(Received for publication, October 23, 1995; and in revised form, November 20, 1995)
From the
Laminin, the major glycoprotein of basement membranes, actively
supports cell migration in development, tissue repair, tumor growth,
metastasis, and other pathological processes. Previously we have shown
that the locomotion of murine skeletal myoblasts is specifically and
significantly enhanced on laminin but not on other matrix proteins. One
of the major laminin receptors of myoblasts is the 7
1
integrin, which was first described in human MeWo melanoma cells and
Rugli glioblastoma cells. In order to investigate and directly test the
role of the
7 integrin in cell migration on laminin, we expressed
the murine
7B splice variant in human 293 kidney cells and 530
melanoma cells which cannot migrate on laminin and are devoid of
endogenous
7. Northern blotting of the transfected cells showed
that the
7 mRNA was expressed efficiently, and the protein was
detected on the cell surface by immunofluorescence and
fluorescence-activated cell sorter analysis. Cell motility measurements
by computer-assisted time-lapse videomicroscopy of the
7-transfected cells revealed an 8-10-fold increase in
motility on laminin-1 and its E8 fragment, but not on fibronectin.
Mock-transfected cells did not migrate significantly on laminin or on
fibronectin. Similarly, transmigration of
7-transfected 293 cells
through laminin-coated filters in a Boyden chamber assay was
significantly enhanced in comparison to mock-transfected cells. These
findings prove that
7 integrin expression confers a gain of
function-motile phenotype to immobile cells and may be responsible for
transduction of the laminin-induced cell motility.
Cells utilize extracellular matrix to migrate during embryonic
development, tissue regeneration, and invasion of tissues in
inflammation and tumor metastasis (reviewed in (1) ).
Laminin-1, a major glycoprotein of basement membranes, has been shown
to promote migration of different cell types including neural crest
cells(2) , skeletal myoblasts(3) , or B16 mouse
melanoma cells(4) . The mechanism of laminin-induced cell
locomotion and the cellular receptors mediating the locomotor signals
are, however, not known. Integrin- and non-integrin receptors are
involved in the specific adhesion of cells to laminin. Dystroglycan, a
156-kDa protein forms a transmembrane linkage together with other
proteins between laminin-2 and dystrophin in the muscle cell
membrane(5) ; a high affinity laminin receptor of 67 kDa has
been isolated from several tumor cells, myoblasts, and other primary
cells (for reviews, see (6, 7, 8) ). Five
laminin-binding integrins of the 1 family recognize different
sites and isoforms of laminin:
6
1 (9, 10) and
7
1 (11, 12) bind to laminin-1, recognizing specifically
the E8 domain;
1
1 binds to a cryptic site in the E1 region of
laminin(13) , but also to types I and IV collagen(14) .
The
3
1 integrin binds to laminin-5 (kalinin) (15) and to other matrix proteins;
2
1 is
predominantly a collagen receptor(16) , but when isolated from
endothelial cells it also binds to laminin-1(17) .
While much has been published on the involvement of these receptors in cell adhesion, the role of the integrins in cell migration on laminin remains to be investigated. When murine skeletal myoblasts are plated on laminin-1-coated dishes, they respond rapidly in a characteristic manner by extending long cell processes and lamellopodia within 1 h, resulting in a dramatic enhancement of cell motility(18, 19) . The functional domain of laminin was restricted to the C-terminal elastase fragment E8; in contrast, fibronectin, collagens or the E1 fragment of laminin-1 do not support migration of murine myoblasts (18, 19) .
The major
laminin-binding integrin isolated from murine skeletal myoblasts is
7
1(11, 20) , which exists in three
cytoplasmic and two extracellular splice
variants(21, 22) . The predominant cytoplasmic splice
variant of
7 integrin in fetal mouse muscle and proliferating
myoblasts is the
7B form;
7A is expressed in adult skeletal
muscle and myotubes(22, 23) . We postulated that the
7
1 complex mediates the multiple responses of myoblasts
to laminin, including cell motility(11) .
In order to
elucidate the role of 7 integrin in cell locomotion, we expressed
the full-length
7B cDNA in two cell lines, human 293 kidney cells
and human 530 melanoma cells (24) which are not motile on
laminin. They do not express
7, but
1 integrin
subunits(25, 26) which are necessary to form a
heterodimeric complex on the cell surface. Here we show that, after
transfection with
7B, both cell types gain motility on laminin and
its E8 fragment severalfold over the mock-transfected or untransfected
cells. Adhesion to laminin, however, was not affected significantly.
Cell migration assays
in Boyden chambers were carried out as described by Albini et
al.(29) ; 7- and vector-transfected cells were
resuspended in serum-free DMEM medium in the presence of 0.5% BSA;
10
cells in 200 µl were added to the upper compartment
of the Boyden chamber. The lower compartment was filled with 200 µl
of DMEM/F12 medium in the presence of 5% fetal calf serum, 0.1% BSA,
250 µg/ml Geneticin, and 300 µg/ml hygromycin. The two
compartments of the Boyden chambers were separated by polycarbonate
filters (5-µm pore size, Nuclepore Costar). The filters were coated
by floating on a solution containing 5 µg/ml laminin-1 or human
plasma fibronectin at 37 °C for 1 h and blocked with 2% BSA
overnight at 4 °C. The cells were allowed to migrate 6 h at 37
°C in a humidified atmosphere containing 7.5% CO
. Cells
remaining on the upper side of the filter were removed mechanically;
cells on the lower surface of the filter were fixed for 3 min in
-20 °C methanol, stained for 30 min with
4,6-diamidino-2-phenylindole), and 5 random fields were counted with a
Zeiss inverted microscope. Each assay was carried out in triplicate and
repeated twice.
A full-length murine 7B integrin cDNA was generated
which included parts of the 3` and 5` untranslated regions and the
complete coding sequence(21, 22, 23) . For
expression in human 293 kidney and 530 melanoma cells, it was
positioned under the control of the cytomegalovirus promoter within the
episomal vector pCEP4 (Fig. 1). Expression of the
7
integrin message was measured by Northern blotting (Fig. 2). The
7-specific probe hybridized to a 4.1-kb
7 mRNA band in the
RNA of murine C2C12 myoblasts, of 293-
7B and 530-
7B cells,
but not to the RNA of mock-transfected cells or HT 1080 fibrosarcoma
cells.
Figure 1:
Construct for eucaryotic expression of
murine 7 integrin. Full-length
7 cDNA was cloned into the
multiple cloning region of the episomal pCEP4 expression vector. The numbers indicated refer to amino acid positions, with 1 being the N terminus after signal peptide (SS) cleavage.
The 10.4-kb vector pCEP4 requires for optimal expression the EBNA-1
antigen which is expressed by the 293 kidney epithelial cells used in
this study.
Figure 2:
Analysis of 7 expression by Northern
hybridization. RNA (12 µg each) from HT1080 fibrosarcoma cells,
from cultured C2C12 myoblasts containing about 30% nuclei in myotubes,
from 293 and 530 cells transfected with
7 cDNA (293EBNA-
7B
and 530-
7B), or with the pCEP4 vector only (293EBNA-pCEP4 and
530-pCEP4) were separated by electrophoresis, blotted onto
nitrocellulose, and hybridized with a murine
7 cDNA probe. Only
C2C12 cells and the
7-transfected 293 and 530 cells show the
4.1-kb band characteristic for
7 mRNA.
Immunofluorescence staining with an affinity-purified
polyclonal antibody raised against the recombinant extracellular domain
of 7
revealed high levels of surface fluorescence on
35% of the transfected 293 cells, a result which was confirmed by FACS
analysis ( Fig. 3and Table 1). This indicated that the
murine
7 chain must have associated with an endogenous human
-integrin chain to form heterodimeric complexes. When plated onto
laminin-coated dishes, about 15% of the
7-transfected 293 cells
assumed spindle shape, in contrast to mock-transfected cells which
stayed round. The adhesion rates on laminin-1, its E8 fragment, or on
fibronectin of
7- and mock-transfected cells did not differ
significantly (Fig. 4). A dramatic difference, however, was seen
when the locomotion of single cells was followed by time-lapse video
microscopy, and paths were measured by a computer- assisted track
analysis program: the motility of 293EBNA-
7B and 530-
7B cells
on laminin or the E8 fragment was 8-10-fold enhanced as compared
to untransfected or pCEP4-transfected cells (Fig. 5). More than
90% of the
7-transfected cells revealed a jerky, nondirected
movement of cells by protruding lamellopodia and rapid retraction of
the cell body into the extended filopodia. The path length of
7-transfected 293 cells after 15 h on laminin was up to 200 nm (Fig. 5). Locomotion over fibronectin was not altered.
Figure 3:
Surface expression of murine
7
1 integrin analyzed in a fluorescence-activated cell sorter
(FACS) after staining with an affinity-purified rabbit antibody raised
against recombinant
7 integrin peptide. 293 kidney cells stably
transfected with the
7B (293EBNA-
7B) or the vector alone
(293EBNA-pCEP4) were stained at 4 °C with the antibody, followed by
fluorescein isothiocyanate-labeled goat anti-rabbit Ig, and analyzed
within 1 h. About 30-35% of the
7-transfected cells show
strong surface fluorescence. - - -, nonimmune serum;
-,
7 immune serum.
Figure 4:
Adhesion of 7- and mock-transfected
293 cells on surfaces coated with fibronectin (FN), laminin-1 (LN), and LN-E8 fragment. 96-well plates (Maxisorb, Nunc) were
coated with serial dilutions of the proteins for 1 h at 37 °C and
blocked with heat-treated 2% BSA overnight. Cells were seeded at a
density of 50,000 cells/well. No significant differences in the
adhesion rate after 1 h in relation to the substrate concentration was
found for both cell lines. Adhesion rates were measured by colorimetric
determination of endogenous hexosaminidase of the attached cells as
described previously(16) .
-
,
293EBNA-
7B;
-
,
293EBNA-pCEP4.
Figure 5:
Videomicroscopy of 7-
(293EBNA-
7B) and mock-transfected 293 kidney cells (293EBNA-pCEP4) (A) and 530 melanoma cells (B), locomoting over LN,
FN, and LN-E8 fragment at 37 °C. The tracks of single cells were
followed and recorded by time-lapse videomicroscopy over 15 h. The
paths of 10 cells each were normalized and converted to wind rose
display (19) where all cells start from the same point in order
to illustrate the average motility.
The
enhanced motility accompanying 7 expression was tolerant to the
cell background. When
7 was transfected into human 530 melanoma
cells, they also showed a significantly enhanced motility on laminin-1
or E8 (Fig. 5), even though the level of expression of
7
was considerably lower in the 530 cells than in the 293 cells that are
stably transfected with EBNA. In comparison to 293 EBNA-
7 cells,
hardly any surface fluorescence of
7 could be detected on
transfected 530 cells, although the locomotory response of these cells
to laminin was as pronounced as that of the transfected 293 cells (Fig. 5).
The results of the 7 transfection on cell
motility on laminin-coated surfaces were confirmed in a transmigration
assay, using Boyden chambers with Nuclepore filter membranes coated
with laminin-1 or fibronectin. About 10
7- or
mock-transfected 293 cells in serum-free medium were placed in the
upper chamber and allowed to migrate through the filters against a
gradient of 5% fetal calf serum in the lower chamber(29) .
After 6 h, the cells appearing on the lower side of the filter were
stained and counted. About 3 times as many
7-transfected cells had
migrated through the laminin-coated filter in comparison to
mock-transfected cells (Fig. 6). On fibronectin-coated filters,
there was no significant migration of both
7- and mock-transfected
cells (Fig. 6); no migration of cells was observed on filters
coated with BSA alone. These results confirmed the ability of
7 to
enhance the motility of 293 cells not only on two-dimensional laminin
surfaces, but also to specifically stimulate transmigration through
laminin-coated filters.
Figure 6:
Transmigration of 7- and mock-
transfected 293 cells through laminin-1 - and fibronectin-coated
nuclepore filters in a Boyden chamber. 10
cells in
serum-free medium were placed in the upper chamber and allowed to
migrate into the lower chamber against a serum gradient. After 6 h, the
cells on the lower side of the filter were stained and
counted.
The ability of laminin to stimulate
migration of cells in development and many pathological processes has
received considerable attention in the past, in particular also in view
of its role in promoting tumor cell invasion and
metastasis(1, 2, 3) . For example, lung
colonization of B16 melanoma cells is enhanced by coinjection with
laminin, and melanoma cells selected for attachment on laminin have a
higher metastatic potential(1, 2) . Each effect could
conceivably be the result of motile induction which we show here can be
triggered by 7 integrin.
Our data indicate that 7
1
integrin is a laminin receptor involved in cell migration on laminin-1
and its E8 fragment, but not on fibronectin. It does not seem to play a
crucial role in the adhesion of the transfected 293 or 530 cells to
laminin-1 as no difference was seen in the adhesion behavior between
7-expressing and nonexpressing cells. In fact, there is no
experimental evidence yet in the literature that
7 integrin is
involved in cell adhesion to laminin. Up to now, no adhesion-blocking
antibodies to
7 integrin are available. The best evidence for
7
1 integrin being a laminin receptor is the fact that it
binds to laminin in vitro; thus, it has been isolated by
affinity chromatography on laminin-1(11, 30) . Other
laminin-binding integrins or non-integrin laminin receptors seem
responsible for the adhesion of 293 and 530 cells to laminin-1.
Candidates are the
6 integrin which has been shown to mediate
adhesion of several human tumor cells (10) and murine B16
melanoma (11) to laminin-1; it is expressed by the kidney
epithelial cell line 293 in small amounts(25, 26) .
Furthermore, embryonic kidney epithelial cells synthesize
-dystroglycan, a major laminin receptor of muscle cells binding to
the E3 fragment of laminin(31) .
The locomotion of the
7-transfected cells shown in the lateral motility assay is not
directed and appears random chemokinesis. Laminin has the ability to
induce outgrowth of neurites (32, 33) and of filopodia
and lamellopodia, e.g. in Schwann cells (34) or
myoblasts(18) . The
7-transfected 293 cells showed
enhanced outgrowth of cell processes and were more elongated on laminin
than their mock-transfected counterparts (Table 1), indicating
that
7 is responsible for this activity of laminin.
Although
7 confers motility to such different cell types as kidney
epithelial cells and melanoma cells, other cell types may utilize
different integrins in migrating over laminin, and certainly when
migrating over other matrix macromolecules (for review, see (41) ). For example, the human melanoma cell BLM migrates over
laminin and expresses
6 but not
7 integrin. (
)Recently, Melchiori et al.(38) have
shown that transmigration of human 2/14 melanoma cells through the
pores of a Boyden chamber coated with basement membrane proteins, using
fibronectin, collagen IV, or laminin as chemoattractants, is inhibited
by antibodies to
3 and
1 integrin chains. Increased
expression of
3
1 integrin has been also associated with
enhanced tumor cell metastasis(39) . Smooth muscle cells
utilize
v
3 for migration on osteopontin(42) , and
human umbilical vein endothelial cells use the same integrin for
transmigration on collagen- or vitronectin-coated filters(43) .
The migration of rhabdomyosarcoma cells through collagen gels was
enhanced when cells were transfected with chimeric
2-chains
carrying the cytoplasmic domain of
4 integrin(44) .
Undirected migration of cells over matrix-coated surfaces, in the
absence of any concentration gradient, was termed
haptotaxis(35, 36) , in contrast to chemotaxis which
was described for cells migrating against a gradient of laminin or
fibronectin fragments(37, 38) . Transmigration of
cells through capillaries, basement membranes of epithelial tumors, or
the matrixcovered filter of a Boyden chamber is, however, a more
complex event than lateral locomotion on laminin-coated surfaces and
may involve also other matrix-induced events such as proliferation and
synthesis of proteases (40) . Interestingly, transfection with
7 cDNA also stimulated the ability of 293 cells to transmigrate
specifically through laminin-coated filters in the Boyden chamber
assay. Migration rates through fibronectin-coated filters were much
lower for both
7- and mock-transfected cells, but there was no
stimulation after
7 transfection.
The 7B splice variant is
prominent in proliferating myoblasts and thus could play a role in
myoblast migration in early embryonic development, e.g. during
migration of myotome cells from the somites into the limb buds (45) or during muscle regeneration by satellite
cells(46) . The rapid response of
7-transfected cells to
laminin, e.g. cell elongation, outgrowth of lamellopodia, and
locomotion within 1 h, offers the possibility to study the signal
transduction mechanism of
7-mediated cell motility. Cell migration
over solid substrates requires continuous membrane reshuffling from the
trailing edge to the leading edge of the cell, and actin polymerization
and depolymerization in the microfilaments in the tips of lamellopodia (47) . All these events involve protein phosphorylation and
dephosphorylation. The cytoplasmic domain of the
7B splice variant
which was used in these experiments contains domains with homology to
protein-tyrosine phosphatases, and two regions similar to a catalytic
phosphotransfer domain and the ATP-binding domain of serine/threonine
kinases(21) . Whether this domain activates the focal adhesion
kinase pp125
-mediated signal transduction pathway of
other integrins (for reviews see (48) and (49) )
remains to be investigated. Comparison of the
7B- and
mock-transfected cells should now enable us to analyze the mechanism of
laminin-induced cell locomotion. It will be interesting to see whether
transfections with the
7A domain (21, 22, 23) also enhance cell motility on
laminin.