Co-regulation and Physical Association of the 67-kDa Monomeric Laminin Receptor and the alpha 6beta 4 Integrin*

(Received for publication, July 17, 1996, and in revised form, October 11, 1996)

Elena Ardini Dagger , Elda Tagliabue Dagger , Alessandra Magnifico Dagger , Simona Butò Dagger , Vincent Castronovo §, Maria I. Colnaghi Dagger and Sylvie Ménard Dagger

From the Dagger  Division of Experimental Oncology E, Istituto Nazionale Tumori, 20133 Milan, Italy and the § Metastasis Research Laboratory, University of Liège, B-400 Liège 1, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 alpha 6 integrin subunit but not with the expression of other laminin receptors. Moreover, cytokine treatment resulted in down-modulated expression of the alpha 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 alpha 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 alpha 6 subunit with an anti-alpha 6 but not an anti-beta 1 monoclonal antibody. Co-regulation of 67LR and alpha 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 alpha 6beta 4 integrin.


INTRODUCTION

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 alpha  and beta  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 alpha 6beta 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 alpha 6 and beta 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.


EXPERIMENTAL PROCEDURES

Cell Lines and Culture Conditions

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 Antibodies

The following monoclonal antibodies were used as purified Ig: MLuC5, directed against the 67-kDa laminin receptor (14); GoH3, directed against alpha 6-chain integrin (Immunotech, Marseille, France); P1E6, directed against alpha 2-chain integrin (Telios, San Diego, CA); P1B5, directed against alpha 3-chain integrin (Telios); 3E1, directed against beta 4-chain integrin (Telios); MAR4, directed against beta 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 Synthesis

Unmodified DNA oligonucleotides corresponding to integrin subunit alpha 6 nucleotides 142-160 (18-mer) were synthesized on an automated synthesizer (Cyclone Plus DNA Synthesizer, Millipore Corp.). The sense and antisense alpha 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, M urea, 45 mM Tris borate buffer, pH 7.0.

Oligonucleotide Treatment of Cells

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 Analysis

Indirect 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 Cultures

Cells 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-alpha (specific activity 5 × 106 units/mg; UBI, Lake Placid, NY) and 20 ng/ml recombinant human interferon-gamma (specific activity 2 × 107 units/mg; UBI) for 48 h at 37 °C.

Immunoprecipitation and Western Blot Analysis

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 beta -mercaptoethanol for 30 min at 65 °C, washed three times in PBS, blocked, and reprobed with the indicated antibodies.


RESULTS

Effect of Laminin Treatment on Laminin Receptor Expression

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 alpha 6 and beta 4 integrin subunits and 67LR was significantly increased, whereas the levels of other laminin receptors such as alpha 2 and alpha 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 beta 1-chain was only slightly increased after long-term treatment.

Table I.

Effect of laminin treatment on laminin receptor expression

A431 cells, treated with exogenous soluble laminin for indicated times, were analyzed by indirect immunofluorescence for the expression of different laminin receptors.
Time of treatment Integrin subunits
67LR
 alpha 2  alpha 3  alpha 6  beta 1  beta 4

0 min 31  ± 5a 97  ± 15 68  ± 7 241  ± 20 73  ± 9 975  ± 47
30 min 27  ± 9 91  ± 10 102  ± 12 250  ± 23 97  ± 10 1150  ± 50
5 days 35  ± 5 102  ± 15 125  ± 14 273  ± 25 152  ± 20 1412  ± 73
30 days 32  ± 7 103  ± 10 130  ± 15 268  ± 20 160  ± 15 1430  ± 68

a  Mean fluorescence intensity ± S.D. obtained from three experiments by immunofluorescence assay and FACScan analysis.

Down-modulation of alpha 6 Integrin Subunit and 67-kDa Laminin Receptor after Cytokine Treatment

Treatment of different tumor cell lines with cytokines has been reported to down-modulate the expression of integrin subunit alpha 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-alpha and 20 ng/ml IFN-gamma for 48 h and analyzed by FACS for the expression of integrins and 67LR. As shown in Fig. 1, down-modulation of alpha 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-alpha and IFN-gamma to the cells and maximal by 48 h (data not shown). No effect on class I HLA expression was observed (Fig. 1).


Fig. 1. Effect of TNF-alpha and IFN-gamma on expression of alpha 6 integrin subunit and 67LR by A431 cells. Cells (1 × 106) were treated with a combination of TNF-alpha (20 ng/ml) and IFN-gamma (20 ng/ml) for 48 h (light line). Expression of alpha 6 integrin subunit (panel A), 67LR (panel B), and HLA (panel C) was analyzed by indirect immunofluorescence using mAbs MAR6, MLuC5, and W6/32 and FITC-conjugated goat anti-mouse Ig. The solid area and bold line show the values of marker expression in the absence of cytokine treatment and background, respectively.
[View Larger Version of this Image (19K GIF file)]


Antisense Oligonucleotide Treatment of A431 Cells

Expression of the alpha 6 integrin subunit was down-modulated by treatment with an antisense oligonucleotide targeted to alpha 6 integrin subunit mRNA; cationic liposome-mediated transfection of A431 cells with the specific antisense led to a marked decrease in alpha 6 cell surface expression, which was detectable after 24 h of treatment (Fig. 2A). Antisense-induced alpha 6 down-modulation was accompanied by a proportional decrease in 67LR cell membrane expression (Fig. 2C). No change in alpha 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 alpha 6 protein (Fig. 3A). By contrast, no difference in the amount of the 67LR and of the 37-kDa precursor was observed (Fig. 3B).


Fig. 2. Effect of antisense oligonucleotide treatment of A431 cells on membrane expression of alpha 6 integrin subunit and 67LR. 24 h after plating, cells (1 × 105) were treated for 5 h with antisense (panels A, C, E) or sense (panels B, D, F) oligonucleotides at a concentration of 40 µg/ml in the presence of cationic liposome DOTMA (30 µg/ml) and maintained in FCS-containing medium for 24 h. Expression of alpha 6 integrin subunit (panels A and B), 67LR (panels C and D), and EGF receptor (panels E and F) was analyzed by indirect immunofluorescence using mAbs MAR6, MLuC5, and MGR1, respectively, and FITC-conjugated goat anti-mouse Ig. Bold and light lines and solid area show the values of background, treated cells, and marker expression in the absence of oligonucleotide treatment, respectively.
[View Larger Version of this Image (31K GIF file)]



Fig. 3. Effect of antisense oligonucleotide treatment of A431 cells on protein expression of alpha 6 integrin subunit and 67LR. 24 h after plating, cells (1 × 105/well) were treated for 5 h with antisense oligonucleotide at a concentration of 40 µg/ml in the presence of cationic liposome DOTMA (30 µg/ml, lane 2) or with cationic liposome DOTMA alone (30 µg/ml, lane 1) and maintained in FCS-containing medium for 24 h. Total lysates were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Western blot analysis was performed using anti-alpha 6 antibody (panel A). The filter was stripped and reprobed with anti-37/67LR MPLR 2 (panel B).
[View Larger Version of this Image (38K GIF file)]


Co-purification of the 67LR with alpha 6 Integrin Subunit

Immunoprecipitation of A431 soluble extracts was performed with mAbs to alpha 6 and beta 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-alpha 6 integrin antibody, whereas no co-immunoprecipitation of the 67LR was found with antibodies directed to the beta 1-chain or the EGF receptor (Fig. 4).


Fig. 4. Co-purification of 67LR with the integrin alpha 6 chain. Non-ionic detergent extracts of A431 cells were subjected to immunoprecipitation with anti-alpha 6, anti-beta 1, or anti-EGF receptor-specific antibodies. The immunoprecipitated proteins were resolved by 7.5% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Western blot analysis was performed using anti-37/67LR mAb MPLR2 (panel A). The filter was stripped and reprobed with anti-alpha 6, anti-beta 1, or anti- EGF receptor-specific antibodies (panel B). Molecular masses (in kDa) are shown on the right.
[View Larger Version of this Image (35K GIF file)]



DISCUSSION

The present study demonstrates that the 67LR and the alpha 6 integrin subunit are physically associated and their expression on the membrane is co-regulated. Indeed, up- or down-modulation of alpha 6 induced the same change in 67LR expression.

Previous studies with small cell lung cancer cell lines demonstrated the co-expression of 67LR and alpha 6beta 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 alpha 6 integrin subunit and 67LR are not only co-expressed but also co-regulated. In A431 cells, despite the presence of the beta 1 subunit, alpha 6 is found exclusively associated with beta 4 integrin (18). Therefore, we can speculate that in this cell line, 67LR is specifically associated with alpha 6beta 4, in keeping with the observation that the alpha 6 subunit can bind either the beta 1 or beta 4 subunit, but when given a choice it preferentially associates with beta 4 (19, 20).

Together, these data suggest that the linkage of 67LR is actually through the alpha 6 subunit independently of the beta -chain to which this alpha -chain is associated, i.e. beta 1 in the case of small cell lung cancer cells and beta 4 in the case of A431 cells.

TNF-alpha and IFN-gamma lead to a decrease in the expression of the alpha 6 integrin subunit (16, 17). Our data show that decreased expression of the alpha 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 alpha 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 alpha 6 mRNA translation followed by inhibition of alpha 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 alpha 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-alpha 6 antisense treatment the lower availability of alpha 6 protein might be responsible for the relevant decrease of the alpha 6-67LR complexes exported to the cell surface.

Consistently, up-modulation of the membrane expression of both alpha 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 alpha 6beta 1 and alpha 6beta 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 beta 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 alpha 6beta 4 or alpha 6beta 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 alpha 6 integrin, forming a complex with this molecule that can interact with laminin with high affinity.


FOOTNOTES

*   This work was supported in part by grants from AIRC/FIRC and CNR ACRO. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Tel.: 39-2-2390573; Fax: 39-2-2362692.
1    The abbreviations used are: 67LR, 67-kDa laminin receptor; FCS, fetal calf serum, EGF, epidermal growth factor; FITC, fluorescein isothiocyanate; DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; PBS, phosphate-buffered saline; mAb, monoclonal antibody; HLA, human leukocyte antigen A, B, C loci; FACS, fluorescence-activated cell sorter; TNF-alpha , tumor necrosis factor-alpha ; IFN-gamma , interferon-gamma .
2    S. Butò, E. Tagliabue, E. Ardini, A. Magnifica, V. Castronovo, M. I. Colnaghi, M. Sobel, and S. Ménard, manuscript in preparation.

Acknowledgments

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.


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