From the Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo, Izumi, Osaka 594-1101, Japan
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ABSTRACT |
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A panel of human tumor cell lines was screened
for selective expression of laminin 5 chain, a newly identified
laminin subunit comprising laminin-10 (
5
1
1) and -11 (
5
2
1). The lung adenocarcinoma cell line A549 was found to
express the
5 chain at relatively high levels but no detectable
amounts of other
chains. The laminin variants containing
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
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-
5 chain
monoclonal antibody capable of recognizing denatured
5 chain on
immunoblots, whereas the 210-kDa chain was recognized by an anti-
1
chain antibody. The purified
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
3 or
1 subunit but not by those
against
2 or
6 subunit, indicating that laminin-10/11 is
specifically recognized by integrin
3
1. Given the wide
distribution of laminin-10/11 in the basement membrane of various
tissue types and dominant expression of integrin
3
1 in most
epithelial cells, specific interaction of laminin-10/11 with integrin
3
1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the
basement membrane.
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INTRODUCTION |
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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 ,
, and
. Nine
genetically distinct laminin chains, i.e.
1-5,
1-3, and
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 (1
1,
2
1,
3
1,
6
1,
6
4,
7
1,
9
1,
v
3, and
?
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
4 and
5 chains, remain to
be defined.
Laminin-10/11 is composed of 5,
1/2, and
1 chains (4). The
laminin
5 chain was cloned initially in mouse and found to be more
related to a Drosophila laminin
chain than to other laminin
chains (5). cDNA clones encoding the G-domain of the
human
5 chain were isolated, and the gene encoding it has been
mapped to chromosome 20q13.2-13.3 (6). In contrast to other laminin
chains,
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 5 chain at
high levels but no detectable amounts of other
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.
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EXPERIMENTAL PROCEDURES |
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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 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 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
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
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
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
5 chain transcript by RT-PCR. The RNA
transcripts encoding the domain IIIb of human laminin
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
1,
2, and
1 chains were amplified using the PCR primers
5'-AACTGTGAGCAGTGCAAGCCGTTT-3' (sense strand for
1 chain;
nucleotides 1054-1077 (15)), 5'-CAACCAAATGGATCTTCACTGCTT-3' (antisense strand for
1 chain; nucleotides 1278-1301),
5'-CACTGTGAGCTCTGTCGGCCCTTC-3' (sense strand for
2 chain;
nucleotides 1153-1176 (16)), 5'-CAAGGAGTGCTCCCAGGCACTGTG-3' (antisense strand for
2 chain; nucleotides 1427-1451),
5'-CACTGTGAGAGGTGCCGAGAGAAC-3' (sense strand for
1 chain;
nucleotides 1033-1056 (17)), and 5'-CATCCTGCTTCAGTGAGAGAATGG-3'
(antisense strand for
1 chain; nucleotides 1203-1226). PCR products
were amplified under the following conditions: 30 cycles at 94 °C
for 1 min, 61 °C (
5 and
2 chains), 57 °C (
1 chain),
55 °C (
1 chain) or 53 °C (
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 5 and
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
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
chains secreted
by human lung carcinoma cells. Monoclonal antibodies against human
laminin
1 chain (4E10) and
1 chain (2E8) were purchased from
Chemicon. Monoclonal antibodies against integrin
5 and
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
2 chain on immunoblots. One of these antibodies, 10G1,
specifically stained ~300-kDa
2 chain but not ~200-kDa
/
chains. Monoclonal antibodies against human integrin
2 and
3
subunits, P1E6 and P1B5, respectively, were purchased from Life
Technologies, Inc., and the monoclonal antibody against human integrin
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 chains by immunoblotting with antibodies
specific to each
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 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.
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RESULTS |
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Screening of Human Tumor Cell Lines for Expression of Laminin 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
5 chain. The PCR primers were designed according to the
nucleotide sequence of human
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
5 cDNA sequence (5). Expression of the laminin
1 chain,
the
chain of the classical laminin-1, was also screened by RT-PCR
in parallel to select cells expressing
5 but not
1. One of the
human lung adenocarcinoma cell lines, A549, was found to express the
5 chain mRNA at relatively high levels but not that of
1
chain, although another lung carcinoma cell line, RERF-LC-AI, expressed
1 but not
5 (Fig.
1A).
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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 1 chain but has recently been shown to recognize the
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-
5 monoclonal antibody 15H5 and also
by monoclonal antibodies specific for the
1 or
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
5 chain (350-kDa band) and for the
1 chain (210-kDa band),
respectively (Fig. 2B). Weak reactivity of the anti-
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
1 or
2
chain, supporting the conclusion that the laminin variants purified
from the conditioned medium of A549 cells were
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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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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DISCUSSION |
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The newest laminin chain identified to date,
5, has been
established as the most widely expressed
chain in mammalian tissues. The anti-human laminin monoclonal antibody 4C7, which was
reported initially to be directed against the
1 chain, has been
shown to recognize the
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
5 chain showed
that the
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
5 chain, however, the biological
functions of the laminin variants containing the
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 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
5 chain on immunoblots. The availability of such a
monoclonal antibody is crucial to identify the
5 chain because final
verification of purified laminin-10/11 requires immunoblotting under
reducing conditions. No such monoclonal antibodies recognizing reduced,
denatured human
5 chain have been reported to date. We also produced
a monoclonal antibody that specifically recognizes
1 chain on
immunoblots. The
1 chain has a molecular mass similar to that of
5 chain, and therefore it is important to distinguish these two
chains with specific antibodies. Previous confusion regarding the
specificity of 4C7 antibody also made it crucial to distinguish
1
and
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 5,
1, or
1 chain on immunoblots, we concluded
that the 350-kDa chain was
5 and the 210-kDa chain was
1. Other
chains including
1,
2, and
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
4 chain-containing laminin variants copurified with laminin-10/11. Weak
reactivity of the anti-
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
1 and
2 chains because both
transcripts encoding
1 and
2 chains were detectable in A549 cells
by RT-PCR. The presence of the
1 chain was verified by positive
staining of the unreduced ~800-kDa band with the anti-
1 antibody.
The relative molecular mass of the 5 chain estimated from SDS-PAGE
(350 kDa) was significantly smaller than the mass of mouse
5 chain
(450 kDa) calculated from the amino acid sequence predicted from the
cDNA (5), raising the possibility that the
5 chain expressed in
A549 cells is processed post-translationally proteolysis as observed
with the
2 and
3 chains (27, 28). Consistent with our
observation, the
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
5 chain was generated by alternative RNA
splicing, as has been reported for the
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 3
1 but not
by
6
1. Integrin
3
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
3
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
3
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 (
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
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-
5 monoclonal
antibody on immunoblots.2 The specific binding of integrin
3
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
3
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
6
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 3
1, although
laminin-5 can also be recognized by integrin
6
4 which plays an
important role in hemidesmosome assembly (37). Furthermore, the
chains of laminin-10/11 and laminin-5, i.e.
5 and
3,
seem to be evolutionally the most related among five different laminin
chains (38). A full sized laminin
3 chain,
3B, was identified
recently in mouse and human, showing the highest homology to the
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
3
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
3
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
6
4, which also bind to substrate-bound laminin variants
(39, 40). Furthermore, integrin
3
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
3
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
3
1-dependent manner. Given that laminin-10/11 are the
predominant laminin variants of most epithelial tissues and that
integrin
3
1 is the most abundant integrin receptor expressed in
epithelial cells of different tissue types, specific interaction of
integrin
3
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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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