(Received for publication, July 17, 1996, and in revised form, October 11, 1996)
From the Division of Experimental Oncology E,
Istituto Nazionale Tumori, 20133 Milan, Italy and the
§ Metastasis Research Laboratory, University of Liège,
B-400 Liège 1, Belgium
The interactions between tumor cells and laminin
or other components of the extracellular matrix have been shown to play
an important role in tumor invasion and metastasis. However, the role
of the monomeric 67-kDa laminin receptor (67LR) remains unclear. We
analyzed the regulation of 67LR expression under different culture
conditions with respect to the expression of other well characterized
laminin receptors. In A431 cells treated with laminin for different
time periods, the regulation of 67LR expression correlated with
expression of the 6 integrin subunit but not with the expression of
other laminin receptors. Moreover, cytokine treatment resulted in
down-modulated expression of the
6 integrin subunit and the 67LR.
Co-regulation of the expression of the two receptors was further
suggested by the observation that specific down-modulation of the
6-chain by antisense oligonucleotides was accompanied by a
proportional decrease in the cell surface expression of 67LR.
Biochemical analyses indicated co-immunoprecipitation of 67LR and the
6 subunit with an anti-
6 but not an anti-
1 monoclonal
antibody. Co-regulation of 67LR and
6 subunit expression, together
with the physical association between the two receptors, supports the
hypothesis that 67LR is an auxiliary molecule involved in regulating or
stabilizing the interaction of laminin with the
6
4 integrin.
The interaction of tumor cells with laminin, the major basement
membrane glycoprotein, is considered a critical determinant of
metastatic dissemination. Cells can bind laminin through different specific receptors, including the integrins, a large family of heterodimeric transmembrane molecules consisting of non-covalently associated and
subunits (1-3).
13 years ago, three independent laboratories isolated a non-integrin protein of 67 kDa, designated 67-kDa laminin receptor (67LR),1 by affinity chromatography on laminin-Sepharose (4-6). To date, the structure of this molecule has not yet been elucidated, and only the cDNA encoding a cytoplasmic precursor of 37 kDa (37LRP) has been identified (7). Although pulse-chase experiments performed on melanoma cells demonstrated that this 37-kDa polypeptide is the precursor of the 67-kDa form, the post-translational mechanism by which the 67LR is synthesized from the precursor is still unknown (8).
Cell surface expression of the 67LR has been shown to correlate with
metastatic potential of solid tumors. Indeed, increased expression of
the 67LR was found in different neoplasias, where it is associated with
poor prognosis (9-11). In a study aimed at elucidating the role of
different laminin receptors in the metastatic process, we found a
strong correlation between the expression of 67LR and the 6
1
integrin on small cell lung carcinoma cells (12). Consistent with these
results, immunoelectron microscopy indicated that the 67LR localized in
the same cytoplasmic structures positive for the
6 and
1 integrin
subunits (13). After a brief exposure to laminin, these cytoplasmic
complexes were exported to the cell surface by a mechanism which is
still unclear.
In the present study, we analyzed whether membrane expression of the 67LR and of integrins involved in laminin recognition is co-regulated and whether the different molecules are physically associated.
Human vulvar epidermoid carcinoma cell line A431 was provided by ATCC. Cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (FCS), penicillin (100 mg/ml), and streptomycin (100 mg/ml). Experiments involving treatment with laminin were performed using murine laminin purified from the mouse Engelbreth-Holm-Swarm tumor (Sigma).
Monoclonal and Polyclonal AntibodiesThe following
monoclonal antibodies were used as purified Ig: MLuC5, directed against
the 67-kDa laminin receptor (14); GoH3, directed against 6-chain
integrin (Immunotech, Marseille, France); P1E6, directed against
2-chain integrin (Telios, San Diego, CA); P1B5, directed against
3-chain integrin (Telios); 3E1, directed against
4-chain integrin
(Telios); MAR4, directed against
1-chain integrin (15); MGR1,
directed against the EGF receptor (12); MPLR2, directed against the
37-kDa laminin receptor precursor and 67LR mature form of the
receptor;2 and W6/32, directed against a
monomorphic epitope on the 45-kDa polypeptide product of the human
leukocyte antigen (HLA) A, B, C loci (Coulte Immunology, Hialeah,
FL).
Fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG and anti-mouse IgG or IgM (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) were used as second-step reagents.
Oligonucleotide SynthesisUnmodified DNA
oligonucleotides corresponding to integrin subunit 6 nucleotides
142-160 (18-mer) were synthesized on an automated synthesizer (Cyclone
Plus DNA Synthesizer, Millipore Corp.). The sense and antisense
6-chain sequences were 5
-GCCCATGGCCGCCGCCGG-3
and
5
-CCGGCGGCGGCCATGGGC-3
, respectively. After cleavage from a
controlled pore glass column and deblocking in concentrated ammonium
hydroxide at 55 °C for 18 h, the oligonucleotides were purified
by ethanol precipitation from a 100 mM NaOAc solution. Analytical gel electrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mM Tris borate buffer, pH 7.0.
Cells plated in 24-well cell culture dishes (1 × 105cells/well) and grown as monolayers for 24 h were gently washed twice with 5 ml of serum-free medium RPMI 1640 (Sigma). Freshly prepared serum-free medium containing 30 µg/ml cationic lipid DOTMA (Lipofectin, Life Technologies, Inc.) was mixed with oligonucleotide (40 µg/ml) and incubated at 37 °C in a humidified incubator for 15 min to allow formation of an oligonucleotide-cationic lipid complex. Cells were cultured in the oligonucleotide-lipid medium for 5 h at 37 °C before addition of FCS to the medium at a final concentration of 10%. After 24 h cells were collected, and protein expression was evaluated by FACS analysis and by Western blot analysis.
Flow Cytometric AnalysisIndirect immunofluorescence assays on live cells were performed using purified monoclonal antibodies (mAbs) at 10 µg/ml or ascitic fluids diluted 1:100 in PBS, 1% bovine serum albumin. Cells were incubated with 100 µl of antibody for 30 min at 37 °C. After washing twice, cells were treated for 30 min at 0 °C with FITC-labeled goat anti-mouse or anti-rat Ig diluted 1:80. After a final wash, cells were suspended in PBS. Fluorescence was evaluated by FACScan using LYSYS II software (Becton Dickinson).
Cytokine Treatment of Cell CulturesCells were plated in
25-cm2 tissue culture flasks at 1 × 106
in 8 ml of 10% FCS-RPMI 1640 (Sigma). After 2 days,
culture medium was replaced by 1% FCS-RPMI 1640 (Sigma), and cells were incubated with 20 ng/ml
recombinant human tumor necrosis factor- (specific activity 5 × 106 units/mg; UBI, Lake Placid, NY) and 20 ng/ml
recombinant human interferon-
(specific activity 2 × 107 units/mg; UBI) for 48 h at 37 °C.
Confluent
cell monolayers were washed with ice-cold PBS and scraped into lysis
buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Lysis was achieved by gentle rocking for 1 h
on ice. Cell lysates were clarified by centrifugation at 16,000 × g for 15 min. For immunoprecipitation, lysates were
incubated with specific antibodies for 2 h at 4 °C with gentle
agitation. Immunocomplexes were collected on Protein G-Sepharose
(Pharmacia) and washed three times with lysis buffer. Bound proteins
were released by heating for 10 min at 95 °C in sample buffer. Total cell lysates (100 µg/lane) or immunoprecipitates were subjected to
7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and
proteins were blotted to nitrocellulose membranes (Amersham, Little
Chalfont, UK). After blocking with Blotto solution (5% dry low fat
milk in PBS), filters were probed with specific antibodies and proteins
were visualized with peroxidase-coupled secondary antibody using the
ECL detection system (Amersham). Filters were stripped in a buffer
containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM -mercaptoethanol for 30 min at 65 °C, washed three times in PBS, blocked, and reprobed with the indicated antibodies.
Modulation of 67LR and the other well characterized
laminin receptors following laminin treatment was investigated using
A431 cells, which express high levels of both 67LR and the integrin laminin receptors. A431 cells were treated with exogenous laminin at a
concentration of 50 µg/ml for different periods of time and analyzed
for cell surface laminin receptor expression by FACS analysis. After 30 min of laminin treatment, membrane expression of the 6 and
4
integrin subunits and 67LR was significantly increased, whereas the
levels of other laminin receptors such as
2 and
3 integrin
subunits remained unchanged. The up-modulation was even more evident
after 5 days of exposure to laminin, but no further increase was
observed after longer treatment (Table I). Expression of
the
1-chain was only slightly increased after long-term
treatment.
|
Treatment of different tumor
cell lines with cytokines has been reported to down-modulate the
expression of integrin subunit 6 (16, 17). We therefore investigated
whether cytokines might also modulate 67LR expression. A431 cells were
treated with a combination of 20 ng/ml TNF-
and 20 ng/ml IFN-
for
48 h and analyzed by FACS for the expression of integrins and
67LR. As shown in Fig. 1, down-modulation of
6
integrin subunit expression was accompanied by a decrease in the level
of 67LR on the cell membrane. Time-course experiments showed that
cytokine-mediated down-regulation of the protein expression was
detectable 24 h after addition of TNF-
and IFN-
to the cells
and maximal by 48 h (data not shown). No effect on class I HLA
expression was observed (Fig. 1).
Antisense Oligonucleotide Treatment of A431 Cells
Expression
of the 6 integrin subunit was down-modulated by treatment with an
antisense oligonucleotide targeted to
6 integrin subunit mRNA;
cationic liposome-mediated transfection of A431 cells with the specific
antisense led to a marked decrease in
6 cell surface expression,
which was detectable after 24 h of treatment (Fig.
2A). Antisense-induced
6 down-modulation
was accompanied by a proportional decrease in 67LR cell membrane
expression (Fig. 2C). No change in
6 and 67LR expression
was observed after transfection with the sense oligonucleotide used as
negative control (Fig. 2, panels B and D), as
well as in the amount of EGF receptor, used as unrelated control, with
either antisense or sense oligonucleotides (Fig. 2, panels E
and F). As expected, Western blot analysis of total cell
lysates obtained from antisense-treated A431 cells revealed a decreased
level of the
6 protein (Fig. 3A). By
contrast, no difference in the amount of the 67LR and of the 37-kDa
precursor was observed (Fig. 3B).
Co-purification of the 67LR with
Immunoprecipitation of A431 soluble extracts was
performed with mAbs to 6 and
1 integrin subunits and EGF
receptor. Western blot analysis of the immunoprecipitated proteins
using a mAb that recognizes the 67LR revealed 67LR in the material
immunoprecipitated with the anti-
6 integrin antibody, whereas no
co-immunoprecipitation of the 67LR was found with antibodies directed
to the
1-chain or the EGF receptor (Fig. 4).
The present study demonstrates that the 67LR and the 6 integrin
subunit are physically associated and their expression on the membrane
is co-regulated. Indeed, up- or down-modulation of
6 induced the
same change in 67LR expression.
Previous studies with small cell lung cancer cell lines demonstrated
the co-expression of 67LR and 6
1 integrin and a correlation between the level of both receptors and cell adhesion to laminin (12).
Moreover, on a human melanoma cell line treated with laminin, a
co-translocation of these two molecules to the cell membrane was
detected (13).
Our experiments demonstrate on a human epidermoid carcinoma cell line
that the 6 integrin subunit and 67LR are not only co-expressed but
also co-regulated. In A431 cells, despite the presence of the
1
subunit,
6 is found exclusively associated with
4 integrin (18).
Therefore, we can speculate that in this cell line, 67LR is
specifically associated with
6
4, in keeping with the observation that the
6 subunit can bind either the
1 or
4 subunit, but when given a choice it preferentially associates with
4 (19, 20).
Together, these data suggest that the linkage of 67LR is actually
through the 6 subunit independently of the
-chain to which this
-chain is associated, i.e.
1 in the case of small cell lung cancer cells and
4 in the case of A431 cells.
TNF- and IFN-
lead to a decrease in the expression of the
6
integrin subunit (16, 17). Our data show that decreased expression of
the
6 chain after cell treatment with a combination of the two
cytokines is accompanied by 67LR down-modulation. Even though this does
not indisputably demonstrate the association of
6 and 67LR, since
cytokine treatment can profoundly perturb the expression of many
different molecules (21-23), the data are in keeping with the results
of the antisense treatment. Indeed, the inhibition of
6 mRNA
translation followed by inhibition of
6 membrane expression, which
also led to inhibition of 67LR membrane expression, strongly indicates
the interaction between the two molecules. The absence of a decrease in
the total amount of the mature form of 67LR as well as in the total
level of the 37-kDa precursor indicates that the antisense treatment
does not influence the 67LR expression. Indeed, the interaction of 67LR
and
6 integrin subunit seems to occur at the cytoplasmic level, when
the two molecules co-translocate from the cytoplasm to the cell
surface. After anti-
6 antisense treatment the lower availability of
6 protein might be responsible for the relevant decrease of the
6-67LR complexes exported to the cell surface.
Consistently, up-modulation of the membrane expression of both 6 and
67LR after exposure to laminin, which is detectable already after 30 min of treatment, suggests an increased delivery of the two associated
receptors from the cytoplasm to the cell surface, as previously
reported (13, 24).
The close association between the two molecules raises the possibility that the two receptors are specifically involved in the same process. This hypothesis is supported by the co-immunoprecipitation experiment demonstrating that the two receptors are not only co-expressed and co-regulated but are also physically associated on the cell membrane.
The 67LR, even though it binds directly to laminin, might be an
accessory molecule for 6
1 and
6
4 integrins, acting in the
regulation of integrin binding to laminin. The interaction of laminin
with 67LR might induce some conformational changes that render the
adhesion molecule more accessible for integrin binding. The finding
that treatment of cells with the peptide YIGSR blocks cell adhesion to
laminin is consistent with this hypothesis (25). Indeed, this
five-amino acid peptide corresponding to the site on the short arm of
the laminin 1
1-chain recognized by 67LR can prevent laminin
interaction with the monomeric receptor, thus inhibiting
integrin-mediated adhesion as well.
The possible requirement for binding of laminin by both the integrin
and 67LR to obtain sufficient affinity is consistent with a recent
study demonstrating that affinity-purified 67LR reapplied to a
laminin-Sepharose column in the same conditions used for initial
purification was recovered mostly in the unbound fraction and that the
ability of the purified protein to bind laminin-Sepharose was restored
only by addition of two fractions of the low affinity eluate (26).
According to our hypothesis, these fractions might contain the 6
4
or
6
1 integrin receptor, and the combination of purified 67LR
with this material allows reconstitution of the complex able to bind
laminin with high affinity. In this light the evaluation of the
affinity of integrins and of the 67LR must take into account the
association of the two receptors, which participate together in the
recognition of laminin.
In conclusion, our data suggest that 67LR is an auxiliary molecule of
the 6 integrin, forming a complex with this molecule that can
interact with laminin with high affinity.
We thank Cristina Ghirelli for excellent technical assistance, Daniela Labadini and Laura Mameli for the preparation of the manuscript, and Mario Azzini for photographic reproduction.