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INTRODUCTION |
Laminins are the major class of basement membrane proteins,
composed of three disulfide-linked subunits,
,
, and
. To
date, five
, three
, and three
chains have been identified,
combinations of which have been shown to give rise to at least 12 different laminin isoforms (1-4). These laminin isoforms are expressed in a tissue-specific and developmentally regulated manner, suggesting that they are functionally distinct (5, 6). The differences in
biological activity among these isoforms, however, have yet to be defined.
Laminins have many biological functions, including promotion of cell
adhesion and migration, control of cell proliferation and gene
expression, maintenance of differentiation phenotypes, and stimulation
of neurite outgrowth. Laminins mediate these functions through binding
to cell surface receptors, particularly the integrin family of cell
adhesion molecules. So far, nine integrin types, including
1
1,
2
1,
3
1,
6
1,
6
4, and
7
1,
have been shown to bind to laminins with distinct specificities (6, 7). For example, cell adhesion to laminin-1, the "classical laminin" purified from mouse Engelbreth-Holm-Swarm tumor, is mainly
mediated by
6
1 integrin, while adhesion
to laminin-5 occurs through
3
1 and
6
4 integrins (8-12). Laminin-2/4 is
recognized by
3
1,
6
1, and
7
1
integrins, depending on the cell type (13, 14). We purified
laminin-10/11 from conditioned medium of lung carcinoma cells and
showed that it interacts with
3
1,
6
1, and
6
4
integrins (15, 16). Recently, the
4 chain-containing laminin
isoform, laminin-8, was reported to bind to
6
1 integrin (17, 18).
The
4 chain is a truncated version of the laminin
chains like
3A (19, 20). The
4 chain is predominantly expressed in
capillaries in brain, muscle, and bone marrow (19, 21-24). In the
kidney, the
4 chain is present in nascent epithelial basement membrane of the renal vesicle and immature glomerular basement membrane, but is absent in the adult kidney (23, 25). The
4 chain is
also localized to muscle basement membrane during muscle formation, but
is absent in adult muscles, except at neuromuscular junctions (26, 27).
Similarly, laminin-8 is the major laminin isoform in developing bone
marrow (24). Despite its restricted expression and localization
in vivo, the biological activity of the
4
chain-containing laminins has not been explored thoroughly due to the
unavailability of sufficient purified laminin-8 for biochemical and
functional analysis. In this study, we purified laminin-8 from
conditioned medium of human glioma cells and characterized its
biological activities, including cell adhesive and cell migration promoting properties and integrin binding specificity in comparison with other laminin isoforms and fibronectin.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human glioblastoma cell line T98G, human lung
adenocarcinoma cell line A549, human gastric cancer cell line AZ-521,
human hepatoma cell line HLF, and human gastric carcinoma cell line MKN-45 were obtained from the Japanese Cancer Research Resources Bank
(Tokyo, Japan). Human lung squamous carcinoma cell line RERF-LC-AI was
purchased from RIKEN Gene Bank (Tsukuba, Japan). These cells were
maintained in DMEM1
containing 10% FBS. K562 human erythroleukemic cells transfected with
cDNAs encoding human integrin
3A or
6A subunits were kindly provided by Dr. Arnoud
Sonnenberg (The Netherlands Cancer Institute) and maintained in RPMI
1640 containing 10% FBS and 1 mg/ml Geneticin (13).
Antibodies--
The mAb 8B12 against the human laminin
4
chain was produced by fusion of Sp2/O mouse myeloma cells with spleen
cells from mice immunized with a glutathione S-transferase
fusion protein containing the G1 domain of the laminin
4 chain
(Ser835-Cys1028 (20)). The mAb 8B12 was capable
of binding to denatured laminin
4 chain on immunoblots, but not to
intact
4 chain-containing laminin assembled with
1 and
1
chains in an enzyme-linked immunosorbent assay, and was therefore
unsuitable for immunoaffinity purification of intact laminins. The mAb
4F5 specific for the human laminin
1 chain was produced similarly to
8B12, except that spleen cells were obtained from mice immunized with
human placental laminin (Chemicon, Temecula, CA). The specificity of
4F5 was determined by isolation and protein sequencing of peptides from
a thermolysin digest of human placenta that selectively bound to
4F5-conjugated Sepharose 4B. The N-terminal amino acid sequence of the
4F5-binding 60-kDa fragment was ARQXDRXLPGHWGFP,
identical to the sequence of the human laminin
1 chain
(Ala848-Pro862 (28)). The mAb 5D6 against the
human laminin
5 chain, was produced by immunizing mice with
commercially available human laminin-10/11 (Chemicon) purified from a
pepsin digest of human placenta by immunoaffinity chromatography using
the anti-
5 chain mAb 4C7 (29, 30). The mAb 5D6 was selected by
positive reactivity with purified human laminin-10/11 (15) and negative
reactivity with commercially available human laminin-2/4 (Chemicon).
The mAb 2B10 against the laminin
3 chain was produced by immunizing mice with human laminin-5 purified from conditioned medium of MKN-45
cells (31). 2B10 was selected by reactivity with purified laminin-5 in
an enzyme-linked immunosorbent assay, followed by specific binding to
the laminin
3 chain as shown on immunoblots. mAbs against the human
laminin
1 (5A3) and
5 (15H5) chains were produced in our
laboratory as described previously (15). A mAb against the laminin
1
chain (DG10) was kindly provided from Dr. Ismo Virtanen (University of
Helsinki, Finland). A hybridoma secreting mAb against the laminin
2
chain (C4), developed by Dr. Joshua Sanes (Washington University School
of Medicine), was obtained from the Developmental Studies Hybridoma
Bank (University of Iowa). MAbs against the human laminin
2 chain
(5H2) and human integrin
2 (P1E6) were purchased from Chemicon. MAbs
against the human laminin
1 chain (2E8 and number 22) were obtained
from Chemicon and Transduction Laboratories (Lexington, KY),
respectively. A mAb against the human integrin
6 (GoH3)
was purchased from Immunotech (Westbrook, ME). MAbs against the human
integrin
3 (3G8),
5 (8F1), and
1 (4G2) chains were produced in
our laboratory as described previously (16, 32). A mAb against integrin
1 (8A2) which activates
1 chain-containing integrins, was a gift
from Dr. Nicholas Kovach (University of Washington, Seattle, WA).
Adhesive Proteins--
Mouse laminin-1 was purified from mouse
Engelbreth-Holm-Swarm tumor tissues by the method of Paulsson
et al. (33). Human laminin-5 was purified from conditioned
medium of MKN45 cells by immunoaffinity chromatography using polyclonal
antibodies against the human laminin
2 chain (31). Human
laminin-10/11 was purified from conditioned medium of A549 cells
according to Kikkawa et al. (15), except that mAb 5D6,
instead of 4C7, was used to conjugate Sepharose 4B in the
immunoaffinity matrix. Human laminin-1 was isolated from conditioned
medium of RERF-LC-AI cells on a 4F5 immunoaffinity column and used only
for immunoblot analysis. Human laminin-2/4 (also referred to as
merosin) was purchased from Chemicon. Human plasma fibronectin was
purified from outdated plasma by gelatin affinity chromatography
(34).
Screening of Cultured Cells for Expression of Laminin
Chains
by RT-PCR--
Total RNA was extracted from 38 human cell lines (13 lung carcinomas, 5 gastric carcinomas, 3 gliomas, 2 kidney carcinomas, 2 cervix carcinomas, 2 tropoblastomas, 2 endothelial cell lines, and
one each of oral carcinoma, salivary grand carcinoma, rhabdomyosarcoma, leukemia, hepatoma, pancreatic carcinoma, epidermoid carcinoma, fibrosarcoma, and lung fibroblast) by the acid guanidinium
isothiocyanate method (35) and used as templates for cDNA
synthesis. cDNAs encoding each of five distinct laminin
chains
were amplified by PCR using the following pairs of primers;
5'-AAGTGTGAAGAATGTGAGGATGGG-3' (forward primer for
1; nucleotides
3020-3043 (36)) and 5'-CACTGAGGACCAAAGACATTTTCCT-3' (reverse primer
for
1; nucleotides 3312-3336); 5'-AAATGTACAGAGTGCAGTCGAGGTCA-3' (forward primer for
2; nucleotides 3314-3339 (37)) and
5'-CAGTGGATGCCTTCCACATTCACCTT-3' (reverse primer for
2; nucleotides
3458-3483); 5'-CACTGTGAACGCTGCCAGGAGGGCTA-3' (forward primer for
3;
nucleotides 280-305 (38)) and 5'-CAGCTACCTCCGAATTTCTGGGGATT-3' (reverse primer for
3; nucleotides 466-491);
5'-CACTGTGAAAAGTGTCTGGATGGT-3' (forward primer for
4; nucleotides
608-631 (20)) and 5'-CAGGTGCTTCCAATGAGGAAGGGG-3' (reverse primer for
4; nucleotides 811-834); 5'-GACTGCCTGCTGTGCCAGC-3' (forward primer
for
5 (15)) and 5'-GGGGTAGCCATGAAAGCCCG-3' (reverse primer for
5); 5'-AACTGTGAGCAGTGCAAGCCGTTT-3' (forward primer for
1;
nucleotides 1054-1077 (39)) and 5'-CAACCAAATGGATCTTCACTGCTT-3' (reverse primer for
1; nucleotides 1278-1301);
5'-CACTGTGAGCTCTGTCGGCCCTTC-3' (forward primer for
2; nucleotides
1153-1176 (40)) and 5'-CAAGGAGTGCTCCCAGGCACTGTG-3' (reverse primer for
2; nucleotides 1427-1451); and 5'-CACTGTGAGAGGTGCCGAGAGAAC-3' (forward primer for
1; nucleotides 1033-1056 (41)) and
5'-CATCCTGCTTCAGTGAGAGAATGG-3' (reverse primer for
1; nucleotides
1203-1226). PCR products were analyzed by electrophoresis using 2%
agarose gels.
Purification of Laminin-8--
T98G cells were grown in
1,700-cm2 roller bottles with DMEM containing 10% FBS.
After the cells reached confluence, the medium was replaced with DMEM
containing 5% FBS and harvested every 6 days. The conditioned medium
(about 4 liters) was clarified by centrifugation and then precipitated
with 45% saturated ammonium sulfate. The precipitates were dissolved
in 10 ml of 25 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, clarified by centrifugation, and subjected to gel
filtration on a Sepharose CL-4B column (2.5 × 120 cm)
equilibrated in PBS containing 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM N-ethylmaleimide, and 1 mM EDTA. The Sepharose CL-4B column was calibrated prior to
use with mouse laminin-1 (~800 kDa), human plasma fibronectin (~450
kDa), and mouse IgG (150 kDa). Fractions containing laminin-8 were
detected by immunoblotting with anti-
4 mAb 8B12. These fractions
were pooled and then applied to a 4F5-conjugated Sepharose 4B column
equilibrated in PBS containing 0.5 mM phenylmethylsulfonyl
fluoride, 0.5 mM N-ethylmaleimide, 5 mM EDTA, 0.5 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. The bound proteins were eluted with 0.1 M glycine-HCl (pH 2.4), immediately neutralized with 1.5 M Tris-HCl (pH 9.0), and dialyzed against PBS containing 1 mM EDTA.
Cell Adhesion Assay--
Cell adhesion assays were performed as
described previously (15) with minor modifications. Ninety-six-well
microtiter plates (Nunc, Wiesbaden, Germany) were coated with 50 µl
of increasing concentrations of cell adhesive proteins overnight at
4 °C, and then blocked with PBS containing 2% BSA at 37 °C for
1 h. 100-µl aliquots of cell suspension (3 × 105 cells/ml in serum-free DMEM containing 10 mM HEPES (pH 7.4) and 0.5% BSA) were added to each well of
the coated plates and incubated at 37 °C for 30 min in a
CO2 incubator. After the plates were washed to remove
unattached cells, attached cells were fixed with 3.7% formaldehyde,
stained with Diff-Quik (International Reagents Corp., Kobe, Japan), and
counted in three independent fields/well. For cell adhesion assays
using K562 cells, cell suspensions (4 × 105 cells/ml
in serum-free RPMI 1640 containing 10 mM HEPES (pH 7.4) and
0.5% BSA) were preincubated with or without 8A2, an integrin
1-activating mAb, for 10 min at room temperature. 50-µl aliquots of preincubated cell suspension were added to 96-well microtiter plates
coated with increasing concentrations of various laminin isoforms and
incubated at 37 °C for 30 min in a CO2 incubator. Attached cells were counted as described above.
Cell adhesion inhibition assays were performed based on the cell
adhesion assays. Cell suspensions (4 × 105 cells/ml
in serum-free DMEM containing 10 mM HEPES (pH 7.4) and 0.5% BSA) were incubated with 20 µg/ml mAbs against different integrin isoforms for 20 min at room temperature. 50-µl aliquots of
preincubated cells were added to wells that had been coated with
different cell adhesive proteins and incubated for 30 min at 37 °C
in a CO2 incubator. Attached cells were then counted as
described above.
Cell Migration Assay--
Cell migration on substrates coated
with laminins or fibronectin was examined by time lapse video
microscopy using the image processing software Image-Pro Plus (Media
Cybernetics, Silver Spring, MD). Glass-bottom culture dishes fitted
with
8-mm coverslips were coated with 250 µl of cell adhesive
proteins overnight at 4 °C, and blocked with PBS containing 2% BSA
at 37 °C for 1 h. 4-ml of cell suspension (1 × 104 cells/ml in DMEM containing 1% FBS) was added to each
coated dish and incubated at 37 °C for 30 min to allow the cells to
attach. The dishes were then placed in a built-in CO2
incubator on the stage of the Zeiss Axiovert 25 microscope, and
subjected to time lapse video microscopy at 10-min intervals for 8 h. Cell migration was quantified by tracing the position of the nucleus
of migration cells using Image-Pro Plus.
For cell migration inhibition assays, cell suspensions (2 × 104 cells/ml in DMEM containing 1% FBS) were incubated
with 20 µg/ml mAbs against different integrin isoforms for 20 min at
room temperature. 2-ml of preincubated cell suspension was added to the
precoated glass-bottom culture dishes and incubated at 37 °C for 30 min to allow cells to attach. Cell migration images were taken as described for cell migration assays for 8 h.
SDS-PAGE and Immunoblotting--
SDS-PAGE was carried out
according to Laemmli (42) using 4 or 5.5% acrylamide gels. Separated
proteins were visualized by silver staining or transferred onto PVDF
membranes. The membranes were probed with mAbs against individual
laminin chains, followed by visualization using the ECL detection kit
(Amersham Pharmacia Biotech).
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RESULTS |
Screening of Human Cell Lines for Selective Expression of the
Laminin
4 Chain--
To purify laminin isoforms containing the
4
chain from conditioned medium of cultured cells, we screened 38 human
cell lines for selective expression of the laminin
4 chain by
RT-PCR. We found that one of the glioma cell lines, T98G, expresses
only one of the five known laminin
chains, the
4 chain (Fig.
1A). Failure to detect RNA
transcripts for other
chains was not due to inappropriate PCR
conditions, since these transcripts were clearly detected in other cell
lines, including the
1 chain in RERF-LC-AI,
2 and
3 chains in
HLF, and
5 chain in AZ521. T98G cells also expressed
1 and
1
chains, but only a low level of the
2 chain (Fig. 1B),
indicating that laminin-8 (
4
1
1) is the major laminin
isoform expressed in T98G cells.

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Fig. 1.
Expression of laminin-8 in human tumor cell
lines. A, transcripts for 1, 2, 3, 4, and
5 chains of human laminin were amplified by RT-PCR from T98G human
glioblastoma cells, RERF-LC-AI human lung carcinoma cells, HLF human
hepatoma cells, and AZ521 human gastric carcinoma cells, using the
primers described under "Experimental Procedures." The predicted
size of the amplified cDNAs were 317 ( 1), 168 ( 2), 212 ( 3), 227 ( 4), and 197 ( 5) base pairs. B,
transcripts for 1, 2, and 1 chains of human laminin were
amplified by RT-PCR from T98G cells, using the primers described under
"Experimental Procedures." The predicted size of the amplified
cDNAs were 248 ( 1), 299 ( 2), and 194 ( 1) base pairs.
C, the 4 chain-containing laminins in conditioned medium
of T98G cells were detected by immunoblot analysis. Conditioned medium
of T98G cells was separated by SDS-PAGE on 4% gels under nonreducing
conditions or on 5.5% gels under reducing conditions. The separated
proteins were transferred onto PVDF membranes followed by
immunostaining with 8B12, a mAb against the laminin 4 chain. Shown
in the margin are the positions of molecular weight markers.
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The expression of laminin-8 in T98G cells was confirmed at the protein
level by immunoblot analysis of conditioned medium of T98G cells using
mAb 8B12 produced by immunizing mice with a recombinant G1 domain of
the human
4 chain. The mAb 8B12 specifically detected a ~650-kDa
band under nonreducing conditions, while reducing conditions resulted
in a major 200-kDa band and a faint 180-kDa band (Fig. 1C).
The 200-kDa band is consistent with the molecular mass of the
laminin
4 chain as predicted by its amino acid sequence (19, 20),
confirming that T98G cells synthesize and secrete the
4 chain as a
disulfide-linked heterotrimer with
1 and
1 chains.
Purification of Laminin-8--
Laminin-8 secreted by T98G cells
was purified from conditioned medium by ammonium sulfate precipitation,
gel filtration, and immunoaffinity chromatography using mAb 4F5
specific for the laminin
1 chain. Conditioned medium of T98G cells
was precipitated with 45% saturated ammonium sulfate, and the
precipitates were subjected to gel filtration on a Sepharose CL-4B
column (Fig. 2). Fractions containing
trimeric laminin-8 were detected by immunoblotting with 8B12, an
anti-
4 chain mAb, pooled, and subjected to immunoaffinity chromatography using 4F5. The eluate from the 4F5-Sepharose column was
found by silver staining to contain a single 650-kDa species under
nonreducing conditions (Fig.
3A). The ~650-kDa band was stained with mAbs specific for the
4,
1, and
1 chains,
confirming that the purified protein was laminin-8. Under reducing
conditions, three bands of 230, 220, and 200 kDa were resolved, each
identified as the
1,
1, and
4 chains, respectively, by
immunoblot analysis (Fig. 3B). A faint 180-kDa band was also
detectable by immunoblotting with anti-
4 mAb. Since the 180-kDa band
was also detectable in fresh conditioned medium (Fig. 1C),
it could be a proteolytically processed
4 chain or an alternatively
spliced variant. Since no bands near 150 kDa were detected by silver
staining under reducing or nonreducing conditions, the purified
laminin-8 seemed to be devoid of nidogen-1.

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Fig. 2.
Size fractionation of conditioned medium of
T98G cells by gel filtration. Conditioned medium of T98G cells
(about 4 liters) was precipitated with 45% saturated ammonium sulfate,
and the precipitates were subjected to gel filtration on a Sepharose
CL-4B column (2.5 × 120 cm). The column was calibrated with mouse
laminin-1 (~800 kDa), human plasma fibronectin (450 kDa), and mouse
IgG (150 kDa) prior to use. Arrows indicate the positions
where laminin-1, fibronectin, and IgG eluted. Fractions containing
laminin-8 were detected by immunoblotting with anti- 4 chain mAb 8B12
under nonreducing conditions. Fractions 37-53 were pooled and
subsequently subjected to immunoaffinity chromatography.
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Fig. 3.
SDS-PAGE and immunoblot analysis of purified
laminin-8. Laminin-8 purified from conditioned medium of T98G
cells was analyzed by SDS-PAGE on 4% gels under nonreducing conditions
(A) or on 5.5% gels under reducing conditions
(B). Separated proteins were visualized by silver staining
or transferred onto PVDF membranes followed by staining with mAbs
against laminin 4 (8B12), 1 (DG10), or 1 (2E8 under
nonreducing conditions and mAb number 22 under reducing conditions)
chains. The positions of molecular size markers are shown in the
left margin.
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To confirm the absence of other laminin isoforms in the purified
laminin-8, we examined the reactivity of the purified laminin-8 with
mAbs specific for each laminin chain by immunoblotting. Purified laminin-8 was strongly stained by a mAb against the
4 chain, but not
by mAbs against the
1,
2,
3,
5, or
2 chains (Fig. 4A), demonstrating the absence
of other
chain-containing laminin isoforms and the
2
chain-containing laminin-9 (
4
2
1). When a mAb against the
1
chain was used as a probe against purified laminin-8, only the 650-kDa
band was labeled, while 700-800-kDa bands were detected in purified
laminin-1, laminin-2/4, and laminin-10/11 samples (Fig. 4B).
The absence of a 700-800-kDa band in laminin-8 purified from
conditioned medium of T98G cells further confirmed the absence of any
contaminating laminin isoforms.

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Fig. 4.
Immunoblot analysis of purified
laminin-8 with mAbs against distinct laminin chains. A,
purified laminin-8 was subjected to immunoblotting with mAbs against
distinct laminin chains or with a mAb against the 2 chain to
confirm the absence of other laminin isoforms. Equal amounts (0.4 µg/lane) of purified human laminin-1 (LN-1), laminin-2/4
(LN-2/4), laminin-5 (LN-5), laminin-8
(LN-8), and laminin-10/11 (LN-10/11) were
separated by SDS-PAGE on 4% gels under nonreducing conditions and
transferred onto PVDF membranes followed by staining with mAbs against
the laminin 1 (5A3), 2 (5H2), 3 (2B10), 4 (8B12), 5
(15H5), or 2 (C4) chains. The mAb against the laminin 2 chain
reacts with the 80-kDa fragment derived from the C-terminal region of
the 2 chain. B, equal amounts (0.4 µg/lane) of purified
human laminin-1 (LN-1), laminin-2/4 (LN-2/4),
laminin-8 (LN-8), and laminin-10/11 (LN-10/11)
were similarly subjected to immunoblotting with anti- 1 mAb 2E8 as
described above. The positions of molecular size markers are shown in
the left margin.
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Cell Adhesive Activity of Laminin-8--
Cell adhesive activity of
laminin-8 was compared with that of other laminin isoforms
(i.e. laminin-1, laminin-2/4, laminin-5, and laminin-10/11)
and fibronectin using T98G cells as substrate. Laminin-8 was a less
potent mediator of cell adhesion than laminin-5 and laminin-10/11, and
was comparable to laminin-1 (Fig. 5).
Half-maximal levels of T98G cell adhesion were achieved at a substrate
coating concentration of 2.5 nM for laminin-5 and
laminin-10/11, and 5 nM for laminin-8 and laminin-1.

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Fig. 5.
Cell adhesive activity of laminin-8.
T98G cells were incubated at 37 °C for 30 min on 96-well microtiter
plates coated with increasing concentrations of laminin-1
(LN-1), laminin-5 (LN-5), laminin-8
(LN-8), laminin-10/11 (LN-10/11), or fibronectin
(FN). The plates were washed with serum-free DMEM three
times to remove unattached cells, after which attached cells were fixed
and counted under a microscope. Bars represent the standard
deviation of triplicate assays.
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Cell adhesion to laminins is mainly mediated by integrins. We examined
the effects of function blocking mAbs against various integrin subunits
on adhesion of T98G cells to laminin-8 to determine the major integrin
type(s) that serves as its adhesion receptor(s) (Fig.
6). As previously demonstrated (9, 10,
15, 31), cell adhesion to laminin-1, laminin-5, and fibronectin is
specifically inhibited by mAbs against integrins
6,
3, and
5, respectively. The mAb against integrin
1 also
inhibit cell adhesion to these proteins. In contrast, the adhesion of
T98G cells to laminin-8 is not inhibited by any single mAb, except for
anti-
1 mAb. When combined, however, mAbs against
3 and
6 were found to strongly inhibit the adhesion of T98G
cells to laminin-8, indicating that T98G cells adhere to laminin-8
through both
3
1 and
6
1 integrins.

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Fig. 6.
Effects of anti-integrin mAbs on adhesion of
T98G cells to laminin-8 and other adhesive proteins.
Ninety-six-well microtiter plates were coated with 8 nM
laminin-1 (LN-1), 2.5 nM laminin-5
(LN-5), 8 nM laminin-8 (LN-8), or 8 nM fibronectin (FN). The coating concentrations
of adhesive ligands were chosen to obtain near-maximal levels of cell
adhesion in the absence of blocking mAbs. T98G cells were preincubated
with the following function-blocking mAbs against integrin subunits at
a concentration of 20 µg/ml IgG for 20 min at room temperature and
then added to the precoated wells: 2, anti-integrin
2 subunit mAb (P1E6); 3, anti-integrin 3 subunit
mAb (3G8); 5, anti-integrin 5 subunit mAb (8F1);
6, anti-integrin 6 subunit mAb (GoH3);
and 1, anti-integrin 1 subunit mAb (4G2). After 30 min incubation
at 37 °C, cells attached to the substrates were counted under a
microscope. The number of adhering cells is expressed as a percentage
of the number of cells adhering in the presence of control mouse IgG.
Each column and bar represents the mean of
triplicate assays and the standard deviation, respectively.
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To confirm the dual integrin specificity of laminin-8, we examined the
laminin-8 adhesion of K562 human erythroleukemic cells transfected with
integrin
3 or
6 in the absence or presence of 8A2, an
anti-
1 activating mAb (Fig. 7).
Untransfected K562 cells, shown to express only the
5 subtype of
1 integrin (13, 43), could not adhere to all laminin isoforms
examined, regardless of the absence or presence of the
1-activating
mAb. K562 cells expressing the integrin
6 subunit
together with endogenous
1 were competent in adhering to laminin-8
and, to a lesser extent, to laminin-5, but not to laminin-1, in the
absence of mAb 8A2. Upon
1 integrin stimulation by 8A2,
6-transfected cells became highly adherent to all these
laminin isoforms with similar potencies. In contrast,
3-transfected
cells expressing
3
1 integrin were only capable of adhering to
laminin-5, and did not respond to laminin-1 or laminin-8 in the absence
of mAb 8A2 stimulation. Upon 8A2 stimulation, however,
3-transfected
cells became adherent to laminin-8. Adhesion of the stimulated
3-transfected cells to laminin-8 was not due to increased
nonspecific adhesiveness of these cells, as they remained nonadherent
to laminin-1. These results confirmed that both
3
1 and
6
1 integrins could serve as the adhesion receptors
for laminin-8, but that the latter may act as the preferred receptor
for laminin-8, since
6-transfected, but not
3-transfected K562 cells adhere to laminin-8 without stimulation by
8A2.

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Fig. 7.
Adhesion of K562 transfectants
expressing
3 1
and
6 1
integrins to different laminin isoforms. Control K562 cells and
those expressing integrin 3 1 (K562/ 3 1) or
6 1 (K562/ 6 1) were incubated for 30 min at 37 °C with or without the stimulatory mAb 8A2 (1:10,000
dilution of ascites) in 96-well microtiter plates precoated with
increasing concentrations of laminin-1 (LN-1), laminin-5
(LN-5), and laminin-8 (LN-8). Cells adhering to
the substrates were fixed, stained, and counted as described under
"Experimental Procedures." Each point and bar
represents the mean of triplicate assays and the standard deviation,
respectively.
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Cell Migration Promoting Activity of Laminin-8--
Laminins have
been shown to stimulate cell migration during development and in many
pathological processes (44-46). We examined the ability of laminin-8
to promote cell migration by using time lapse video microscopy to track
T98G cells migrating on dishes coated with different concentrations of
laminin-8 or other adhesive proteins. Among the six different adhesive
ligands examined, laminin-8 was most potent in promoting cell
migration, attaining maximal activity at coating concentrations of >10
nM without a significant decline up to 80 nM
(Fig. 8). Laminin-10/11 was also very
potent in promoting cell migration, attaining maximal activity at
coating concentrations of 5-10 nM. Laminin-2/4 and
laminin-5 were of roughly equal potency, lower than laminin-8 and
laminin-10/11, while laminin-1 was weaker still. Fibronectin was barely
active, if at all, in promoting migration of T98G cells.

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Fig. 8.
Cell migration promoting activity of
laminin-8. Glass-bottom culture dishes were precoated with
increasing concentrations of laminin-1 (LN-1), laminin-2/4
(LN-2/4), laminin-5 (LN-5), laminin-8
(LN-8), laminin-10/11 (LN-10/11), or fibronectin
(FN). T98G cells were added to the precoated dishes and the
migrating cells were tracked by time lapse video microscopy at 37 °C
for 8 h. Cell migration paths were traced and quantified using the
Image-Pro Plus image-processing software. Each point
represents the mean of migration distances of 10 different migrating
cells.
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Cell migration promoting activity of all six adhesive ligands except
laminin-8 declined at higher coating concentrations, consistent with
previous observations (47-49) that cell migration speed was dependent
on the adhesive strength of cells to substratum, giving a biphasic
dose-response curve when substrates are coated with increasing
concentrations of adhesive ligands. Laminin-8 also strongly promoted
migration of other cell types, including A549 human lung adenocarcinoma
cells, to a similar degree as was demonstrated with T98G cells (data
not shown).
T98G cells migrating on laminin-8 assumed an elongated morphology with
frequent pseudopod extension in the direction of cell migration (Fig.
9). Small lamellipodia-like structures
were observed at the tip of the pseudopods. The cells plated on
laminin-8-coated substrates were capable of easily detaching from the
substratum at the rear, allowing them to crawl smoothly. A similar
elongated morphology with pseudopods extended in the direction of cell
migration was observed with cells plated on substrates coated with
laminin-2/4 and laminin-10/11, but not with laminin-1, laminin-5, or
fibronectin. Cells on laminin-1, laminin-5, or fibronectin-coated
substrates assumed rather round morphologies with wide lamellipodia
extending in multiple directions from time to time, thereby preventing
them from becoming polarized. There was a close correlation between the
ability of the substrates to polarize cells and their potency in
promoting cell migration.

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Fig. 9.
Morphology of T98G cells migrating on the
substrates coated with laminin-8 and other adhesive proteins. T98G
cells were plated onto glass-bottom culture dishes precoated with 10 nM laminin-1 (LN-1), laminin-2/4
(LN-2/4), laminin-5 (LN-5), laminin-8
(LN-8), laminin-10/11 (LN-10/11), or fibronectin
(FN). Cells were incubated on the substrates for 30 min to
allow them to attach, after which migrating cells were recorded by time
lapse video microscopy at 30-min intervals for 2.5 h.
Arrowheads indicate pseudopods extended in the direction of
cell migration. Bar, 100 µm.
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To explore whether both
3
1 and
6
1 integrins are
involved in cell migration on laminin-8, we examined the effects of
function-blocking mAbs against different integrin subunits on T98G cell
migration on laminin-8-coated substrates. The mAb against
3 integrin
strongly inhibited laminin-8-mediated cell migration, but an
anti-
6 integrin mAb was only marginally inhibitory (Fig.
10). The combination of
3 and
6 integrin mAbs completely inhibited cell migration.
These results indicate that cell migration on laminin-8 is
predominantly driven by the interaction of
3
1 integrin with
laminin-8.

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Fig. 10.
Effects of anti-integrin mAbs on migration
of T98G cells adhering to laminin-8. Glass-bottom culture dishes
were precoated with 20 nM laminin-8. T98G cells were
preincubated with the following function blocking mAbs against integrin
subunits at a concentration of 20 µg/ml IgG for 20 min at room
temperature and then added to the precoated dishes: IgG, control mouse
IgG; 3, anti-integrin 3 subunit mAb (3G8); and 6,
anti-integrin 6 subunit mAb (GoH3). Cell migration
images were acquired by time lapse video microscopy at 10-min intervals
for 8 h. Cell migration was quantified as described under
"Experimental Procedures." Each column represents the mean of
migration distances of 10 different migrating cells. Bars
represent the standard deviation.
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DISCUSSION |
In this study, we purified laminin-8 from conditioned medium of
T98G cells by affinity chromatography using a mAb specific for the
laminin
1 chain. T98G cells were used as a source of laminin-8
because these cells express only the
4 subtype of laminin
chain.
Selective expression of the
4 chain was confirmed at both the RNA
and protein level by RT-PCR and immunoblot analysis. Although mAb 8B12
recognizes only denatured, and not intact,
4 chains, and cannot be
used as an affinity ligand for purification, selective expression of
the
4 chain in T98G cells allowed us to purify laminin-8 by affinity
chromatography using mAb against the laminin
1 chain. The identity
of the purified laminin-8 was confirmed by its positive reactivity with
mAbs against the
4,
1, and
1 chains. The absence of other
contaminating laminin isoforms was established by negative reactivity
to a panel of mAbs recognizing each one of the other four known
chains. Furthermore, laminin-9 (
4
2
1) was shown to be absent
from the purified laminin-8, as evidenced by a lack of reactivity with
a mAb against the
2 chain. Immunoblotting of purified laminin-8 with
an anti-
4 chain mAb detected a major 200-kDa band, together with a
minor 180-kDa band. The 180-kDa band was also detectable in conditioned
medium of T98G cells, suggesting that the 180-kDa
4 chain could be a proteolytically processed form or an alternatively spliced form. A
similar 180-kDa
4 chain was also detected in laminin-8 purified from
platelets (17).
Both
3
1 and
6
1 integrins function as the major
cell surface receptors for laminin-8, based on the following
observations: 1) adhesion of T98G cells to laminin-8 was not inhibited
by any single mAb against the integrin
2,
3,
5, or
6 chain, but inhibited by an anti-
1 integrin mAb or a
combination of anti-
3 and anti-
6 mAbs. 2) Wild-type
K562 cells could not adhere to laminin-8 even after stimulation of
1
integrins by 8A2, whereas K562 cells transfected with
3 or
6 integrin subunits could adhere to laminin-8.
6-Transfected cells adhered to laminin-8 without
1
integrin stimulation, but
3-transfected cells needed
1 integrin
activation to adhere to laminin-8, indicating that although both
3
1 and
6
1 could serve as adhesion receptors for
laminin-8,
6
1 might be the preferred laminin-8
receptor. The role of
6
1 integrin as the laminin-8 receptor was recently reported using platelet laminin-8 (17) and
recombinant laminin-8 (18). Geberhiwot et al. (17)
showed that platelet adhesion to laminin-8 is inhibited by
anti-
6 mAb alone, in contradiction to our present
observations. This could be due to a very low level, if at all, of
expression of
3
1 integrin in platelets (50), while platelets have
been shown to express
6
1 integrin that is
functionally active in mediating platelet adhesion to laminin-1
(51).
Recently, Kortesmaa et al. (18) produced recombinant
laminin-8 in a mammalian expression system by triple transfection of human laminin
4 and
1, and mouse
1 chains, with an acidic
FLAG-tag attached to the C terminus of the
4 chain. They showed that
K562 cells expressing
6
1 integrin could adhere to
recombinant laminin-8 without integrin stimulation. They also reported
that K562 cells expressing
3
1 integrin adhere poorly to
recombinant laminin-8, although they did not show the data, nor did
they indicate whether the assays were done with or without integrin
stimulation. Their results are consistent with our observations, except
that they did not indicate whether
3-transfected cells could adhere
to recombinant laminin-8 with integrin stimulation. They also reported, however, that adhesion of HT1080 cells to recombinant laminin-8 was
strongly inhibited by the anti-integrin
6 mAb alone,
while our data show that both anti-
3 and anti-
6 mAbs
are necessary to inhibit adhesion of T98G cells to laminin-8. This
apparent discrepancy is not due to contamination of our laminin-8
preparation, which has been determined by immunoblot analysis to be
free of laminin isoforms containing
1,
2,
3, or
5 chains,
as well as the
2 chain-containing laminin-9. Laminins have been
shown to undergo post-translational modifications such as proteolytic processing and N- and O-linked glycosylation (52,
53), which may explain the discrepancy in the integrin-binding
specificity between the recombinant laminin-8 and the laminin-8 that we
purified. The recombinant laminin-8 was produced in human embryonic
kidney cells, while our laminin-8 was produced in human glioma cells. Cell type-dependent glycosylation and/or proteolytic
processing of subunit chains may therefore modify the integrin-binding
specificity of laminin-8. Differences in elution protocols for
immunoaffinity purification of laminin-8 may also explain the apparent
discrepancy in integrin-binding specificity between the recombinant
laminin-8 and our laminin-8 preparation. Although the recombinant
laminin-8 was eluted from an anti-FLAG immunoaffinity column under
nondenaturing conditions using the FLAG peptide, our laminin-8 was
eluted from an anti-
1 mAb column under denaturing conditions using
0.1 M glycine-HCl (pH 2.4). It should be also noted that
the recombinant laminin-8 is a hybrid protein consisting of human
4
and
1 chains assembled with a mouse
1 chain, with an acidic
FLAG-tag attached to the C terminus of the
4 chain. It is possible
that such non-physiological features associated with the recombinant
expression system also modify the integrin-binding specificity of
laminin-8.
Although both anti-
3 and anti-
6 integrin mAbs were
needed to inhibit adhesion of T98G cells to laminin-8, cell migration on laminin-8-coated substrate was strongly inhibited by anti-
3 mAb
alone. It seems, therefore, that
3
1, but not
6
1, integrin is the major adhesion receptor through
which laminin-8 mediates cell migration. In support of this notion,
3
1 integrin has been shown to serve as the principal adhesion
receptor in migration of various cell types. For example, migration of
neurons along radial glial fibers during development of the cerebral
cortex is dependent on
3
1 integrin (54). Migration of
keratinocytes during wound healing is also mediated primarily by
3
1 integrin with concomitant deposition of laminin-5 (46).
Integrin
3
1 has also been shown to serve as the major integrin
receptor operating in the migration and invasion of various tumor cells
(31, 55). The prevalence of
3
1 integrin in cell migration
processes may explain the varied responses to different laminin
isoforms. The observation that laminin-1 is a much weaker promoter of
cell migration than laminin-8, even though they have similar
cell-adhesive activity, may be because laminin-1 does not act through
3
1 integrin as an adhesion substrate. In contrast, laminin-2/4,
laminin-5, and laminin-10/11, all of which are ligands for
3
1
integrin (12, 15, 31), were more potent than laminin-1 in promoting
cell migration, further supporting the role of
3
1 integrin as a
preferred receptor for this specific activity.
For a cell to migrate, the cell needs to extend membrane ruffles in the
direction of cell migration and become polarized. T98G cells adhering
to laminin-8 assumed a highly polarized morphology with extension of
multiple pseudopods in the direction of cell migration. A similar
polarized morphology was also observed in cells adhering to
laminin-10/11, and, to lesser extent, in cells adhering to laminin-2/4.
In contrast, laminin-1 and fibronectin, both of which were very weak
promoters of cell migration, did not induce morphological polarization,
consistent with the close correlation between the ability of cells to
become polarized and to migrate (48). Although the mechanisms of cell
polarization are not well understood, it seems likely that the Rho
family of small GTPases, particularly Rac and Cdc42, play critical
roles in this phenomenon through promoting lamellipodia and filopodia formation (56, 57). It has been established that activation of Rac and
Cdc42 is involved in extension of lamellipodia and filopodia,
respectively. Thus, the prominent cell polarization with multiple
pseudopod extension observed to be induced by laminin-8 may be
associated with activation of Rac and/or Cdc42. Ligation of
3
1
integrin by anti-
3 mAbs has been shown to induce curtain-like lamellipodia in skin fibroblasts adhering to laminin-1, which usually
is associated with the adoption of a typical fibroblastic morphology
with dense focal contacts (58). This suggests that ligation of
3
1
integrins transduces signals that activate Rac. Consistent with this
view,
3
1 integrin has been shown to negatively regulate actin
cytoskeletal reorganization. For example, ablation of
3
1 by gene
targeting in keratinocytes and kidney epithelial cells resulted in
enhanced stress fiber formation and denser focal contacts (59, 60).
Since Rac has been shown to down-regulate Rho activity (61, 62), it
seems likely that
3
1 integrin transduces signals that activate
Rac, which in turn down-regulates Rho and suppresses formation of
stress fibers and focal contacts. For a cell to migrate, it needs to
detach from the substratum at its rear edge. Formation of focal
contacts stabilized by stress fibers is disadvantageous for this cell
detachment. Enhanced cell migration on laminin-8 may be due to the
suppression of this stabilization mechanism. Indeed, cells migrating on
laminin-8-coated substrates were not only highly polarized, but
also frequently became rounded during migration (data not shown),
consistent with this proposed mechanism.
Cell migration speed has been shown to exhibit a biphasic dependence on
adhesive ligand concentration, regardless of integrin expression level
or integrin-ligand binding affinity (49). Our data also show that cell
migration on various types of laminin isoforms, except laminin-8,
follows a biphasic curve as the substratum was coated with increasing
concentrations of the adhesive ligands. The reason why cell migration
speed did not decline when laminin-8 was used at high coating
concentrations (i.e. up to 80 nM) is not clear,
but it is conceivable that formation of focal contacts and stress
fibers, the major suppressor of cell detachment at the rear, was not
induced under these conditions, possibly due to the signals transduced
by the
3
1 integrin. The relatively low binding affinity of
laminin-8 toward
3
1/
6
1 integrins, as revealed
by the relatively weak cell adhesive activity of laminin-8, may also
contribute to the facilitation of rear cell detachment, even at high
coating concentrations. Further studies are needed, however, to better
understand the molecular mechanisms of signaling pathways through
3
1 and
6
1 integrins on laminin-8 and other laminin isoforms.
Laminin isoforms containing the
4 chain have been shown to be
expressed strongly in embryonic and regenerating tissues, where cells
are actively migrating. For example, in developing kidney, invading
vessels that are destined to generate the capillary loops of the
glomeruli are surrounded by a basement membrane rich in
4- but not
5-containing laminin isoforms (23). During the late stages of kidney
development, arteriolar basement membranes lose
4 and acquire the
5 chain (23). In the adult, expression of
4 chain-containing
laminins is restricted to the capillary basement membrane (22, 23, 26).
Given the remarkable potency of laminin-8 to promote cell migration,
selective expression of
4 chain-containing laminins in invading
blood vessels during organogenesis and in capillary basement membranes
in the adult suggests that laminin-8 is involved in endothelial cell
migration during these processes. The role of laminin-8 as a potent
stimulator of cell migration is consistent with the expression pattern
of the
4 chain in muscle, where it is expressed exclusively during development and in the neuromuscular junctions of adults (26, 27). In
addition, the expression of the
4 chain is up-regulated in
regenerating muscle following a traumatic crushing injury (63). Furthermore, laminin-8 is one of the major components of neural crest
cell migration pathways. These pathways have been shown to be blocked
by an antibody against a laminin-8-agrin complex (45, 64), indicating
that laminin-8 is also involved in migration of neural crest cells.
Since these cells do not express
6 subunit-containing integrins, the primary candidate for an integrin operating during neural crest cell migration may be
3
1 (50), the laminin-8 receptor most prominent in promoting cell migration. Further studies on
the tissue distribution of laminin-8 under both normal and pathological
conditions, as well as the effects of laminin-8 on many other
biological processes involving vast cell migration, should shed light
on the roles of laminin-8 in cell migration during development and
tissue regeneration.