From the Institute of Molecular Pathology, University
of Copenhagen, Frederik V's vej 11, DK-2100, Copenhagen, Denmark, the
§ Division of Molecular Medicine, Lund University, SE-20502
Malmö, Sweden, the ¶ Department of Vascular Biology VB-1,
The Scripps Research Institute, La Jolla, California 92037, the
Department of Pathology, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, Massachusetts 02215, and the
** Division of Biomedical Sciences, Imperial College London,
SW7 2AZ, London, United Kingdom
Received for publication, September 3, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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The ADAMs (a disintegrin
and metalloprotease) comprise a large family of
multidomain proteins with cell-binding and metalloprotease activities.
The ADAM12 cysteine-rich domain (rADAM12-cys) supports cell
attachment using syndecan-4 as a primary cell surface receptor that
subsequently triggers The extracellular environment profoundly influences cell
shape. Following an initial cell attachment event, cells may or may not
spread depending on cell type and the nature of the molecular signal
they receive. Cell spreading is a fundamental cellular process required
for cell migration, survival, proliferation, and differentiation (1).
Cell spreading on the extracellular matrix requires reorganization of
the actin cytoskeleton and activation of integrins (2), resulting in
stable adhesion through formation of stress fibers and focal adhesions.
Several signaling proteins, including
PKC,1 phosphatidylinositol
3-kinase and R-Ras, have been shown to regulate cell spreading in
different cell types (3-6).
ADAMs (a disintegrin and
metalloprotease) constitute a recently characterized family
of metalloproteases that also mediates cell adhesion. The prototype
ADAM is a multidomain protein composed of pro-, metalloprotease,
disintegrin-like, cysteine-rich, epidermal growth factor-like repeat,
transmembrane, and cytoplasmic tail domains (7-9). The disintegrin
domain of several different ADAMs including ADAM 2, 9, 12, 15, and 23 has been shown to support cell attachment (10-14). For example, the
disintegrin domain of both ADAM 2 and 9 supports cell adhesion through
the Syndecan-4 is a member of a family of four (syndecans 1-4)
transmembrane heparan sulfate proteoglycans (19-22). The core protein of syndecans is characterized by divergent extracellular domains and
highly conserved cytoplasmic tails that contain two constant regions
(C1 and C2) separated by a variable region (V) unique to each family
member (23). Specifically, the V-region of syndecan-4 contains a unique
seven-residue binding site for phosphatidylinositol 4,5-bisphosphate
(PIP2) involved in PKC In the present study, we determined how cell spreading is
regulated by ADAM12/syndecan-4 signaling. Our data indicate a
critical role for syndecan-4 as a primary receptor for rADAM12-cys
and demonstrate that ADAM12/syndecan- 4 regulates cell spreading in a
Cell Lines--
The cell lines used in this study were: CHO-K1
(CCL-61 ATCC), CHO-pgsD677 (CRL-2244 ATCC), and CHO cells stably
transfected with full-length rat syndecan-4 (S4) (27), a mutant form of syndecan-4 terminated at isoleucine 191 in the center of the V (variable) region of the cytoplasmic tail (S4 Antibodies and Matrix Proteins--
The mAb against activated
cDNAs and Transfections--
A bicistronic vector
(pIRES2-EGFP) containing rat full-length syndecan-4 (20) was kindly
provided by Dr. A. Horowitz and E. Tkachenko (Harvard Medical School,
Boston, MA). cDNAs encoding full-length rat syndecan-2 and a mutant
form of syndecan-4 terminated at isoleucine 191 in the center of the V
(variable) region of the cytoplasmic tail (syn4 Cell Attachment Assays--
The cell attachment assay was
performed as described previously (16, 17). Briefly,
Nunc-ImmunoTM 96-well plates with MaxiSorpTM
surface (Nunc A/S) or bacterial culture dishes (35 × 10 mm;
Corning Incorporate Ltd., Corning, NY) were coated with 20 µg/ml of
rADAM12-cys or fibronectin (10 µg/ml) in 0.1 M
NaHCO3 buffer, pH 9.5, overnight at 4 °C. In some
experiments, Mn2+ (1 mM), heparin (10 µg/ml),
or suramin (10 µM) was added, or cells were pretreated
for 15 min with PKC inhibitors: 10 µM GF109203X or 2 µM Gö6976 (Calbiochem-Novobiochem GmbH, Bad
Soden/TS, Germany), with 300 ng/ml of CNF1 toxin for 16 h or a
mixture of 7 µg/ml of C3 exoenzyme (Cytoskeleton Inc., Denver, CO)
and 5 µg/ml of LipofectAMINE for 12 h at 37 °C in culture
medium. Each assay point was derived from 3-6 separate wells and
repeated at least three times.
Morphological and Immunocytochemical Analysis--
Adherent
cells were rinsed in phosphate-buffered saline (PBS), fixed with 3.5%
paraformaldehyde, and permeabilized with 0.25% Triton X-100 in PBS for
5 min at room temperature. Cells were then washed and incubated with
TRITC-phalloidin for 30 min for F-actin staining, and washed and
mounted with fluorescent mounting medium (DAKO). For detection of focal
adhesions, cells were incubated with mAbs for vinculin or paxillin for
1 h, followed by rinsing and incubation with secondary antibodies.
Activated Overexpression of Syndecan-4 in Carcinoma and CHO-K1 Cells
Promotes Cell Spreading in Response to ADAM12--
We have shown
that RKO colon carcinoma cells attach to rADAM12-cys via syndecan(s) as
the primary cell surface receptor, whereas additional activation of
When CHO-K1 cells are plated on fibronectin they attach and spread
(Fig. 1, A, E, and
F). However, when plated on rADAM12-cys they attach but
remain round (Fig. 1, B and F). Overall, less than 2% of cells spread following attachment to rADAM12-cys, while close to 100% spread when plated on fibronectin. Heparin and suramin, which interfere with the function of cell surface heparan sulfate glycosaminoglycan (GAG) chains completely inhibited cell attachment to
rADAM12-cys, but did not inhibit cell attachment or spreading on
fibronectin (Fig. 1E). CHO-K1 pgsD-677 cells, which are
deficient in the synthesis of GAG chains, did not attach to rADAM12-cys in a cell attachment assay (Fig. 1C). Together, results
shown in Fig. 1, B and C indicate that, similar
to carcinoma cells (16, 17), the attachment of CHO-K1 cells to
rADAM12-cys is through the GAGs of syndecans. Spreading of CHO-K1 cells
on ADAM12 could be induced by addition of Mn2+ (Fig.
1D) but spreading was not as extensive as that seen when cells attached and spread on fibronectin (Fig. 1, D and
F). Well-developed stress fibers and focal adhesions were
observed in CHO-K1 cells attaching on fibronectin as revealed by
phalloidin and vinculin staining, respectively (not shown); however,
when cells attached and spread on rADAM12-cys in the presence of
Mn2+, stress fibers developed but focal adhesions were not
apparent (not shown). These results indicate that CHO cells, like
several carcinoma cell lines including RKO colon carcinoma cells (16, 17) attach to rADAM12-cys through syndecans as the primary attachment receptor and require activation of
Based on the reports that syndecans are down-regulated in certain
carcinoma cells (42, 43), we hypothesized that the level of syndecans
in carcinoma cells might be insufficient to allow efficient cross-talk
with and activation of
We next asked whether overexpression of syndecan-4 promoted activation
of
These results demonstrate that transfection of full-length syndecan-4
into CHO-K1 and RKO colon carcinoma cells is sufficient to promote cell
spreading on rADAM12-cys in a PKC
To determine the consequences of PKC
To determine whether the effect of Myr-PKC
We next plated the mesenchymal human MG-63 cells on rADAM12-cys, on
which they attach and spread, and on fibronectin as a control, and then
stained for both activated
These findings (Figs. 6-8) together with experiments using
function-blocking Activation of Both PKC
Since activated RhoA alone did not induce cell spreading or stress
fiber formation in RKO colon carcinoma or CHO-K1 cells, we asked
whether activating both RhoA and PKC ADAM12 supports cell attachment and cell spreading of a variety of
cells in a process that is syndecan-4-initiated but ADAM12 has been demonstrated to be a potent modulator of cell function
in vivo and in vitro (55-58). It is up-regulated
in carcinoma cells (16) and may therefore dramatically influence cell-cell interactions as well as the local microenvironment at the
interface between the tumor cells and the surrounding stroma. To
determine the function of ADAM12, we and others have performed cell
attachment assays (11, 12, 16, 17, 59). We found that rADAM12-cys is a
substrate for cell attachment using syndecan(s) as the primary receptor
(16, 17). Subsequent cell spreading depends on cross-talk between
syndecan and ADAM12/syndecan-4 signaling observed in the present study was dependent
on specific sequence information residing in the V-region of
syndecan-4, as evidenced by the failure of syn4 Although expression of Myr-PKC1
integrin-dependent cell spreading, stress fiber assembly,
and focal adhesion formation. This process contrasts with cell adhesion
on fibronectin, which is integrin-initiated but
syndecan-4-dependent. In the present study, we investigated ADAM12/syndecan-4 signaling leading to cell spreading and stress fiber
formation. We demonstrate that syndecan-4, when present in significant
amounts, promotes
1 integrin-dependent cell
spreading and stress fiber formation in response to rADAM12-cys. A
mutant form of syndecan-4 deficient in protein kinase C (PKC)
activation or a different member of the syndecan family, syndecan-2,
was unable to promote cell spreading. GF109203X and Gö6976,
inhibitors of PKC, completely inhibited ADAM12/syndecan-4-induced cell
spreading. Expression of syndecan-4, but not syn4
I, resulted in the
accumulation of activated
1 integrins at the cell
periphery in Chinese hamster ovary
1 cells as revealed by 12G10
staining. Further, expression of myristoylated, constitutively active
PKC
resulted in
1 integrin-dependent cell
spreading, but additional activation of RhoA was required to induce
stress fiber formation. In summary, these data provide novel
insights into syndecan-4 signaling. Syndecan-4 can promote cell
spreading in a
1 integrin-dependent fashion
through PKC
and RhoA, and PKC
and RhoA likely function in
separate pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6
1 integrin (13, 15). For ADAM12 both
the disintegrin and the cysteine-rich domains provide molecular
information for cell attachment. Thus, cell attachment to the
recombinant ADAM12 disintegrin domain is mediated primarily through
9
1 integrin in an RGD-independent manner
but cell spreading does not occur (11). The cysteine-rich domain of
ADAM12 also possesses important cell binding activities (16, 17). We
have previously demonstrated that cell attachment of several different
cell lines and primary muscle cell cultures to rADAM12-cys (recombinant
ADAM12 cysteine-rich domain) is mediated through syndecans,
specifically syndecan-4. Notably, mesenchymal cells attach, spread, and
form stress fibers and focal adhesions upon attaching to rADAM12-cys
(17). Carcinoma cells also attach through syndecans, but only form
cellular actin-containing projections rather than being spread fully.
Cell spreading is only obtained upon further integrin activation by the
addition of either Mn2+ or activating monoclonal antibodies
(16, 17). The importance of the cysteine-rich domain in cellular
interactions in vivo was recently underscored by the studies
of Gaultier et al. (18), which demonstrated the
importance of the interaction of ADAM13 disintegrin-cysteine rich
domains with extracellular matrix proteins like fibronectin. The
molecular mechanisms of the downstream events following binding of
ADAMs and syndecan-4, however, are completely unknown.
activation (23-25, 66) and
multimerization (23, 26), which is critical for cell spreading and
cytoskeletal reorganization (27). Syndecan-4 is a widespread component
of focal adhesions and appears to be a co-receptor in cell adhesion to
many extracellular matrix ligands, modifying the integrin-mediated
responses (23, 28, 29). Syndecan-4 has been implicated in the
pathogenesis of numerous diseases. Syndecan-4 levels were shown to be
up-regulated in fibroblasts and endothelial cells during wound repair
(30). Delayed wound repair and impaired angiogenesis were demonstrated
in syndecan-4-deficient mice (31). Syndecan-4 was up-regulated in
proliferative renal disease (32) and mice deficient in syndecan-4 were
more susceptible to
-carrageenan induced renal damage (33)
indicating that syndecan-4 plays an important role in renal diseases.
Most of these studies suggest that syndecan-4 plays a role in cell
adhesion to extracellular matrix substrates and in the regulation of
cell migration, but the mechanism of syndecan-4 signaling during cell
adhesion, spreading, and migration is not clearly understood.
1 integrin-dependent manner through PKC
and
RhoA, which function in separate pathways.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
I) (27) or with human
1 integrin (CHO
1) (34), RKO colon carcinoma (35), and
MG-63 osteosarcoma (CRL-1427 ATCC). CHO cells were grown in Dulbecco's
modified Eagle's medium (DMEM)/F12 medium and the other cell lines
were grown in DMEM, supplemented with glutamax I and 4,500 mg/liter
glucose and 10% fetal bovine serum. Stably transfected cell lines were
cultured in the presence of G418. All media used in this study were
supplemented with 50 units/ml penicillin and 50 µg/ml streptomycin,
and the cells were grown in Nunc tissue culture flasks (Nunc A/S,
Roskilde, Denmark) at 37 °C in a 5% CO2 humidified
atmosphere. Cell culture reagents were obtained from Invitrogen.
1 integrin (clone 12G10) was from Serotec Ltd (Oxford,
UK) (36, 37). The
1 integrin function blocking mAb,
AIIB2, was obtained from the Developmental Studies Hybridoma Bank
maintained by the University of Iowa, Department of Biological
Sciences, and polyclonal antibodies against
1 integrin (M-106) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Monoclonal antibodies to vinculin were kindly provided by Dr. M. Glukhova, Institut Curie, Paris, France and the mAb to paxillin was
from Chemicon International (Harrow, UK). The monoclonal antibody
(150.9) against the ectodomain of syndecan-4 was described previously
(27). Secondary antibodies conjugated to tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were purchased from DAKO A/S (Glostrup, Denmark). TRITC-phalloidin was
obtained from Molecular Probes (Leiden, The Netherlands). Human fibronectin was purchased from Invitrogen, and recombinant human
rADAM12-cys was produced in Escherichia coli and purified as
described (16, 17). Cytotoxic Necrotizing Factor 1 (CNF1) toxin
was kindly provided by Drs. Anita Sjölander (Lund
University, Malmö, Sweden) and Gianfranco Donelli
(Istituto Superiore di Sanita, Rome, Italy).
I) cloned into the
pcDNA3 were described previously (27). Another mutant form of
syndecan-4, syn4
E, truncated at glutamate 199, removing the
C-terminal FYA motif that binds PDZ (postsynaptic density 95, disk
large, zona occludens-1) proteins, was constructed essentially as
described previously (27). The eukaryotic expression vector for
Myr-PKC
-EGFP was described previously (38). The cDNA for PKC
(BglII/SalI fragment from full-length PKC
in
the pEGFP-N1 vector (39) was subcloned into the same Myr-EGFP vector.
The cDNAs for RhoGTPases, L63RhoA, and N19RhoA were kindly provided
by Dr. Alan Hall (University College, London, UK). Except for the
pEGFP-bicistronic constructs, the pEGFP-N1 vector was co-transfected
with the signaling constructs in order to visualize transfected cells.
Transient transfections were performed using LipofectAMINE plus reagent
(Invitrogen) and 3-10 µg of plasmid DNA/ml in serum-free medium,
according to the manufacturer's protocol. For the transfection of
MG-63 cells, FuGENE 6 transfection reagent (Roche Molecular
Biochemicals, Hvidovre, Denmark) was used.
1 integrin was detected by using the
monoclonal antibodies 12G10. To detect activated and total
1 integrins, respectively, MG-63 cells were incubated
with
1 integrin antibodies (12G10 and M-106,
respectively) and double-stained by incubating with FITC-conjugated
goat anti-mouse and rhodamine-conjugated swine anti-rabbit secondary
antibodies. Cells were examined using an inverted microscope (Zeiss
Axiovert) equipped with phase contrast optics and connected to a
PentaMAX chilled charge-coupled device camera (Princeton Instruments). Images were processed using Metamorph Software Program. Spread cell
areas were measured electronically using the Metamorph program. The
images shown were representive from at least three separate experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 integrin with Mn2+ or the activating mAb
12G10 is required for cell spreading to occur (16, 17). In contrast,
mesenchymal cells, such as MG-63 osteosarcoma cells, attach, spread,
and form stress fibers and focal adhesions spontaneously on
rADAM12-cys. These results suggest that depending on cell type,
rADAM12-cys can activate cross-talk between syndecan(s) and
1 integrin, resulting in cell spreading. In the present
study, we have characterized the downstream signaling pathway activated
in rADAM12-cys mediated cell attachment and spreading. In
addition to human MG-63 osteosarcoma cells and human RKO colon
carcinoma cells (17), we also analyzed hamster CHO-K1 ovarian
epithelial cells. CHO-K1 cells express endogenous syndecans 1, 2, and 4 as well as
1 integrins (27, 40, 41) and have been
extensively used to define the molecular mechanisms of cell attachment
and spreading on fibronectin (27).
1 integrins for cell
spreading to occur.
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Fig. 1.
CHO-K1 cell attachment and spreading on
ADAM12 and fibronectin. CHO-K1 (A, B,
D, E, and F) or CHOpgsD-677
(C) were plated on rADAM12-cys (B-D) or
fibronectin (A), and cell attachment was examined.
E, effect of heparin (10 µg/ml) and suramin (10 µM) on CHO cell attachment (control) to
rADAM12-cys (white bar) and fibronectin (black
bar). F, cell area for CHO cells plated on fibronectin
(FN) or on rADAM12-cys alone (A12) and
treated with 1 mM Mn2+ (A12 + Mn2+).
1 integrin upon binding of
rADAM12-cys. To test this hypothesis, CHO-K1 cells were transfected with expression vectors encoding full-length and mutant forms of
syndecans (Fig. 2A,
schematic representation) and 40 h later cells were
plated on rADAM12-cys for 4 h. As shown in Fig. 2, B
and C, cells transfected with full-length syndecan-4
underwent spreading, whereas non-transfected cells remained round. The
syndecan-4-expressing cells were large, flattened, and exhibited a
well-spread morphology. F-actin staining of CHO-K1 cells showed stress
fiber formation in the spread cells only (Fig. 2C), and
focal adhesions were not formed as revealed by vinculin or paxillin
staining (not shown). Transfection of CHO-K1 cells with a mutant form
of syndecan-4, syn4
E, which lacks the C-terminal FYA sequence shown
to bind PDZ domain-containing proteins (44), also induced cell
spreading and stress fiber formation in CHO cells plated on rADAM12-cys (Fig. 2, D and E). By contrast, transfection with
a different syndecan-4 mutant, syn4
I, which lacks the domain of the
V-region required for PKC
binding, did not induce spreading on
rADAM12-cys (Fig. 2, F and G). Expression of a
different member of the syndecan family, syndecan-2, failed to induce
cell spreading on rADAM12-cys (Fig. 2, H and I).
Both syn4
I- and syndecan-2-expressing cells were round in morphology
and exhibited only peripheral F-actin staining (Fig. 2, G
and I). An estimate of cell spreading revealed that around
15% of syn4- and 10% of syn4
E-expressing cells were spread,
whereas only 2% of the EGFP-, 3% of the syn4
I-, and 3% of the
syn2-expressing cells were spread (Fig. 2J). Next, we used CHO cells that are stably expressing full-length wild type syndecan-4 (S4) and a mutant form lacking the PKC
binding site, syn4
I
(S4
I). These cells have been FACS-selected and shown to express high levels of syndecan-4 at the cell surface (27). Labeling for syndecan-4
using an ectodomain-specific monoclonal antibody (150.9) revealed
distinct dot-like staining at the cell periphery and at the peripheral
ruffles in S4 cells (Fig. 2J, inset) An estimate of spreading assays using these cells revealed that spreading was
increased from 15 to 26% in S4 stably transfected cells compared with
syn4 transiently transfected cells. In contrast, the spreading of cells
stably transfected with S4
I was still only around 2% (Fig.
2J). Transfection of RKO colon carcinoma cells with
full-length syndecan-4 similarly induced spreading (Fig.
3A), but cells did not form
typical stress fibers (not shown).
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Fig. 2.
Syndecan-4 overexpression restores cell
spreading on ADAM12. A, schematic representation of the
cytoplasmic domain amino acid sequences of rat full-length syndecan-4
(syn4) and forms of the protein truncated at the C terminus
(syn4 E) and in the center of the V-region
(syn4
I) and the corresponding sequence of
syndecan-2 (syn2). EC, extracellular domain;
TM, transmembrane domain; C1, cytoplasmic tail:
membrane proximal region; V, central unique variable region;
and C2, distal region (C2). CHO-K1 cells were transfected
with expression vectors encoding syndecan-4-EGFP (B and
C), syn4
E (D and E), syn4
I
(F and G) or syndecan-2 (H and
I) and plated on rADAM12-cys for 4 h. Transfected cells
were visualized by EGFP fluorescence (B, D,
F, and H), and phalloidin staining was used to
stain F-actin (C, E, G, and
I). J, the effect of transient (white
bars) or stable (black bars) transfection of different
syndecan constructs (full-length syndecan-4 (S4), syn4
I
(S4
I), syn4
E
(S4
E), or syndecan-2 (S2)) on CHO
cell spreading on rADAM12-cys was estimated by counting the number of
spread cells. For Syn4
E and Syn2 only transiently transfected cells
were evaluated. Transiently transfected cells were visual- ized by co-expression of EGFP. Data are presented as percentage
of spread cells and are mean ± S.D. from three separate
experiments. Inset in J shows syndecan-4
immunostaining of the CHO cells stably transfected with full-length
syndecan-4 (S4).
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Fig. 3.
Syndecan-4-induced cell spreading requires
activation of 1 integrins.
RKO colon carcinoma cells were transfected with the syndecan-4-EGFP
expression construct (A-D) and plated on rADAM12-cys for
4 h. In C and D, the cells were pretreated
with function-blocking
1 integrin antibodies, AIIB2 (10 µg/ml), for 15 min.
1 integrin following binding to rADAM12-cys. For this
experiment, we used a function-blocking antibody to
1
integrin (AIIB2). Only RKO colon carcinoma cells were used because this antibody is specific for human
1 integrin. As expected,
pretreatment of RKO cells with AIIB2, but not with isotype-matched
control antibodies, completely abolished cell attachment to fibronectin (data not shown). Pretreatment of syndecan-4-expressing RKO colon carcinoma cells with AIIB2 completely inhibited cell spreading on
rADAM12-cys (Fig. 3, C and D) without affecting
cell attachment.
1
integrin-dependent manner. Furthermore, the fact that
deletion of the PKC
-activating domain from syndecan-4 abrogates this
effect indicates a role for PKC
in this process.
Is Downstream of Syndecan-4 in Response to ADAM12--
To
characterize the potential role of PKC
downstream of syndecan-4, we
first examined MG-63 osteosarcoma cells, mesenchymal cells that are
known to attach and spontaneously spread on rADAM12-cys (Ref. 17 and
Fig. 4A). These cells were
treated with a general PKC inhibitor, GF109203X, and a compound that
only inhibits classical PKC isoforms, Gö6976, and the response of
cells to rADAM12-cys was analyzed. As shown in Fig. 4, B-F,
both compounds significantly inhibited cell spreading on rADAM12-cys
but not on fibronectin. Moreover, treatment with PKC inhibitors
completely inhibited formation of stress fibers in MG-63 cells plated
on rADAM12-cys (Fig. 4, G, I, and K)
but not on fibronectin (Fig. 4, H, J, and
L). These results suggests that activation of a conventional
PKC isoform occurs downstream of the syndecan-4, leading to mesenchymal
cell attachment and spreading on rADAM12-cys. This observation supports the finding that syndecan-4 binds to and activates PKC
(45).
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Fig. 4.
PKC activity is essential for cell
spreading. MG-63 osteosarcoma cells untreated (A,
D, G, and H) or pretreated with the
PKC inhibitors GF109203X (B, E, I, and
J) or Gö6976 (C, F,
K, and L) as described under "Materials and
Methods" and plated on either rADAM12-cys (A-C,
G, I, and K) or fibronectin
(D-F, H, J, and L) for
1 h. A-F, phase contrast micrographs; G-L,
stained for F-actin with TRITC-phalloidin.
activation, RKO and CHO-K1
cells were subsequently transfected with a constitutively active,
myristoylated PKC
(Myr-PKC
) construct. Transient expression of
Myr-PKC
resulted in cell spreading (Fig.
5, A and B), while transient transfection with a different myristoylated isoform of
PKC
, Myr-PKC
, known to restore cell spreading in cells deficient in integrin
1 cytoplasmic signaling (1), did not result in spreading
(Fig. 5, C and D). Myr-PKC
-transfected cells
were clearly spread but less so than syndecan-4-transfected cells.
F-actin staining was observed only in the periphery of both
Myr-PKC
-expressing and control cells, and no stress fibers were
formed (Fig. 5B).
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Fig. 5.
Expression of Myr-PKC
restores cell spreading on ADAM12. CHO-K1 cells were
transfected with expression vectors encoding Myr-PKC
-EGFP
(A, B, E, and F),
Myr-PKC
-EGFP (C and D) or syndecan-4-EGFP
(G and H) and plated on rADAM12-cys for 4 h.
EGFP fluorescence was used to visualize transfected cells
(A, C, E, and G) and
TRITC-phalloidin was used to stain F-actin (B, D,
F, and H). In E-H, cells were
pretreated with 2 µM Gö6976 for 15 min before
plating on rADAM12-cys.
on cell spreading
requires its catalytic activity, we treated Myr-PKC
-transfected CHO-K1 cells with PKC inhibitors GF109203X (not shown) and Gö6976 (Fig. 5, E and F). As expected, both inhibitors
completely abolished cell spreading on rADAM12-cys. In addition,
treatment of syndecan-4 transfected CHO-K1 cells with Gö6976
completely abolished cell spreading (Fig. 5, G and
H). Together, these findings indicate that activation of
PKC
is critical for cell spreading. Furthermore a signal other than
or in addition to PKC
is required for cells to form stress fibers on
rADAM12-cys.
1 Integrin Activation Is Downstream of PKC
in
Syndecan-4 Signaling--
We next investigated the molecular mechanism
through which PKC
mediates cell spreading in response to
rADAM12-cys. Syndecan-4-mediated cell spreading is dependent on
1 integrin activation (Fig. 3), and PKC has been shown
to regulate
1 integrin trafficking (37, 46). We
therefore studied the activation of
1 integrin itself during cell spreading, using CHO cells stably transfected with human
1 integrin (CHO
1) and a monoclonal
1 integrin antibody, 12G10, which specifically
recognizes the activated form of
1 integrin (36, 37,
47-49). These cells were transiently transfected with syn4 or syn4
I
expression vectors and were thereafter plated on rADAM12-cys and
stained for activated
1 integrin using mAb 12G10. As
shown in Fig. 6A, syndecan-4
transfection resulted in cell spreading and accumulation of activated
1 integrins (12G10). Activated
1 integrin
could be detected in peripheral ruffles and cell edges (Fig.
6A, arrows). 12G10 labeling showed a typical dot-like staining and closely resembled that of syndecan-4 staining in
S4 cells (Fig. 2J, inset). In contrast, no
typical 12G10 staining was observed at cell edges and peripheral
ruffles in cells expressing syn4
I that lack PKC
binding site
(Fig. 6B). These findings indicate that syndecan-4 activates
1 integrin through PKC
. CHO
1 cells plated on fibronectin served as positive controls for
1
integrin staining (Fig. 6C). To further confirm the role of
PKC
in the activation of
1 integrins, we stained
Myr-PKC
-expressing CHO
1 cells (Fig. 6D)
with 12G10 antibodies. As shown in Fig. 6E, Myr-PKC
expression resulted in prominent accumulation of 12G10 immunostaining along the cell membranes (arrows).
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Fig. 6.
1 integrins are
activated downstream of PKC
in
ADAM12/syndecan-4-mediated cell spreading. CHO
1
cells were either transfected with an expression vector encoding
syndecan-4-EGFP (A) or co-transfected with vectors encoding
both EGFP and syn4
I (B) or Myr-PKC
-EGFP
(D-E) and plated on rADAM12-cys for 4 h.
The activated
1 integrins were detected using 12G10
monoclonal antibody (A, B, C, and
E). In C, the non-transfected CHO
1
cells (control) were plated on fibronectin as a positive
control for
1 integrin staining. The transfected cells
were identified with EGFP fluorescence. Myr-PKC
was present at the
cell membrane (arrows in D) at the same sites as
activated
1 integrin was shown (arrows in
E).
1 integrins (12G10 mAb) and
total
1 integrins with a polyclonal
1
integrin antibody (M-106). Staining with the 12G10 antibody
demonstrated the presence of activated
1 integrins at
the cell membranes in MG-63 cells plated on both rADAM12-cys and
fibronectin (Fig. 7, A and
C), whereas the polyclonal antibodies to
1
integrins stained throughout the cytoplasm (Fig. 7, B,
D, and F). Pretreatment with the PKC inhibitor Gö6976,
completely inhibited the activated integrin staining at the membrane as
well as cell spreading (Fig. 7E). These findings indicate
that syndecan-4 indeed activates
1 integrins through
PKC
. To determine a direct role for syndecan-4 in the activation of
1 integrins, we transfected MG-63 cells with the syn4
I construct and determined its ability to inhibit cell spreading and
1 integrin activation. As shown in Fig.
8 expression of syn4
I (C
and D) but not EGFP alone (A and B)
significantly inhibited 12G10 staining and cell spreading in MG-63
cells plated on rADAM12-cys. The 12G10 staining demonstrated the
presence of activated
1 integrins at the cell membrane
in EGFP-expressing cells (Fig. 8, A and B) but no
staining of 12G10 was observed at the cell membrane in syn4
I-transfected cells (Fig. 8, C and D).
Syn4
I transfection resulted in ~60% inhibition of MG-63 cell
spreading on rADAM12-cys (Fig. 8E).
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Fig. 7.
Syndecan-4 mediates
1 integrin activation and cell
spreading through PKC
in mesenchymal MG-63
cells. MG-63 osteosarcoma cells were plated on either rADAM12-cys
(A, B, E, and F) or
fibronectin (C and D). In E and
F the cells were pretreated with 2 µM
Gö6976 for 15 min before plating on rADAM12-cys. The cells were
fixed and double stained for occupied (12G10) and total
1 integrins (M-106) as described under "Materials and
Methods."
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Fig. 8.
Syndecan-4 mutant lacking
PKC binding site inhibits
1 integrin activation and cell
spreading in MG-63 cells. MG-63 cells transfected with vectors
encoding EGFP (A and B) or co-transfected with
EGFP and syn4
I expression vectors (C and D)
were plated on rADAM12-cys and stained for activated
1
integrins (12G10) (A and C). E,
analysis of cell spreading from EGFP and syn4
I-transfected MG-63
cells that were plated on rADAM12-cys. The percentage of cells spread
(white bar) and non-spread (gray bar) are shown.
Values are mean ± S.D. from three separate experiments.
1 integrin antibodies, such as AIIB2
(Fig. 3) strongly suggest that syndecan-4 activates
1
integrin through PKC
.
and RhoA Is Required for Stress Fiber
Formation in Response to ADAM12--
Since RhoA is the principal
regulator of stress fiber assembly (50), we asked whether modulation of
RhoA activity might alter rADAM12-cys-induced cell spreading and stress
fiber formation. CHO-K1 cells were transfected with vectors encoding
constitutively active (L63RhoA) or dominant negative (N19RhoA) forms of
RhoA (Fig. 9). Forty hours after
transfection cells were plated on rADAM12-cys and stained for F-actin.
Neither active RhoA (Fig. 9B, arrow) nor dominant
negative RhoA (not shown) restored cell spreading or stress fiber
formation.
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Fig. 9.
RhoA activation by CNF1 treatment restores
stress fiber formation in Myr-PKC -expressing cells plated
on ADAM12. CHO-K1 cells were untreated (Control,
A) or transfected with expression vectors encoding L63RhoA
(B), Myr-PKC
-EGFP (C and D), or
syndecan-4-EGFP (E), or co-transfected with both
syndecan-4-EGFP and N19RhoA vectors (F) and plated on
rADAM12-cys for 4 h. In D, cells were pretreated with
CNF1 toxin (300 ng/ml) for 16 h before plating on rADAM12-cys.
F-actin staining was performed using TRITC-phalloidin
(A-F).
would do so. We used CNF1 toxin
to activate RhoA (51-53). Similar to the findings with the L63RhoA
construct, treating CHO-K1 cells with CNF1 alone did not induce cell
spreading in response to rADAM12-cys (data not shown). However, upon
treatment of Myr-PKC
-expressing CHO-K1 cells (Fig. 9C)
with CNF1, the cells spread, and phalloidin staining revealed formation
of prominent actin stress fibers (Fig. 9D), but focal
adhesions were not formed (not shown). Morphologically these flattened
and well-spread cells resembled those of syndecan-4 overexpressing
CHO-K1 cells (compare Fig. 9, D and E). In
agreement with these results, CHO-K1 cells transfected with both
syndecan-4 and the dominant-negative N19RhoA constructs did not exhibit
impaired cell spreading, but stress fiber formation was significantly
inhibited (Fig. 9F). Likewise, inhibition of RhoA with C3
exoenzyme in mesenchymal cells (MG-63) completely abolished
syndecan-4-mediated stress fiber formation without inhibiting cell
spreading in response to rADAM12-cys (data not shown). These findings
suggest that PKC
and RhoA function in separate pathways and that
RhoA requires prior cell spreading mediated by PKC
-
1
integrin to induce stress fiber formation in response to
rADAM12-cys.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 integrin-dependent (17). In contrast, cell attachment and
spreading on fibronectin are integrin-initiated events, but stress
fiber and focal adhesion formation are syndecan-4-dependent
(29, 54). Therefore, by using rADAM12-cys as a substrate, it is
possible to obtain direct and novel information about syndecan-4
signaling. We show here that ADAM12/syndecan-4 signaling through PKC
and RhoA activates
1 integrin-dependent cell
spreading and stress fiber formation. PKC
and RhoA appear to be
activated in an ordered way and in two distinct pathways.
1 integrin. In carcinoma and CHO-K1
epithelial cells the pathway leading to activation of
1
integrin is not functional but can be mimicked by activating
1 integrin activity with exogenous Mn2+ or
activating monoclonal antibodies (Ref. 17 and this study). We postulate
that in carcinoma cells, cross-talk between syndecan and
1 integrin is uncoupled, potentially influencing tumor
cell behavior and/or survival. In support of this hypothesis,
syndecan-4 levels have been shown to be down-regulated in certain
carcinoma cells (42, 43). In the present study, we investigated
downstream signaling pathways following ADAM12-syndecan engagement and
found that overexpression of syndecan-4 in carcinoma and CHO cells
promoted spreading on rADAM12-cys in a
1
integrin-dependent manner. Overexpression of syndecan-4
could conceivably induce this effect either by promoting dimerization
with endogenous syndecan-4 or through accumulation of high levels of
syndecan-4 in the cell, or both.
I and of syndecan-2 to induce cell spreading. The V-region of syndecan-4 contains the motif
for binding to PIP2, an essential cofactor for activation of PKC
by syndecan-4 (25). Further evidence that the ADAM12/syndecan effect is mediated via PKC
is that (a) cell spreading
induced by syndecan-4 transfection could be completely inhibited by
both a general PKC inhibitor (GF109023X) and a specific inhibitor of classical PKC isoforms (Gö6976) and (b) transient
expression of a constitutively activated form of PKC, Myr-PKC
,
induced spreading of carcinoma cells on rADAM12-cys.
could restore cell spreading, it did
not lead to stress fiber assembly. However, activation of RhoA with
CNF1 toxin in cells expressing Myr-PKC
induced reorganization of
actin into stress fibers. On the other hand, isolated activation of
RhoA by CNF-1 toxin or by constitutively active RhoA failed to induce
spreading and stress fiber assembly, indicating that RhoA activation
alone is not sufficient to mimic the syndecan-4 pathway. Finally, a
dominant negative mutant of RhoA, N19RhoA, inhibited syndecan-4-induced
stress fiber formation but not cell spreading. These results indicate
that ADAM12/syndecan signaling activates PKC
and RhoA through
separate pathways (Fig. 10,
scheme).
View larger version (16K):
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Fig. 10.
A model for syndecan-4-mediated signaling
events leading to cell spreading and stress fiber formation in cells
plated on ADAM12. Cell attachment to rADAM12-cys through
syndecan-4 results in oligomerization of syndecan-4 and activation of
PKC , which in turn increases the accumulation of activated
1 integrins at the cell membranes. Activated
1 integrins intitate cell spreading perhaps by
recruiting active Rac1 to the cell membrane (see "Discussion").
After initial cell spreading, RhoA is activated probably through a
co-operative signals from syndecan-4 and
1 integrins and
induces stress fiber formation.
The observation that ADAM12/syndecan signaling activates PKC and
RhoA in apparently separate pathways in response to rADAM12-cys is
novel. When fibroblasts are plated on the cell-binding domain of
fibronectin, full spreading and stress fiber formation can be restored
by activating either PKC or Rho, suggesting that both PKC and Rho are
situated in the same pathway downstream of integrin and syndecan-4 (29,
60, 61). In fact in the latter study (29), addition of syndecan-4
antibodies induced full spreading, stress fibers and focal adhesions in
fibroblasts attached to the integrin-binding domain of fibronectin.
Defilippi et al. (62) demonstrated that cells can bind to
integrin antibodies but require exogenous activation of both PKC and
Rho for stress fiber formation, suggesting that integrins cooperate
with another molecule to activate PKC and Rho. Intriguingly, syndecan-4
overexpression in our present study led to activation of all three
signaling molecules, PKC
, integrins, and RhoA, suggesting that
syndecan-4 is a plausible candidate acting as a co-receptor for
integrins. Interestingly, while this manuscript was in preparation,
Goetinck's group (63) suggested that syndecan-4-induced FAK activation
depends on activation of Rho but not PKC, also implying that Rho and
PKC function in separate pathways.
In our study, overexpression of syndecan-4 or Myr-PKC resulted in
1 integrin-dependent cell spreading,
indicating that the cross-talk between syndecan-4 and ADAM12 leads to
activation of
1 integrin. This observation was confirmed
by the finding that activated
1 integrin accumulated on
cell surfaces of these cells. On the other hand, a mutant form of
syndecan-4 lacking the PKC
binding site failed to induce activation
of
1 integrin. To our knowledge, this is the first
report demonstrating activation of
1 integrins by a
proteoglycan, syndecan-4. PKC
and
isoforms have been implicated
in the trafficking of
1 integrins (37, 46), supporting a
role for PKC
downstream of syndecan-4 in the activation and
accumulation of
1 integrins at the cell membrane. The
exact downstream effectors activated following
syndecan-
1 integrin cross-talk observed in the present
study are not known. Rac has been implicated in
1
integrin-mediated cell spreading (64), and it was recently shown that
1 integrin activation is required for dissociation of
GTP-Rac from Rho-GDI and its recruitment to the plasma membrane where
it can interact with effectors (65). Carcinoma cell attachment to
rADAM12-cys decreased the levels of GTP-Rac1 compared with their
attachment to fibronectin. Co-transfection of N17Rac1 and syndecan-4
inhibited syn4-induced cell spreading on rADAM12-cys, indicating that
Rac1 is downstream of
1 integrin in syndecan-4-mediated
cell spreading.2 The findings
that dominant negative N19RhoA inhibited only stress fiber formation
but not cell spreading and that RhoA activation was unable to induce
cell spreading argue against the participation of RhoA in the
activation of
1 integrins by syndecan-4.
Finally, we emphasize the differences and similarities between cell
adhesion on fibronectin and rADAM12-cys. The cell-binding domain of
fibronectin supports cell attachment via 1 integrins and
requires the syndecan-4 binding-hep II domain of fibronectin for full
spreading and cytoskeletal reorganization (29, 54). In contrast
rADAM12-cys supports cell attachment through syndecan-4 but requires
1 integrins for cell spreading and stress fiber formation (Fig. 10).
Irrespective of their choice of primary attachment receptors, both
fibronectin and rADAM12-cys require cooperation between syndecan-4 and
1 integrins in controlling the integral process of cell adhesion.
In conclusion, our study provides novel insights into
rADAM12-cys/syndecan-4 signaling. Our findings suggest that
rADAM12-cys/syndecan-4 can promote cell spreading and stress fiber
formation by activating two separate pathways at different stages of
cell adhesion. One involves the sequential activation of PKC and
1 integrins and the other involves RhoA.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Brit Valentin and Helle Stiller for technical assistance. We thank Bent Børgesen for photographic assistance and Elise Lamar for editorial assistance. We thank Drs. Alan Hall and Arie Horowitz and Eugene Tkachenko for providing plasmids for RhoGTPases and syndecan-4 EGFP, respectively. We thank Drs. Gianfranco Donelli and Anita Sjölander for providing CNF1 toxin and Dr. Maria Glucova for providing vinculin antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Danish Cancer Society, The Danish Medical Research Council, The Neye-Foundation, Novo Nordisk, Haensch, Munksholm, Velux, and Dansk Kraeftforsknings Fond (to U. W.), Wellcome Trust Program Grant 065940, and National Institutes of Health Grants GM50194 (to J. R. C.) and CA80789 (to A. M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute of
Molecular Pathology, University of Copenhagen, Frederik V's vej 11, DK-2100, Copenhagen, Denmark. Tel.: 45-3532-6056; Fax: 45-3532-6081; E-mail: ullaw@pai.ku.dk.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M208937200
2 C. K. Thodeti and U. M. Wewer, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; mAb, monoclonal antibody; ADAM, a disintegrin and metalloprotease; GAG, glycosaminoglycan; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; CHO, chinese hamster ovary cells; CNF1, cytotoxic necrotizing factor 1; rADAM12-cys, recombinant ADAM12 cysteine-rich domain.
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