Correspondence to: Guerrino Meneguzzi, U385 INSERM, Faculté de Médecine, Cedex 2, 06107 Nice, France. Tel:(33) 493-37-77-78 Fax:(33) 493-81-14-04 E-mail:meneguzz{at}unice.fr.
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Abstract |
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Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the 2 chain that removes the NH2-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the
2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the
2 chain short arm, we expressed mutated
2 cDNAs into immortalized
2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the
2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the
2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.
Key Words: keratinocyte, epithelial adhesion, cell migration, basement membrane, epidermolysis bullosa
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Introduction |
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The epithelial basement membranes consist of a complex network of extracellular matrix (ECM)1 molecules that mediate tissue integrity and homeostasis, and control morphogenesis as well as tissue repair and tumorigenesis. Laminins are multifunctional glycoproteins of the ECM that contribute to the architecture of the basement membranes and play a crucial role in cell adhesion, growth, migration, and differentiation (, ß, and
chains on the basis of their primary structure deduced from sequence data. Laminin isoforms are expressed at various stages of development and in specific tissue locations in different species. In the human epidermis, basal keratinocytes express laminin 5 (
3ß3
2), laminin 6 (
3ß1
1), and laminin 10 (
5ß1
1) (
Laminin 5 is found in the basement membrane of stratified and transitional epithelia, prevalently associated with the extracellular anchoring filaments of the lamina lucida that connect the hemidesmosomes to the anchoring fibrils of the underlying stroma (
Current models propose that laminin 5 mediates epithelial cell adhesion via integrin 3ß1 in focal adhesions and integrin
6ß4 in hemidesmosomes (
3, ß3, and
2 chains are substantially truncated and do not conserve the domains known to direct integration of laminins into the basement membrane architecture (
6ß4 to type VII collagen and provide the significant force that promotes cohesion of the dermis and epidermis, whereas laminin 5 complexed to laminins 6 and 7 interacts with integrin
3ß1 and contributes to assembly and stability of the basement membrane (
2 short arms (
3 chain (
2 chain associates mouse laminin 5 to the matrix proteins (
In the ECM, laminin 5 is found in two most abundant forms of 440 and 400 kD. The 440-kD heterotrimer is generated by proteolytic cleavage of the 3 chain present in the 460-kD cell-associated form of laminin 5 (
3 that excises the COOH terminus at a cleavage site within the subdomain G4, and proteolytic cleavage within the EGF-like repeat 2 of domain IIIa, have also been reported (
3 chain G domain is thought to modulate the interactions of laminin 5 with the integrin cell receptors and to govern cell anchorage and motility (
Extracellular processing also removes the globular domain IV and the EGF-like rich domain V of the laminin 2 short arm. Excision of the 434 NH2-terminal amino acids shortens the
2 chain from 155 to 105 kD, and reduces the size of laminin 5 to 400 kD (
3, 165 kD; ß3, 140 kD;
2, 105 kD). Therefore, this processed 400-kD form of the laminin 5 lacks most of the
2 chain short arm, but leaves the ß3 short arm intact. The functional role of the
2 cleavage is unknown and the biological functions specific to each extracellular form of laminin 5 remain obscure.
Extracellular 440-kD laminin 5 has so far been considered a precursor form of the fully processed laminin 5. To define the functions of this molecule, and to identify the possible biological role of the 2 short arm excised by the proteolytic processing, we have transferred mutated laminin
2 cDNAs into immortalized
2-null keratinocytes. In this study, we report that the laminin
2 short arm is required for deposition of laminin 5 into the ECM, and we provide evidence that the unprocessed 440-kD form of laminin 5 sustains cell adhesion and inhibits cell migration.
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Materials and Methods |
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cDNA Constructs
Mutated laminin 2 constructs were obtained by site-directed mutagenesis of a full length
2 cDNA cloned in the expression vector p
WT (
2 cDNA (see Fig 1 B). The presence of the T7-Tag does not affect the laminin 5 function (data not show). To construct mutant p
NC, two
2 cDNA fragments of 1,308 and 476 bp (comprising nt 11293 and 13051767 of the laminin
2 cDNA, respectively; EMBL/GenBank/DDBJ accession no.
X73920) were PCR amplified using plasmid p
WT as a template. The 1,308-bp cDNA fragment was amplified using primers A, left and B, right (Table 1). The reaction mixture (25 µl) contained 10 ng of the template, 400 nM of each primer, 20 mM Tris-HCl, pH 8.8, 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 100 µg/ml nuclease-free BSA, 200 mM dNTP, and 2.5 U of Pfu DNA polymerase (Stratagene). For amplification of the 476-bp cDNA fragment, primers were: C, left, and D, right. The primary PCR products were mixed and used as templates for a secondary PCR program using primers A and D in a reaction mixture of 25 µl. The PCR products were electrophoresed on a 1% agarose gel, eluted, mixed together, cleaved with the restriction endonucleases BsmBI and EcoRI, and cloned into a BsmBI-EcoRIdigested p
WT vector. To generate p
C, a
2 cDNA with an internal deletion of 1,208 bp (nt 130294) was prepared by PCR amplification of two
2 cDNA fragments: a 107-bp
2 cDNA fragment (nt 193) using primer A, left, and E, right, and a 478-bp
2 cDNA fragment (nt 1,3031,766) using primers F, left, and D, right. The amplification products were subjected to a secondary PCR amplification using primers A and D. The resulting cDNA products were cloned into a BsmBI-EcoRIdigested p
WT plasmid as described above. To obtain plasmid p
M, a 1,081-bp (nt 11065) and a 496-bp (nt 12861766) cDNA fragments were amplified using the primer pair A, left, and G, right and pair H, left and D, right, respectively. After a secondary PCR amplification using primers A and D, the resulting amplimers were cloned into plasmid p
WT as described above. To generate the mutant p
III, which bears an internal deletion of 101 bp, two
2 cDNA fragments of 1,159 and 536 bp were PCR amplified using the primers pair A, left and I, right, respectively, and primer pair J, left and D, right. After a secondary PCR amplification using primers A and D, the resulting amplimers were cloned into plasmid p
WT as described above. To construct p
V, which contains an internal deletion of 242 bp, p
WT was double digested with Bsu36I and BspEI (equals AccIII), blunted, and then religated. Plasmid p
F1 was generated using the QuikChangeTM site-directed mutagenesis kit (Stratagene) and primers K, left and L, right to substitute the amino acid residues SVHKI (residues 203207) with five alanines. Plasmid p
F2 was generated from p
F1, using the QuikChangeTM kit and the oligonucleotide pair M, left and N, right to subsitute the peptidic sequence SAEYSVHKI (residues 199207) with nine alanines. Plasmid p
GP was generated using the QuikChangeTM kit and primers O, left and P, right to delete the amino acid residues YS (residues 432433) and substitute the amino acid residues GD (residues 434435) with the amino acid residues GP. The conformational changes of the
2 chain introduced by the GP substitution were assessed using the programs Biopolymer (Molecular Simulations, Inc.) and Antheprot (http://www.ibcp.fr).
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To generate plasmid p50, the 1,302-bp
2 cDNA fragment (nt 11302) coding for the NH2-terminal domains of the laminin
2 chain was subcloned into the BamH1-EcoRV sites of the expression vector pcDNA3, upstream the T7-Tag. To construct plasmid pß60 the 1,574-bp cDNA sequence encoding the NH2-terminal domains of the laminin ß3 chain (nt 11574; EMBL/GenBank/DDBJ accession no.
L25541) was subcloned into pcDNA3 upstream the hemagglutinin (HA) Tag sequence (YPYDVPDYA).
The plasmids were amplified in Escherichia coli XL1blue (Stratagene), purified using a plasmid purification kit (QIAGEN), and analyzed by nucleotide sequencing using an ABI Prism 310 genetic analyzer.
Antibodies
mAbs used in the study were: K140, specific to laminin ß3 (
Expression of laminin 2 chain was examined using polyclonal antibody (pAb) SE144 (
2 short arm. To prepare the fusion protein, the
2 cDNA fragment was excised from plasmid p
50 and subcloned into the EcoRI site of the procaryotic expression vector pGEX-5X-3 from Amersham Pharmacia Biotech. The purified GST fusion protein was used for rabbit immunization. pAb SE1097 was then purified by immunoaffinity chromatography against the corresponding fusion protein immobilized on nitrocellulose filters after SDS-PAGE. The second antibodies coupled to tetramethylrhodamine isothiocyanate or to fluoresceine isothiocyanate were obtained from Dako. Actin cytoskeleton was labeled using phalloidin Texas red (Molecular Probes, Inc.).
Cells and Organotypic Cell Cultures
The human keratinocytes cell lines HKSV and LSV5 were grown in a 3:1 mixture of DME and Ham's F-12 medium (Life Technologies and GIBCO BRL) containing FCS (10%), hydrocortisone (0.4 µg/ml), cholera toxin (0.1 nM), and EGF (10 ng/ml) (
To construct artificial epithelia, cultured keratinocytes (1.6 x 107 cells) were seeded in stainless steel rings (0.6 cm2), laid on acetate cellulose filters (Millipore), and maintained immersed in growth medium changed every other day. 4 d later, the medium in the rings was removed and the keratinocyte culture was brought at the airliquid interface for 4 d to induce differentiation (
DNA Transfections
For transient expression, subconfluent keratinocyte cultures were transfected using the polycationic lipid Dosper (1,3-Di-Oleoyloxy-2,6-[carboxy-spermyl]-propyl-amide) from Roche. In brief, before transfection the cell cultures were incubated at 37°C with medium deprived of serum and growth factors. DNA and Dosper were separately diluted in Hepes-buffered saline (20 mM Hepes, 150 mM NaCl, pH 7.4), then mixed at a final concentration of 0.2 and 0.1 µg/ml, respectively, and added dropwise to the cell layers. After 6 h at 37°C, cells were fed with medium containing serum and growth factors two times concentrated. Transfection efficiency and expression of the transfected genes was monitored 48 h later using indirect immunofluorescence assays.
To establish cell lines LNC, L
C, L
GP, L
F1, and L
F2 using plasmids p
NC, p
C, p
GP, p
F1, and p
F2, respectively, calcium-phosphate transfection of LSV5 cells was performed using the MBSTM mammalian transfection kit (Stratagene). 1.5 x 105 cells were seeded in 100-mm petri dishes and incubated for 24 h at 37°C. 20 µg of plasmid DNA precipitated for 15 min at room temperature in BES buffer (50 mM N,N-Bis2hydroxyethyl-2-aminoethan sulfonic acid, 250 mM NaCl, 1.5 mM Na2HPO4, pH 6.95) containing 125 mM CaCl2 was added dropwise to the cell layers. The cell cultures were incubated for 3 h in DME supplemented with 5% modified bovine serum provided with the transfection kit, then rinsed and fed with DME/Ham's F12 medium. Selection for neoresistance to geneticin (400 µg/ml; Sigma-Aldrich) was started 48 h later.
Immunofluorescence Microscopy
Immunofluorescence analysis of subconfluent cell layers grown on glass coverslips and fixed in PBS, pH 7.4, containing 1 mM CaCl2, 1 mM MgCl2, and 3% formaldehyde were processed as reported (
Immunoprecipitation and Immunoblotting Studies
Immunoblotting and immunoprecipitation analysis of the cell extracts and samples of culture medium were as already described (
Cell Detachment Assay
Cells (2 x 104/cm2) were seeded in tissue culture flasks and incubated for either 12 or 48 h at 37°C to reach 50 and 80% confluence, respectively. The monolayers were then treated with a solution of trypsin/EDTA (Versene and BioWhittaker) diluted 1:70 in PBS. The number of cells detached at increasing time of incubation was determined by collecting the supernatants and direct cell counting. Each experiment was repeated six times. A representative experiment is shown.
Quantification of Laminin 5 Deposited in the ECM by Cultured Keratinocytes
Laminin 5 deposition was quantified both on plastic cell culture substrate and on plastic coated with ECM components. To coat the plastic substrate, multiwell plates (96 wells; TPP) were incubated with solutions of collagen type I or IV, vitronectin, fibronectin, laminin 1, or ECM (all from Sigma-Aldrich) dissolved in PBS at a concentration of 10 µg/ml. 104 cells per well were seeded and incubated for either 12 or 48 h at 37°C in humidified atmosphere in the presence of 5% CO2. The cells were then washed twice in PBS and detached as devised by NC cells.
Adhesion Assay
Multiwell plates containing the ECM secreted by the different mutant cell lines were prepared as described. Wells were saturated for 1 h at room temperature with a solution of 0.5% heat-denatured BSA. Wild-type HNKs (3 x 104 cells per well) were plated and fed with serum-deprived medium containing 0.25% heat-denatured BSA. After 1 h at 37°C, the medium and the cells in suspension were removed and the wells were washed with PBS. Adherent cells were fixed in 3.7% formaldehyde, stained with 0.5% crystal violet dissolved in 20% methanol, and washed three times in PBS. The dye was eluted with 50% ethanol/0.1 M sodium citrate, pH 4.2. Absorbance at 540 nm was determined using a microplated reader. Values were expressed as a percentage of the values obtained with mutant LNC cells.
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Results |
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The functional role of the short arm domains of the laminin 2 chain was investigated by transfecting a series of mutant
2 cDNAs into keratinocyte cell line LSV5. Cell line LSV5 is derived from the keratinocytes of an H-JEB patient with a homozygous nonsense mutation (R95X) in the gene (LAMC2) coding for the laminin
2 chain (
2 chain, but express the full repertoire of the laminin chains found in HNKs, including the laminin
3 and ß3 chains (
2 cDNA restores production of functional
3ß3
2 laminin 5 molecules (
2 chains were generated by directed mutagenesis of the expression vector p
WT which encodes the full length
2 polypeptide (
NC expresses a recombinant
2 polypeptide with an internal deletion encompassing the four amino acids (YSGD) within domain III that constitute the proteolytic cleavage site of the
2 chain (
GP, the GlyPro residues substitute the YSGD cleavage site and introduce a structural modification from "sheet" to "coil" configuration of the
2 chain domain III. Mutants p
III and p
V carry a deletion affecting the egf-like repeat 1 of domain III and repeats 2 and 3 of domain V, respectively. To investigate the functional role of the laminin
2 chain short arm, we generated a cDNA clone encoding a polypeptide lacking the NH2-terminal domains IV and V that are excised in the extracellular processing of laminin 5 (plasmid p
C). Further, a polypeptide with an internal deletion encompassing the EGF-like repeat 1 of domain III and the COOH-terminal portion of domain IV (plasmid p
M) was also constructed. Plasmid p
M corresponds to a mutated
2 chain missing 73 amino acids of the polypeptide sequence detected in a patient suffering from JEB (
The different mutant cDNAs were transiently transfected into actively growing LSV5 keratinocytes. Immunocytochemical analysis of the transfected cultures using pAb SE144 (not shown) and mAb GB3 indicated that all the mutant 2 polypeptides were actively synthesized and incorporated into laminin 5 heterotrimers (Fig 2). The ECM deposited on the culture support by cells L
WT, L
NC, L
III, L
V, and L
GP transfected with plasmids p
WT, p
NC, p
III, p
V, and p
GP was immunoreactive, whereas the ECM deposited by the cell cultures L
C and L
M transfected with plasmids p
C and p
M was not labeled. These results indicate that deletions within the globular domain IV of the
2 chain prevent secretion and/or deposition of laminin 5 into the ECM, but do not limit laminin 5 synthesis.
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To confirm the role of the globular domain of the 2 polypeptide in deposition of laminin 5 to the cell culture substratum, plasmids p
NC, p
GP, and p
C were stably transfected into LSV5 keratinocytes. Cell lines L
NC and L
GP were generated that are expected to secrete a mutated 440-kD form of laminin 5 with a full length
2 chain (155 kD), whereas cell line L
C is expected to produce a 400-kD molecule corresponding to laminin 5 with a processed
2 chain (105 kD). Consistent with the results obtained with transiently transfected LSV5 cells, immunocytochemical analysis of L
NC, L
GP, and L
C cells using pAb SE144 and mAb GB3 detected a strong cytoplasmic labeling in all cell lines. These experiments also confirmed that only the ECM laid down by L
NC and L
GP keratinocytes contains immunoreactive laminin 5 epitopes (not shown). These results indicate that presence of the
2 short arm is required for deposition of laminin 5 to the culture substratum, but do not rule out the possibility that laminin 5 harboring a
C recombinant chain is secreted into the media without being incorporated into ECM. Expression and secretion of the recombinant
NC,
GP, and
C polypeptides were examined by Western blot analysis of spent medium of L
NC, L
GP, and L
C cells. A unique 105-kD migration band was detected in the L
C medium using pAb SE144, whereas a specific 155-kD band was observed with L
NC and L
GP cells (Fig 3 A). The intensity of these bands was comparable and was estimated to be threefold weaker than the intensity of the corresponding bands detected in the medium conditioned by wild-type keratinocytes. Thus, although the
2 short arm is required for incorporation into the ECM, it is not needed for secretion of
2 chain.
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Incorporation of the recombinant 2 polypeptides into extracellular laminin 5 molecules was further verified by immunoprecipitation of L
C and L
NC cell culture medium using the mAb K140 and the antiT7-Tag mAb. As shown in Fig 3 B, comparable amounts of 400- and 440-kD laminin 5 molecules were immunoprecipitated from the L
NC and L
C cell medium, respectively, which attests to the assembly of the 105- and 155-kD recombinant
2 polypeptides into laminin 5. These data demonstrate that the absence of the
2 NH2-terminal domains does not hinder the intracellular processing of laminin 5 and its secretion into the culture medium. They also confirm that the tetrapeptide YSGD is the unique physiological cleavage site of the extracellular processing of the
2 chain.
Artificial epithelia constructed either with HNKs or LSV5 cells expressing a recombinant wild-type 2 chain have been shown previously to lay down laminin 5 at the interface between the basal cells and the cell culture support (
2 short arm appeared to prevent deposition of laminin 5 on monolayer submersed cultures, L
C and L
NC keratinocytes were grown to confluence on cellulose acetate filters and exposed to air to obtain stratification into multilayered epithelia. Immunofluorescence analysis of the artificial epithelia using mAb GB3 detected a strong reactivity in the case of L
NC and HKSV cells (Fig 3 C, b and d), and no reactivity with LSV5 and L
C keratinocytes (Fig 3 C, a and c). Therefore, these observations confirm that the 400-kD form of laminin 5 produced by L
C keratinocytes is not incorporated into the ECM, and underscore the importance of the
2 short arm plays in the deposition of laminin 5 at the epithelialECM interface.
Because our results suggested that the laminin 2 short arm is essential for the incorporation of laminin 5 into the ECM secreted by LSV keratinocytes, we verified whether human skin and the matrix secreted by wild-type keratinocytes contain the NH2-terminal
2 polypeptide generated by the extracellular processing of the 440-kD laminin 5. Specific antiserum pAb SE1097 was generated against recombinant fragment
50, which is cleaved from the short arm of the
2 chain (Fig 1 B). The antibody stained the human epidermal basement membrane in a strong linear fashion (Fig 4A and Fig B), comparable to the labeling observed with antibody SE144 (not shown). Samples of the ECM produced by cultures of wild-type keratinocytes were then collected from culture dishes and analyzed by immunoblotting using pAb SE144 and pAb SE1097. As shown in Fig 4 C, pAb SE144 recognized the 155- and 105-kD migration bands corresponding to the unprocessed and processed
2 polypeptide, respectively. pAb SE1097 identified the unprocessed 155-kD
2 chain and an additional fast-migrating band with the expected mass (50 kD) of the NH2-terminal domains, which are extracellularly excised from the
2 chain (Fig 4 D). The finding that only unprocessed
2 chain is found intracellularly and that the processed
2 chain and its cleavage fragment are found in the ECM indicate that the 440-kD laminin 5 molecules are proteolytically cleaved into the 400-kD form after incorporation into the ECM. This strengthens the idea that the
2 short arm plays a role in the integration of laminin 5 in the matrix.
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To verify this hypothesis, wild-type keratinocytes were transfected with the construct p50 encoding the NH2-terminal domains IV and V of
2 carrying a T7-Tag peptide (Fig 1). Control cultures were transfected with an expression vector (pß60) that encodes the NH2-terminal domains III, V, and VI of the laminin ß3 chain tagged with the HA epitope (Fig 1). As shown by immunofluorescence microscopy, mAb T7-Tag reacted with the cytoplasm of the keratinocytes transfected with plasmid p
50 and labeled the ECM deposited by these cells (Fig 4 E, a and b). Conversely, the anti-HA mAb stained the cytoplasm of the keratinocytes expressing the recombinant cDNA pß60, but not the ECM they deposited (Fig 4 E, c and d). Similar results were obtained by transfection experiments performed using LSV5 cells (not shown). These observations suggest that the short arm of the laminin
2 chain carries domains essential to the incorporation of laminin 5 into the matrix.
It has been suggested that the globular domain IV of the mouse laminin 2 chain binds to the ECM protein fibulin 2 (
2 amino acid sequence, which is essential to the interaction, is not conserved in humans. In addition, we were unable to demonstrate interactions between human laminin 5 and fibulin 2 in our experimental conditions (data not shown). Intriguingly, the amino acid sequence of the NH2-terminal region of the
2 domain IV is highly conserved in mammals (Table 2), which may reflect a relevant physiological role of this portion of the polypeptide. Indeed, it was suggested that disruption of the fibulin 2 binding site could hamper the proteolytic processing of
2 (
F1 and p
F2 that encode
2 polypeptides in which the amino acid sequence SADFSVHKI (residues 199207) homologous to the active site of the mouse fibulin 2 binding site was partially (p
F1) and totally (p
F2) substituted by alanine residues (Fig 1). LSV5 keratinocytes transfected with constructs p
F1 and p
F2 were examined by immunofluorescence analysis using mAb GB3. Expression of the mutant laminin 5 molecules resulted in a strong staining of the cytoplasm, and also of the ECM deposited onto the tissue culture support (Fig 5 A, a and c). Western blot analysis of medium collected from cultures of L
F2 keratinocytes using pAb SE144 detected hybridization bands corresponding to the uncleaved (155 kD) and the proteolytically cleaved (105 kD) p
F2 chain (Fig 5 B). According to these observations, immunofluorescence examination of frozen sections of artificial epithelia constructed using L
F1 and L
F2 keratinocytes detected a strong reactivity of the basement membrane zone to mAb GB3 (Fig 5 C, a and b). These results attest to the incorporation of the mutant laminin 5 molecules into the ECM deposited on the cell culture substrate and show that disruption of the region homologous to the putative fibulin 2 binding site of the mouse
2 short arm does not interfere with the processing and deposition of laminin 5 to the matrix.
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Reexpression of wild-type laminin 5 restores adhesion of LSV5 cells and H-JEB keratinocytes (C keratinocytes are not deposited into the ECM, L
C cells are expected to retain the poor adhesion capacity of the parental cell line LSV5. Indeed, epidermal sheets of stratified epithelium generated by confluent cultures of L
C, L
GP, and L
F1 keratinocytes spontaneously detached from the culture vessel. In contrast, when L
NC keratinocytes became confluent and stratified, the epidermal sheet firmly adhered to the plastic dish and detachment required enzymatic treatment (not shown). Therefore, attachment of L
C keratinocytes was quantified in detachment kinetic assays in the presence of trypsin/EDTA (
NC keratinocytes was also compared with that of wild-type keratinocytes. Cell suspensions were seeded on petri dishes to obtain exponentially growing cultures. 12 h after plating, the percentage of the adhering L
NC and L
C cells was similar to that of parental LSV5 cells (Fig 6 A). Although the number of L
NC and L
WT cells resistant to trypsinization increased 48 h after seeding (50% of L
NC cells were dislodged after 14 min), that of L
C and LSV5 cells remained low (50% of dislodged cells after 8 min; Fig 6 B). Therefore, the progressive enhancement of adhesion of L
NC and L
WT keratinocytes appeared to correlate with accumulation of laminin 5 molecules harboring the
2 short arm in the matrix. Detachment of L
GP and L
F1 keratinocytes was also assessed in similar experimental conditions. As demonstrated in Fig 6 B, in all these cells other than L
NC synthesis of mutated laminin 5 molecules had no appreciable effect on the strength of cell adhesion.
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To confirm that resistance to trypsinization of LNC cells correlates with laminin 5 incorporation in the ECM, the amount of laminin 5 layered down by the different
2 mutant keratinocytes was determined by ELISA assay using mAb GB3. As shown in Fig 6 C, laminin 5 produced by L
NC cells was efficiently deposited on the plastic culture substrate and accumulated with increasing time, whereas mutant
C laminin 5 was inefficiently layered down. L
WT, L
GP, or L
F1 cells layered down comparable amounts of laminin 5. In addition, by seeding the mutant keratinocytes in a plastic vessel coated with different components of the ECM, we assessed the deposition rate of the wild-type and mutant laminin 5 molecules to be independent from the nature of the cell culture substrate on which the keratinocytes are grown (Fig 7). Plating on a feeder of irradiated mouse 3T3-J2 cells did not modify the deposition pattern of laminin 5 (not shown).
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Because the trypsinization assay provides information on the effect of laminin 5 on the strength of cell attachment, the functional role of the laminin 2 short arm in cell adhesion was further assessed by seeding wild-type primary human keratinocytes on the ECM deposited by the different LSV5
2 mutants. The mutant cells were seeded in a range of concentrations leading to deposition of equivalent amounts of laminin 5 48 h after plating. The ECM was prepared after detachment of the cells by EDTA treatment, and concentration of laminin 5 in the ECM was checked by ELISA assays using mAb GB3. Primary wild-type keratinocytes were then seeded and allowed to adhere for 60 min at 37°C. Results show that adhesion of keratinocytes on the matrix secreted by L
NC and L
WT cells was comparable, whereas adhesion on the matrix produced by L
C cells was strongly reduced and equivalent to the values obtained with matrix produced by the
2-null LSV5 cells (Fig 8). Adhesion on the matrix produced by the L
F1 and L
GP mutant cells was also reduced to a lesser extent. This result suggests that deposition of these laminin 5 molecules allows interaction of their COOH-terminal G domain with the adhesive integrin receptors at the cell surface, although participation of other cell receptors in adhesion cannot be excluded. However, the cell detachment assay shows that the adhesion strength of mutant L
F1 and L
GP cells is affected, which may reflect a lower binding capacity between laminin 5 secreted by these cells and the culture substrate (Fig 7 B).
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Because keratinocyte adhesion was measured on a matrix produced by the different LSV mutant cells, we could not determine whether the adhesive effect we observed was directly or indirectly sustained by laminin 5. Nevertheless, our observations demonstrate that the laminin 5 molecules harboring an unprocessed 2 chain are biologically active and indicate that structural changes in the NH2-terminal region of the globular domain IV affect the biological function of laminin 5.
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Discussion |
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Transfection of a wild-type laminin 2 cDNA in LSV5 keratinocytes rescues expression of functional laminin 5 molecules (
2 short arm. In this study, we examined the functional role of the laminin
2 short arm by complementing the genetic defect of LSV5 cells by transfer of mutant
2 cDNAs. Expression of the mutant cDNAs demonstrated that all the different recombinant
2 chains associate with the endogenous laminin
3 and ß3 polypeptides to assemble into
3ß3
2 heterotrimers, and that the NH2-terminal domains of the
2 chain are required for deposition and incorporation of laminin 5 in the ECM.
Our results, for the first time, define a physiologic function for the short arm of the 2 chain. We demonstrate that mutant laminin 5 molecules lacking the
2 chain NH2-terminal sequences that are excised in the extracellular processing are found in the spent media of L
C cells, but are not layered on the cell culture substrate. Specifically, deposition of laminin 5 in the ECM produced by the LSV5 keratinocytes requires integrity of the
2 globular domain IV, whereas preservation of the tightly folded structure of the EGF-like rich domains III and V is not essential. Indeed, transfection of LSV5 cells with
2 cDNA mutants carrying a deletion within the EGF-like repeats of either domain III or V results in deposition of laminin 5. Consistent with these observations, laminin 5 molecules carrying a deletion of the
2 domain V EGF-like repeat 2 has been found in the dermalepidermal junction of a patient with mild epidermolysis bullosa consequent to in-frame skipping of LAMC2 exon 4 (Castiglia, D., P. Posteraro, M. Pinola, C. Angelo, P. Puddu, and A. Zambruno, manuscript submitted for publication). Therefore, removal of EGF-like repeats weakens the functionality of laminin 5, but may not prevent the polarized secretion and deposition of the protein to the basement membrane. Conversely, transfection of mutant p
M, in which the deleted fragment encompasses the
2 domains III and IV, results in lack of deposition of laminin 5 that correlates well with the epidermolysis bullosa phenotype of the patient carrying this mutation (
M chain was consequent to lack of secretion. Conversely, by deletion of the proteolytic cleavage site YSGD of the
2 polypeptide, we show that laminin 5 with an unprocessed
2 chain (mutant plasmid p
NC) is efficiently layered down and enhances adhesion of LSV5 and wild-type keratinocytes. These finding imply that the extracellular 440-kD form of laminin 5 is an active adhesion ligand and not a mere precursor of the processed 44-kD form.
Basement membrane components are coordinately deposited in the ECM and subsequently assemble to form a supramolecular network sustaining multiple functions, including cell adhesion (2 chain. Apart from Phe-202, which is required for the full binding activity of the mouse
2 chain, the remaining active residues of the consensus sequence mediating interaction between the laminin
2 chain and fibulin 2 are conserved in humans and other mammals (Table 2). We could not demonstrate a direct interaction between the
2 chain and fibulin 2 in human keratinocytes. In addition, our results show that in mutants
F1 and
F2, substitution of the putative fibulin 2 binding site with alanine residues that modify the nH2-terminal region of the
2 short arm domain IV does not affect the cleavage of the
2 chain and deposition of laminin 5. This argues against the participation of this region of the
2 globular domain IV in the extracellular processing of the
2 polypeptide (
F1 or the
F2 chain is diminished, as attested by the weak resistance to trypsinization of the cells expressing these molecules. The relevance of the
2 short arm in cell adhesion is confirmed by the fact that substitution of the YSGD site with the amino acids GlyPro, where the proline residue modifies the orientation on the plane of the NH2-terminal domains, also results in efficient deposition of 440-kD laminin 5 molecules exerting a reduced adhesive function. Although further investigations at the molecular level are needed to clarify the mechanisms underlying these observations, it is likely that integrity of the globular domain IV and the correct orientation of the
2 NH2 terminus determine the appropriate arrangement of laminin 5 in the ECM produced by LSV5 cells and contribute the functional activity of this adhesion ligand.
Laminin 5 purified from mouse epidermis is a mixture of molecules harboring a cleaved (105 kD) or an uncleaved (155 kD) 2 chain (
2 chain to 105 kD was not observed, whereas laminin 5 isolated from amnion was found to contain the 105-kD
2 chain only (
2 short arm may facilitate the interaction between the ß3 short arm of laminin 5 and other basement membrane components, such as laminins 6 and 7 and collagen type VII, and consequently may activate the adhesion function of laminin 5 (
2 short arm are secreted in the culture medium, but are not found in the matrix produced by LSV5 cells. Conversely, we demonstrate that the progressive accumulation of the 440-kD form of laminin 5 on tissue culture plastic and the dermal equivalent of organotypic cultures enhances adhesion of LSV5 cells. Our data confirm previous observations showing that a threshold level of laminin 5 accumulation is required for efficient cell adhesion (
2 chain. However, these results do not argue against a role of the processed 400-kD form of laminin 5 in cell adhesion. Proteolysis of the
2 chain, which may occur after interaction of laminin 5 with laminins 6 and 7 or collagen type VII, could trigger additional or distinct adhesion functions to laminin 5.
Proteolysis regulates ECM assembly, editing the excess ECM components and release of active polypeptides during morphogenesis, growth, tissue repair, and pathological processes (for review see
Proteolytic digestion of the laminin 2 chain by BMP1 at the physiologic YSGD cleavage site has recently been demonstrated in vitro (
2 chain is cleaved in vitro by the matrix metalloprotease (MMP) MT1-MMP to yield the shortened 105-kD
2 polypeptide and a 80-kD
2 chain with a further truncated NH2 terminus (
2 chain is not found in skin extracts and in cultures of human keratinocytes, whereas the 105-kD
2 polypeptide and the excised 50-kD NH2-terminal domain are easily detectable. In this study we demonstrate that the
2 chains missing the tetrapeptide YSGD do not undergo proteolytic steps, which confirms in vitro studies and suggests the involvement of BMP-1 in the processing of laminin 5 at this cleavage site (
2 chain is not essential to keratinocyte adhesion, or that other proteases, including MT1-MMP, can compensate for the absence of BMP-1 in the extracellular processing of the laminin
2 chain. Indeed, cleavage of the laminin
2 chain by MT1-MMP has been associated with the migratory behavior of a variety of transformed epithelial cell lines, and inhibition of MT1-MMP partially downregulates cell migration induced by laminin 5 in tumoral cells. Therefore, MT1-MMP may cleave the
2 short arm and release the interaction between laminin 5 and the ECM components. Since the proteases of the MMPs family are involved in tissue remodeling in various physiological and pathological conditions, it is tempting to speculate that MT1-MMP cleaves laminin 5 and activates cell migration in pathologic circumstances, whereas BMP-1 governs proteolysis of laminin 5 in a physiologic context.
Several proteolytic fragments of ECM proteins maintain a biological function (2 chain and its proteolytic fragments are found at the invasion front of tumors, where they may play positive roles in neoplastic invasion (
2 chain is also secreted by carcinoma cells in vitro, but the biological relevance of these observations is unclear (
2 chain short arm is detected in the epithelial basement membrane and in the matrix produced in vitro by normal keratinocytes. Deposition of the recombinant
2 short arm (
50) in ECM by cultured keratinocytes confirms the adhesive potential of the polypeptide. Therefore, it is possible that after cleavage from laminin 5 the
2 short arm sustains interactions with specific basement membranes components and cell receptors and participates in basement membrane assembly. In this regard it has been reported that keratinocyte-specific knock-out of mouse integrin ß1 results in disorganization of the cutaneous basement membrane and induces skin blistering that correlates with a reduced immunoreactivity and an altered staining pattern of a pAb antibody directed against the
2 short arm (
Laminin 5 promotes adhesion, migration, and scattering of several types of cultured cells more efficiently than all the other ECM proteins. We demonstrate that the extracellular deposition of laminin 5 is mediated by the short arm of the 2 chain that then steers intermolecular interactions with the basement membrane components and promotes cell adhesion. The functional role of the proteolytic processing of the laminin
2 chain in the formation of the epithelial basement membranes remains unclear. The construction of knock-in mice expressing either a mutant
NC or a truncated
C chain will clarify the specific biological functions of the extracellular forms of laminin 5.
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Footnotes |
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1 Abbreviations used in this paper: ECM, extracellular matrix; HA, hemagglutinin; JEB, junctional epidermolysis bullosa; MMP, matrix metalloprotease; pAb, polyclonal antibody.
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Acknowledgements |
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We acknowledge A. Spadafora for technical assistance, A. Mastrogiacomo for the protein structural analyses, and A. Charlesworth for critical reading of the manuscript.
This work was supported by grants from EEC BIOMED 2 (BMH4-97-2062), the Programme Hospitalier de Recherche Clinique (France), the DEBRA Foundation (UK), and the Association Francaise contre les Myopathies (Crowthorne, Evry, France).
Submitted: 26 February 2001
Revised: 30 March 2001
Accepted: 30 March 2001
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References |
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