©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Specific Induction of Cell Motility on Laminin by 7 Integrin (*)

(Received for publication, October 23, 1995; and in revised form, November 20, 1995)

Frank Echtermeyer Stefan Schöber Ernst Pöschl Helga von der Mark Klaus von der Mark (§)

From the Institute of Experimental Medicine, Friedrich-Alexander University, Erlangen-Nuremberg D-91054, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha7beta1 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 alpha7 integrin in cell migration on laminin, we expressed the murine alpha7B splice variant in human 293 kidney cells and 530 melanoma cells which cannot migrate on laminin and are devoid of endogenous alpha7. Northern blotting of the transfected cells showed that the alpha7 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 alpha7-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 alpha7-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 alpha7 integrin expression confers a gain of function-motile phenotype to immobile cells and may be responsible for transduction of the laminin-induced cell motility.


INTRODUCTION

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 beta1 family recognize different sites and isoforms of laminin: alpha6beta1 (9, 10) and alpha7beta1 (11, 12) bind to laminin-1, recognizing specifically the E8 domain; alpha1beta1 binds to a cryptic site in the E1 region of laminin(13) , but also to types I and IV collagen(14) . The alpha3beta1 integrin binds to laminin-5 (kalinin) (15) and to other matrix proteins; alpha2beta1 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 alpha7beta1(11, 20) , which exists in three cytoplasmic and two extracellular splice variants(21, 22) . The predominant cytoplasmic splice variant of alpha7 integrin in fetal mouse muscle and proliferating myoblasts is the alpha7B form; alpha7A is expressed in adult skeletal muscle and myotubes(22, 23) . We postulated that the alpha7bulletbeta1 complex mediates the multiple responses of myoblasts to laminin, including cell motility(11) .

In order to elucidate the role of alpha7 integrin in cell locomotion, we expressed the full-length alpha7B 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 alpha7, but beta1 integrin subunits(25, 26) which are necessary to form a heterodimeric complex on the cell surface. Here we show that, after transfection with alpha7B, 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.


MATERIALS AND METHODS

Construction of Full-length alpha7 cDNA

Rat alpha7 PCR (^1)clones Ralpha7-PCR-2 and -5 were prepared by reverse transcription-PCR from total RNA isolated from Rugli glioblastoma cells(11) ; they map to the nucleotide position 937-1843 of the extracellular domain and position 3006-3448 in the intracellular domain(22, 23) , respectively. A random- and oligo(dT)-primed cDNA library in gt11 from mouse muscle (Clontech, Catalog No. Ml 1041b) was screened with the two rat PCR clones and the oligonucleotide R33 (5`-TTCAACCTGGATGTGATGGGTGCCATACGCAAG) specific for the alpha7 N terminus. Three non-overlapping cDNA clones were isolated and identified as mouse alpha7 clones by sequence analysis. The gaps between the three mouse alpha7 cDNA clones were filled by the two mouse PCR clones Malpha7-PCR-1 and -2, synthesized by reverse transcription-PCR from total RNA isolated from C2C12 myoblasts with the following primer pairs. R33.UP, 5`-TTCAACCTGGATGTGATGGGTGCCATACGCAAG/1029.DW, 5`-GGCTGAGACACCGAAGGGACTAAGGTACAAACCC; 2226.UP, 5`-GAGCTGGAGGTGAAATTGCTGTTAGCCACG/2987.DW, 5`-CCAACACCTTCCTCAGGGGACCACCCAGTA. The complete alpha7 cDNA then was cloned using the endogenous restriction sites StuI, EcoRV, BamHI, and NsiI in the overlapping regions of cDNA and PCR clones. The full-length alpha7B cDNA construct contains parts of the 3` and 5` untranslated regions, the complete signal sequence, and the extracellular, transmembrane, and intracellular domain of the mature alpha7B chain.

Generation of alpha7-transfected Cell Lines

10^6 human embryonic kidney cells 293EBNA (Invitrogen, Catalog No. R620-07) and human melanoma cells 530 (24) were stably transfected by the calcium phosphate method (27) with either 10 µg of alpha7B cDNA cloned into the episomal expression vector pCEP4 (Invitrogen, Catalog No. V044-50) or with 10 µg of the vector pCEP4 only. alpha7-transfected cells (293EBNA-alpha7B and 530-alpha7B) and mock-transfected cells (293EBNA-pCEP4 and 530-pCEP4) were selected with 300 or 50 µg/ml hygromycin, respectively, and the expression of alpha7 mRNA and protein was analyzed by Northern blotting and FACS analysis, respectively.

Northern Blot Analysis

RNA was prepared by guanidinium thiocyanate/acid phenol extraction procedure (28) from murine C2C12 myoblasts, human HT1080 fibrosarcoma cells, alpha7- and mock-transfected human kidney cells 293 and 530 cells. 12 µg of total RNA each was size-fractionated on 1.3% agarose/formaldehyde gels and transferred to nylon membranes. The 1050-base pair PCR fragment Malpha7-PCR-1 was synthesized by PCR, gel-purified, and labeled by random-primed synthesis with [P]dCTP. Nylon membranes were hybridized in a solution consisting of 5 times SSC and 1% N-lauroylsarcosine at 65 °C and washed first in 3 times SSC, 0.5% N-lauroylsarcosine at 65 °C and second in 3 times SSC at 65 °C.

FACS Analysis

Surface expression of alpha7 was analyzed in a fluorescence-activated cell sorter (FACS). 2 times 10^5 cells each were incubated for 30 min at 4 °C with a 1/100 dilution of an affinity-purified rabbit antibody raised against a recombinant alpha7-integrin peptide. (^2)After washing with phosphate-buffered saline containing 5% fetal calf serum and 0.01% sodium azide cells were counterstained with a 1/200 dilution of fluorescein isothiocyanate-labeled goat anti-rabbit Ig (Dianova); fluorescence intensity was measured within 1 h using a FACS (Epic, Inc.).

Attachment Assay

96-well plates (Maxisorb, Nunc) were coated with serial dilutions of laminin, E8, and fibronectin starting with 10 µg/ml for 1 h at 37 °C. Residual protein binding sites were then blocked with heat-treated (10 min at 80 °C) 2% BSA overnight at 4 °C. Cells were seeded at a density of 5 times 10^4 cells per well in 100 µl of DMEM, 0.5% BSA. After a 1-h incubation at 37 °C, unattached cells were removed by phosphate-buffered saline washing, and the attached cells were assayed by colorimetric determination of endogenous hexosamidase activity(11, 13) .

Cell Migration Assays

Cell migration was measured by time-lapse videomicroscopy and in the Boyden chamber. For videomicroscopy, 15-cm^2 tissue culture flasks (Falcon labware) were coated with FN, LN-1 (5 µg/ml), or E8 (3 µg/ml) for 1 h at 37 °C and blocked with 2% BSA overnight at 4 °C. Cells were seeded at a density of 2000 cells/cm^2 in DMEM/F12 medium containing 20 mM HEPES, 5% fetal calf serum, 250 µg/ml Geneticin and 300 µg/ml hygromycin. After adhesion and spreading for 4 h at 37 °C, flasks were sealed to maintain CO(2) atmosphere. The cells were filmed by time-lapse video microscopy (1 picture/2 min) using a JVC BR-900V recorder and a Zeiss inverted microscope placed in a 37 °C tempered hood for 15 h. The tracks of single, randomly chosen cells were evaluated by exporting video signals to a graphics program (McDraw Pro, Claris). For each plot, the paths of 10 cells were measured, normalized, and converted to a wind rose display where all cells start from the same point in order to illustrate the average motility(19) .

Cell migration assays in Boyden chambers were carried out as described by Albini et al.(29) ; alpha7- and vector-transfected cells were resuspended in serum-free DMEM medium in the presence of 0.5% BSA; 10^4 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(2). 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.


RESULTS AND DISCUSSION

A full-length murine alpha7B 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 alpha7 integrin message was measured by Northern blotting (Fig. 2). The alpha7-specific probe hybridized to a 4.1-kb alpha7 mRNA band in the RNA of murine C2C12 myoblasts, of 293-alpha7B and 530-alpha7B cells, but not to the RNA of mock-transfected cells or HT 1080 fibrosarcoma cells.


Figure 1: Construct for eucaryotic expression of murine alpha7 integrin. Full-length alpha7 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 alpha7 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 alpha7 cDNA (293EBNA-alpha7B and 530-alpha7B), or with the pCEP4 vector only (293EBNA-pCEP4 and 530-pCEP4) were separated by electrophoresis, blotted onto nitrocellulose, and hybridized with a murine alpha7 cDNA probe. Only C2C12 cells and the alpha7-transfected 293 and 530 cells show the 4.1-kb band characteristic for alpha7 mRNA.



Immunofluorescence staining with an affinity-purified polyclonal antibody raised against the recombinant extracellular domain of alpha7^2 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 alpha7 chain must have associated with an endogenous human beta-integrin chain to form heterodimeric complexes. When plated onto laminin-coated dishes, about 15% of the alpha7-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 alpha7- 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-alpha7B and 530-alpha7B 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 alpha7-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 alpha7-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 alpha7beta1 integrin analyzed in a fluorescence-activated cell sorter (FACS) after staining with an affinity-purified rabbit antibody raised against recombinant alpha7 integrin peptide. 293 kidney cells stably transfected with the alpha7B (293EBNA-alpha7B) 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 alpha7-transfected cells show strong surface fluorescence. - - -, nonimmune serum; -, alpha7 immune serum.






Figure 4: Adhesion of alpha7- 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) . circle-circle, 293EBNA-alpha7B; bullet-bullet, 293EBNA-pCEP4.




Figure 5: Videomicroscopy of alpha7- (293EBNA-alpha7B) 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 alpha7 expression was tolerant to the cell background. When alpha7 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 alpha7 was considerably lower in the 530 cells than in the 293 cells that are stably transfected with EBNA. In comparison to 293 EBNA-alpha7 cells, hardly any surface fluorescence of alpha7 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 alpha7 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 10^4 alpha7- 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 alpha7-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 alpha7- and mock-transfected cells (Fig. 6); no migration of cells was observed on filters coated with BSA alone. These results confirmed the ability of alpha7 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 alpha7- and mock- transfected 293 cells through laminin-1 - and fibronectin-coated nuclepore filters in a Boyden chamber. 10^4 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 alpha7 integrin.

Our data indicate that alpha7beta1 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 alpha7-expressing and nonexpressing cells. In fact, there is no experimental evidence yet in the literature that alpha7 integrin is involved in cell adhesion to laminin. Up to now, no adhesion-blocking antibodies to alpha7 integrin are available. The best evidence for alpha7beta1 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 alpha6 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 alpha-dystroglycan, a major laminin receptor of muscle cells binding to the E3 fragment of laminin(31) .

The locomotion of the alpha7-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 alpha7-transfected 293 cells showed enhanced outgrowth of cell processes and were more elongated on laminin than their mock-transfected counterparts (Table 1), indicating that alpha7 is responsible for this activity of laminin.

Although alpha7 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 alpha6 but not alpha7 integrin. (^3)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 alpha3 and beta1 integrin chains. Increased expression of alpha3beta1 integrin has been also associated with enhanced tumor cell metastasis(39) . Smooth muscle cells utilize alphavbeta3 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 alpha2-chains carrying the cytoplasmic domain of alpha4 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 alpha7 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 alpha7- and mock-transfected cells, but there was no stimulation after alpha7 transfection.

The alpha7B 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 alpha7-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 alpha7-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 alpha7B 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 alpha7B- 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 alpha7A domain (21, 22, 23) also enhance cell motility on laminin.


FOOTNOTES

*
This work was supported by the generous financial support of Wilhelm-Sander Foundation Grant 92.057.1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Experimental Medicine, Friedrich-Alexander-University, Schwabachanlage 10, D-91054 Erlangen, Germany. Tel.: 49-9131-85-9104; Fax: 49-9131-85-6341.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; FACS, fluorescence-activated cell sorter; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; kb, kilobase(s); EBNA, Epstein-Barr nuclear antigen.

(^2)
H. von der Mark, F. Echtermeyer, E. Pöschl, H. Moch, and K. von der Mark, manuscript in preparation.

(^3)
S. Schöber et al., manuscript in preparation.


ACKNOWLEDGEMENTS

We wish to thank Helga Moch for expert help in the cell cultures, Dr. Peter Rohwer (Institute for Clinical Immunology, Friedrich-Alexander-University, Erlangen-Nuremberg) for introducing us into the technique of FACS analysis, and Dr. Simon Goodman for correcting the English.


REFERENCES

  1. Liotta, L. A. (1983) Invest. 49, 636-649
  2. Liotta, L. A. (1986) Cancer Res. 46, 1-7 [Medline] [Order article via Infotrieve]
  3. Albelda, S. M. (1993) Lab. Invest. 68, 4-16 [Medline] [Order article via Infotrieve]
  4. Goodman, S. L. (1993) in Molecular & Cell Aspects of Basement Membranes (Timpl, R., ed) pp. 345-358, Academic Press, New York
  5. Gee, S. H., Blacker, R. W., Douville, P. J., Provost, P. R., Yurchenco, P. D., and Carbonetto, S. (1993) J. Biol. Chem. 268, 14972-14980 [Abstract/Free Full Text]
  6. Liotta, L., Rao, C. N., and Wewer, U. M. (1986) Annu. Rev. Biochem. 55, 1037-1057 [CrossRef][Medline] [Order article via Infotrieve]
  7. von der Mark, K., and Risse, G. (1987) Methods Enzymol. 144, 490-507 [Medline] [Order article via Infotrieve]
  8. Sobel, M. (1993) Semin. Cancer Biol. 4/5, 311-318
  9. Sonnenberg, A., Modderman, P. W., and Hogervorst, F. (1988) Nature 336, 487-489 [CrossRef][Medline] [Order article via Infotrieve]
  10. Sonnenberg, A., Linders, C. J. T., Modderman, P. W., Damsky, C. H., Aumailley, M., and Timpl, R. (1990) J. Cell Biol. 110, 2145-2155 [Abstract]
  11. von der Mark, H., Dürr, J., Sonnenberg, A., von der Mark, K., Deutzmann, R., and Goodman, S. L. (1991) J. Biol. Chem. 266, 23593-23601 [Abstract/Free Full Text]
  12. Kramer, R. H., McDonald, K. A., and Wu, M. P. (1989) J. Biol. Chem. 264, 15642-15649 [Abstract/Free Full Text]
  13. Goodman, S. L., Deutzmann, R., and von der Mark, K. (1987) J. Cell Biol. 105, 589-598 [Abstract]
  14. Kern, A., Eble, J., Golbik, R., and Kühn, K. (1993) Eur. J. Biochem. 215, 151-159 [Abstract]
  15. Delwel, G. O., deMelker, A. A., Hogervorst, F., Jaspars, L. H., Fles, D. L., Kuikman, I., Lindblom, A., Paulsson, M., Timpl, R., and Sonnenberg, A. (1994) Mol. Biol. Cell 5, 203-215 [Abstract]
  16. Gullberg, D., Terracio, L., Borg, T. K., and Rubin, K. (1989) J. Biol. Chem. 264, 12686-12694 [Abstract/Free Full Text]
  17. Languino, L. R., Gehlsen, K. R., Wagner, E., Carter, W., Engvall, E., and Ruoslahti, E. (1989) J. Cell Biol. 190, 2455-2462
  18. Öcalan, M., Goodman, S. L., Kühl, U., and Hauschka, S. D. (1986) Dev. Biol. 125, 158-169
  19. Goodman, S. L., Risse, G., and von der Mark, K. (1989) J. Cell Biol. 109, 799-809 [Abstract]
  20. Song, W. K., Wang, W., Foster, R. F., Bielser, D. A., and Kaufmann, S. J. (1992) J. Cell Biol. 117, 643-659 [Abstract]
  21. Song, W. K., Wang, W., Sato, H., Bielser, D. A., and Kaufmann, S. J. (1993) J. Cell Sci. 106, 1139-1152 [Abstract/Free Full Text]
  22. Ziober, B., Wu, M. P., Waleh, N., Crawford, J., Lin, C. S., and Kramer, R. H. (1993) J. Biol. Chem. 268, 26773-26783 [Abstract/Free Full Text]
  23. Collo, G., Starr, L., and Quaranta, V. (1993) J. Biol. Chem. 268, 19019-19024 [Abstract/Free Full Text]
  24. Klein, C. E., Dressel, D., Steinmayer, T., Mauch, C., Eckes, B., Krieg, T., Bankert, R. B., and Weber, L. (1991) J. Cell Biol. 115, 1427-1436 [Abstract]
  25. Bodary, S. C., and McLean, J. W. (1990) J. Biol. Chem. 265, 5938-5941 [Abstract/Free Full Text]
  26. Yokosaki, Y., Palmer, E. L., Prieto, A. L., Crossin, K. L., Bourdon, M. A., Pytela, R., and Sheppard, D. (1994) J. Biol. Chem. 269, 26691-26696 [Abstract/Free Full Text]
  27. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752 [Medline] [Order article via Infotrieve]
  28. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  29. Albini, A., Allavena, G., Melchiori, A., Giancotti, F., Richter, H., Comoglio, P. M., Parodi, S., Martin, G. R., and Tarone, G. R. (1987) J. Cell Biol. 105, 1867-1872 [Abstract]
  30. Kramer, R. H., Vu, M. P., Cheng, Y.-F., Ramos, D. M., Timpl, R., and Waleh, N. (1991) Cell Regul. 2, 805-817 [Medline] [Order article via Infotrieve]
  31. Durbeej, M., Larsson, E., Ibraghimov-Beskrovnaya, O., Roberds, S. L., Campbell, K. P., and Ekblom, P. (1995) J. Cell Biol. 130, 79-91 [Abstract]
  32. Van Evercooren, A., Kleinman, H. K., Ohno, S., Merangos, P., Schwartz, J. P., and Dubois-Dalq, M. E. (1982) J. Neurosci. Res. 8, 179-194 [Medline] [Order article via Infotrieve]
  33. Rogers, S. L., Letourneau, P. C., Palm, S. L., McCarthy, J., and Furcht, L. F. (1984) J. Cell Biol. 98, 212-220
  34. McGarvey, M. L., van Evercooren, A., Kleinmann, H. K., and Dubois-Dalq, M. (1984) Dev. Biol. 105, 18-28 [Medline] [Order article via Infotrieve]
  35. McCarthy, J. B., Palm, S. L., and Furcht, L. T. (1983) J. Cell Biol. 97, 772-777 [Abstract]
  36. McCarthy, J. B., and Furcht, L. T. (1984) J. Cell Biol. 98, 1474-1480 [Abstract]
  37. Mensing, H., Albini, A., Krieg, T., Pontz, B. F., and Müller, P. K. (1984) Int. J. Cancer 33, 43-48 [Medline] [Order article via Infotrieve]
  38. Melchiori, A., Mortarini, R., Carlone, S., Marchisio, P. C., Aninchini, A., Noonan, D., and Albini, A. (1995) Exp. Cell Res. 219, 233-242 [CrossRef][Medline] [Order article via Infotrieve]
  39. Natali, P. G., Nicotra, M. R., Bartolazzi, A., Cavaliere, R., and Bigotti, A. (1993) Int. J. Cancer 54, 68-72 [Medline] [Order article via Infotrieve]
  40. Werb, I, Tremble, P. M., Berendtzen, O., Crowley, E., and Damsky, C. H. (1989) J. Cell Biol. 109, 877-889 [Abstract]
  41. Huttenlocher, A., Sandberg, R. R., and Horwitz, A. F. (1995) Curr. Opin. Cell Biol. 7, 697-706 [CrossRef][Medline] [Order article via Infotrieve]
  42. Liaw, L., Skinner, M. P., Raines, E. W., Ross, R., Cheresh, D. A., Schwartz, S. M., and Giachelli, C. M. (1995) J. Clin. Invest. 95, 713-724 [Medline] [Order article via Infotrieve]
  43. Leavesley, D. I., Schwartz, M. A., Rosenfeld, M., and Cheresh, D. A. (1993) J. Cell Biol. 121, 163-170 [Abstract]
  44. Chan, B. M., Kassner, P. D., Schiro, J. A., Byers, H. R., Kupper, T. S., and Hemler, M. E. (1992) Cell 68, 1051-1060 [Medline] [Order article via Infotrieve]
  45. Christ, B., Jacob, M., and Jacob, H. J. (1983) Anat. Embryol. 166, 87-101 [Medline] [Order article via Infotrieve]
  46. Bischoff, R. (1986) Dev. Biol. 115, 129-139 [Medline] [Order article via Infotrieve]
  47. Trinkaus, J. P. (1985) Neurosci. Res. 13, 1-19
  48. Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-239 [Medline] [Order article via Infotrieve]
  49. Yamada, K. M., and Miyamoto, S. (1995) Curr. Opin. Cell Biol. 7, 681-689 [CrossRef][Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.