in mediating adhesion of A549 cells, exhibiting maximal activity at concentrations as low as 3 nM. At this concentration, other laminin variants as well as fibronectin were barely capable of supporting cell adhesion (Fig. 4). The coating concentrations for half-maximal adhesion were ~2 nM for laminin-10/11 and laminin-5, 4 nM for laminin-2/4, 5 nM for fibronectin, and 6 nM for laminin-1. Similar results were also obtained with other cell types including A172 human glioma cells and A431 human epidermoid carcinoma cells (data not shown).


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Fig. 3.   Attachment and spreading of A549 cells on surfaces coated with laminin-10/11 and other adhesive proteins. A549 cells (2 × 104) were seeded onto 96-well microtiter plates coated with laminin-10/11 (LN10/11), mouse laminin-1 (LN1), laminin-2/4 from human placenta (LN2/4), laminin-5 (LN5), or fibronectin (FN) and incubated for 60 min at 37 °C. The protein concentration used for coating was 5 nM. The cells were rinsed with DMEM, fixed in methanol, and stained with Diff-Quik. Bar, 50 µm.


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Fig. 4.   Adhesion of A549 cells onto surfaces coated with laminin-10/11 and other adhesive proteins. 96-well microtiter plates were coated with increasing concentrations of laminin-10/11 (LN10/11, bullet ), mouse laminin-1 (LN1, black-square), laminin-2/4 from human placenta (LN2/4, triangle ), laminin-5 (LN5, square ), or fibronectin (FN, open circle ) and incubated with A549 cells at 37 °C for 1 h. After incubation, cells attached to the surfaces were quantified by crystal violet staining as described under "Experimental Procedures." Each point represents the mean of triplicate assays.

Because cell adhesion onto the laminin-coated substratum is mediated predominantly by the integrin family of adhesion receptors, we examined the effects of function-blocking monoclonal antibodies against various integrin subunits on adhesion of A549 cells onto the surfaces coated with laminin-10/11 or other adhesive proteins (Fig. 5). Adhesion onto surfaces coated with laminin-1, laminin-5, and fibronectin was specifically inhibited by antibodies against integrin alpha 6, alpha 3, and alpha 5 subunit, respectively, and also by the antibody against integrin beta 1 subunit, consistent with previous reports (12, 21, 22). Interestingly, adhesion of A549 cells onto laminin-10/11-coated surfaces was inhibited completely by anti-integrin alpha 3 antibody and by anti-integrin beta 1 antibody, but not by antibodies against other alpha  subunits including anti-alpha 6 antibody. These results indicated that cell adhesion onto laminin-10/11 is mediated by integrin alpha 3beta 1, as is the case with laminin-5. Specific inhibition of the laminin-10/11-mediated cell adhesion by anti-integrin alpha 3 subunit antibody was also observed with A172 glioma cells (data not shown).


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Fig. 5.   Effects of anti-integrin monoclonal antibodies on adhesion of A549 cells to plastic substrates coated with laminin-10/11 and other adhesive proteins. Wells of microtiter plates were coated with 4 nM laminin-10/11 (LN10/11), 30 nM laminin-1 (LN1), 4 nM laminin-5 (LN5), or 12 nM fibronectin (FN). A549 cells were preincubated with the following function-blocking monoclonal antibodies against integrin subunits at a 100 × dilution for ascites or at a concentration of 10 µg/ml IgG for 15 min at room temperature and were then added to the precoated wells: alpha 2, anti-integrin alpha 2 subunit antibody (P1E6); alpha 3, anti-integrin alpha 3 subunit antibody (P1B5); alpha 5, anti-integrin alpha 5 subunit antibody (8F1); alpha 6, anti-integrin alpha 6 subunit antibody (GoH3); beta 1, anti-integrin beta 1 subunit antibody (4G2). After a 30-min incubation, cells attached to the substrates were quantified by crystal violet staining as described under "Experimental Procedures." The numbers of adhering cells are expressed as percentages of the number of cells adhering in the absence of monoclonal antibodies. Each column represents the mean of triplicate assays. Bars, standard deviation.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The newest laminin alpha  chain identified to date, alpha 5, has been established as the most widely expressed alpha  chain in mammalian tissues. The anti-human laminin monoclonal antibody 4C7, which was reported initially to be directed against the alpha 1 chain, has been shown to recognize the alpha 5 chain (19), resolving the previous discrepancy in histological distribution between mouse laminin-1 and its human counterpart defined by 4C7 (2). Immunohistochemical studies using 4C7 and other antibodies specific for mouse alpha 5 chain showed that the alpha 5 chain is localized in basement membranes of a wide variety of epithelial tissues and of blood vessels (4, 23, 24). Despite the ubiquitous distribution of the alpha 5 chain, however, the biological functions of the laminin variants containing the alpha 5 chain remain to be determined, mainly because these laminin variants have not been purified in intact form. This study was performed to characterize the biological activities of laminin-10/11 using purified, intact proteins.

The strategies employed to purify laminin-10/11 were as follows. First, we selected a human cell line that expresses only alpha 5 chain by screening more than 30 different human tumor cell lines by RT-PCR and immunoblotting. Conditioned medium of cultured cells is superior to tissue extracts as a source of intact laminins because the laminin variants reactive with the 4C7 antibody can be solubilized only after proteolytic digestion (e.g. pepsin digestion) but not by neutral salt or EDTA extraction, which instead solubilizes laminin-2/4 (25, 26). Second, we produced a monoclonal antibody that specifically recognizes human alpha 5 chain on immunoblots. The availability of such a monoclonal antibody is crucial to identify the alpha 5 chain because final verification of purified laminin-10/11 requires immunoblotting under reducing conditions. No such monoclonal antibodies recognizing reduced, denatured human alpha 5 chain have been reported to date. We also produced a monoclonal antibody that specifically recognizes alpha 1 chain on immunoblots. The alpha 1 chain has a molecular mass similar to that of alpha 5 chain, and therefore it is important to distinguish these two alpha  chains with specific antibodies. Previous confusion regarding the specificity of 4C7 antibody also made it crucial to distinguish alpha 1 and alpha 5 chains on immunoblots. Third, we employed affinity chromatography with 4C7-Sepharose to ensure the authenticity of the purified protein. Based on these strategies, we selected A549 cells as a source for human laminin-10/11 and purified them on a 4C7-Sepharose column. In a separate experiment, we also purified laminin-10/11 by conventional procedures for purification of laminins from tissues, i.e. size fractionation on Sepharose 4B-CL, heparin-Sepharose chromatography, and ion exchange chromatography with HiTrap Q-Sepharose. The resulting laminin-10/11 preparation contained some contaminant proteins but exhibited essentially identical cell adhesive and integrin binding activities as observed with those purified by 4C7 immunoaffinity chromatography.2

The laminin-10/11 thus purified gave a single band migrating at ~800 kDa under nonreducing conditions and consisted of three chains of 350, 220, and 210 kDa. Based on the reactivity with monoclonal antibodies specific for alpha 5, beta 1, or gamma 1 chain on immunoblots, we concluded that the 350-kDa chain was alpha 5 and the 210-kDa chain was gamma 1. Other alpha  chains including alpha 1, alpha 2, and alpha 3 were not detectable in the purified laminin-10/11. The absence of a 500-600-kDa protein in the purified laminin-10/11 also made it unlikely that the alpha 4 chain-containing laminin variants copurified with laminin-10/11. Weak reactivity of the anti-beta 1 monoclonal antibody with reduced, denatured protein failed to identify the 220-kDa chain, but the 220-kDa chain is considered to be a mixture of beta 1 and beta 2 chains because both transcripts encoding beta 1 and beta 2 chains were detectable in A549 cells by RT-PCR. The presence of the beta 1 chain was verified by positive staining of the unreduced ~800-kDa band with the anti-beta 1 antibody.

The relative molecular mass of the alpha 5 chain estimated from SDS-PAGE (350 kDa) was significantly smaller than the mass of mouse alpha 5 chain (450 kDa) calculated from the amino acid sequence predicted from the cDNA (5), raising the possibility that the alpha 5 chain expressed in A549 cells is processed post-translationally proteolysis as observed with the alpha 2 and alpha 3 chains (27, 28). Consistent with our observation, the alpha 5 chains expressed in human choriocarcinoma cells (19) and in mouse endothelial cells (24) were also found to be significantly smaller than the predicted mass. It is also possible that the 350-kDa form of alpha 5 chain was generated by alternative RNA splicing, as has been reported for the alpha 3 chain (29).

Using purified laminin-10/11, we demonstrated that laminin-10/11 is a highly adhesive protein, as potent as laminin-5 in mediating cell attachment and spreading onto the substratum. Cell adhesion onto laminin-10/11-coated surfaces was mediated by integrin alpha 3beta 1 but not by alpha 6beta 1. Integrin alpha 3beta 1, once thought to be a promiscuous receptor recognizing laminin-1, collagen, and fibronectin with low affinities, has been shown to recognize laminin-5 preferentially (21, 22, 30-32). Our results indicate that integrin alpha 3beta 1 is a dominant surface receptor recognizing both laminin-5 and laminin-10/11, both of which are major constituents of basement membranes of a wide variety of epithelial tissues. Although our results provide the first clear evidence demonstrating the integrin binding specificity of laminin-10/11, there have been previous reports on identification of integrin types binding to human laminin. Gehlsen et al. (33) reported that integrin alpha 3beta 1 was specifically bound by an affinity column of human laminin which was prepared from placenta after pepsin digestion followed by immunoaffinity chromatography with a B1 (beta 1) chain-specific monoclonal antibody (34). Although the laminin used in their study appeared to be a mixture of truncated forms of laminin variants containing the beta 1 chain, its strong reactivity with 4C7 (25) indicated that the human laminin from pepsinized placenta contained a significant amount of laminin-10/11 in truncated form. In support of this, we found that human placental laminins obtained from different commercial sources, either purified after pepsin digestion or EDTA extraction, were strongly reactive with our anti-alpha 5 monoclonal antibody on immunoblots.2 The specific binding of integrin alpha 3beta 1 to an affinity column of human laminin from pepsinized placenta is therefore consistent with our conclusion that laminin-10/11 is specifically recognized by integrin alpha 3beta 1. Essentially an identical approach was also taken by Sonnenberg et al. (35) to identify laminin-binding integrin types, resulting in a similar conclusion except that human laminin could also bind to integrin alpha 6beta 1 with lower affinity. The apparent discrepancy between these two previous reports may have been caused by the differences in the proportion of laminin-10/11 relative to other contaminant laminin variants.

Among three other laminin variants examined in this study, laminin-10/11 seems to be more related to laminin-5 than to other laminin variants (i.e. laminin-1 and laminin-2/4) in adhesive properties. Laminin-5 has been reported to be most potent in mediating adhesion of keratinocytes (32), endothelial cells (36), and glioma cells (12) among various adhesive proteins including laminin-1, laminin-2/4, fibronectin, and vitronectin. Our results showed that laminin-10/11 has potency comparable to that of laminin-5 in mediating cell adhesion to the substratum. Furthermore, both laminin variants seem to be specifically recognized by integrin alpha 3beta 1, although laminin-5 can also be recognized by integrin alpha 6beta 4 which plays an important role in hemidesmosome assembly (37). Furthermore, the alpha  chains of laminin-10/11 and laminin-5, i.e. alpha 5 and alpha 3, seem to be evolutionally the most related among five different laminin alpha  chains (38). A full sized laminin alpha 3 chain, alpha 3B, was identified recently in mouse and human, showing the highest homology to the alpha 5 chain at the amino acid level (4, 38). Despite these similarities, however, it should be noted that morphologies of cells adhering onto surfaces coated with either laminin-10/11 or laminin-5 were significantly different. Cells on laminin-10/11-coated surfaces assumed an elongated morphology with multiple thin processes, and those adhering to laminin-5-coated surfaces assumed a cobblestone-like morphology. This clear distinction in adhering cell morphology suggests that the signals transduced from substrate-adsorbed laminin-10/11 and laminin-5 through integrin alpha 3beta 1 are functionally different. The differences in signaling events could be either quantitative, i.e. simply the result of differences in the binding affinity of these laminin variants with integrin alpha 3beta 1, which in turn determines the magnitude of cytoplasmic signals elicited by the ligand-ligated integrin, or qualitative, i.e. the result of differences in the involvement of coreceptors such as dystroglycan and integrin alpha 6beta 4, which also bind to substrate-bound laminin variants (39, 40). Furthermore, integrin alpha 3beta 1 has been shown to associate with transmembrane-4 superfamily proteins (41, 42) and EMMPRIN (43). These integrin-associated membrane proteins could be involved in the regulation of signaling events mediated by ligand-ligated integrin alpha 3beta 1, leading to different cell morphologies on substrata coated with different laminin variants.

In summary, we purified laminin-10/11 from the conditioned medium of A549 cells and demonstrated that it is highly competent in mediating cell adhesion to the substratum in an integrin alpha 3beta 1-dependent manner. Given that laminin-10/11 are the predominant laminin variants of most epithelial tissues and that integrin alpha 3beta 1 is the most abundant integrin receptor expressed in epithelial cells of different tissue types, specific interaction of integrin alpha 3beta 1 with laminin-10/11 may play a central role not only in the adhesion of epithelial cells to underlying basement membranes but also in the regulation and maintenance of the differentiated phenotypes of epithelial cells in vivo.

    FOOTNOTES

* This work was supported by special coordination funds from the Science and Technology Agencies of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-6-879-8617; Fax: 81-6-879-8619; E-mail: sekiguch{at}protein.osaka-u.ac.jp.

1 The abbreviations used are: EHS, Engelbreth-Holm-Swarm; DMEM, Dulbecco's modified Eagle's medium; RT-PCR, reverse transcription-polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

2 Y. Kikkawa, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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