Originally published In Press as doi:10.1074/jbc.M001326200 on May 2, 2000
J. Biol. Chem., Vol. 275, Issue 29, 22495-22502, July 21, 2000
Structural Requirement of Carboxyl-terminal Globular Domains of
Laminin 
3 Chain for Promotion of Rapid Cell Adhesion and Migration
by Laminin-5*
Tomomi
Hirosaki
§¶,
Hiroto
Mizushima¶,
Yoshiaki
Tsubota§,
Kayano
Moriyama, and
Kaoru
Miyazaki§
From the Division of Cell Biology, Kihara Institute
for Biological Research and § Graduate School of Integrated
Sciences, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku,
Yokohama 244-0813, Japan
Received for publication, February 16, 2000, and in revised form, April, 18, 2000
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ABSTRACT |
The basement membrane protein laminin-5, a
heterotrimer of laminin 
3, 
3, and 
2 chains, potently promotes
cellular adhesion and motility. It has been supposed that the
carboxyl-terminal globular region of the 3 chain consisting of five
distinct domains (G1 to G5) is important for its interaction with
integrins. To clarify the function of each G domain, we transfected
cDNAs for the full-length (wild type (WT)) and five deletion
derivatives (
Gs) of the 3 chain into human fibrosarcoma cell line
HT1080, which expressed and secreted the laminin 3 and 2 chains
but not the 3 chain. The transfectants with the 3 chain cDNAs
lacking G5 (G5), G4-5 (G4-5),
G3-5 (G3-5), and G2-5 (G2-5) secreted
laminin-5 variants at levels comparable to that with WT cDNA.
However, the transfectant with the cDNA without any G domains
(G1-5) secreted little laminin-5, suggesting that the G
domains are essential for the efficient assembly and secretion of the
heterotrimer 332. The transfectants with WT,
G5, and G4-5 cDNAs survived in
serum-free medium longer than those with G3-5,
G2-5, and G1-5 cDNAs. The
transfectants with WT, G5, and G4-5
cDNAs secreted apparently the same size of laminin-5, which lacked
G4 and G5 due to proteolytic cleavage between G3 and G4, and these
laminin-5 forms potently promoted integrin
31-dependent cell adhesion
and migration. However, the laminin-5 forms of G3-5 and
G2-5 hardly promoted the cell adhesion and motility.
These findings demonstrate that the G3 domain, but not the G4 and G5
domains, of the 3 chain is essential for the potent promotion of
cell adhesion and motility by laminin-5.
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INTRODUCTION |
Laminins are a family of extracellular matrix proteins that are
localized mainly in basement membranes and regulate various cellular
functions such as adhesion, motility, growth, differentiation, and
apoptosis through interaction with specific integrins on the cell
surface (1, 2). The three subunits of laminins, designated , ,
and chains, form the well known cross-shaped structure linked
together by disulfide bonds. Five , three , and three chains
and at least 12 structural isoforms of laminin (laminin-1 to -12)
having distinct chain combinations have been identified in human thus
far (3, 4). Among these laminin isoforms, laminin-5, which consists of
the laminin 3, 3, and 2 chains, is unique in the structure and
biological activity. Laminin-5 was originally found as a
keratinocyte-derived matrix protein named epiligrin, kalinin, or nicein
(5-7) and a laminin-like cell scattering factor, ladsin, secreted by
human gastric carcinoma cells (8). Laminin-5 has a feature lacking some
domains found in the amino-terminal regions (or the short arms) of the
three subunits of other laminin isoforms (2). The laminin 3 chain is
found in laminin-6 (311) and laminin-7 (321) besides laminin-5, but the laminin 3 and 2 chains are found only in laminin-5. More interestingly, laminin-5 has much higher activity to
promote adhesion, migration, and scattering of various types of cells
than laminin-1, fibronectin, and vitronectin (8-10).
Most cultured cell lines utilize integrin
31 as a major receptor to adhere and
migrate on the laminin-5 substrate, but integrins 64 and 61
also act as the additional receptors of laminin-5 depending on cell
types (5, 9, 10). Laminin-5 is an important component of basement
membranes of the skin and many other epithelial tissues (5, 6, 11). The
interaction of laminin-5 with integrin 64
in the hemidesmosome structures is essential for the stable anchorage
of basal epithelial cells to the underlying connective tissues. Defects
of laminin-5 genes cause Herlitz-type junctional epidermolysis bullosa
(H-JEB), which is characterized by splitting of epidermal/dermal
junctions (12, 13). Similarly, targeted disruption of the laminin-5
genes or integrin 64 genes in mice causes
severe junctional blisters and abnormal hemidesmosomes, resulting in
neonatal lethality (14-16). On the other hand, the potent cell
motility activity of laminin-5 has been suggested to contribute to
wound healing (17) and tumor invasion (18).
For understanding the molecular basis for the unique bifunctional
properties of laminin-5, the stable adhesion and motility, it seems
important to clarify its structural and functional relationship. All
laminin chains have a large carboxyl-terminal globular domain consisting of a tandem array of five small globular domains (or modules) (G1 to G5) (1, 2). These G domains are autonomous folding
units (19). They contain binding sites for 1 integrins (20) and heparin (21), as well as -dystroglycan in some laminin isoforms (22, 23). Our previous study with recombinant G domains of the
laminin 3 chain has shown that the G2 domain contains an integrin
31-binding site, and the G4 and G5
domains weakly interact with heparan sulfate proteoglycans (24). To
clarify the functions of the G domains of the laminin 3 chain, we
have prepared recombinant laminin-5 proteins serially lacking G domains of the 3 chain, and we examined their activities to promote cell adhesion and motility.
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MATERIALS AND METHODS |
Antibodies--
Mouse monoclonal antibodies against human
laminin 3 chain were established in our laboratory with the support
of Eiken Chemical (Tokyo, Japan). These antibodies were raised against
the glutathione S-transferase fusion protein of the
amino-terminal region of human laminin 3 chain (amino acid residues
109-331). One of these antibodies, LS3c4, was used for
immunoaffinity purification of recombinant laminin-5 proteins.
Monoclonal antibody against human laminin 2 chain (D4B5) was
described previously (11, 25). Rabbit polyclonal antibody (3G4) was
raised against the glutathione S-transferase fusion protein
of the G4 domain of human laminin 3 chain (amino acid residues
1335-1552), which included the carboxyl-terminal 19 amino acid
residues of the G3 domain besides the complete G4 sequence. Monoclonal
antibody against human laminin 3 chain was purchased from
Transduction Laboratories (Lexington, KY). Function-blocking antibodies
against integrins used are anti-2-integrin antibody P1E6, anti-3-integrin antibody P1B5, and
anti-1-integrin antibody P4C10 from Life Technologies,
Inc., (Gaithersburg, MD) anti-5-integrin antibody P1D6
from Chemicon (Temecula, CA), and anti-6-integrin antibody G0H3 from PharMingen (San Diego, CA).
Cell Culture--
Human fibrosarcoma cell line HT1080 and human
tongue squamous adenocarcinoma cell line C-4I were obtained from
Japanese Cancer Resources Bank. Buffalo rat liver-derived epithelial
cell line has been used in previous studies (8, 9). These cell lines were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium
and Ham's F12 medium
(DME/F12)1 (Life
Technologies, Inc.) supplemented with 10% fetal calf serum (FCS) (JRH
Biosciences, Lenexa, KS), penicillin, and streptomycin sulfate.
cDNA Construction and Transfection--
Human laminin 3
chain cDNA has been cloned from a cDNA library of gastric
cancer cells (25). pGEM-LS/CX, a plasmid vector pGEM3Zf(+) (Promega,
Madison, WI) encoding a full-length laminin 3 chain, was prepared in
this study. A series of carboxyl-terminal cDNA fragments of the
laminin 3 chain were amplified by polymerase chain reaction (PCR)
using the laminin 3 chain cDNA as a template and ligated into
the pGEM-3zf(+) vector. PCR primers used were as follows: G4-5',
5'-CTGGATCCCTGTTGCAGGACACA-3' (nucleotides 4003-4017,
sense); G4X-3', 5'-CGTCTAGATCAGTGAGCCAAGACGAC-3'
(nucleotides 4642-4656, antisense); G3-5',
5'-CAGGATCCAGTGGTGTCGTTAGA-3' (nucleotides 3445-3459,
sense); G3X-3', 5'-GGTCTAGATCATCCATGATTGGCCTG-3'
(nucleotides 4081-4095, antisense); G2-5',
5'-CAGGATCCGTTCTGAGCTTGTAC-3' (nucleotides 2851-2865,
sense); G2X-3', 5'-CCTCTAGATCAGGAGAATGAGGCAGA-3'
(nucleotides 3445-3459, antisense); G1-5',
5'-CGGAATTCTGCCAAATGACCTG-3' (nucleotides 2347-2361,
sense); G1X-3', 5'-GGTCTAGATCAAGCATAGCCCGTACC-3'
(nucleotides 2953-2967, antisense); LSp-14, 5'-GATGAGCTGGTGCGCTG-3'
(nucleotides 1636-1652, sense); and I/II X-3',
5'-GGTCTAGACTATCCAAGGTACATCAC-3' (nucleotides 2446-2460,
antisense), in which the underlines indicate restriction sites for
subcloning. Each reverse primer included a stop codon followed by a
XbaI site. The sense primers except for LSp-14 were used in
a previous study (24). The carboxyl-terminal fragments G4X, G3X, G2X,
G1X, and I/II X were obtained by the following primer-combinations:
G4-5'/G4X-3', G3-5'/G3X-3', G2-5'/G2X-3', G1-5'/G1X-3' and
LSp-14/I/II X-3', respectively. pGEM-G5,
pGEM-G4-5, pGEM-G3-5,
pGEM-G2-5, and pGEM-G1-5 were generated
by the insertion of a BstXI/SphI fragment of
pGEM-G4X, a ClaI/SphI fragment of G3X, a
XhoI/SphI fragment of G2X, a
SalI/SphI fragment of G1X, and an
Eco52I/SphI fragment of I/II X into the corresponding restriction sites of pGEM-LS/CX, respectively. The plasmid constructs thus obtained encoded the following domains of the
laminin 3 chain: G5 (amino acid residues 1-1552,
from IIIa to G4), G4-5 (amino acid residues 1-1365,
from IIIa to G3), G3-5 (amino acid residues 1-1153,
from IIIa to G2), G2-5 (amino acid residues 1-989,
from IIIa to G1), and G1-5 (amino acid residues 1-820,
from IIIa to I/II). All nucleotide sequences of the plasmid constructs
were confirmed by sequence analysis. These cDNAs were subsequently
cloned into the XbaI site of mammalian expression plasmid
vector pEF-BOS-CITE-NEO2, a modified version of pEF-BOS-CITE-NEO (27).
Before cDNA transfection, HT1080 cells were cloned by the limiting
dilution method. The cDNA expression plasmid vectors were
transfected into a HT1080 cell clone by the calcium-phosphate
precipitation method. Selection of stable transfectants and subsequent
amplification of the introduced cDNAs were carried out with 500 µg/ml geneticin (Life Technologies, Inc.).
Electrophoretic Analyses--
Northern blotting analysis of the
laminin 3, 3, and 2 chains were performed by the method
described previously (26). SDS-polyacrylamide gel electrophoresis
(PAGE) was performed on 6% gels under nonreducing or reducing
conditions. Separated proteins were stained with Coomassie Brilliant
Blue R-250. For immunoblotting, proteins separated by SDS-PAGE were
transferred onto nitrocellulose membranes. The three subunits of
laminin-5 were detected by the alkaline phosphatase method with
chain-specific antibodies.
Assay of Cell Adhesion--
Adhesion of BRL cells or C-4I cells
to purified laminin-5 proteins and extracellular matrices deposited by
various HT1080 transfectants was assayed as described previously (24).
To prepare the matrices, each transfectant clone of HT1080 cells
(5 × 103 cells) was inoculated and incubated on
96-well plastic plates (Sumibe Medical, Tokyo, Japan) in DME/F12 medium
containing 10% FCS at 37 °C for 4 days. Then the transfectant cells
were detached from the plates by incubating with 100 µl of 10 mM EDTA in Ca2+- and Mg2+-free
phosphate-buffered saline. Complete detachment of cells was confirmed
under a microscopy. The plates were washed twice with
phosphate-buffered saline and blocked with 200 µl of 1.2% (w/v)
bovine serum albumin in phosphate-buffered saline at 37 °C for
1.5 h. These plates were used as the matrix-coated plates for the
assay of cell adhesion. Cells adhered to the laminin-5 proteins or the
HT1080 matrices were stained with Hoechst 33342 for 1.5 h, and the
fluorescent intensity of each well was measured using a CytoFluor 2350 fluorometer (Millipore, Bedford, MA).
Assay of Cell Migration and Cell Scattering--
Migration and
scattering of BRL cells on purified laminin-5 proteins and
extracellular matrices deposited by various HT1080 transfectants were
assayed as reported previously (9). For the cell migration assay, the
matrices of HT1080 transfectants were prepared in 25-cm2
tissue culture flasks (Beckton Dickinson, Franklin Lakes, NJ) as
described above. Cell migration on these substrates was monitored at
37 °C with time-lapse video equipment in which a video camera (MKC-385; Olympus, Tokyo) was mounted on an inverted microscope (IX-70;
Olympus) and connected to a time-lapse control unit (LVR-3000AN; Sony,
Tokyo, Japan). The length of cell migration was measured with a video
micrometer (VM-30; Olympus).
Purification of Recombinant Laminin-5--
Four transfectants of
HT1080 cells were grown to confluence in roller bottles (Beckton
Dickinson) with DME/F12 medium containing 10% FCS (200 ml/bottle). The
confluent cultures were washed twice with and incubated in serum-free
DME/F12 medium. The serum-free conditioned medium was harvested every 2 days, clarified by centrifugation, and subjected to protein
precipitation with 80%-saturated ammonium sulfate. The precipitated
proteins were dissolved in and dialyzed against 20 mM
Tris-HCl buffer containing 0.5 M NaCl, 0.01% (w/v) Brij35,
and 0.1% (w/v) CHAPS and then applied to molecular sieve chromatography on a Sepharose 4B (Amersham Pharmacia Biotech) (8).
Fractions containing laminin-5 were pooled and subjected to
immunoaffinity chromatography with the anti-laminin-3 monoclonal antibody (LS3c4). Bound proteins were eluted from the affinity column with 0.05% (v/v) trifluoroacetic acid and immediately
neutralized with 1/3 volume of 1 M Tris-HCl (pH 7.5). The
eluted laminin-5 fractions were further purified by immunoaffinity
chromatography with the anti-laminin-2 monoclonal antibody (D4B5).
The recombinant laminin-5 proteins thus purified were stored at 4 °C
in the presence of 0.005% Brij35 and 0.1% CHAPS.
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RESULTS |
Expression of Full-length and Carboxyl-terminally Deleted
Derivatives of Laminin 3 Chain in HT1080 Cells--
To assess the
contribution of the five G domains of the laminin 3 chain to the
cell adhesion and cell motility activities of laminin-5, we tried to
prepare recombinant laminin-5 variants serially lacking G domains.
Human fibrosarcoma cell line HT1080, which expresses the laminin 3
and 2 chains but not the 3 chain (26), was chosen as the
recipient cells to transfect the 3 chain cDNAs.
Expression vectors containing the cDNAs for wild type (WT) and the
following five deletion derivatives of human laminin 3 chain were
constructed: the 3 chains without G5 (G5); without G4
and G5 (G4-5); without G3, G4, and G5
(G3-5); without G2, G3, G4, and G5
(G2-5); and without any G domains (G1-5) (Fig. 1). These
expression vectors and the control vector without any 3 chain
cDNA were transfected into HT1080 cells, and stable transfectant
clones were isolated.
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Fig. 1.
Schematic diagram of cDNAs for
full-length laminin 3EpA
(3) chain and its carboxyl-terminally deleted
derivatives. The full-length cDNA (WT) and
partially deleted cDNAs (G5, G4-5,
G3-5, G2-5, and G1-5)
are shown by closed bars. The scale in base pairs
(bp) is shown on the top. The domain structures
of laminin 3 chain (open column) are indicated by IIIa,
I/II, and G1 to G5. c, conserved cysteine residues in I/II
domain. The shaded portion indicates the signal peptide.
Numbers in parentheses indicate the length of the cDNAs
in base pairs.
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When cell morphology and growth rate were compared among the HT1080
transfectants, the difference is not evident between the control and
cDNA transfectants under usual serum-containing culture conditions.
However, when incubated in serum-free culture medium, HT1080
transfectants of WT, G5, and G4-5
survived much longer than the other cDNA transfectants and the
control transfectant (data not shown). This suggested that the
laminin-5 forms with G1-3 might prevent HT1080 cells from apoptosis in
serum-free medium.
Next, we examined gene expression of the three laminin-5 subunits in
the control and cDNA transfectants of HT1080 cells by Northern
blotting. As expected, all of the five transfectants of the 3 chain
cDNAs expressed the 3 chain mRNA with different sizes at
comparable levels, whereas the control transfectant expressed no
positive signal (Fig. 2A). In
contrast, the laminin 3 and 2 chain mRNAs were expressed in
the control transfectant as well as the cDNA transfectants (Fig. 2,
B and C). The transfection of the laminin 3
chain cDNAs did not affect the transcriptional levels of the 3
and 2 chain genes in any clones.
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Fig. 2.
Expression of three laminin-5 subunits in
control and laminin 3 chain cDNA
transfectants of HT1080 cells. Total cellular RNAs were isolated
from the indicated transfectants of HT1080 cells and subjected to
Northern blotting for laminin 3 chain (LNa3A), 3 chain
(LN3), 2 chain
(LN2), and glyceraldehyde-3-phosphate
dehydrogenase (G3PDH). The RNA from the gastric
adenocarcinoma cell line STKM-1, which secretes a high level of
laminin-5, was included as a positive control. Different sizes of the
3 chain mRNAs are detectable in the cDNA transfectants but
not in the control transfectant (Control), which was
transfected with the control vector without laminin 3 cDNA. The
laminin 3 cDNA transfectants of WT and Gs are shown in Fig.
1. Experimental conditions are described in the text.
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Secretion of Laminin-5 Variants by HT1080 Transfectants--
To
examine the secretion of laminin-5 mutant proteins from HT1080 cells
transfected with different laminin 3 chain cDNAs, the three
laminin-5 subunits in the conditioned media of the transfectants were
analyzed by Western blotting with the specific antibodies (Fig.
3). When probed with the anti-3 chain
antibody, the conditioned media of the cDNA transfectants, but not
the control transfectant, showed immunoreactive bands different in size
(Fig. 3A). HT1080/WT (transfectants of the full-length
laminin 3) secreted a 190-kDa full-length 3 chain and a 160-kDa
3 chain (Fig. 3A, lane 2), whereas
HT1080/G5 secreted 175- and 160-kDa 3 chains (Fig.
3A, lane 3). HT1080/G4-5,
HT1080/G3-5, and HT1080/G2-5 secreted a
single 3 chain of 160, 140, and 120 kDa, respectively (Fig.
3A, lanes 4-6). On the other hand,
HT1080/G1-5, which lacked all G domains, secreted only
a trace amount of 100-kDa 3 chain (Fig. 3A, lane 7). We
could not detect deposition of the 3 chain in the extracellular
matrix or cell lysate of HT1080/G1-5 (data not shown).
As shown in Fig. 2A, this transfectant significantly expressed the 3 chain message. Therefore, it is considered that the
G domains of the 3 chain are required for the assembly and subsequent secretion of the heterotrimer 332.
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Fig. 3.
Western blotting analysis of three laminin-5
subunits secreted from control and cDNA transfectants of HT1080
cells. Concentrated protein samples from 0.5 ml of serum-free
conditioned medium were run on 6% gels under reducing conditions,
transferred onto nitrocellulose membranes, and then probed with
monoclonal antibodies against laminin 3 chain (A), 3
chain (B), and 2 chain (C).
Ordinate, molecular weights in thousands. A,
arrowheads indicate the 3 chains of 190, 175, 160, 140, and 120 kDa. B, an arrowhead indicates laminin 3 chain
of 135 kDa. C, arrowheads indicate the unprocessed laminin
2 chain (150 kDa) and the processed laminin 2 chain (105 kDa).
Other experimental conditions are described in the text.
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As described above, the HT1080 transfectants of WT, G5,
and G4-5 secreted apparently the same size (160 kDa) of
the 3 chain. It is known that the mature 3 chain of 190 kDa is
proteolytically processed to the 160-kDa form (28). When the
conditioned media of the control HT1080, HT1080/WT, and
HT1080/G5 were analyzed by Western blotting with a
rabbit polyclonal antibody against a recombinant G4 protein, the
190-kDa band in HT1080/WT and the 175-kDa band in
HT1080/G5 were clearly detected, whereas the 160-kDa
band in both transfectants was scarcely detected by the antibody (Fig.
4). Therefore, we judged the 160-kDa band
as the 3 chain that had been proteolytically cleaved between the G3 and G4 domains.
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Fig. 4.
Western blotting analysis with two
anti-3 chain antibodies of conditioned media
from control and two cDNA transfectants of HT1080 cells.
Laminin 3 chains secreted by the control (CT), wild type
(WT), and G5 (G5)
transfectants of HT1080 cells were analyzed as described in Fig. 3.
Left panel, a monoclonal antibody (LS3c4) that recognizes
the amino-terminal region of the 3 chain; right panel, a
polyclonal antibody (Ab) that recognizes the G4 domain and
the carboxyl-terminal sequence of 19 amino acid residues of the G3
domain. Arrowheads indicate the 3 chains of 190, 175, and
160 kDa. Note that the 160-kDa band intensely detected with LS3C4
antibody (left panel) is scarcely detected with the anti-G4
antibody (right panel). In the right panel, all
bands visible in CT are nonspecific bands, and it is not clear whether
a faint 160-kDa band in G5 is a nonspecific band or an
immunoreactive band containing the short carboxyl-terminal sequence of
the G3 domain.
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Western blotting analysis with the anti-3 chain antibody detected
almost a single band of the 135-kDa mature 3 chain in the
conditioned media of all transfectants (Fig. 3B). On the
other hand, analysis with the anti-2 chain antibody detected the
mature 2 chain of 150 kDa and its proteolytically processed form of 105 kDa at relatively irregular intensity (Fig. 3C). It is
noteworthy that the control transfectant secreted significant amounts
of the 3 and 2 chains. This indicates that the secretion of the laminin 3 and 2 chains does not depend on the co-expression of
the 3 chain.
To confirm the secretion of laminin-5, immunoprecipitation with the
anti-laminin 2 chain monoclonal antibody was carried out with the
conditioned media of some HT1080 transfectants. The anti-2 chain
antibody precipitated both the laminin 3 and 3 chains, as well as
the laminin 2 chain, from the conditioned media of transfectants of
the laminin 3 cDNAs, indicating the formation of the
heterotrimer of the 3, 3, and 2 chains (data not shown).
Interestingly, the non-processed forms of the laminin 3 chains were
hardly precipitated from the conditioned media of HT1080/WT and
HT1080/G5, as compared with their processed forms (data
not shown).
Cell Adhesion Activity of Matrices Deposited by HT1080
Transfectants--
To examine the cell adhesion activity of the
laminin-5 deletion forms, the matrices deposited by HT1080
transfectants were tested with Buffalo rat liver cell line BRL, which
has been used for the assays of cell scattering activity and cell
adhesion activity of laminin-5 (8, 9). This cell line adheres not only
to laminin-5 but also to fibronectin. When the relative amounts of the
laminin 3 chain proteins deposited by HT1080 transfectants were
determined using the anti-laminin-3 antibody, all of the matrices
except for the control and G1-5 contained the laminin
3 chains at similar levels (data not shown). When the cell adhesion
activity was assayed at 20 min after seeding, BRL cells attached and
spread on the matrices of HT1080/WT, HT1080/G5, and
HT1080/G4-5 but not on the matrices of the other
transfectants (Fig. 5, closed
columns). When assayed at 1 h after seeding, all of the
matrices supported the adhesion of BRL cells at almost the same
activity (Fig. 5, open columns). However, there was a morphological difference of BRL cells between the matrices from the
transfectants of laminin 3 cDNAs with and without the G3 domain.
BRL cells spread more on the matrices of WT, G5, and G4-5 than on those of the others (data not shown). It
has previously been reported that the RGD peptide inhibits adhesion of
BRL cells to fibronectin by about half but does not inhibit that to
laminin-5 at all (9). When BRL cells were treated with a RGD-containing
peptide (GRGDSP) or a control peptide (GRGESP), the cell adhesion to
the G3-5 matrix, but not that to the G4-5 matrix, was effectively inhibited by the GRGDSP
peptide (Fig. 6). This suggests that BRL
cells preferentially attach to and spread on laminin-5 in the matrices
of G4-5, G5, and WT, whereas they slowly
attach to fibronectin or other cell adhesion proteins in the matrices
of the other transfectants including G3-5. This
possibility was confirmed using the adhesion assay with human cervix
epidermoid carcinoma cell line C-4I, which is able to adhere to
laminin-5 but not fibronectin, vitronectin, or
laminin-1.2 When C-4I cells
were seeded on the matrices of HT1080 transfectants, they effectively
attached to and spread on only the matrices of WT, G5,
and G4-5 even at 1.5 h after seeding (Fig.
7). When effect of function-blocking
anti-integrin antibodies was examined, antibodies to integrins
3 and 1 strongly inhibited the cell
adhesion to the G4-5 matrix and to purified laminin-5, indicating that C-4I cells had adhered to the laminin-5 deposited on
the matrix through integrin 31
(Table I). The
anti-6-integrin antibody weakly inhibited both cell
adhesion to the G4-5 matrix and to the purified
laminin-5, but antibodies to integrins 2 and
5 rather stimulated the cell adhesion. All these results strongly suggested that the G3 domain of the laminin 3 chain is
essential for the potent cell adhesion activity of laminin-5.
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Fig. 5.
Adhesion of BRL cells to extracellular
matrices deposited by control and cDNA transfectants of HT1080
cells. Extracellular matrices were prepared from the confluent
culture of each transfectant of HT1080 cells. BRL cells were plated and
incubated on the matrices in serum-free medium for 20 min (open
columns) and 60 min (closed columns), and the relative
numbers of adherent cells were determined by measuring fluorescent
intensity. Each point represents the mean ± S.D. for
triplicate cultures. Other experimental conditions are described in the
text.
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Fig. 6.
Effect of GRGDSP peptide on adhesion of BRL
cells to extracellular matrices of
HT1080/G4-5 and
HT1080/G3-5. BRL cells were
preincubated with 10 µg/ml the GRGDSP peptide (shaded
columns) or the control GRGESP peptide (open columns)
in a plastic tube for 5 min, and then the cell suspension was
transferred onto the matrices of G4-5 and
G3-5. After incubation for 60 min, the relative numbers
of adherent cells were determined by measuring fluorescent intensity.
The averaged fluorescent intensity of the control cultures (GRGESP) was
taken as 100%. Each point represents the mean ± S.D. for
triplicate cultures. Other experimental conditions are the same as in
Fig. 5. GRGDSP and GRGESP peptides were purchased from Iwaki Glass,
Tokyo.
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Fig. 7.
Adhesion of C-4I cells to extracellular
matrices deposited by control and cDNA transfectants of HT1080
cells. BRL cells were plated and incubated on the matrices of
HT1080 transfectants in serum-free medium for 90 min, and the relative
numbers of adherent cells were determined by measuring fluorescent
intensity. Each point represents the mean ± S.D. for
triplicate cultures. Other experimental conditions are described in the
text.
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Table I
Effect of function-blocking antibodies specific to various integrin
subunits on adhesion of C-4I cells to matrix of
HT1080/G4-5 and to laminin-5
C-4I cells were incubated with the indicated anti-integrin antibodies
(1:100 dilution), mouse IgG (15 µg/ml), or PBS at 37 °C for 30 min, and then they were seeded on plates precoated with the
G4-5 matrix or with 0.3 µg/ml of laminin-5, which had
been purified from STKM-1 cells (26). The numbers of adherent cells
were measured after a 1.5-h incubation. The averaged value of the PBS
controls was taken as 100%. Each value represents the mean ± S.D. for triplicate assays.
|
|
Cell Migration Activity of Matrices Deposited by HT1080
Transfectants--
Laminin-5 has potent cell scattering- and cell
migration-stimulating activities toward BRL cells (8, 9). The cell
scattering activity of the matrices deposited by each HT1080
transfectant was analyzed using BRL cells. BRL cells showed marked cell
scattering on the matrices of WT, G5, and
G4-5 but not on the others (data not shown).
To compare the cell migration activity of laminin-5 deletion forms, BRL
cells were incubated on the matrix deposited by each HT1080
transfectant. The cell migration on the matrix was monitored for
12 h using a time-lapse video recorder (Fig.
8). The migration speed was about 5-10
times higher on the matrix of WT than on the control matrix, suggesting
that laminin-5 was responsible for the high motility of BRL cells.
Similar elevated cell motility was observed on the matrices of
G5 and G4-5, whereas this activity was
remarkably decreased by losing the G3 domain. The matrix of
G3-5 slightly stimulated the migration of BRL cells as
compared with those of the control transfectants, G2-5 and G1-5. These results indicate that the G3 domain of the laminin 3 chain is indispensable for the strong cell motility activity of laminin-5 and that the G2 domain also has a low cell motility activity.
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Fig. 8.
Migration of BRL cells on extracellular
matrices deposited by control and cDNA transfectants of HT1080
cells. Extracellular matrices were prepared from the confluent
cultures of the indicated transfectants of HT1080 cells on culture
flasks, and BRL cells were plated and incubated on the culture flasks
in serum-free medium The migration of BRL cells was monitored by video
microscopy and then quantitated for the total migration of each cell
over a 12-h period. Each point represents the mean ± S.D.
for 10 cells. Other experimental conditions are described in the
text.
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|
Purification of Recombinant Laminin-5 Deletion Forms and Their
Subunit Composition--
HT1080 cells are expected to secrete some
cell adhesion proteins such as fibronectin and collagens. To rule out
the effects of these intrinsic matrix proteins, we purified recombinant
laminin-5 variants and investigated their biological activities. Four
types of laminin-5, WT, G5, G3-5, and
G2-5, were prepared from the conditioned media of the
respective cDNA transfectants. Each recombinant laminin-5 was
separated by molecular sieve chromatography followed by immunoaffinity
chromatography using the anti-laminin-3 monoclonal antibody
LS3c4. The laminin-5 preparations slightly contained the laminin
1 chain (220 kDa) and the laminin 1 chain (210 kDa), besides the
laminin-5 subunits, suggesting that they contained laminin-6.
Therefore, these preparations were finally applied to an
anti-laminin-2 antibody (D4B5) column to remove laminin-6. The total
amount of laminin-5 and laminin-6 in conditioned medium was estimated
to be about 100 µg/liter in WT, 60-80 µg/liter in
G5 and G3-5, and less than 50 µg/liter
in G2-5. The recovery of laminin-5 ranged between 25 and 50% in the final laminin-5 preparations. The purified materials
contained the laminin 3 chains (160-120 kDa), the 3 chain (135 kDa), the 2 chain (150 kDa), and the proteolytically processed
laminin 2 chain (105 kDa) (Fig. 9,
left). The approximate size of the laminin 3 chain was
160 kDa in WT and G5, 140 kDa in G3-5,
and 120 kDa in G2-5 (Fig. 9, right). The
laminin-5 forms of WT and G5 were considered to be
essentially identical to the laminin-5 form of G4-5
(see Figs. 3A and 4). When the laminin-5 of WT was analyzed
by Western blotting with the anti-G4 antibody, no immunoreactive band
was detected at a low molecular weight region, indicating that the
cleaved fragment of G4-5 was not associated with the purified
laminin-5 (data not shown).
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Fig. 9.
SDS-PAGE analysis of four recombinant forms
of laminin-5. Recombinant laminin-5 proteins were purified from
the HT1080 transfectants of WT, G5,
G3-5, and G2-5. Left panel,
1 µg of each sample was run on a 6% SDS-PAGE gel after disulfide
bond reduction and visualized by Coomassie Brilliant Blue staining.
Right panel, 100 ng of each sample was separated by SDS-PAGE
under the same conditions and immunoblotted with the laminin 3
chain-specific monoclonal antibody. Right panel, arrowheads
indicate the 3 chains of 160 kDa in WT and G5, 140 kDa in G3-5, and 120 kDa in G2-5.
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|
The fractions eluted from the anti-laminin-3 antibody column
slightly contained the non-processed 3 chains (190 kDa in WT and 175 kDa in G5) and the laminin 1 and 1 chains, but
these proteins passed through the anti-laminin-2 antibody column.
This suggested that the non-processed 3 chains belonged to
laminin-6.
Biological Activity of Purified Recombinant Laminin-5 Deletion
Forms--
The purified recombinant laminin-5 deletion forms were
assayed for cell adhesion activity using BRL cells as the indicator. When each recombinant laminin-5 preparation was precoated on plastic plates at different concentrations, WT and G5 promoted
the cell adhesion in a concentration-dependent manner, but
neither G3-5 nor G2-5 supported the
cell adhesion even at the maximum concentration tested (Fig.
10). This confirmed that the G3 domain is essential for the potent cell adhesion activity of laminin-5. As
expected from the above SDS-PAGE analysis, WT and G5
showed essentially the same activity.
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Fig. 10.
Effects of various concentrations of four
recombinant forms of laminin-5 on adhesion of BRL cells. 96-Well
plates were coated with the indicated concentrations of each laminin-5
protein: WT ( ), G5 ( ), G3-5 ( ),
or G2-5 ( ). BRL cells were plated on the plates in
serum-free medium and incubated at 37 °C for 1 h. After the
incubation, relative numbers of cells attached to the plastic surface
were determined by measuring fluorescent intensity. Each point
represents the mean ± S.D. for four wells. Other experimental
conditions are described in the text. The decrease of cell adhesion at
the highest concentration of WT and G5 seems due to the
effect of the increased concentration of detergents included in the
sample solutions.
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|
The purified recombinant laminin-5 forms were also assayed for
cell-scattering activity using BRL cells (Fig.
11). When BRL cells were incubated with
each recombinant protein in 1% FCS-containing medium for 2 days,
typical cell scattering was observed with WT and G5.
G3-5 induced weak cell scattering of BRL cells as
compared with the negative control, but G2-5 did not at
all. These results were very consistent with the previous experiments with matrices deposited by HT1080 transfectants (Fig. 8). It is clear
that the G3 domain of the laminin 3 chain is indispensable for the
potent cell motility activity of laminin-5, as well as its cell
adhesion activity.
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Fig. 11.
Cell scattering of BRL cells on recombinant
laminin-5 proteins. Five hundred microliters of BRL cell
suspension (1.4 × 104 cells/ml in DME/F12 medium
containing 1% FCS) was inoculated into each well of 24-well plates,
and a purified recombinant laminin-5 form of WT, G5,
G3-5, or G2-5 was added to a final
concentration of 0.3 µg/ml and incubated at 37 °C for 2 days. The
cell morphology was examined under a phase-contrast microscope.
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|
| |
DISCUSSION |
The present study demonstrated that the laminin-5 lacking both the
G4 and G5 domains in the 3 chain had potent activity to promote
adhesion and migration of BRL cells, but deletion of the G3 domain
caused marked decrease of the biological activity of laminin-5. This
implies that the G3 domain plays an indispensable role in the
expression of biological activity of laminin-5. HT1080 cells expressing
laminin-5 forms with the G3 domain were more resistant to apoptosis in
serum-free culture medium than those without the G3 domain. This is
consistent with the previous report that laminin-5-deficient
keratinocytes exhibit reduced survival as compared with normal cells
(14).
Laminin-1 has been reported to have several integrin-binding sites in
the G domains of the laminin 1 chain. Two or more integrin recognition sequences, which are adjacently located in distinct G
domains, are likely to cooperate in ligand binding (20, 29). However,
our previous study with four recombinant G domains of the laminin 3
chain showed that only the G2 domain contains an integrin
31 binding activity, although the
activity of the G3 domain was not examined (24). The recombinant G2
protein and the integrin 31-binding
peptide (3G2A) have a very low cell adhesion activity compared with
intact laminin-5 (24). In addition, the G2 domain and intact laminin-5
lose their cell adhesion activity by heating (8, 24). These imply that
a specific conformation of the G2 domain produced by the interaction
with the G1, G3, and some other parts of the 3, 3, and 2
chains might be important for the high affinity binding of laminin-5 to
integrins. Correspondingly, Talts et al. (19, 23) have
recently shown that recombinant G1-3 protein of laminin 2 chain has
cell adhesion activity similar to that of native laminin-2/4, although
each of G1, G2, and G3 recombinant proteins does not. However, any
natural or recombinant laminin-2/4 forms without G4 and G5 domains has
not been reported.
Laminin-5 is synthesized initially as a high molecular weight precursor
protein that undergoes specific processing to smaller forms after being
secreted (28, 30, 31). The size reduction is a result of proteolytic
processing of the 3 and 2 subunits from 190 to 160 and from 150 to 105 kDa, respectively (8, 28, 31). In this study, we first
demonstrated that the laminin 3 chain was proteolytically cleaved
between the G3 and G4 domains in HT1080/WT cell line, producing the
160-kDa 3 chain. Both the naturally processed laminin-5 with the
160-kDa 3 chain and the recombinant laminin-5 lacking G4 and G5
(G4-5) showed high cell adhesion and cell motility
activities. This clearly indicates that the G4 and G5 domains are not
essential for the stimulation of cell adhesion and motility. We have
recently found that the G4-G5 fragment of laminin 3 chain is
secreted from some human carcinoma cell
lines.3 The G4-G5 fragment
appeared not to be associated with laminin-5 after the proteolytic
cleavage. It has been reported that recombinant G4 and G5 domains bind
to heparan sulfate proteoglycans as major cell surface receptors, and
the G5 recombinant protein has some activity to stimulate cell
migration (24). Therefore, it is conceivable that the secreted G4-G5
fragment acts on cells in cooperation with or independently of the
laminin-5 with the 160-kDa 3 chain.
Goldfinger et al. (32) have compared biological activities
of extracellular matrices containing different forms of laminin-5 and
found that the laminin-5 with the unprocessed, 190-kDa 3 chain has
high cell motility activity, whereas one with the processed, 160-kDa
3 chain supports stable cell adhesion. However, we have observed the
high motility activity of the laminin-5 with the 160-kDa 3 chain
toward BRL cells and many human carcinoma cell lines (8). It is evident
that the laminin-5 with the 160-kDa 3 chain exhibits potent cell
motility activity toward some cell types. It has also been reported
that the processing of the laminin 2 chain by the matrix
metalloproteinases gelatinase A and MT1-MMP regulate the cell motility
activity of laminin-5 (33, 34). For clarifying the structure-function
relationship of laminin-5, it seems essential to isolate the laminin-5
forms with processed and unprocessed 3 and 2 chains. In this
respect, it should be noted that HT1080/WT cells secreted both the 160- and 190-kDa 3 chains, but only the 160-kDa 3 chain was purified
as a laminin-5 complex. Our recent attempt to isolate the 190-kDa 3
chain has shown that this 3 chain exists as a laminin-6 but not
laminin-5 form.4 This
suggests that the proteolytic processing of the laminin 3 chain
occurs specifically in laminin-5.
The present study showed that the control HT1080 cells, which do not
express the 3 chain, secreted the laminin 3 and 2 chains into
culture medium. Various laminin subunits are assembled in the rough
endoplasmic reticulum. Several groups have proposed the mechanism for
the assembly of laminin subunits. In laminin-1 and laminin-5, a
disulfide-linked heterodimer is formed as a presumed
intermediate, and chain is added at a subsequent stage (35-37). In
laminin-1, the 1 chain can be secreted as a single subunit, whereas
the 1 and 1 chains cannot (37). When the 1 and 1 chains are
overexpressed separately or together, they remain intracellular as the
disulfide-linked dimer of 11 or 11. We recently found that
the laminin 2 chain is solely overexpressed at the invasion front of
gastric carcinomas, and the 2 chain monomer is secreted from gastric
carcinoma cells in vitro (25). These results clearly
indicate that the laminin-5 subunits are secreted differently from the
laminin-1 subunits. Furthermore, the present study indicated that
HT1080/G1-5, which lacked all G domains, secreted only
a trace amount of the laminin 3 chain into culture medium and
contained little 3 protein in the cytoplasm despite the high
expression of its mRNA. Therefore, the G domains appear to be
essential for the formation of stable heterotrimer of laminin-5. The
failure of subunit assembly may cause the prompt degradation of the
3 chain inside the cells.
In conclusion, we first demonstrated that the G3 domain, but not G4 and
G5 domains, of the laminin 3 chain is required for the potent
activity of laminin-5 to promote cell adhesion and migration. The
physiological meaning of the proteolytic cleavage and the biological
activity of the G4-G5 fragment are currently under investigation.
| |
ACKNOWLEDGEMENTS |
We thank to Drs. H. Yasumitsu and S. Higashi
for helpful discussion.
| |
FOOTNOTES |
*
This work was supported by a Grant-in-aid from the Ministry
of Education, Science, Sports and Culture 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.
¶
Both authors contributed equally to this work.
To whom correspondence should be addressed: Division of Cell
Biology, Kihara Institute for Biological Research, Yokohama City University, 642-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813. Tel.:
81-45-820-1905; Fax: 81-45-820-1901; E-mail:
miyazaki@yokohama-cu.ac.jp.
Published, JBC Papers in Press, May 2, 2000, DOI 10.1074.jbc.M001326200
2
Y. Kikkawa and K. Miyazaki, unpublished data.
3
Y. Tsubota, H. Mizushima, T. Hirosaki, S. Higashi, H. Yasumitsu, and K. Miyazaki, unpublished data.
4
T. Hirosaki, H. Mizushima, K. Moriyama, and K. Miyazaki, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
DME/F12, Dulbecco's
modified Eagle's medium/Ham's F12 medium;
FCS, fetal calf serum;
G
domains, carboxyl-terminal globular domains of laminin 3 chain;
PCR, polymerase chain reaction;
Gx, laminin 3 chain lacking
Gx domain;
WT, wild type of laminin 3 chain;
PAGE, polyacrylamide gel electrophoresis;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
| |
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TOP
ABSTRACT
INTRODUCTION
