* Department of Cell and Molecular Biology, and Department of Obstetrics and Gynecology, Northwestern University Medical
School, Chicago, Illinois 60611
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Abstract |
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The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial
cell MCF-10A is capable of nucleating assembly of cell-
matrix adhesive devices called hemidesmosomes when
other cells are plated upon them. These matrices also
impede cell motility. In contrast, cells plated onto the
laminin-5-rich matrices of pp126 epithelial cells fail to
assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126
cells, using a panel of laminin-5 subunit-specific antibodies. The 3 subunit of laminin-5 secreted by pp126
cells migrates at 190 kD, whereas that secreted by 804G
and MCF-10A cells migrates at 160 kD. The pp126 cell
190-kD
3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5
and
chains. After plasmin treatment, pp126 cell
laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported
by immunofluorescence analyses that demonstrate
colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells.
Whereas tissue-type plasminogen activator (tPA),
which converts plasminogen to plasmin, codistributes
with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5-rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5
3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that
laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as
an adhesive factor. In cells in culture, this functional
conversion appears dependent upon and is regulated by
tPA and plasminogen.
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Introduction |
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EPITHELIAL cells are separated from connective tissue
by a basement membrane that is composed of a variety of extracellular matrix molecules including
proteoglycans, collagen, and laminin isoforms. Together,
these proteins create a framework that is essential for
maintaining tissue integrity. However, extracellular matrix
proteins play more than just a structural role; they also display a diverse set of biological functions that regulate adhesion, migration, proliferation, differentiation, and gene
expression of adjacent cells (Roskelly et al., 1995).
Laminin, of which there are at least 10 isoforms, is a major component of basement membranes, and has been
shown to mediate cell-matrix attachment, gene expression, tyrosine phosphorylation of cellular proteins, and
branching morphogenesis (Tryggvason, 1993; Timpl and
Brown, 1994
; Streuli et al., 1995
; Malinda and Kleinman,
1996
; Stahl et al., 1997
). The expression patterns of the
laminin isoforms are tissue specific. The laminin-5 isoform (nicein, epiligrin, and kalinin) is abundant in transitional
epithelium, stratified squamous epithelia, lung mucosa,
and other epithelial glands (Kallunki et al., 1992
; Stahl et
al., 1997
). Laminin-5 is a heterotrimer consisting of
3,
3,
and
2 subunits that associate via large
helical regions to
produce a cruciform-shaped molecule (Rousselle et al.,
1991
; Baker et al., 1996a
). Laminin-5 is synthesized initially as a 460-kD molecule that undergoes specific processing to a smaller form after being secreted into the extracellular matrix (Marinkovich et al., 1992
; Vailly et al.,
1994
; Matsui et al., 1995a
). The size reduction is a result of
processing the
3 and
2 subunits from 190-200 to 160 kD
and from 155 to 105 kD, respectively (Marinkovich et al.,
1992
; Vailly et al., 1994
; Matsui et al., 1995a
). The identity
of the proteases involved in such proteolytic events has
not been described previously.
In a number of studies, it has been demonstrated that
laminin-5 produced by 804G cells can nucleate the assembly of hemidesmosomes in squamous cell carcinoma
(SCC)112, human adult calcium-transformed (HaCaT),
pp126, and normal human keratinocyte (NHEK) cells as
well as in corneal epithelial cells maintained in vitro (Langhofer et al., 1993; Hormia et al., 1995
; Baker et al., 1996a
,b;
Tamura et al., 1997
; El-Ghannam et al., 1998
). However,
SCC12, HaCaT, pp126, and NHEK cells themselves secrete laminin-5, which is incapable of supporting the assembly of hemidesmosomes, suggesting that structural differences between laminin-5 molecules may regulate their
functions. The current data demonstrate that the electrophoretic mobility of the
3 chain of laminin-5 in the matrix
of these cell types is distinct. Furthermore, proteolytic processing of the
3 subunit of laminin-5 in the extracellular matrix is crucial for hemidesmosome formation. We also
present evidence that processing of the
3 subunit of laminin-5 can be mediated by a plasmin-dependent mechanism
involving tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation. A model is proposed that
could explain the role of laminin-5 processing in hemidesmosome assembly.
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Materials and Methods |
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Protein Preparations and Other Chemicals
Plasminogen and plasmin were purified as previously described (Stack et al.,
1993, 1994
; Stack and Pizzo, 1994
). Mast cell tryptase was provided by D. Johnson (East Tennessee State University, Johnson City, TN; Little and
Johnson, 1995
). Purified two-chain tPA was the gift of H. Berger
(Wellcome Research Laboratories, Research Triangle Park, NC). Urinary-type plasminogen activator (uPA) was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). The serine proteinase inhibitor,
dichloroisocoumarin, was purchased from Sigma Chemical Co. (St. Louis,
MO). Matrix metalloproteinase-2 (MMP-2, gelatinase A) and MMP-9
(gelatinase B) were obtained from the serum-free conditioned medium of
epithelial ovarian carcinoma cells as previously described or were the gift
of H. Nagase (University of Kansas, Lawrence, KA) (Young et al., 1996
).
Laminin-1 was a gift from N. Chilukuri (Northwestern University Medical
School, Chicago, IL).
For affinity purification of pp126 laminin-5, tissue culture plastic was
coated with 50 µg/ml GB3 antibody, a mouse monoclonal antibody
against the 2 chain of human laminin-5, in 10 mM Tris, pH 7.4, overnight
at 4°C (Verrando et al., 1987
; Matsui et al., 1995b
). Dishes were rinsed
briefly and then incubated with conditioned medium from pp126 cells for
1 h at 37°C. The dishes were then washed in 20 mM Tris, pH 7.4, containing 250 mM NaCl, and then followed by three washes in 10 mM Tris, pH
7.4. To assess the purity of the laminin-5 prepared by this procedure, confluent dishes of pp126 cells were radiolabeled overnight with 50 µCi/ml of
[35S]PRO-MIX cell label (Amersham Corp., Arlington Heights, IL). The
labeled, conditioned medium of the pp126 cells was then overlaid on the
GB3 antibody-coated dishes as above. After washing, the material "captured" by the antibody was solubilized in SDS-PAGE sample buffer, proteins were resolved by SDS-PAGE, and then the resulting gel was prepared for autoradiography (see below). For overlay studies, human
laminin-5 was provided by Desmos Inc. (San Diego, CA).
Cell Culture and Preparation of Laminin-5 Matrices
Michigan Cancer Foundation MCF-10A cells and 804G cells were maintained as described previously (Riddelle et al., 1991; Stahl et al., 1997
).
NHEK (purchased from Clonetics Corp., San Diego, CA), SCC12, and
pp126 cells, a gift from D. Oda (University of Washington, Seattle, WA)
were maintained in the serum-free growth medium Keratinocyte-SFM
(GIBCO BRL, Gaithersburg, MD) supplemented with 20 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 ng/ml EGF, and 50 µg/
ml bovine pituitary extract (Oda et al., 1996
). MCF-10A, pp126, SCC12,
NHEK, and 804G cell matrix were prepared as described previously (Gospodarowicz, 1984
; Langhofer et al., 1993
). In experiments where various
proteases were used, pp126 cells were removed from their matrix with ammonium hydroxide as previously described. Then the matrix was extensively washed in PBS and ~50 µg of matrix was incubated with 1 ml of
PBS containing plasmin at concentrations of 0.01, 0.1, and 1 µg/ml for 90 min at 37°C or 50 µg of matrix was treated with 1 ml of PBS containing 5 µg/ml of either MMP-2 or MMP-9 overnight at 37°C. In some studies, 50 µg of pp126 cell matrix was treated overnight at 37°C with 1 ml of PBS
containing either 5 or 10 µg/ml tPA. In all cases, dichloroisocoumarin was
then added to the treated matrix at 10 µg/ml for 15 min at room temperature. After washing with PBS, the treated matrices were solubilized in
sample buffer consisting of 8 M urea, 1% sodium dodecyl sulfate in 10 mM Tris-HCl, pH 6.8, and 15%
-mercaptoethanol.
SDS-PAGE, Western Immunoblotting, and Overlay Assays
Protein preparations were separated by the method of Laemmli (1970) in
6% acrylamide gels. In cases where radiolabeled proteins were resolved
by SDS-PAGE, gels were dried and then exposed to X-Omat Imaging
Film (Eastman Kodak Co., Rochester, NY). For immunoblotting, separated proteins were transferred to nitrocellulose according to standard
procedures (Harlow and Lane, 1988
). The nitrocellulose membranes were
processed for blotting as described in Zackroff et al. (1984)
. Blots were
developed either using chloronaphthol as a colorimetric reagent, or using
the LumiGlo chemiluminescence kit (Kirkegaard and Perry Laboratories,
Inc., Gaithersburg, MD). For overlay assays, 1 ng of protein was dotted
onto nitrocellulose, which was then blocked in PBS containing 0.2% fish
gelatin. The membrane was subsequently incubated overnight with 20 µg/ml
of either tPA, uPA, or plasminogen, at 4°C with vigorous shaking. The nitrocellulose was blocked in 5% milk in PBS and then processed for immunoblotting.
Antibodies
GB3 antibody against the 2 chain of human laminin-5 was purchased
from Harlan Sprague Dawley Inc. (Indianapolis, IN) (Verrando et al.,
1987
; Matsui et al., 1995b
). For production of a rabbit serum against the
human
2 chain, a clone encoding amino acids 522-722 of human laminin-5
2 chain was identified in a
gt11 keratinocyte expression library (Clontech Laboratories Inc., Palo Alto, CA) as detailed in Langhofer et al.
(1993)
. A 25-ml culture of Y1090 was grown at 37°C in shaking suspension
to an OD of 0.7 and was subsequently inoculated with a 108 phage containing the laminin-5
2 insert. After 1 h, isopropyl B-p-thiogalactopyranoside was added to a concentration of 1 mM and then incubated for an
additional 3 h. Cells were pelleted and then resuspended in gel sample
buffer (8 M urea, 1% SDS in 10 mM Tris-HCl, pH 6.8, 5%
-mercaptoethanol). The resulting protein sample was then simultaneously processed
for SDS-PAGE with a protein sample derived from non-isopropyl B-p-thiogalactopyranoside-induced cells. After staining of the gel, a prominent
protein migrating at ~140 kD (i.e., a portion of laminin-5
2 chain fused
with
-galactosidase) was observed exclusively in the induced cells. This
polypeptide was excised, rinsed in PBS, and then used to immunize a rabbit for polyclonal antibody production (Harlow and Lane, 1988
). Blood was collected from the rabbit at 3-wk intervals.
For production of serum antibodies against the COOH-terminal region
of the human laminin-5 3 subunit, a 535-base pair HindIII/XhoI cDNA
fragment encoding amino acid residues 1561-1713 of the G5 domain of
the
3 subunit was subcloned into the HindIII and XhoI sites of the
pET32b vector (Novagen, Inc., Madison, WI) and then transfected into
DE3
cells (Ryan et al., 1994
). A histidine (His) fusion protein was induced
and then the cells expressing the fusion protein were extracted in SDS
buffer, as described above. The fusion protein was identified using a His-HRP probe (SuperSignal HisProbe Western blotting kit; Pierce Chemical
Co., Rockford, IL) and on an SDS-PAGE gel after protein staining in
Coomassie brilliant blue (Sigma Chemical Co., St. Louis, MO). The protein was excised from the gel and then the gel pieces were homogenized and subsequently injected into BALB/C mice for generation of mouse antisera. One such serum (Cta3) was used in the course of these studies, although all of the sera showed the same immunoblotting reactivities.
Clone 17, a mouse monoclonal antibody specific for the 3 chain of human laminin-5, was purchased from Transduction Laboratories (Lexington, KY). The mouse monoclonal antibody 10B5, which recognizes the
3
subunit of rat and human laminin-5, was generated using 804G laminin-5
as immunogen as described in Langhofer et al. (1993)
. The mouse monoclonal inhibitory antibody P3H9-2 against human laminin-5 and monoclonal antibody MAB 1921 against the
1 laminin subunit were purchased
from Chemicon International, Inc. (Temecula, CA). PAM-3, a mouse
monoclonal antibody against human tPA, and monoclonal antibody 394 against human uPA, were purchased from American Diagnostica Inc.
(Greenwich, CT). Antihuman plasminogen rabbit antiserum was described in Stack et al. (1993
, 1994
) and Stack and Pizzo (1994)
.
Immunofluorescence
MCF-10A and pp126 cells were maintained on glass coverslips for 24-48 h,
permeabilized in acetone at 20°C for 2 min, and then air dried. Coverslips were incubated with a mix of primary antibodies diluted in PBS at
37°C in a humid chamber for 1 h, washed three times in PBS, and then incubated for an additional hour at 37°C with the appropriate mix of secondary antibodies conjugated to rhodamine and FITC. Mounted glass
coverslips were viewed using a laser scanning confocal microscope (model
LSM10; Zeiss Inc., Thornwood, NY). Images were stored on optical discs (Sony Corp., Park Ridge, NJ) and printed on a Tektronix printer (model
Phaser IISDX; Tektronix, Wilsonville, OR).
Electron Microscopy
Cells maintained on tissue culture plastic or on matrix for 36 h were fixed
in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for a
minimum of 30 min. Fixed preparations were washed three times in 0.1 M
sodium cacodylate buffer, pH 7.2, and then postfixed in 1% osmium
tetroxide containing 0.8% potassium ferricyanide. Samples were then
stained with uranyl acetate, dehydrated six times in ethanol, and then embedded in Epon-Araldite resin (Tousimis Corp., Rockville, MD). Sections
were cut perpendicular to the substratum and viewed on an electron microscope (model 1220; JEOL USA, Inc., Peabody, MA) at 60 kV (Riddelle et al., 1991). Morphometric analyses were performed using the software of this microscope.
Motility Assay
Cells were plated in media containing 20 mM Hepes onto various substrata for 1 h. They were subsequently maintained at 37°C and then viewed by phase-contrast microscopy using a Nikon Diaphot system (Tokyo, Japan). The field was photographed every 5 min over a 2-h period with a chilled charge-coupled device camera (Hamamatsu Photonic Systems Corp., Park Ridge, IL). The location of each cell was translated to numerical coordinates using the public domain National Institutes of Health Image Program (Bethesda, MD), and motility for each cell was calculated as displacement in micrometers from the starting point to the ending point. An average of 30 cells was monitored for each trial.
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Results |
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The 3 Subunit of Laminin-5 Is Processed Differently
in Diverse Cell Types
The rat epithelial cell line 804G and the human breast epithelial cell line MCF-10A are unusual in that both assemble cell-matrix attachment devices called hemidesmosomes when maintained in tissue culture (Riddelle et al.,
1991; Stahl et al., 1997
). Recent data indicate that this
property is dependent upon the laminin-5 secreted by
these cells (Baker et al., 1996a
; Stahl et al., 1997
; Tamura et al., 1997
). However, laminin-5 is expressed by many
other cell types, such as the squamous cell carcinoma line
SCC12 and the transformed oral epithelial cell line called
pp126, which do not assemble hemidesmosomes under
normal conditions (Langhofer et al., 1993
; Baker et al.,
1996a
; Tamura et al., 1997
). In addition to expressing laminin-5, both SCC12 and pp126 cells express all the major
known protein components of hemidesmosomes, yet only assemble hemidesmosomes when plated onto laminin-5
secreted by 804G cells or MCF-10A cells (Langhofer et al.,
1993
; Baker et al., 1996a
; Tamura et al., 1997
; Jones,
J.C.R., unpublished observations). The latter finding suggests the possibility that distinct structural and functional
forms of laminin-5 may be expressed by different cell
types. Thus, we have analyzed the subunit composition of laminin-5 secreted by 804G, MCF-10A, pp126, and SCC12
cells, as well as a "normal" cell population (NHEK), by
Western immunoblotting using a panel of antibodies
against specific laminin-5 subunits. For our studies, we
prepared the matrix secreted by these cell types using a
procedure developed by Gospodarowicz (1984)
. Laminin-5 subunits are the major constituents of these matrices (Fig.
1 shows the protein profiles of 804G, MCF-10A, and
pp126 matrices).
|
|
An antibody against the 3 subunit of laminin-5 recognizes a band migrating at 145 kD present in MCF-10A as
well as SCC12, NHEK, and pp126 cell matrices (Fig. 2 a).
This particular monoclonal antibody does not recognize
rat material, hence the lack of reactivity with the 804G cell
matrix preparation. A polyclonal antibody recognizing the
2 subunit of human laminin-5, which displays cross-reactivity with the rat homologue, detects 155- and 105-kD
polypeptides in the matrices of MCF-10A, 804G, pp126,
and NHEK cells but only a 105-kD species in the matrix of
SCC12 cells (Fig. 2 b). For the analysis of the
chain of
laminin-5, we made use of a monoclonal antibody, 10B5,
which was prepared against laminin-5 in the extracellular matrix of 804G cells (Langhofer et al., 1993
; Baker et al.,
1996a
). 10B5 specifically recognizes the
3 chain of rat
laminin-5 and displays cross-reactivity with the
3 subunit
of the human homologue (Fig. 2 c). The
3 subunits of
pp126, SCC12, and NHEK cell laminin-5 migrate at 190 kD,
identical to the reported size of the unprocessed
3 chain
of laminin-5 (Fig. 2 c) (Marinkovich et al., 1992
; Matsui et al., 1995a
). The appearance of the unprocessed
3 chain
of laminin-5 in NHEK matrix has not previously been
noted (Marinkovich et al., 1992
; Matsui et al., 1995a
). In
contrast, in the matrices of MCF-10A and 804G cells the
3 chain migrates at 160 kD, similar to the molecular
weight of the processed
3 chain subunit (Fig. 2 c)
(Marinkovich et al., 1992
; Matsui et al., 1995a
). Similar results are obtained using two other monoclonal antibodies
against the human
3 subunit (results not shown).
These results indicate that there is no obvious correlation between the sizes of the 2 or
3 subunits of laminin-5
and the ability of a cell to assemble a hemidesmosome. In
contrast, the matrix of those cells (MCF-10A and 804G)
that assemble hemidesmosomes contains processed
3
subunits, whereas matrix of those cells (pp126, SCC12, and
NHEK) that do not assemble hemidesmosomes contains unprocessed
3 subunits. Thus, we next tested several proteinases that are known to cleave extracellular matrix proteins for their ability to alter the electrophoretic mobility
of the laminin-5
3 subunit of pp126, SCC12, and NHEK
cells. For these studies, we chose proteinases that are associated with these cells (i.e., plasmin, MMP2, and MMP9)
(Stack, M.S., unpublished observations). We describe only
those results using pp126 cells since the results are identical to those using the other cell lines. MMP-2 and MMP-9 at enzyme/substrate ratios of ~1:10 exhibit no obvious effect on the
3 subunit of pp126 matrix (data not shown).
We also treated ~50 µg of pp126 matrix with 1 ml of PBS
containing the proteinase plasmin at concentrations of
0.01, 0.1, and 1 µg/ml for 90 min. Plasmin at concentrations
of 0.01 and 0.1 µg/ml has no obvious effect on the subunits of
laminin-5 (result not shown). However, after treatment of
pp126 matrix with plasmin at 1 µg/ml, the
3 subunit is
converted to a 160-kD species, as shown by Western immunoblotting with 10B5 antibody (Fig. 2 c). This treatment does not induce proteolysis of the
3 and
2 subunits
(Fig. 2, a and b). In addition, a 90-min treatment of pp126
matrix with 1 µg/ml of mast cell tryptase, which, like plasmin, is a trypsin-like serine proteinase, also converts the
190-kD
3 subunit to a 160-kD species (result not shown).
Where Does Plasmin Cleave the 190-kD Form of the
3 Subunit?
To test the possibility that plasmin cleavage occurs towards the COOH terminus of the 3 subunit of laminin-5,
we prepared an antiserum (Cta3) against residues 1561-
1713 at the COOH terminus of the
3 subunit. The Cta3
serum contains antibodies that show reactivity with the
190-kD unprocessed form of the
3 subunit present in
pp126 matrix, but which fail to show reactivity with any
species in MCF-10A matrix (Fig. 3). In contrast, the 10B5 monoclonal antibody recognizes the unprocessed 190-kD
3 subunit in pp126 matrix as well as the processed 160-kD
species in MCF-10A matrix in a comparable immunoblot
(Fig. 3). Although this study does not rule out the possibility there may be a plasmin cleavage site close to the NH2
terminus of the molecule, it provides direct evidence that
plasmin can cleave the 190-kD
3 subunit towards its
COOH terminus.
|
Functional Consequences of Processing of the 3
Subunit of Laminin-5
The specific, limited proteolytic cleavage of
the 3 chain of pp126 cell laminin-5 from 190 to 160 kD
suggests that plasmin-mediated proteolysis may have functional consequences for this matrix ligand. Thus, we assessed the motility of SCC12 cells plated on the laminin- 5-rich matrix of pp126 cells relative to MCF-10A matrix.
SCC12 cells plated onto MCF-10A matrix exhibit significantly lower motility after 2 h compared with SCC12 cells
plated onto pp126 matrix (Fig. 4). However, SCC12 cells
plated onto plasmin-modified pp126 matrix display a 2.5-
3-fold decrease in motility, similar to that observed on
MCF-10A matrix (Fig. 4).
|
Since there is evidence that laminin-5 is complexed with
other laminin isoforms such as laminin-6, we were concerned that other laminin variants may play a role in the
motility phenomena we detail (Champliaud et al., 1996).
However, pp126 cell matrix contains little or no detectable
laminin-6 as assessed by immunoblotting with a monoclonal antibody probe against the 1 laminin subunit (Fig.
5). In addition, we have assessed the motility of SCC12 cells on affinity-purified pp126 cell laminin-5. To prepare
the purified laminin-5, GB3 antibody, immobilized to a
cell culture dish, was used to capture laminin-5 from the
conditioned medium of radiolabeled pp126 cells (Fig. 6).
For some studies, the affinity-purified material was treated
for 90 min with plasmin at 1 µg/ml. By SDS-PAGE, the affinity-purified untreated pp126 cell laminin-5 consists of
three distinct polypeptides of 190, 145, and 105 kD, representing the
3,
2, and
2 chains, respectively (Fig. 6). In
the plasmin-modified material, the
3 chain migrates at
~160 kD, whereas the
2 and
2 chains show no obvious
change in their electrophoretic mobility (Fig. 6). These results were confirmed by immunoblotting (data not shown).
|
|
SCC12 cells were plated onto both untreated, affinity-purified pp126 cell laminin-5 as well as the plasmin-modified purified laminin-5, and their motility was compared. The 2.4-fold higher motility of SCC12 cells on affinity-purified laminin-5 compared with that on plasmin-modified purified laminin-5 shows significance at P < 0.008 as determined using the nonparametric analysis of variance Mann-Whitney U test (Fig. 4).
Hemidesmosome Assembly.Laminin-5 is the extracellular
ligand of the integrin pairs 6
4 and
3
1 (Carter et al.,
1991
; Niessen et al., 1994
). The precise physiological role
of
3
1 integrin-laminin-5 ligation is unknown, whereas
the
6
4 integrin-laminin-5 complex forms the core of
hemidesmosomes (Stepp et al., 1990
; Jones et al., 1994
;
Borradori and Sonnenberg, 1996
; Green and Jones, 1996
). Therefore, we investigated whether SCC12 cells are induced to assemble hemidesmosomes on four distinct
pp126 cell-derived laminin-5 substrates: untreated pp126
cell laminin-5-rich matrix, plasmin-modified pp126 laminin-5-rich matrix, affinity-purified pp126 laminin-5, and
plasmin-modified affinity-purified pp126 laminin-5. SCC12 cells were plated onto these matrices and then after 24 h
the samples were processed for electron microscopy. For
each matrix, we evaluated hemidesmosome assembly in at
least 15 cells in at least two different trials. The results for
one complete trial are presented in Table I and representative images are provided in Fig. 7. SCC12 cells maintained on unmodified pp126 matrix and affinity-purified
pp126 laminin-5 assemble few hemidesmosomes at their
basal surface and those that occur appear immature (Table I). We define an immature hemidesmosome as an electron-dense structure, located along the cell-substrate interface, that has a poorly developed cytoplasmic plaque
lacking a trilayered appearance and obvious keratin bundle association (Langhofer et al., 1993
; Fig. 7). SCC12 cells
plated onto plasmin-modified laminin-5 preparations readily
assemble mature hemidesmosomes (Fig. 7, a and b; Table I).
These hemidesmosomes possess triangular-shaped, trilayered, electron-dense cytoplasmic plaques and are associated with the keratin intermediate filament cytoskeleton
(Fig. 7, a and b). These data support the conclusion that
plasmin processing of pp126 cell-derived laminin-5 activates
the ability of laminin-5 to nucleate assembly of hemidesmosomes. To provide further confirmation that laminin-5
is involved in nucleation of mature hemidesmosome assembly in this system, we also assessed hemidesmosome
assembly in SCC12 cells maintained for 24 h on plasmin-modified pp126 matrix as well as plasmin-modified affinity-
purified pp126 laminin-5, both of which had been treated
with the laminin-5-blocking antibody 1947. Antibody treatment considerably reduces the formation of mature hemidesmosomes in SCC12 cells maintained on such antibody-treated substrates (Fig. 7 c; Table I).
|
|
Plasminogen and tPA Interaction with Laminin-5
In Vivo Association.The apparent ability of plasmin to
modify the structure of laminin-5 and the resulting functional consequences on cell behavior raise the question of
how these events may be regulated in our culture systems.
Plasmin is generated by cleavage of the proenzyme plasminogen, which is present in serum and found in association with extracellular matrices. The cleavage of plasminogen to produce the functional enzyme plasmin is mediated
by either of two so-called plasminogen activators designated tPA and uPA (Wun, 1988). Since colocalization of
enzyme and substrate is an important regulatory property
governing matrix remodeling, we determined whether
plasminogen and either tPA or uPA are associated in vivo
with laminin-5 (Ranby, 1982
; Stack et al., 1995
).
MCF-10A and pp126 cells were processed for double-
label immunofluorescence microscopy using an antiserum
raised against purified plasminogen or tPA in combination
with antibodies against human laminin-5 (Fig. 8). Laminin-5 antibodies generate staining patterns in circles and
arcs at the basal aspect of the cells (Fig. 8, a, d, g, and j). In
addition, some staining is also seen in areas of the substrate not covered by cells, as reported previously (Rousselle et al., 1991; Baker et al., 1996a
; Stahl et al., 1997
). Interestingly, plasminogen staining in both MCF-10A and
pp126 cell cultures colocalizes almost exactly with laminin-5
(Fig. 8, a, b, d, and e). It should be noted that plasminogen
associated with pp126 cell matrix is likely derived from the
bovine pituitary extract that is added to the serum-free
growth medium in which the pp126 cell cultures are maintained. The bovine pituitary extract contains a high level
of plasminogen as seen by Western immunoblotting using
plasminogen antibody preparations (result not shown).
|
In MCF-10A cells, a monoclonal antibody against tPA shows an almost identical staining pattern to that generated by an antiserum against laminin-5 (Fig. 8, g and h). In contrast, tPA does not localize to the matrix of pp126 cells, although it shows weak staining of cell bodies (Fig. 8, j and k). The cell bodies of pp126 cells but not matrix are also stained by a uPA antibody, whereas the cells show no staining with an antibody against uPA receptor (result not shown). MCF-10A cells and underlying matrix do not stain positively for uPA or the uPA receptor, suggesting that uPA is not involved in plasmin-mediated laminin-5 processing in vivo, at least in this cell type (result not shown).
The above results suggest the possibility that lack of tPA
in the matrix of pp126 cells may preclude conversion of
plasminogen to plasmin and the subsequent plasmin-mediated 3 chain processing in pp126 cell matrix. Therefore,
we determined whether addition of purified tPA to pp126
matrix could induce cleavage of the pp126 cell
3 subunit.
Western immunoblotting of the tPA-treated pp126 matrix
reveals that the laminin-5
3 subunit is processed to a 160-kD
species that comigrates with the laminin-5
3 subunit in MCF-10A matrix (Fig. 9). In addition, SCC12 cells show
less motility on the tPA-treated pp126 matrix (Fig. 4).
|
The observed colocalization of tPA and plasminogen with laminin-5 in fixed MCF-10A cell matrix suggests that both might bind directly to laminin-5. This was confirmed using a dot blot overlay assay. Purified human laminin-5 and the control proteins fibronectin and BSA were blotted onto nitrocellulose. Subsequently, tPA, uPA, or plasminogen were incubated in solution with the membranes overnight. The blots were then processed by immunoblotting using antibodies against tPA, plasminogen, or uPA. tPA binds to laminin-5 and to fibronectin (Fig. 10). Plasminogen binds only to laminin-5, whereas uPA binds both BSA and fibronectin (Fig. 10). uPA binds poorly to laminin-5 (Fig. 10).
|
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Discussion |
---|
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---|
Laminin-5 has been reported to function in the nucleation
of hemidesmosome assembly and as an adhesive factor
that retards cell motility (Baker et al., 1996a; O'Toole et al.,
1997
). In contrast, some authors have provided evidence
that laminin-5 enhances cell motility and is expressed at
the migrating edges of certain tumor cell populations
(Kikkawa et al., 1994
; Pyke et al., 1994
, 1995
; Zhang and
Kramer, 1996
). The data presented here indicate that posttranslational processing of the
3 subunit of laminin-5 may
modulate laminin-5 function. In the case of laminin-5 derived from pp126 cells, the
3 subunit is ~190-kD, similar
to the size of the unprocessed laminin-5
chain identified
by others (Marinkovich et al., 1992
; Matsui et al., 1995a
).
Laminin-5-rich matrix that contains this unprocessed
3
chain supports keratinocyte cell motility and does not induce hemidesmosome assembly. Upon plasmin-mediated proteolytic cleavage of the
3 subunit to 160 kD, laminin-5-rich matrix becomes competent to trigger the assembly
of hemidesmosomes, leading to decreased cell motility.
The latter suggestion is in conflict with data presented by
Kikkawa (1994), who have shown that buffalo rat liver
cells scatter on processed laminin-5. However, one likely
explanation for this apparent anomaly is that liver cells do
not assemble hemidesmosomes and therefore, may not be capable of establishing stable anchorage sites on laminin-5.
Our initial experiments were performed using the laminin-5-rich matrix of pp126 cells. This matrix contains no
detectable 1 laminin subunit suggesting that there is little, if any, laminin-6 in the material that could contribute
to the phenomena we detail. However, using this material,
we could not rule out the possibility that a minor nonlaminin-5 component of pp126 matrix is involved in the nucleation of hemidesmosome formation. Thus, to confirm a
specific role for plasmin-treated pp126 laminin-5 in hemidesmosome assembly, we have made use of a function-
inhibiting laminin-5 antibody. Our data reveal that this antibody inhibits the ability of the plasmin-modified pp126
laminin-5 to nucleate hemidesmosome formation in SCC12
cells. Furthermore, as final confirmation of the importance
of laminin-5 and its processing in determining its function,
we have shown that SCC12 cells migrate less and assemble
more hemidesmosomes on plasmin-modified purified laminin-5 compared to untreated purified laminin-5.
Based on the observation that laminin-5 is expressed at
the leading edge of migrating tumor cells, one might hypothesize that this laminin-5 includes the 190-kD 3 subunit rather than the 160-kD processed form (Pyke et al.,
1994
, 1995
). Of course, the actively migrating buds of tumors are likely to be rich in a variety of proteinases that
could further degrade laminin-5 and impact its function. In
this regard, it has been recently shown that the
2 chain of
laminin-5 is proteolyzed to an 80-kD species by MMP-2
and that laminin-5 containing the truncated
2 chain induces cell motility (Giannelli et al., 1997
).
The specific structural and functional impact of plasmin
treatment on the 3 chain of pp126 laminin-5 in our in
vitro assays led us to investigate the possibility that the
plasminogen activator/plasmin system is involved in processing laminin-5 in our cultured cell models. Indeed, our
data support the hypothesis that codistribution of enzyme
(plasmin) and substrate (laminin-5) facilitates modification of laminin-5 structure with a resulting impact on its
function. We have shown that plasminogen is associated
with laminin-5 matrix in cultured epithelial cells at the
morphological level. Furthermore, tPA is also associated
only with the matrix of those cells whose laminin-5 contains a processed
3 chain. tPA is not apparently present
in matrix of pp126 cells, in which the
3 chain appears in
its unprocessed form. Moreover, we show using a dot blot
overlay assay that both plasminogen and tPA can bind
laminin-5. Intriguingly, we have been able to induce processing of the
3 chain of pp126 cell laminin-5 as well as
conversion from a high to low motility matrix by simply
adding tPA to pp126 cell extracellular matrix. Since tPA is
a proteinase with extremely limited substrate specificity, it
is unlikely that tPA alone could proteolyze the
3 chain
(Stack et al., 1995
). Together, these data suggest that addition of tPA to the pp126 cells is able to catalyze the conversion of plasminogen to plasmin, which can then target
the
3 subunit.
The role of plasmin and tPA in generating a truncated
laminin-5 3 subunit in this system has striking parallels to
the relationship of plasmin and tPA to laminin-1. For example, extracellular tPA secreted by B16F10 melanoma
cells and human colon carcinoma cells induces hydrolysis
of laminin-1 in a plasminogen-dependent manner (Stack
et al., 1993
; Tran-Thang et al., 1994
; Sordat et al., 1995). Both tPA and plasminogen exhibit high-affinity binding to
the
1 subunbit of laminin-1 (Moser et al., 1993
). Moreover, full-length laminin-1, as well as a short peptide of
the laminin-1
1 subunit containing the sequence SRARKQAASIKVAV, is able to stimulate the tPA-catalyzed
activation of plasmin from plasminogen (Stack and Pizzo,
1993
; Stack et al., 1994
a). In this regard, the
3 subunit of
the laminin-5 isoform contains a similar sequence (IQQARDAASKVAV) just upstream of the putative start of
its globular or G domain. As we have previously shown
that the G domain of the laminin-5
3 chain is essential for
nucleating hemidesmosomes (Baker et al., 1996a
), tPA/
plasmin-mediated cleavage of laminin-5 may generate a
truncated, functional G domain that is capable of triggering hemidesmosome assembly. This possibility is supported by our finding that plasmin cleavage occurs at the
COOH terminus. Furthermore, a stimulatory effect of
laminin-5 on tPA-induced plasminogen activation, similar
to that observed with laminin-1, would support a feedback mechanism, whereby epithelial cells indirectly influence
their own behavior by affecting the structure and function
of their own matrix molecules in their extracellular environment (Roskelly et al., 1995
).
Based on our results, we propose the following model
for nucleation of hemidesmosomes in cultured epithelial
cells. Laminin-5, containing a 190-kD 3 subunit, is secreted into the extracellular environment by epithelial
cells. Plasminogen associates directly with the matrix by
binding laminin-5. Only certain epithelial cell types such as
MCF-10A secrete tPA which, like plasminogen, also associates with laminin-5. The spatial colocalization of plasminogen and tPA on the laminin-5 molecule allows for efficient production of plasmin from plasminogen, catalyzed
by tPA. The newly generated plasmin then initiates cleavage of the
3 subunit of laminin-5. After cleavage of its
3
subunit, laminin-5 is then able organize the appropriate integrins and other cell surface-associated proteins to nucleate assembly of a hemidesmosome (Baker et al., 1996a
).
Those cell types, such as pp126 cells that do not express tPA in their extruded matrix, are thus incapable of forming hemidesmosomes because the
3 chain of the laminin-5
molecule is incompletely processed.
In summary, we have elucidated a regulatory enzymatic cascade which, in cells secreting the appropriate enzymes, appears to result in proteolysis of laminin-5 and subsequent nucleation of hemidesmosome assembly. Many investigators have recognized the ability of extracellular matrix to alter cellular behavior. Our data, showing the downstream effects of secretion of specific enzymes that bind to and modify laminin-5, indicate that epithelial cells indirectly regulate their own behavior, via changes in the surrounding matrix.
![]() |
Footnotes |
---|
Received for publication 7 May 1997 and in revised form 22 December 1997.
Address all correspondence to Jonathan C.R. Jones, Department of Cell and Molecular Biology, Morton 4-616, Northwestern University School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611. Tel.: (312) 503-1412. Fax: (312) 503-6475. E-mail: j-jones3{at}nwu.eduWe are grateful to M. Ravosa for performing the statistical analyses of our motility assay data and to T.-L. Chen for help in using the video microscope. We thank X. He for technical assistance (all three from Northwestern University Medical School, Chicago, IL).
This work is supported by National Institutes of Health grants to J.C.R. Jones (GM38470 and PO1 DE12328) and M.S. Stack (CA58900). L.E. Goldfinger is supported by training grant (T32 CA09560) from the National Cancer Institute.
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Abbreviations used in this paper |
---|
ECM, extracellular matrix; His, histidine; MMP, matrix metalloproteinase; NHEK, normal human keratinocyte; SSC, squamous cell carcinoma; tPA, tissue-type plasminogen activator; uPA, urinary-type plasminogen activator.
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