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J Biol Chem, Vol. 273, Issue 44, 28633-28641, October 30, 1998
From the Craniofacial Developmental Biology and Regeneration
Branch, NIDR, National Institutes of Health,
Bethesda, Maryland 20892-4370
The culture of human submandibular gland (HSG)
cells on laminin-1 induces acinar differentiation. We identified a site
on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin Laminins, a family of heterotrimeric glycoproteins found in
basement membranes, promote cell adhesion, migration, differentiation, proliferation, neurite outgrowth, and tumor growth (1, 2). Such diverse
biological functions suggest multiple cellular interactions with the
laminin molecule. Current reports identify 11 isoforms of laminin
displaying tissue-specific expression patterns at different times
during development (3, 4). Cell type-specific interactions between
different laminins and multiple receptors and signaling pathways
provide mechanisms for regulating the complex events of morphogenesis
and the maintenance of differentiated adult tissues.
Integrins are well characterized laminin receptors (5, 6). Different
cell types can use different integrins to attach to laminin. Studies
with recombinant and elastase fragments of laminin-1 indicate
interactions of Syndecans, a family of transmembrane heparan sulfate proteoglycans,
bind a wide range of components through their heparan sulfate side
chains. All cells and tissues except B stem cells express syndecans in
cell-specific, tissue-specific, and developmentally specific patterns
(10, 11). Syndecans-1 and -4 mediate cell-cell adhesion, which occurs
by interaction with a heparan sulfate-binding ligand present on an
adjacent cell surface (12). Antisense experiments show that maintenance
of an epithelial phenotype requires syndecan-1 (13). Syndecan-1
expressed by transfected NIH-3T3 cells binds to laminin-1. The E3
fragment from the G-domain of laminin-1 revealed the highest binding
for syndecan-1 compared with the other elastase fragments (14).
Syndecans have been proposed to function as co-receptors for components
in the extracellular microenvironment, the syndecan utilizing a heparan
sulfate-protein interaction and the other receptor a classical
protein-protein interaction (10).
We have identified biologically active sites on the laminin molecule
using a synthetic peptide approach. YIGSR on the We have shown that laminin-1 promotes acinar differentiation of the
human submandibular gland
(HSG)1 cell line, and other
components such as transforming growth factor- Cell Culture--
The HSG cell line (24) was cultured in
Dulbecco's modified Eagle's medium/Ham's F-12 (1:1), containing 5%
fetal bovine serum (Biofluids, Rockville, MD), 100 units/ml penicillin,
and 100 µg/ml streptomycin (Life Technologies, Inc.). The cells were
maintained at 37 °C in a humidified, 5% CO2, 95% air
atmosphere.
Acinar Formation Assay--
Laminin-1 and growth factor-reduced
Matrigel were prepared as described previously (25-27). Either
Matrigel (135 µg in 50 µl of media) or laminin-1 (155 µg in 50 µl of media) was added to 96-well tissue culture dishes. HSG cells
(1.0 × 104 cells in 100 µl of media) were added and
incubated for 48 h and stained with Diff-Quick (American
Scientific Products, McGaw Park, IL). An image analysis program (NIH
Image) was used to measure the number and area of acini formed in the
matrix. We assayed three fields/well in triplicate wells (70-150 acini
total) and determined the percentage of acini with a surface area
either less than 800 µm2 or greater than 800 µm2.
Preparation of Peptides--
All peptides were manually
synthesized using the Fmoc (9-fluorenylmethoxycarbonyl)-based solid
phase strategy with a C-terminal amide form and purified by
reverse-phase high performance liquid chromatography as described
previously (18).
Inhibition of Acinar Formation and Culture of Cells with
Peptides--
Peptides (100 µg/ml) and cells (1.0 × 104 cells) were added to wells containing laminin-1 (155 µg in 50 µl of serum-free media), and after 48 h the cells
were stained and the sizes of acini were measured. HSG cells were also
cultured with the laminin-derived peptides (100 µg/ml) in 96-well
dishes for 48 h. The peptides were incubated in the wells with 50 µl of media for 2 h before the cells (in a volume of 150 µl)
were added; unbound peptide was not removed from the wells. Cell
proliferation was measured using a nonradioactive cell proliferation
assay (Cell Titer 96TM; Promega, Madison, WI), according to the
manufacturer's instructions.
Function-perturbing integrin antibodies were added to cells cultured on
laminin-1. Antibodies to Integrin Immunostaining--
Acini were immobilized on
microscope slides using a Cytospin centrifuge (Shandon, Pittsburgh,
PA), fixed with 3.7% formaldehyde for 10 min, and permeabilized by
treatment with ice-cold methanol for 10 min. Cells were blocked by
overnight incubation at 4 °C in blocking buffer (5% goat serum,
0.5% bovine serum albumin (BSA), and 0.075% saponin) and then
incubated for 2 h at room temperature with antibodies diluted in
blocking buffer. The primary antibodies recognizing integrin subunits
were Rotary Cell Culture--
A rotary cell culture system with
10-ml-high aspect ratio vessel bioreactors was used for suspension
cultures (Synthecon, Houston, TX). 5 × 105 cells/ml
were cultured in Dulbecco's modified Eagle's medium/F-12 (1:1),
containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The bioreactors were rotated at 22 rpm at
37 °C in an humidified, 5% CO2, 95% air atmosphere.
Cells were cultured for up to 24 h with 0.5 mg/ml Matrigel; 0.2 mg/ml laminin-1; 0.1 mg/ml AG73, AG73T, and MG73; or no peptide. In later experiments, heparin (0.1 mg/ml) was added to the bioreactors alone and in combination with laminin-1 and the peptides.
Cell Adhesion Assays--
A 50-µl volume containing either
laminin-1 (0.1 µg/well), AG73 (0.05 µg/well), MG73 (2 µg/well),
or E3 (1.5 µg/well) was dried overnight onto round-bottomed 96-well
plates. The wells were blocked with 3% BSA for 1 h at 37 °C
and then washed twice with 0.1% BSA. 25,000 cells in 100 µl of 0.1%
BSA in Dulbecco's modified Eagle's medium/F-12 were added per well
for 45 min at 37 °C. The medium was gently removed from the wells,
and the cells were stained with crystal violet for 10 min and washed
twice with water. The cells were lysed with 50 µl of 10% SDS, and
the optical density (600 nm) was measured.
Inhibition of Cell Adhesion--
Antibodies and cells were
rotated for 15 min at 4 °C and then added to the adhesion assay.
Function perturbing antibodies to the Treatment of the Cell Surface with GAG-degrading
Enzymes--
Cells were cultured for 2 h with 2 µg/ml
cycloheximide to inhibit new protein synthesis (23), washed, and
resuspended in 100 µl of medium consisting of Dulbecco's modified
Eagle's medium/F-12, 0.1% BSA, 2.0 µg/ml cycloheximide, 2.0 mM aminoethylbenzesulfonyl fluoride, 1.5 mM
aprotinin, and either 0.05 units/ml heparitinase or 0.02 units/ml
heparinase (Seikagaku, Rockville, MD). Cells were incubated at 37 °C
for 90 min, with mixing every 15 min. After enzyme treatment, the cells
were used in a cell adhesion assay as described above. Cells were also
treated with inactivated enzymes (100 °C for 10 min and the addition
of 1 mM ZnCl2) as negative controls.
Peptide and Laminin-1 Affinity Chromatography--
Affinity
columns (1 ml) were prepared using Affi-Gel 10 (Bio-Rad) according to
the manufacturer's instructions. Laminin-1, AG73, MG73 affinity
columns, and negative control columns were run in parallel. An AG73T
column was used as a peptide negative control, a BSA column was used as
a protein negative control column, and an Affi-Gel control column was
used to detect nonspecific binding to the resin. The columns were
equilibrated in running buffer containing 6.0 M urea, 1%
Triton X-100, 2.0 mM phenylmethylsulfonyl fluoride in TBS,
pH 7.4. Cells were surface-biotinylated using sulfo-NHS-biotin (Pierce,
IL) as described in the manufacturer's instructions. A crude cell
membrane fraction was prepared by hypoosmotic lysis in 10 mM KCl, 20 mM Tris, pH 7.4, 0.1%
GAG Digestion and Western Analysis--
Material eluted from the
peptide affinity columns with 1.0 M NaCl was precipitated
with acetone and digested for 4 h with both heparitinase (0.1 units/ml) and chondroitinase ABC (1.0 units/ml) containing 1.5 mM aprotinin, 2.0 mM aminoethylbenzesulfonyl
fluoride, 5.0 mM EDTA, 1.0 mM leupeptin, and
0.36 mM pepstatin A. The digested material was separated by
4-20% SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose filters. The filters were incubated with HSE-1 (1:1000)
a polyclonal serum to the recombinant ectodomain of human syndecan-1
(gift of M. Bernfield, Boston, MA). Antibody-reactive material was
detected with a horseradish peroxidase-conjugated secondary antibody
and visualized by ECL.
Laminin Peptides Decrease the Size of Acini Formed on
Laminin-1--
We examined the effects of various laminin-derived
synthetic peptides on HSG cells cultured in an acinar formation assay
on laminin-1 (Table II). The assay is a
competitive inhibition assay where peptides compete for cell binding
with intact laminin-1. The addition of AG73 resulted in a significant
reduction in the size of acini formed (Fig.
1a). AG73 had the greatest
effect, whereas AG73T, a scrambled version of AG73, had no effect (Fig. 1b) and was used as a negative control. Other active
peptides from the G-domain of the laminin Laminin Peptides Promote Morphological Organization of HSG
Cells--
Most laminin peptides tested had no effect on the
morphological organization of HSG cells, and the cells grew in a
monolayer (Fig. 2a).
Surprisingly, cells incubated with AG73 (Fig. 2b) and MG73
(Fig. 2c) formed multicellular three-dimensional structures. Hematoxylin- and eosin-stained sections of the structures formed with
the peptides (Fig. 2, d and e) revealed they were
not well organized. The cell nuclei were not polarized to the basal
surface of the structures and lumens did not form. In contrast, cells incubated with laminin-1 formed acinar structures with basally polarized nuclei, lumens, and cystatin immunoreactivity (23). When the
peptides were added to collagen gels, acinar formation was not observed
(data not shown).
The major problems of culturing cells with peptides were that cell
adhesion, spreading, and formation of multicellular structures all
occur in the same well. Also, high concentrations of peptides were
required to see morphological effects. We therefore used suspension
cultures to study the effect of the peptides in solution utilizing a
rotary cell culture system. Acinar formation in the bioreactor occurred
within 24 h in the presence of laminin-1 (Fig. 3, a and e). The
cells formed both spherical and multilobulated structures (Fig.
3a) as observed by light microscopy. In cross-sections, stained with hematoxylin and eosin (Fig. 3e), the cell
nuclei were clearly located at the basal surface of the structures and lumen formation occurred. Similar to our previous results (23), the
formation of acinar structures was a cell density-dependent event occurring within 24-48 h. When cells were cultured for 24 h
in a bioreactor either with AG73T (Fig. 3, b and
f) or without peptide (not shown), the cells remained mainly
as a single cell suspension. In contrast, both AG73 and MG73 stimulated
HSG cells to form large multicellular structures with a smooth
lobulated surface, appearing similar by light microscopy to those
formed with laminin-1 (Fig. 3, c and d). However,
in hematoxylin and eosin-stained cross-sections of the structures that
formed with AG73 and MG73 (Fig. 3, g and h), it
is apparent that they do not organize morphologically as well as those
that formed with laminin-1 (Fig. 3e). The structures that
formed with the peptides do not have cell nuclei polarized to the basal
surface, and there was no lumen formation. These results suggested that
the peptides may be providing some but not all of the signals for the
morphological changes in the process of acinar differentiation. It is
likely that multiple sites on laminin, including integrin and
nonintegrin interactions and/or conformational requirements of the
intact laminin molecule are required for complete differentiation.
Since integrins are well characterized laminin receptors, we wanted to
define their role in HSG cell acinar formation. We also wanted to
determine whether the mechanism of action of the peptides involved integrin or nonintegrin interactions.
We studied the effect of anti-integrin antibodies on cell adhesion to
peptides and to laminin-1 using a solid phase adhesion assay (Fig.
4c). Dose-response curves and time courses of adhesion determined the amount of each substrate and the time of the assay. We
coated the wells with the least amount of substrate that gave a maximal
cell adhesion response. The cells attached to the peptides more quickly
than to laminin-1; i.e. 50% of total adhesion to AG73
occurred in 10 min, whereas 50% of total adhesion occurred to
laminin-1 in 30 min (data not shown). None of the integrin antibodies
used inhibited cell adhesion to AG73 or MG73. The
anti-
HSG cells interact with multiple sites on the laminin-1 molecule, and
adhesion to a peptide sequence may be affected by the conformation of
the surrounding protein. We compared the ability of AG73 to inhibit
cell adhesion to laminin-1, AG73, MG73, and the E3 fragment, which
contains the AG73 sequence. AG73 slightly inhibited HSG cell adhesion
to laminin-1 and inhibited adhesion to E3, AG73, and MG73 (Fig.
5). Therefore, AG73 may be an important adhesion site in the E3 fragment. AG73T did not inhibit cell adhesion to any substrate.
The addition of EDTA inhibited cell adhesion to laminin-1 and partially
reduced cell adhesion to E3, AG73, and MG73 (Fig. 5). Taken together
with the anti- Adhesion of HSG Cells to Laminin Peptides and E3 Is Inhibited by
Heparin--
Since the E3 fragment contains a heparin binding region,
we tested the effects of GAGs on cell adhesion to the peptides, E3, and
laminin-1. None of the GAGs tested (all at 50 µg/ml) inhibited cell
adhesion to laminin-1 (Fig.
6a). Heparin inhibited cell
adhesion to E3, and chondroitin sulfate C had a slight inhibitory
effect, suggesting that cell adhesion to E3 was mediated by both GAGs. Heparin inhibited cell adhesion to both AG73 and MG73. Heparan sulfate
inhibited cell adhesion to AG73 but only partially to MG73 (Fig. 6,
c and d). At higher doses (100 µg/ml), heparan
sulfate inhibited adhesion to both MG73 and AG73 (data not shown).
De-N-sulfated heparin; chondroitin sulfates A, B, and C;
keratan sulfate; and hyaluronic acid had no effect on cell adhesion to
the peptides. These data suggest that the adhesion to the peptides may
be mediated by regions of the GAG chains with different sulfation
patterns and that N-sulfation may be involved.
Cell adhesion to the peptides still occurred when cells were
preincubated with heparin and washed before being used in an adhesion
assay. However, cell adhesion did not occur when heparin was
preincubated in the peptide-coated well and then washed out of the well
before the addition of the cells (Table
III). Thus, heparin was not binding to
the cell surface but was binding to the peptide and blocking cell
adhesion to the peptide.
We added heparin (0.1 mg/ml) to the bioreactors in combination with
laminin-1 and the peptides. Heparin inhibited the morphological organization of the cells cultured with AG73 and MG73 (data not shown);
they appeared as a single cell suspension, similar to the cells
cultured with the scrambled peptide (Fig. 3b). The addition of both heparin and laminin-1 in the bioreactor had no effect on the
formation of acinar-like structures (similar to Fig. 3a). These results are consistent with the acinar formation assay, where
heparin did not affect the size of acini formed on laminin-1 (data not
shown), and with the finding that heparin did not inhibit cell adhesion
to laminin-1 (Fig. 6a). As a control, we added heparin to
cells in the bioreactor without peptides or laminin and also found no
effects on the morphology of the cells.
Treatment of the Cell Surface with Glycosaminoglycan-degrading
Enzymes--
Heparitinase treatment slightly decreased cell adhesion
to laminin-1 and to AG10 (p < 0.05) when compared with
cells treated with inactive enzyme (Fig.
7). This suggested that cell adhesion to
intact laminin may be in part mediated by heparan sulfate, although we
could not significantly inhibit adhesion to laminin-1 with heparan
sulfate in the previous adhesion assay. AG10, another active peptide
from the G-domain, was included as a positive control for cell adhesion
to a peptide. Cell adhesion to AG10 is mediated by
Laminin-1 and Peptide Affinity Chromatography and Western Blot
Analysis with HSE-1, an Antiserum Recognizing Syndecan-1--
The
major species bound to the AG73 and MG73 peptide columns eluted with
1.0 M NaCl and appeared as a high molecular weight smear,
with a molecular mass greater than 250 kDa (Fig.
8a and b,
lanes 5). Columns prepared with a scrambled
peptide, AG73T (Fig. 8c), with BSA (not shown), or with
uncoupled Affi-Gel resin (not shown) allowed identification of
nonspecific interactions and gave similar results. As expected,
laminin-1 affinity chromatography (Fig. 8d) identified
multiple species binding to the column including a high molecular
weight smear with a similar appearance to that eluted from the peptide
columns. We were unable to inhibit adhesion of the high molecular
weight smear to the laminin-1 column by preincubating the cell lysate
with AG73 (data not shown). We interpret this result as meaning there
are other syndecan binding sites on the laminin molecule. It is well
documented that other heparin binding sites exist on laminin. This is
also supported by our cell adhesion experiments to the intact laminin-1
molecule (Fig. 5), where we were unable to completely inhibit cell
adhesion to laminin-1 with AG73. Another species (~150 kDa) also
eluted from the laminin-1 column and corresponded to the expected size
of dystroglycan, another proteoglycan known to bind E3 (31). However, in a Western blot (data not shown) with an anti-
Treatment of the material bound to the peptide affinity columns with
heparitinase and chondroitinase ABC resulted in a shift in molecular
weight of the smear and enriched for a core species at ~67 kDa (Fig.
9). Enzyme treatment of the material
eluted from the laminin-1 column also showed a similar sized core
protein, although multiple other species were also present (data not
shown). Western blot analysis with HSE-1, a polyclonal antibody to the core protein of human syndecan-1, revealed an antibody-reactive band
that co-migrated with the major band that appeared after heparitinase
and chondroitinase ABC treatment (Fig. 9). The other minor species that
appeared after GAG removal have not yet been identified. These data
demonstrate that syndecan-1 binds to the AG73 sequence in the G-domain
of laminin-1 and to the homologous laminin-2 peptide, MG73.
Our previous studies identified a role for laminin-1 in the
morphological differentiation of human salivary gland cells cultured on
Matrigel, a basement membrane substrate (23, 32). Others demonstrated
that laminin-1 played an important role in salivary gland morphogenesis
(9, 33, 34). The E3 fragment from the G-domain of the laminin AG73 and MG73 have been tested in both in vitro and in
vivo assays with a number of cell types. They played a role in
melanoma cell adhesion and metastases (21) and promoted neuronal cell line adhesion and neurite outgrowth (20). Other peptides from the
G-domain of laminin-1 were active with neuronal cells in a cell
type-specific manner distinct from that observed with HSG cells. A high
degree of homology exists between AG73, MG73, and the corresponding
regions in the AG73 is located in E3, a fragment of laminin-1 that mediates cell
binding and has heparin binding activity. AG73 inhibited cell adhesion
to itself and to E3 but not to laminin-1. Interestingly, the addition
of heparin (0.1 mg/ml) inhibited the morphological effects of the
peptides in the bioreactor, and the cells remained as a single cell
suspension. However, heparin did not inhibit the morphological effects
of the entire laminin-1 molecule. Clearly, this site is not the only
active site on laminin for HSG cells, but it does influence cell-cell
interactions and morphological organization.
Our studies begin to define the role of certain cell-matrix
interactions in acinar differentiation. The inhibition of acinar formation on laminin-1 with Syndecans, by way of their GAG side chains, have been proposed to
function as co-receptors with integrins for fibronectin (10). Laminin
has multiple heparin binding sites, which are found at the ends of the
molecule and co-localize with integrin binding sites. It has been
suggested that integrin binding and cell surface proteoglycan binding
may be involved in laminin-mediated cell adhesion and cell signaling
(7). Our data are consistent with this hypothesis, since both integrin-
and syndecan-mediated adhesion are involved in acinar formation.
Recently, a direct interaction between cell surface chondroitin sulfate
GAG and Interaction between laminin and syndecans has been previously shown.
NIH-3T3 cells transfected with syndecan-1 produced a laminin binding
form of syndecan-1 (14). The E3 fragment of laminin-1 had the highest
binding to syndecan-1 among the elastase-derived fragments of laminin.
Furthermore, overexpression of syndecan-1 resulted in an increase in
cell adhesion to laminin. Analysis of two cell lines with identical
amounts of syndecan-1 revealed that the syndecan-1 from one cell line
does not bind collagen, whereas the other does (36). Analysis of the
side chains showed that the fine structure of heparan sulfate differed
and that these differences could control fundamental cell properties
such as cell matrix adhesion. Studies of the syndecan-1 side chain
variability between mammary gland cells and cell lines revealed
differences in the number of highly sulfated domains in the GAG side
chains (13). These data support the concept that variability of
syndecan-1 GAG side chains may affect the interaction between different
laminin isoforms and syndecan-1.
Our data show differences between the interaction of HSG cells with
AG73 and MG73. Although both peptides bound syndecan-1, they had
different effects on HSG cells. Our in vitro data are supported by collaborative studies with Kadoya's group (22) that
showed AG73 inhibited branching morphogenesis of day 13 embryonic salivary gland explants but MG73 had no effect. In our in
vitro acinar formation assay, AG73 decreased the size of the acini
formed, but MG73 had no effect. Furthermore, both heparin and heparan sulfate inhibited HSG cell adhesion to AG73, but heparan sulfate decreased cell adhesion to MG73 by ~50%. Heparitinase treatment of
HSG cells, which digests less sulfated regions of heparan sulfate, inhibited cell adhesion to AG73. However, heparinase treatment, which
digests more sulfated, "heparin-like" domains of heparan sulfate,
did not inhibit cell adhesion to AG73. Taken together, these data
suggest that MG73 might bind to a more highly sulfated region of
heparan sulfate than AG73. We have not analyzed the syndecan-1 heparan
sulfate side chains that bind AG73 as compared with those that bind
MG73. The differential adhesion to heparan sulfate would allow cells to
respond distinctly to the heparan sulfate binding effectors (in our
case the laminin isoforms) in the cellular microenvironment (13).
During early stages of salivary gland morphogenesis (embryonic day 13),
laminin In summary, here we have identified a syndecan-1 binding site in the
G-domain of laminin. The laminin We thank Dr. M. Bernfield for the
anti-syndecan-1 antibody HSE-1, Dr. K. Campbell for the
anti-dystroglycan antibody, Dr. S Akiyama (NIH) for the anti-integrin
antibodies, and Naomi Amichay for helping to set up the rotary cell
culture system system.
*
The studies were supported in part by the NASA-National
Institutes of Health Center for Tissue Culture.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
HSG, human
submandibular gland; GAG, glycosaminoglycan; BSA, bovine serum
albumin.
Laminin-1 and Laminin-2 G-domain Synthetic Peptides Bind
Syndecan-1 and Are Involved in Acinar Formation of a Human
Submandibular Gland Cell Line*
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 and 2 chains. The 1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size
of acini formed on laminin-1. Cells cultured with either AG73 or the
homologous 2 chain peptide MG73 (KNRLTIELEVRT) form structures that
appear acinar-like, but the cell nuclei are not polarized to the basal
surface and no lumen formation occurs, indicating that additional sites
on laminin are required for complete differentiation. The G-domain of
laminin-1 contains both integrin and heparin binding sites, and
anti-
1-integrin antibodies disrupt acinar
formation. Cell adhesion to the peptides and to E3, an elastase digest
fragment of laminin-1 containing AG73, is specific, since other laminin
peptides or EDTA do not compete the binding. Heparin and heparan
sulfate decrease cell adhesion to AG73 and MG73 but
anti-1-integrin antibodies have no effect. Treating the
cell surface with heparitinase inhibits adhesion to both AG73 and MG73.
We isolated cell surface ligands using both peptide affinity
chromatography and laminin-1 affinity chromatography. Treating the
material bound to the affinity columns with heparitinase and
chondroitinase enriches for a core protein identified as syndecan-1 by
Western blot analysis, thus identifying a syndecan-1 binding site in
the globular domain of laminin-1 and laminin-2. In summary, multiple
interactions between laminin and HSG cells contribute to acinar
differentiation, involving both 1-integrins and
syndecan-1.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
61, 31, 71 and 64 within the
C-terminal G-domain of the 1 and 2 chains (7). Laminin-1 also
contains multiple heparin binding sites including one in the E3
fragment, although the exact epitope is undefined (2, 8). The E3
fragment is important in early stages of branching morphogenesis of the
salivary gland, since antibodies to E3 inhibit branching of salivary
gland buds in culture (9).
1 chain promotes
cell adhesion and migration and inhibits angiogenesis and tumor
metastasis (15). IKVAV, on the 1 chain, promotes cell adhesion,
neurite outgrowth, experimental metastasis, collagenase IV secretion,
angiogenesis, and tumor growth (16, 17). Systematic screening of the
G-domain of the laminin 1 chain has identified five active peptides
with cell adhesion and spreading activities (18, 19). Testing these
peptides for activity with neuronal cell lines identified peptide
sequences from corresponding regions of the 1 and 2 laminin
chains with cell type specificity for neurite outgrowth (20). These two
active sequences, AG73 (RKRLQVQLSIRT) from laminin-1 and MG73
(KNRLTIELEVRT) from laminin-2, are in the E3 fragment of the G-domain
and are highly conserved between murine laminin chains (Table
I). Conservation of these sequences among
different species suggests a critical biological role (18, 19). AG73
promotes malignant behavior of melanoma cells in vitro and
tumor growth and metastases in vivo (21). AG73 also inhibits branching morphogenesis of cultured embryonic mouse submandibular glands (22).
Protein sequence comparison of the highly conserved mouse laminin chain isoforms homologous to AG73
3 also contribute to
acinar formation (23). Acinar differentiation is a multistep process
involving multiple cell-matrix interactions, but here we focus on the
interactions between HSG cells and laminin. Integrins clearly play an
important role, since function-perturbing anti-1-integrin antibodies disrupt acinar formation. We
have used synthetic peptides from the G-domain of laminin 1 and 2 chains to identify sites involved in acinar formation. We have identified the heparan sulfate proteoglycan syndecan-1 as a cell surface ligand for one of the peptides and for laminin-1. Our results
suggest multiple interactions between laminin-1 and HSG cells occur
during acinar formation involving both 1-integrins and
syndecan-1.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1- and
5-integrin subunits (M13 and M16, gifts of Dr. S. K. Akiyama, National Institutes of Health) and nonimmune rat IgG
(Sigma) as a negative control were used at a 1:200 dilution. Antibodies
to 2, 3 (P1E6 and P1B5; Life
Technologies), and 6 (GoH3, AMAC Inc., Westbrook, ME)
integrin subunits and normal mouse ascities (NS-1; Sigma) were used
1:40. After 48 h, the cells were fixed in 3.7% paraformaldehyde, embedded in paraffin, and sectioned and stained with hematoxylin and
eosin.
1 (M13, 1:400 dilution), 5 (M16,
1:400), 2 (P1E6, 1:100), v (VNR147
1:100), and 6 (GoH3, 1:100). The cells were incubated
with fluorescein-labeled secondary antibodies in blocking buffer. After
washing, immunofluorescence was detected using a confocal
laser-scanning microscope (Leica, Heidelburg, Germany).
6, (GoH3),
1 (M13), 5 (M16), 2
(P1E6), and 3 (P1B5) integrin subunits were used.
Nonimmune rat IgG and mouse ascities (Sigma) were used as negative
controls. Antibodies were used in a range of dilutions (1:20, 1:33, and
1:100). Cells were also preincubated for 10 min at room temperature
with peptides (1 µg/well), EDTA (2 mM), or GAGs (50 µg/ml) and then added to the adhesion assay. Heparin;
de-N-sulfated heparin; heparan sulfate; chondroitin sulfates
A, B, and C; keratan sulfate; and hyaluronic acid (Sigma) were all
tested. In other experiments, heparin was incubated with the
peptide-coated wells for 30 min, the wells were washed twice, and the
cells were added. Conversely, heparin was incubated with the cells for
30 min, and the cells were washed twice and then added to the
peptide-coated wells.
-mercaptoethanol, 1 mM EDTA. After Dounce
homogenization, the nuclei were removed by centrifugation (1500 × g for 5 min). The NaCl concentration of the remaining
supernatant was increased to 150 mM, and the cell membranes
were pelleted at 50,000 × g for 30 min. The cell membrane pellet was solubilized in 2 ml of 8.0 M urea, 1%
Triton X-100, 0.5 M KCl, 2.0 mM
phenylmethylsulfonyl fluoride in TBS, pH 7.4, and insoluble material
was removed by centrifugation at 14,000 × g for 20 min. The volume was increased to 10 ml with running buffer. A 500-µl
aliquot of the crude cell membrane fraction (~700 µg of total
protein) was incubated with the peptide affinity column for 2-4 h at
4 °C. The columns were washed with running buffer and then
sequentially eluted with 2-ml aliquots of running buffer containing
either 20 mM EDTA, 250 mM NaCl, 1.0 M NaCl, or 2.0 M NaCl. Bound material was
precipitated with acetone, washed in 80% ethanol, and air-dried.
Samples were separated by SDS-polyacrylamide gel electrophoresis
(4-20% gels) and transferred to nitrocellulose filters (Novex, San
Diego, CA). The filters were blocked in 3% nonfat milk in PBS-T (Tween
20, 0.1%), washed, incubated with streptavidin-horseradish peroxidase
in PBS-T for 1 h and then washed again three times for 10 min in
PBS-T. The biotinylated material was visualized by ECL (Amersham
Pharmacia Biotech). In separate experiments to try to inhibit syndecan
binding to laminin-1, the crude cell membrane fraction was preincubated
with 1 mg/ml AG73 or AG73T for 1 h before incubating with
laminin-1 affinity columns. The columns were then eluted, and the
fractions were analyzed as described above.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 chain and their 2
homologues (19) as well as other previously identified sequences such
as IKVAV, YIGSR, RGD, and SINNNR (28) had no effect on the size of
acini formed except for AG10, which had a small but consistent effect.
Effect of laminin peptides on acinar-like morphological development of
HSG cells
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Fig. 1.
AG73 decreases the size of acini formed on
laminin-1 compared with other peptides. Peptides (100 µg/ml) and
cells were added to wells containing laminin-1, and after 48 h the
cells were stained and the sizes of acini were measured. a,
the numbers of acini with surface area greater or less than 800 µm2 are shown. Each bar represents three
fields measured/well, and three wells are used for each time point
(70-150 acini). The graph is representative of at least three similar
experiments, and the S.E. are indicated. *, p = 0.0011;
**, p = 0.0011. b, light micrographs of the
acini after 48 h incubation with AG73T (scrambled control peptide)
and AG73. Bar, 50 µm.
View larger version (126K):
[in a new window]
Fig. 2.
HSG cells form multicellular structures when
cultured with two laminin derived peptides. The peptides or
laminin-1 were incubated in 96-well dishes for 2 h before the
cells were added; unbound peptide was not removed from the wells. Cells
cultured on either plastic or AG73T (a) grow as a monolayer.
Cells cultured on a gel of laminin-1 form spherical structures
(b). HSG cells cultured with AG73 (c) and MG73
(d) form three-dimensional structures, although some cells
spread on the culture dish. Hematoxylin and eosin-stained sections of
HSG cells cultured on laminin-1 (e) show the cell nuclei are
polarized to the basal surface of the acini. In comparison, sections of
the structures formed when cells are cultured with AG73 (f)
and MG73 (g) show less organized structures where the cell
nuclei are not polarized to the basal surface of the cells.
View larger version (92K):
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Fig. 3.
Effect of laminin-1 and laminin-derived
peptides on HSG cells cultured in a in rotary cell culture system.
3 × 105 cells/ml cultured for 24 h with 0.2 mg/ml laminin-1 (a), 0.1 mg/ml AG73 (c), and 0.1 mg/ml MG73 (d) all formed multicellular lobulated and
spherical structures that appeared similar by light microscopy. Cells
cultured with the scrambled peptide (b), 0.1 mg/ml AG73T,
appeared as a predominately single cell suspension. Hematoxylin and
eosin-stained sections showed that cells cultured with laminin-1
(e) contained nuclei polarized to the basal surface of the
structures and lumens beginning to form. In comparison, cells cultured
with AG73 (g) and MG73 (h) were not as well
organized. The cell nuclei were not polarized to the basal surface of
the structures, and lumens were not apparent. Cells cultured with AG73T
remained as a single cell suspension (f). Bar, 20 µm.
1-Integrins Are Involved in Acinar Formation and
Adhesion to Laminin but Not in Adhesion to the Synthetic
Peptides--
Immunofluorescent analysis of integrins on the cell
surfaces of acini showed differential distribution (Fig.
4a). Anti-1 and
v stained the cell matrix surfaces and the cell-cell
contacts, whereas anti-5 only stained the cell-cell
contacts. Anti-6 and anti-2 stained
mainly the cell matrix surface, although 6 also weakly
stained the cell-cell contacts and 2 had mainly an
intracellular localization. When function-blocking antibodies were
tested with HSG cells on laminin-1, only the
anti-1-integrin antibody inhibited acinar formation.
Paraffin sections of these cells stained with hematoxylin and eosin
showed a lack of organization into acinar structures and no
polarization of cell nuclei (Fig. 4b).
View larger version (69K):
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Fig. 4.
Localization and role of integrins in acinar
formation. a, immunostaining showed differential
localization of integrin subunits within acini. Acini were stained with
antibodies against integrin subunits 1 (M13),
5 (M16), 2 (P1E6), v
(VNR147), and 6 (GoH3). Immunofluorescence was detected
with 1-µm optical sections using a confocal laser-scanning
microscope. b, anti-1-integrin antibodies
(M13) disrupted acinar formation. Antibody or nonimmune rat IgG was
incubated with cells for 48 h, and the cells were sectioned and
stained with hematoxylin and eosin. c, integrin antibodies
do not inhibit cell adhesion to the peptides. Antibodies and cells were
preincubated for 15 min at 4 °C, added to the wells coated with
either laminin-1 or the peptides, and incubated for 30 min at 37 °C.
The cells were stained and lysed, and the A560
was measured. Nonimmune rat IgG and mouse ascities were used as
negative controls. *, p < 0.005.
1-integrin antibody inhibited adhesion of HSG cells
to laminin-1. The -subunit antibodies alone (or in combinations,
data not shown) were unable to inhibit adhesion to either laminin-1 or
the peptides. Taken together, these data suggest that
1-integrins were important for laminin-1-mediated acinar
formation but did not bind to AG73 and MG73.
View larger version (26K):
[in a new window]
Fig. 5.
Effect of peptides and EDTA on HSG cell
adhesion. Cell adhesion assays are described under "Materials
and Methods." AG73 (1 µg/well) only partially inhibited cell
adhesion to laminin-1, whereas EDTA (2 mM) inhibited most
cell adhesion. AG73 specifically inhibited cell adhesion to E3, AG73,
and MG73, whereas EDTA only partly inhibited and AG73T (1 µg/well)
had no effect. Triplicate wells are used for each condition, and the
graph is representative of three similar experiments. S.E. values are
indicated. *, p < 0.015; **, p < 0.0009.
1-integrin results (Fig. 4c), these data suggest that HSG cells attach to laminin-1 by multiple interactions, including integrin, divalent cation-independent, and
heparin-mediated interactions. The data suggest that cell adhesion to
AG73 and to E3 is by a similar mechanism.
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Fig. 6.
Effect of glycosaminoglycans on HSG cell
adhesion to laminin-1, E3, and laminin-derived peptides. Cell
adhesion assays are described under "Materials and Methods."
Heparin inhibited HSG cell adhesion to AG73, MG73, and E3 but not to
laminin-1. Lane 1, no GAG; lane
2, heparin; lane 3, heparan sulfate;
lane 4, chondroitin sulfate A; lane
5, chondroitin sulfate B; lane 6,
chondroitin sulfate C; lane 7, keratan sulfate.
GAGs were all 50 µg/ml. Triplicate wells are used for each condition,
and the graph is representative of three similar experiments, and S.E.
values are indicated. **, p < 0.0001; *,
p < 0.005
Heparin inhibits cell adhesion to AG73 by binding to the peptide
substrate not to the cell surface
6-integrin (18). The decrease in cell adhesion to AG10
with GAG removal suggests that GAGs may affect ligand binding
properties of the integrin, as recently reported with the
41-integrin (29). Heparitinase treatment
inhibited cell adhesion to AG73 and to MG73 compared with cells treated
with inactive enzyme. However, heparinase treatment of the cell surface
inhibited the adhesion to MG73 but did not significantly decrease the
adhesion to AG73. Heparinase digests more highly sulfated or
"heparin-like" regions of the GAG chain, whereas heparitinase
digests less sulfated or "heparan sulfate-like" regions of the GAG
chain. Our earlier finding showed that heparan sulfate decreased cell
adhesion to AG73 more than to MG73 (Fig. 6, b and
c). Taken together, these results suggested that AG73 and
MG73 bind to regions of the heparan sulfate chains with different
sulfation patterns i.e. MG73 binds to a more sulfated (or
heparin-like) region of the heparan sulfate side chain than AG73. Also,
these results suggested that the cell surface receptor for AG73 could
be a heparan sulfate proteoglycan.
View larger version (14K):
[in a new window]
Fig. 7.
Effect of heparitinase or heparinase
treatment of the cell surface on cell adhesion to AG73 and MG73.
Heparitinase treatment decreased cell adhesion to AG73 and MG73 by 75%
but decreased adhesion to laminin-1 by only 20%. Heparinase treatment
inhibited cell adhesion to MG73 but not to AG73. Enzyme treatments are
described under "Materials and Methods." Cells were also treated
with inactivated enzymes as negative controls, and the percentage of
cell adhesion and p values were calculated by comparing
adhesion to cells treated with inactive enzymes. Triplicate wells were
used for each condition, and the graph is representative of
at least three similar experiments. S.E. values are indicated. *,
p < 0.05; **, p < 0.001.
-dystroglycan antibody (IIH6 C4, a kind gift of K. Campbell), the band did not stain.
Our attempts to isolate an AG73 receptor using octyl glucoside or
Triton X-100 alone were unsuccessful at identifying specific binding
molecules. Syndecans are insoluble in these lysis buffers and
precipitate with the cytoskeleton (30).
View larger version (82K):
[in a new window]
Fig. 8.
Identification of potential cell surface
receptors by AG73, MG73, and laminin-1 affinity chromatography of
biotinylated HSG cell membranes. Surface-biotinylated HSG cell
membranes were incubated with the columns, and after collecting the
flow-through (lane 1), the columns were washed
(lane 2) and then sequentially eluted with 20 mM EDTA (lane 3), 250 mM
NaCl (lane 4), 1.0 M NaCl (lane
5), or 2.0 M NaCl (lane
6). Bound material was blotted and detected with
streptavidin-horseradish peroxidase/ECL. AG73 (a) and MG73
(b) affinity columns gave similar results; the major species
bound appeared as a high molecular weight smear > 250 kDa that
was eluted with 1.0 or 2.0 M NaCl (arrow). AG73T
(c) and BSA (not shown) affinity columns were similar to
each other. Laminin-1 affinity chromatography (d) revealed
multiple components bound the column including a high molecular weight
smear (arrow), which was also eluted in the 1.0 NaCl
eluate.
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[in a new window]
Fig. 9.
GAG removal from material bound to the AG73
and MG73 peptide affinity columns and subsequent Western blot analysis
with an antibody recognizing syndecan-1. Biotinylated material
from the 1.0 M NaCl eluate was digested with heparitinase
and chondroitinase ABC. The digested material was detected with
streptavidin-horseradish peroxidase/ECL. Lane 1,
enzymes only; lane 2, undigested sample;
lane 3, heparitinase/chondroitinase ABC digest;
lane 4, Western analysis with HSE-1 of digested
material from lane 3.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1
chain was involved, since salivary gland explants treated with an
anti-E3 antibody were unable to form branches (9). We identified a
number of biologically active peptides in the G-domain of laminin-1
(18, 19, 35). The AG73 peptide, from the E3 fragment of the G-domain,
decreased the size of acini that formed on laminin-1, suggesting that
this site of interaction was important for acinar formation.
Furthermore, of the active peptides identified, only AG73 and the
corresponding homologous peptide MG73 from the 2 laminin chain,
stimulated multicellular morphological organization of HSG cells in
suspension cultures. The peptides may bind to the cell surface and
promote cell-cell adhesion and/or stimulate the cells to begin
morphological organization. However, in hematoxylin and eosin-stained
sections of the peptide-induced structures, the cells were not as well organized as those cultured with laminin-1. The cell nuclei were not
polarized to the basal surface of the cells, and lumens within the
structures were not apparent, suggesting that other sites on laminin-1
were necessary for complete differentiation.
3a, 3b, 4, and 5 laminin chains (Table I).
These conserved sequences may have an important role in cell surface
receptor interactions. Taken together, these studies in a number of
biological systems and the highly conserved sequence suggest an
important role for AG73 and for MG73 in mediating some of the
biological functions of laminin.
1-integrin antibodies
suggests an important role for 1-integrins in the
multistep process of acinar differentiation. Furthermore, integrin
subunits showed differential localization during acinar formation.
Inhibition of HSG cell adhesion to laminin-1 with the
1-integrin antibody and with EDTA indicated that
integrins were important HSG cell laminin receptors. However, neither
integrin antibodies nor EDTA inhibited cell adhesion to the active
peptides. Rather, heparin and heparan sulfate inhibited cell adhesion
to the peptides and to E3. Furthermore, treatment of the cell surface
with heparitinase reduced cell adhesion to the peptides but not to
laminin-1. These data indicate that the cell surface ligand for the
peptide contains heparan sulfate. AG73 peptide affinity chromatography
of cell membrane extracts identified a high molecular weight species
comparable in size with the syndecans. Heparitinase and chondroitinase
treatment of this material resulted in a major core protein (~64
kDa), which was identified by subsequent Western blot analysis as
syndecan-1. We were unable to identity the other minor species at ~36
kDa (Fig. 9, lane 3). Our data suggest that
syndecan-1 is the major AG73 and MG73 binding ligand on HSG cells,
although other heparan sulfate-containing ligands may exist. Laminin-1
affinity chromatography also showed a similar high molecular weight
species bound to intact laminin-1. We attempted with no success to
address the issue of binding specificity by eluting syndecan from the
laminin-1 and peptide columns with peptides. These types of experiments
have been used to elute integrins from RGD columns with RGD-containing peptides. The interaction between integrins and RGD is relatively weak,
and integrins are eluted with EDTA or 1 mM peptide. With AG73 columns, however, the interaction of syndecans with the peptide column is mediated by heparan sulfate and is relatively strong, as
evidenced by the need to elute material with more than 250 mM NaCl.
41-integrin was shown to affect
the ligand binding properties of the integrin (29). Whether a direct
interaction occurs between syndecan-1 GAG and an integrin subunit on
the HSG cell surface remains to be determined.
1 chain was found in the basement membrane surrounding the
entire submandibular gland rudiment (22). By day 17, the staining
pattern of 1 was still faintly detectable around the ducts, while
2 staining was found around the terminal tubules and developing
acinar cells. In situ analysis of laminin chain
expression in the developing mouse salivary glands showed barely
detectable 1, 2, and 4 at day 15.5, compared with high levels
of 3 and 5 (4). We have not yet studied the conserved 3 and
5 laminin chain homologues to AG73, but they may also be important
in salivary gland development. These results suggest that different
laminin chains may be involved in different morphological events
during development.
1 chain peptide AG73 (RKRLQVQLSIRT)
and its laminin 2 chain homologue MG73 (KNRLTIELEVRT) are
biologically active in a variety of in vitro and in
vivo systems. The identification of their cell surface ligand as
the heparan sulfate side chains of syndecan-1 provides new insights
into the mechanism of their activity. The interaction between laminin
and heparan sulfate side chains of syndecans could provide another mechanism for laminin isoform involvement in specific biological processes during development.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: NIDR, NIH, 30/433, 30 Convent Dr., MSC 4370, Bethesda MD 20892-4370. Tel.: 301-496-6069; Fax:
301-402-0897; E-mail: kleinman{at}yoda.nidr.nih.gov.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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P. K. Nielsen, Y. S. Gho, M. P. Hoffman, H. Watanabe, M. Makino, M. Nomizu, and Y. Yamada Identification of a Major Heparin and Cell Binding Site in the LG4 Module of the Laminin alpha 5 Chain J. Biol. Chem., May 5, 2000; 275(19): 14517 - 14523. [Abstract] [Full Text] [PDF]
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J. Muschler, A. Lochter, C. D. Roskelley, P. Yurchenco, and M. J. Bissell Division of Labor among the alpha 6beta 4 Integrin, beta 1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and beta -Casein Expression in Mammary Epithelial Cells Mol. Biol. Cell, September 1, 1999; 10(9): 2817 - 2828. [Abstract] [Full Text]
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M Grassi, G Moens, P Rousselle, J. Thiery, and J Jouanneau The SFL activity secreted by metastatic carcinoma cells is related to laminin 5 and mediates cell scattering in an integrin-independent manner J. Cell Sci., January 8, 1999; 112(15): 2511 - 2520. [Abstract] [PDF]
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M. P. Hoffman, J. A. Engbring, P. K. Nielsen, J. Vargas, Z. Steinberg, A. J. Karmand, M. Nomizu, Y. Yamada, and H. K. Kleinman Cell Type-specific Differences in Glycosaminoglycans Modulate the Biological Activity of a Heparin-binding Peptide (RKRLQVQLSIRT) from the G Domain of the Laminin alpha 1 Chain J. Biol. Chem., June 15, 2001; 276(25): 22077 - 22085. [Abstract] [Full Text] [PDF]
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A. Utani, M. Nomizu, H. Matsuura, K. Kato, T. Kobayashi, U. Takeda, S. Aota, P. K. Nielsen, and H. Shinkai A Unique Sequence of the Laminin alpha 3 G Domain Binds to Heparin and Promotes Cell Adhesion through Syndecan-2 and -4 J. Biol. Chem., July 27, 2001; 276(31): 28779 - 28788. [Abstract] [Full Text] [PDF]
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