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INTRODUCTION |
The recent emergence of the CCN family of angiogenic regulators
has called attention to their functional versatility and mechanisms of
actions (1). This family of secreted proteins consists of six members:
Cyr61,
CTGF1,
Nov, WISP-1, WISP-2, and WISP-3 (1, 2), with the first three members of the family identified providing the acronym CCN. These
structurally conserved proteins share four modular domains with
sequence similarities to insulin-like growth factor-binding proteins,
von Willebrand factor type C repeat, thrombospondin type 1 repeat, and
growth factor cysteine knots (1, 3). Each of these domains is encoded
by a separate exon, suggesting that CCN genes arose through exon
shuffling of preexisting genes to form proteins with multiple
functional domains.
Cyr61 and CTGF are both encoded by immediate early genes and are
coinduced by serum, bFGF, platelet-derived growth factor, and TGF-
1
in fibroblasts (4-7). Cyr61 and CTGF share ~45% amino acid sequence
identity (4, 8), and both proteins bind heparin, associate with the
ECM, and exhibit remarkable functional versatility (9, 10). Purified
Cyr61 and CTGF mediate cell adhesion, stimulate cell migration, and
augment growth factor-induced DNA synthesis (10-12). Cyr61 and CTGF
can promote chondrogenic differentiation, consistent with their
expression in prechondrogenic mesenchyme during embryogenesis (13-15).
Both Cyr61 and CTGF stimulate chemotaxis in endothelial cells through
an integrin
V
3-dependent
pathway and induce neovascularization in vivo (16, 17).
Expression of Cyr61 in tumor cells enhances tumorigenicity by
increasing tumor size and vascularization (16), whereas expression of
CTGF has been correlated with both systemic and localized fibrotic diseases (18-20). Both proteins are also coinduced in granulation tissues during cutaneous wound healing (19, 21). Thus, Cyr61 and CTGF
may participate in wound repair by acting as angiogenic inducers upon
endothelial cells and by acting as chemotactic, proliferative, and
matrix remodeling factors upon fibroblasts.
Through what mechanism(s) might Cyr61 and CTGF act to execute such a
diversity of biological functions? Inasmuch as CTGF was first
identified as a growth factor (8), it has been tempting to postulate
that it might function as a classical growth factor, although a cell
surface receptor for CTGF that resembles a classical growth factor
receptor has not been identified to date. By contrast, both Cyr61 and
CTGF share characteristics of ECM-associated signaling proteins in
several compelling ways (1, 22): 1) they are heparin-binding,
ECM-associated proteins; 2) they contain sequence similarities to
matrix proteins including von Willebrand factor and thrombospondin; and
3) they regulate cell adhesion, migration, proliferation,
differentiation, and survival, all functions that can be modulated
through cell and matrix interactions, especially through
integrin receptors (23, 24). Indeed, we have demonstrated that Cyr61
and CTGF are ligands of, and bind directly to, the integrins
V
3 and
IIb
3
(25, 26). Recently, we showed that Cyr61 supports the attachment, or
the early phase of cell adhesion, of human skin fibroblasts through
integrin
6
1 and HSPGs (27).
To understand their mechanism of actions, we have investigated the
cellular responses to and signaling consequences of cell adhesion to
immobilized Cyr61 and CTGF. We present herein the first conclusive
evidence that Cyr61 and CTGF function as adhesive signaling molecules.
In the absence of any other stimulus, fibroblast adhesion to either
protein is sufficient to generate signaling events resulting in
morphological changes, activation of intracellular kinases, and
activation of gene expression consistent with the biological activities
of these CCN proteins. These findings establish Cyr61 and CTGF as
inducible, ECM-associated adhesive substrates capable of signaling
through integrin-mediated pathways, provide a mechanistic
interpretation for the chemotactic and mitogenic activities of these
proteins, point to unique signaling capabilities of integrin
6
1 and HSPGs, and indicate a potential
function for Cyr61 and CTGF in matrix remodeling through the activation of metalloproteinases during angiogenesis and wound healing.
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MATERIALS AND METHODS |
Cell Culture and Adhesion Assay--
Normal human fibroblasts
(1064SK) from skin biopsy of healthy newborn were obtained from the
American Type Culture Collection (ATCC; number CRL-2076). The culture
was maintained in IMDM (Life Technologies, Inc.) with 10% fetal bovine
serum (Intergen, Purchase, NY) at 37 °C with 5% CO2 and
used for experiments before passage 8. Cell adhesion assays were
carried out largely as described (10, 25). Briefly, 96-well microtiter
plates (Becton Dickinson, Franklin Lakes, NJ) were coated with test
proteins diluted in PBS (137 mM NaCl, 2.7 mM
KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3) at 50 µl/well,
incubated at 4 °C for 16 h, and blocked with 1% BSA at room
temperature for 1 h. 1064SK fibroblasts were harvested in PBS with
2.5 mM EDTA and resuspended in IMDM with 0.5% BSA at
5 × 105 cells/ml and allowed to adhere to
protein-coated wells at 50 µl/well. After incubation at 37 °C for
30 min, wells were washed twice with PBS. Adherent cells were fixed
with 10% formalin, stained with methylene blue, and quantified by dye
extraction and measurement of absorbance at 620 nm (28). Where
indicated, EDTA or peptides were mixed with cells prior to plating.
Antibodies were incubated with cells at room temperature for 1 h
before plating. Inhibition of glycosaminoglycan sulfation was achieved
by growing cells in medium containing sodium chlorate (40 mM) for 24 h; cells were then detached and plated for
cell adhesion assays as described above (29). To show the specificity
of sulfation blockage, 10 mM sodium sulfate was included in
the culture medium together with sodium chlorate.
Proteins, Antibodies, Peptides, and Reagents--
Murine Cyr61
and CTGF proteins were synthesized in a Baculovirus expression system
(Invitrogen Corp., Carlsbad, CA) using Sf9 cells and purified
from serum-free insect cell conditioned medium on Sepharose-S columns
as described (10, 11). Fibronectin, laminin, vitronectin, and type I
collagen were purchased from Collaborative Biomedical (Bedford, MA).
BSA, heparinase I (EC 4.2.2.7), chondroitinase ABC (EC 4.2.2.4), and
heparin (sodium salt, from porcine intestinal mucosa) were from Sigma.
Synthetic peptides GRGDSP and GRGESP were purchased from Life
Technologies, Inc. Function-blocking mAbs against various integrins
were purchased from Chemicon, Inc. (Temecula, CA), including JB1A
(anti-
1), FB12 (anti-
1), P1E6
(anti-
2), P1B5 (anti-
3), P1H4
(anti-
4), JBS5 (anti-
5
1),
GoH3 (anti-
6), and LM609
(anti-
v
3). For immunofluorescence microscopy studies, mAbs against talin (clone 8d4) and paxillin (clone
349) were obtained from Transduction Laboratories (San Diego, CA); mAbs
against phosphotyrosine (clone PY-20) and human
1-integrin (clone
DE9) were from Upstate Biotechnology Inc. (Lake Placid, NY); and mAb
against human integrin
6-subunit (clone 4F10) was from
Chemicon. mAbs against human MMP-1 (clone 41-1ES), MMP-2 (clone
42-5D11), and MMP-3 (clone 55-2A4) were from Oncogene Research Products
(Cambridge, MA). Anti-Rac mAb (cone 23A8), anti-FAK mAb (clone 2A7),
and anti-phosphotyrosine mAb used in immunoblotting studies (clone
4G10) were obtained from Upstate Biotechnology, Inc.
Fluorescence Microscopy--
Immunofluorescence microscopy was
carried out as described (30). Cells adhered to coverslips were
incubated with monoclonal antibodies against phosphotyrosine (clone
PY-20), anti-
6 integrin (4F10), anti-
1
integrin (clone DE9), anti-talin (clone 8d4), or anti-paxillin (clone
349) at 10 µg/ml, 50 µl/slip, and incubated at 37 °C for 1 h. Samples were washed four times with PBS plus 0.5% BSA and then
incubated with fluorescein-conjugated horse anti-mouse IgG (20 µg/ml)
at 37 °C for 30 min. To visualize actin cytoskeleton, cells were
stained with rhodamine-conjugated phalloidin (Sigma) at 10 µg/ml, 100 µl per slip, and washed four times with PBS plus 0.5% BSA after
incubation at room temperature for 1 h.
Immunoprecipitation, SDS-PAGE, and Immunoblotting--
For
studies on intracellular signaling (see Figs. 5 and 6), 100-mm
nontissue culture plastic dishes were precoated with various proteins
at 10 µg/ml, 4 ml per dish, at 4 °C for 16 h, followed by
blocking with 1% BSA at room temperature for 1 h. Cells were serum-starved and collected as described above. Protein-coated 100-mm
dishes were kept at 37 °C and cells were plated at 2-3 ×106 cells/dish in 4 ml of 0.5% BSA-IMDM. Dishes and
media were prewarmed at 37 °C, which helped to ensure consistent
results with respect to cell adhesion and spreading within 5-15 min
after plating.
After incubation, cells were washed and lysed in a buffer (50 mM Tris·Cl, pH 7.5, 135 mM NaCl, 1% Triton
X-100, 0.1% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, 2 mM sodium orthovanadate, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride); immunoprecipitation and immunoblotting were carried out
according to standard protocols (31). To determine total FAK or
paxillin in each sample, blots were stripped in a buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM
-mercaptoethanol) at 60 °C and then reprobed with mAbs against
FAK or paxillin.
To study protein secreted by cells adhered on various substrates (as in
Fig. 8), the conditioned media were collected after 24 h of cell
incubation. The media were first centrifuged to remove cellular debris.
Protein in the media was concentrated using Centricon YM-10
(molecular mass cut-off 10 kDa). An equal amount of conditioned media was loaded on SDS-PAGE and analyzed by immunoblotting with monoclonal antibodies against human MMP-1 (clone 41-1ES), MMP-2 (clone
42-5D11), and MMP-3 (clone 55-2A4).
p42/p44 MAPK Activation--
1064SK fibroblasts were
serum-starved for 24 h, harvested, and plated on 35-mm dishes
precoated with proteins as described above. Total cell lysates were
prepared and applied on SDS-PAGE, and immunoblotting was carried out
using rabbit polyclonal antibodies against the dually phosphorylated
active form p42/p44 MAPK
(Thr(P)183/Tyr(P)185) at a 1:5000
dilution as suggested by the manufacturer (Promega, Madison, WI).
Rac Activation--
1064SK fibroblasts were serum-starved for
24 h, harvested, and plated on plastic dishes precoated with
various proteins as described above. The Rac activation assay was done
using Rac activation kit according to the manufacturer's protocol
(Upstate Biotechnology, Inc.).
RNA Analysis--
1064SK fibroblasts were serum-starved 24 h, mildly trypsinized, and replated on protein-coated 100-mm plastic
dishes in serum-free IMDM as described above. After incubation at
37 °C for various times, total cellular RNA was isolated and
subjected to RNA blot analysis using various
[32P]dCTP-labeled cDNA probes (32). Human MMP-1 and
MMP-2 full-length cDNA clones were obtained from the American Type
Culture Collection. MMP-3 probe was generated using reverse
transcriptase-polymerase chain reaction with a primer pair
corresponding to human MMP-3 cDNA nucleotides 1493-1521 and
1736-1763. The blots were washed at high stringency (0.2× SSC, 0.1%
SDS at 65 °C) and analyzed by a PhosphorImager (Molecular Dynamics,
Inc., Sunnyvale, CA).
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RESULTS |
Adhesion of Primary Human Skin Fibroblasts to CTGF Requires both
Integrin
6
1 and Cell Surface
HSPGs--
We previously showed that adhesion of primary human skin
fibroblasts to Cyr61 is mediated through both integrin
6
1 and cell surface HSPGs (27). Given
that CTGF is also a heparin-binding protein with strong sequence
homology with Cyr61, we surmised that CTGF may also support fibroblast
adhesion through a similar mechanism. To test this hypothesis, we
prepared microtiter wells coated with purified recombinant CTGF onto
which primary human skin fibroblasts were allowed to adhere under
serum-free conditions. Fibroblast adhesion to CTGF was
dose-dependent and saturable and was completely abrogated
by the presence of 2 µg/ml heparin in the plating medium (Fig.
1A). These results suggest
that occupancy of the CTGF heparin-binding site by soluble heparin may
prevent its interaction with cell surface HSPGs, thus inhibiting cell adhesion. To test this possibility, we cultured human fibroblasts in
the presence of sodium chlorate, an inhibitor of 3-phosphoadenosine 5'-phosphosulfate synthesis, to block sulfation of proteoglycans (29).
Adhesion to CTGF was inhibited under this condition, whereas adhesion
of the same cells to fibronectin and vitronectin was unaffected (Fig.
1B). The inhibitory effects of sodium chlorate on cell
adhesion to CTGF was reversed by the inclusion of 10 mM Na2SO4 in the culture medium, verifying that
this inhibitory effect is mediated through a sulfation block (29). To
substantiate the finding that cell surface sulfated proteoglycans are
required for cell adhesion to CTGF, fibroblasts were treated with
heparinase I, an enzyme that targets highly sulfated heparan sulfate
proteoglycans (33, 34). Heparinase I-treated cells were unable to
adhere to CTGF, whereas the same treated cells adhered to fibronectin or vitronectin indifferently (Fig. 1C). Treatment of
fibroblasts with chondroitinase ABC had no effect. These results show
that CTGF, like Cyr61, requires cell surface HSPGs to mediate adhesion of fibroblasts.

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Fig. 1.
Adhesion of human skin fibroblasts to CTGF
requires cell surface HSPGs. A, washed 1064SK
fibroblasts were detached with 2.5 mM EDTA and resuspended
in serum-free IMDM at 2.5 × 105 cells/ml. 50 µl of
cell suspension was plated on each microtiter well coated with the
indicated amounts of CTGF. After incubation at 37 °C for 30 min,
adherent cells were fixed and stained with methylene blue. Extracted
dye was quantified by absorbance at 620 nm. Where indicated, soluble
heparin (2 µg/ml) was added to the plating medium. B,
cells were treated with 40 mM sodium chlorate or with
sodium chlorate plus 10 mM sodium sulfate, for 24 h
before plating on wells coated with CTGF (1 µg/ml), vitronectin
(VN, 0.4 µg/ml), or fibronectin (FN, 2 µg/ml), and adhesion experiments were performed as above.
C, cells were treated with 2 units/ml heparinase I or 2 units/ml chondroitinase ABC at 37 °C for 30 min, washed, and then
plated on wells coated with CTGF (1 µg/ml), fibronectin
(FN, 2 µg/ml), or vitronectin (VN, 0.4 µg/ml). Data shown for all panels are mean ± S.D. of
triplicate determinations and are representative of three
experiments.
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Fibroblast adhesion to CTGF was inhibited by the presence of EDTA or
Ca2+ in the assay media, but not by Mg2+ (Fig.
2A). In control experiments,
cell adhesion to type I collagen showed the same divalent cation
sensitivity as to CTGF, whereas cell adhesion to vitronectin was not
inhibited by Ca2+, as previously observed (27). This
divalent cation sensitivity of fibroblasts adhesion to CTGF is
consistent with the involvement of an integrin receptor. To help
distinguish the specific integrin involved, we examined the inhibitory
effects of RGD-containing peptides. The peptide GRGDSP was unable to
inhibit fibroblast adhesion to CTGF even when present at 2 mM, a concentration that abrogated cell adhesion to either
fibronectin or vitronectin, ligands of the integrin
5
1 and the
V integrins,
respectively (Fig. 2B). The control peptide GRGESP was
unable to inhibit cell adhesion to any substrate. Thus, adhesion of
fibroblasts to CTGF is unlikely to be mediated through the major
RGD-sensitive integrins in these cells, namely the
V
integrins and integrin
5
1.

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Fig. 2.
Adhesion of human fibroblasts to CTGF is
mediated through integrin
6 1.
1064SK human fibroblasts were plated on a microtiter well coated with
either CTGF (1 µg/ml), type I collagen (Coll-I, 0.4 µg/ml), fibronectin (FN, 2 µg/ml), laminin
(LN, 5 µg/ml), or vitronectin (VN, 0.4 µg/ml), and adhesion assays were performed as above. A,
where indicated, cells were suspended in medium containing EDTA (2.5 mM), Ca2+, or Mg2+ (5.0 mM each) before plating, and the adhesion assays
were performed. B, where indicated, synthetic peptide
GRGDSP or GRGESP was present in the plating medium at 2 mM.
C, cells were preincubated with either control mouse IgG or
mAb against integrin 6 (GoH3) at 50 µg/ml for 60 min
before the adhesion assay. D, as indicated, cells were
preincubated with either mAb against integrin 1 subunit
(JB1A) or control normal mouse IgG at 50 µg/ml at room temperature
for 30 min before the adhesion assay. Data shown for all
panels are mean ± S.D. of triplicate determinations
and are representative of three experiments.
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To define the specific integrin that mediates cell adhesion to CTGF, we
tested the ability of a panel of function-blocking mAbs to inhibit
adhesion. Preincubation of cells with mAbs against integrin subunits
1,
2,
3, or
4 or the integrins
5
1
(JBS5) and
V
3 (LM609) had no effect on
fibroblast adhesion to CTGF (data not shown). By contrast, mAb against
integrin
6 subunit (GoH3) completely abrogated cell
adhesion to CTGF, whereas adhesion to fibronectin was not affected
(Fig. 2C). Adhesion to laminin, a known ligand for integrin
6
1, was only minimally affected. This is
most likely because skin fibroblasts utilize
2
1, rather than
6
1, as the major receptor for laminin
(35). Likewise, mAb against the integrin
1 subunit
strongly inhibited cell adhesion to CTGF but not to vitronectin (Fig.
2D). Similar inhibitory effect was observed in adhesion to
type I collagen, a known substrate for
1 integrins.
Together, these results show that adhesion of human skin fibroblasts to
CTGF, like adhesion to Cyr61, requires both integrin
6
1 and cell surface HSPGs.
Fibroblast Adhesion to Cyr61 or CTGF Induces Extensive and
Prolonged Formation of Filopodia and Lamellipodia--
Interaction of
integrins and their adhesive ligands leads to intracellular signaling,
resulting in a range of cellular responses including cytoskeletal
reorganization, focal adhesion complex formation, and cell spreading
(36, 37). Fibroblasts adhered to matrix proteins such as fibronectin or
laminin are known to transiently form filopodia, lamellipodia, and
pseudopod extensions indicative of cell motility, although these
structures usually retreat within 30 min after plating, both in reports
by others and in our own observations (38).
To understand the signaling responses as cells adhere to Cyr61 and
CTGF, we have examined the morphological changes and signaling responses in cells adhered to these proteins. More than 90% of cells
attached to and spread on coverslips coated with Cyr61 or CTGF within
10 min after plating, as was the case when cells were allowed to adhere
to fibronectin or laminin, whereas cells plated on coverslips coated
with BSA never spread even after 1 h of incubation (Fig.
3). Adherent fibroblasts were fixed with
3% paraformaldehyde and stained with rhodamine-conjugated phalloidin
to reveal their actin cytoskeleton structures. Fluorescence microscopy
showed that 30 min after plating, fibroblasts adhered to Cyr61 or CTGF had numerous extended pseudopods (Fig. 3). Arrays of actin filaments existed inside the cells, and microspikes of actin filaments protruded from the cell surface in actin-containing membrane ruffling, typical of
filopodia and lamellipodia (39, 40). These structures became even more
prominent at later times, and by 60 min after plating, cells displayed
complex contours due to their abundance. In contrast, filopodia and
lamellipodia formed in fibroblasts plated on fibronectin or laminin are
fewer in number and more transient in nature, giving the cells a
smoother overall shape by 30-60 min after plating (Fig. 3). Thus,
fibroblast adhesion to Cyr61 or CTGF resulted in cytoskeleton changes
and cell spreading including formation of filopodia and lamellipodia,
consistent with the chemotactic activities of both proteins (11,
17).

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Fig. 3.
Human skin fibroblast adhesion to Cyr61 and
CTGF induces actin cytoskeleton reorganization. 1064SK fibroblasts
were washed with PBS and harvested with 2.5 mM EDTA and
0.025% trypsin. The enzyme was neutralized immediately, and cells were
resuspended in serum-free IMDM at 5 × 105 cells/ml.
Cells were plated on coverslips precoated with 50 µg/ml each of
Cyr61, CTGF, fibronectin, or laminin at 104
cells/cm2 of coverslip area. Cells were incubated at
37 °C for either 30 min or 1 h, and adherent cells were fixed
with 3% paraformaldehyde in PBS followed by permeabilization with
0.5% Triton X-100 in PBS. Actin filaments were stained with
rhodamine-conjugated phalloidin. Data shown are typical of three
experiments. Bar, 20 µm.
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Formation of Focal Complexes upon Adhesion to Cyr61 or
CTGF--
Ligation of integrins to their adhesive substrates can lead
to their association with actin cytoskeleton through their
intracellular cytoplasmic domains. Depending on the nature of F-actin
organization, integrins may be clustered into focal adhesions or focal
complexes. Focal adhesions are large integrin-containing protein
complexes at the end of prominent actin stress fibers, whereas focal
complexes are smaller, integrin-containing protein complexes at the
tips of filopodia and lamellipodia (41). To investigate whether focal adhesions or focal complexes are formed upon adhesion to Cyr61 or CTGF,
human skin fibroblasts were plated on coverslips coated with various
proteins. Since both Cyr61 and CTGF mediate fibroblasts adhesion
through integrin
6
1, the resultant focal
complexes should include this integrin. Consistent with this
expectation, indirect immunofluorescence microscopy revealed that
fibroblasts adhered to either Cyr61 or CTGF form focal complexes that
contain both integrin
6 and
1 subunits
(Fig. 4A). Moreover,
6
1 integrin was localized in needle
head-shaped structures at the tips of filopodia and lamellipodia
characteristic of focal complexes. Cell adhesion to fibronectin, which
binds integrin
5
1, resulted in focal
adhesions that included integrin
1 but not integrin
6, as expected. Likewise, cells adhered to laminin,
which interacts with integrins
2
1 and
6
1, formed focal adhesions involving both
integrins
6 and
1 (42-44).
Immunofluorescence with control IgG gave only weak and diffused
background staining and did not reveal any discrete structure (Fig.
4A).

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Fig. 4.
Cyr61 and CTGF induce formation of focal
adhesion complexes in fibroblasts. A, 1064SK
fibroblasts were collected and resuspended in serum-free IMDM. Cells
were plated on coverslips precoated with Cyr61, CTGF, fibronectin, or
laminin (50 µg/ml each) at 104 cells/cm2 of
coverslip and incubated at 37 °C for 30 min. Adherent cells were
fixed, permeabilized, and stained with mAb against integrin
6 (clone 4F10) or 1 subunit (clone DE9)
at 20 µg/ml. For control, normal mouse IgG (20 µg/ml) was used in
place of primary antibody. B, cells adhered on the indicated
substrates were fixed, permeabilized, and stained with mAb against
phosphotyrosine (clone PY-20), paxillin, or talin at 20 µg/ml. Data
shown are representative of three experiments. Bar, 20 µm.
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Associated with focal adhesions and focal complexes are numerous
signaling proteins including FAK, and structural proteins including
paxillin and vinculin (41, 45, 46). When cells adhered to fibronectin
or laminin, paxillin and talin are found in typical focal adhesion
structures distributed over the cell bodies, as previously reported
(30, 44, 47) (Fig. 4B). When cells adhered to Cyr61 or CTGF,
however, both paxillin and talin were also localized at the tips of
filopodia and lamellipodia characteristic of focal complexes.
Focal complexes are sites of intracellular signaling and are rich in
protein kinases and tyrosine-phosphorylated proteins (36, 37).
Immunofluorescence staining with mAb against phosphotyrosine revealed a
pattern of numerous needle head-shaped protein complexes in cells
adhered to Cyr61 or CTGF, similar to the pattern of paxillin and talin
localization (Fig. 4B). As expected, tyrosine-phosphorylated proteins were detected in protein complexes in cells adhered to fibronectin or laminin. Together, these data show that fibroblast adhesion to Cyr61 or CTGF is mediated through integrin
6
1 and cell surface HSPGs, resulting in
cell spreading and actin cytoskeleton reorganization, as well as
formation of focal complexes involving integrin
6
1, paxillin, and talin at the leading
edges of resultant filopodia and lamellipodia.
Cell Adhesion to Cyr61 or CTGF Activates FAK, Paxillin, and
Rac--
Of the many protein kinases activated by integrins and
localized in focal complexes, FAK plays a central role in
integrin-mediated signaling (48). To examine whether Cyr61 and CTGF can
activate FAK, human skin fibroblasts were allowed to adhere on plastic dishes coated with either protein for various durations and harvested. FAK was immunoprecipitated from the resulting cell lysates with mAb
against FAK and analyzed by immunoblotting with mAb recognizing phosphotyrosine residues. Increased levels of tyrosine phosphorylation on FAK were observed in cells adhered on either Cyr61 or CTGF as
compared with cells plated on poly-L-lysine (Fig.
5A), indicating activation of
FAK. The induction occurred within 30 min and was maintained for 1 h at a level comparable with those induced by other ECM proteins such
as fibronectin, laminin, and type I collagen (Fig. 5A).
These data indicate that Cyr61 and CTGF are both capable of activating
FAK signaling, a property that is consistent with their ability to
support cell adhesion and spreading.

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Fig. 5.
Fibroblasts adhesion to Cyr61 or CTGF leads
to activation of FAK and paxillin. Serum-starved and washed 1064SK
fibroblasts were detached with 2.5 mM EDTA plus 0.025%
trypsin in PBS and resuspended in serum-free IMDM at 5 × 105 cells/ml. 4 ml of cell suspension was plated on each
100-mm plate coated with 10 µg/ml each of poly-L-lysine
(B), Cyr61, CTGF, fibronectin (FN), laminin
(LN), or type I collagen (Col.I). Cells were
incubated at 37 °C for either 30 min or 1 h, lysed, and
immunoprecipitated with mAb against either FAK or paxillin as described
under "Materials and Methods." A, FAK was
immunoprecipitated from lysates, electrophoresed on 6% SDS-PAGE, and
immunoblotted with mAb (clone 4G10) against phosphotyrosine
(Pi-Tyr). The blot was then stripped of antibodies and
reblotted with mAb against FAK. B, paxillin was
immunoprecipitated from cell lysates, electrophoresed on 8% SDS-PAGE,
and immunoblotted sequentially with anti-phosphotyrosine mAb and
anti-paxillin mAb. Data shown are representative of at least three
independent experiments.
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To establish that FAK signaling pathway is activated by Cyr61 and CTGF,
we tested whether paxillin, a major substrate of FAK, is
tyrosine-phosphorylated upon cell adhesion to either protein. Paxillin
is a structural protein having multiple tyrosine phosphorylation sites
and plays integral roles in the assembly and regulation of
integrin-mediated signaling complexes (37). Once
tyrosine-phosphorylated, paxillin is recruited into focal adhesions or
focal complexes and binds to various signaling protein molecules
including FAK, Src, and Csk (49-51). In experiments similar to those
performed to examine FAK activation, we found that paxillin was also
tyrosine-phosphorylated upon fibroblasts adhesion to Cyr61 or CTGF
(Fig. 5B). The time course of paxillin phosphorylation was
similar to that of FAK activation, and the level was also comparable
with that induced by other ECM proteins tested, including fibronectin,
laminin, and type I collagen (Fig. 5B). This result is
consistent with localization of paxillin in focal complexes upon cell
adhesion to Cyr61 or CTGF (Fig. 4B). Thus, fibroblast
adhesion to Cyr61 or CTGF activates the FAK signaling pathway.
The Rho family GTPase Rac regulates actin dynamics, leading to
formation of lamellipodia and clustering of ligand-bound integrins into
focal complexes (52, 53). Of the effector protein kinases regulated by
Rac, p21-activated kinases bind directly to the active form of Rac,
leading to autophosphorylation and kinase activation (54). To study if
Rac is activated in cells adhered on Cyr61 and CTGF, an affinity
precipitation assay using PDB (p21 binding domain from human
PAK-1)-glutathione S-transferase fusion protein was employed
in a pull-down assay to capture activated Rac from cell lysates (55). A
similar approach has been used to show that 3T3 fibroblast adhesion to
fibronectin promotes transient activation of Rac and more prolonged
activation of Rho (56, 57), consistent with the actin cytoskeletal
organization and lamellipodia formation that follow adhesion (58, 59).
As shown in Fig. 6, the levels of
activated Rac captured by the pull-down assay were minimal 5-10 min
after replating, during which cell spreading had just begun to occur.
By 30 min after plating and when most cells were actively spreading,
Rac was activated to maximal levels that were maintained through 60 min
after plating and then declined thereafter (Fig. 6). The level of
activated Rac at the zero time point was higher than during the first
5-10 min after replating (Fig. 6), which may be due to perturbation of
actin cytoskeleton by the physical events of detaching and attaching
cells. A similar pattern of Rho activation was also reported when Swiss
3T3 cells were detached and replated on fibronectin-coated surfaces
(57). Together, these results are consistent with the role of Rac in
cell spreading and in the formation of lamellipodia.

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Fig. 6.
Cyr61 and CTGF activate the small GTPase Rac
in human skin fibroblasts. 1064SK fibroblasts were serum-starved,
collected, and resuspended in serum-free IMDM and plated at 2-3 × 106 cells/100-mm dish precoated with Cyr61 (10 µg/ml)
or CTGF (10 µg/ml) for various times as indicated. The dishes were
prewarmed to 37 °C immediately before use. Time
0 indicates cells kept in suspension. Adherent cells were
lysed at the indicated times, and activated Rac was
affinity-precipitated and then immunoblotted with mAb against Rac
protein. 50 µl of the clarified lysate from each sample was reserved
for immunoblotting of total Rac protein. Data shown are representative
of two independent experiments.
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Sustained Activation of MAPKs Induced by Cell Adhesion to Cyr61 or
CTGF--
MAPKs are known to be transiently activated by
integrin-mediated signaling upon cell adhesion to matrix proteins such
as collagen, fibronectin, vitronectin, and laminin (60-62). To address
whether cell adhesion to Cyr61 or CTGF can activate MAPKs,
serum-starved human skin fibroblasts were allowed to adhere to these
proteins or control ECM proteins for various durations in serum-free
medium. Cell lysates were electrophoresed and immunoblotted with
phosphospecific antibodies against p42/p44 MAPKs. When fibroblasts
adhered to fibronectin, laminin, or type I collagen, p42/p44 MAPK
activation was rapid and transient, reaching maximal levels by 15 min
and declining thereafter to background levels within 1 h (Fig.
7A). In contrast, p42/p44 MAPK
activation in cells adhered to Cyr61 or CTGF was maintained at a high
level for at least 9 h after cell plating. The same blots were
stripped and probed with antibodies against p42/p44 MAPKs, and the
results indicated that the total amount of MAPK proteins was unchanged
(Fig. 7B). Thus, fibroblasts adhered to Cyr61 or CTGF
exhibit strong and prolonged activation of p42/p44 MAPKs observed 3-9
h after plating, in sharp contrast to the rapid and transient
activation in cells plated on fibronectin, laminin, or type I collagen
(Fig. 7). This sustained activation of MAPKs upon fiboblast adhesion to
Cyr61 or CTGF appears to be unique among immobilized matrix substrates
and may reflect distinct signaling capabilities mediated through
integrin
6
1.

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Fig. 7.
Adhesion of human skin fibroblasts leads to
prolonged MAPK activation. A, 1064SK fibroblasts were
serum-starved and resuspended in serum-free IMDM at 6 × 105 cells/ml. Cells were plated at 1 ml/dish (35-mm dishes)
precoated with 10 µg/ml each of Cyr61, CTGF, fibronectin
(FN), laminin (LN), or type I collagen
(Col.I) for various times as indicated. Clarified lysates
were loaded on SDS-PAGE at 50 µl/sample. Immunoblotting of activated
p42/p44 MAPKs was done using affinity-purified polyclonal antibodies
against dually phosphorylated (pTEpY) p42/p44 MAPKs. B, the
same blots as in A were stripped and reprobed with
antibodies against the indicated proteins. Data shown are
representative of three independent experiments.
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Adhesion of Human Skin Fibroblasts to Cyr61 or CTGF Induces
Expression of MMP-1 and MMP-3--
Expression of both
cyr61 and CTGF is highly induced in
granulation tissues during cutaneous wound healing (21, 63), leading to
the possibility that Cyr61 and CTGF may act to facilitate matrix degradation and remodeling. To test this hypothesis, serum-starved primary fibroblasts were harvested and allowed to adhere on immobilized Cyr61 or type I collagen in serum-free medium for various durations, and steady state levels of MMP-1 and MMP-2 mRNAs were analyzed by
RNA blots. As shown in Fig.
8A, MMP-1 mRNA level was
minimal 2 h after replating but increased to a high level between
6 and 12 h after replating on Cyr61 or collagen. By 24 h
after plating, MMP-1 mRNA level in cells plated on Cyr61 was 5-fold
higher than cells plated on type I collagen. By contrast, MMP-2
mRNA level remained the same under all conditions. We next examined
if CTGF can also lead to elevated MMP-1 mRNA level in fibroblasts
by acting as an adhesion substrate. As shown in Fig. 8B,
cells adhered to either CTGF or Cyr61 displayed 5-7-fold higher steady
levels of MMP-1 mRNA 24 h after plating compared with cells
adhered to fibronectin, laminin, or type I collagen. Likewise, MMP-3
mRNA levels were 7-9-fold higher in cells adhered to either Cyr61
or CTGF compared with cells adhered to fibronectin, laminin, or type I
collagen (Fig. 8B). It is noteworthy that in normal diploid
fibroblasts, MMP-1 and MMP-3 genes are regulated coordinately by growth
stimuli, whereas MMP-2 expression is inducible by phorbol ester but not responsive to growth factors (64).

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Fig. 8.
Fibroblasts adhesion on Cyr61 and CTGF
enhances expression of MMP-1 and MMP-3. A, 1064SK
fibroblasts were serum-starved for 24 h before being collected and
resuspended in serum-free IMDM at 5 × 105 cells/ml.
2 × 106 cells were plated on 100-mm dishes precoated
with Cyr61 (10 µg/ml) or type I collagen (Col.I, 10 µg/ml) for various times as indicated. Total cellular RNA was
extracted and analyzed by Northern blotting using full-length human
MMP-1 and MMP-2 cDNA as probes as described under "Materials and
Methods." Data shown are representative of two independent
experiments. B, cells were plated on dishes precoated with
10 µg/ml each of Cyr61, CTGF, fibronectin (FN), type I
collagen (Col.I), or laminin (LN) for 24 h
before lysis and total RNA extraction. Data shown are representative of
three independent experiments. C, conditioned media from
cells plated on various substrates as indicated above were collected
24 h after plating and resolved by SDS-PAGE followed by
immunoblotting with antibodies against MMP-1, MMP-2, and MMP-3.
Lane B, conditioned medium collected from cells
not subjected to detachment and replating. Results were representative
of two independent experiments.
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To show that MMP-1 and MMP-3 protein synthesis is actually induced upon
cell adhesion to Cyr61 or CTGF, we examined the conditioned media of
cells plated on various substrates 24 h after plating by
immunoblotting. As shown in Fig. 8C, both MMP-1 and MMP-3
are readily detected in conditioned media when human fibroblasts were plated on Cyr61 or CTGF, but none was detectable when these cells were
plated on fibronectin, laminin, or collagen. The level of MMP-2 protein
in the conditioned media remains the same in cells plated on all
substrates, consistent with the levels of mRNAs expressed in these
cells (Fig. 8B). Since serum-starved normal skin fibroblasts
express minimal levels of MMP-1 and MMP-3 (65) (Fig. 8, A
and C), we conclude that adhesion of fibroblasts to Cyr61 or
CTGF results in the up-regulation of these metalloproteinases at the
levels of both mRNA and protein synthesis (Fig. 8).
 |
DISCUSSION |
Although a multitude of activities has been described for Cyr61
and CTGF, the mechanisms of their actions have not been elucidated. Results in this study present the first conclusive evidence that Cyr61
and CTGF can function through adhesive signaling. The adhesion of
fibroblasts to immobilized Cyr61 or CTGF, in the absence of any other
stimulus, is sufficient to induce distinct cellular responses
consistent with the activities of both proteins and different from
those induced by other known matrix ligands. Based on the high degree
of homology among members of the CCN family, we anticipate that other
proteins in this family may also function through adhesive signaling.
Several unexpected findings emerged through examination of Cyr61 and
CTGF as ECM-associated adhesive ligands. First, the absolute requirement for cell surface HSPGs for cell attachment to Cyr61 or CTGF
appears to be unique. Although increasing evidence indicates that HSPGs
can function with integrins as coreceptors in cell and matrix
interactions (66, 67), HSPGs thus far have been observed to influence
only the cell spreading phase of adhesion following cell attachment
(27, 68, 69). Second, fibroblast adhesion to Cyr61 and CTGF induces
persistent formation of filopodia and lamellipodia with focal complexes
rather than focal adhesions. Third, cell adhesion to Cyr61 or CTGF
induces signaling responses with unusual kinetics, including sustained
activation of MAPKs and prolonged induction of MMP-1 and MMP-3
expression. Together, these findings provide new insight into the
biological functions of Cyr61 and CTGF as well as a mechanistic
interpretation of these activities through integrin-mediated signaling.
Both Cyr61 and CTGF have been shown to support the attachment phase of
cell adhesion through integrin receptors, and the specific integrins
involved appeared to be cell type-dependent. Thus, Cyr61 and CTGF support the attachment of endothelial cells through integrin
V
3 (17, 25), blood platelets through
integrin
IIb
3 (26), and fibroblasts
through integrin
6
1 and cell surface
HSPGs (27) (Figs. 1 and 2). The present study extends these
observations to document the cellular responses as a result of cell
attachment to Cyr61 and CTGF. One of the most prominent responses upon
integrin engagement to Cyr61 or CTGF is actin cytoskeleton
reorganization, leading to cell spreading and formation of filopodia
and lamellipodia (Fig. 3). Whereas fibroblast adhesion to matrix
proteins such as fibronectin and laminin also results in formation of
filopodia and lamellipodia, these structures are transient in nature,
and they largely retreat within 30 min after plating. By contrast, these pseudopods persist for at least 1 h after cell plating on Cyr61 or CTGF and in fact grew more prominent with time (Fig. 3). In
agreement with recent studies indicating that Rac is a critical
activator for lamellipodia (41, 45), cell adhesion on Cyr61 and CTGF
also leads to activation of Rac (Fig. 6). Lamellipodia are required for
cell motility, while filopodia are indispensable for migration toward a
chemogradient (70, 71). Inasmuch as the chemotactic activities of
soluble Cyr61 and CTGF have been previously documented in a Boyden
chamber assay (16, 17), these observations suggest that both proteins
may function as haptotactic factors when tethered to the ECM to
stimulate cell migration within tissues.
Concomitant with cell spreading and actin cytoskeleton reorganization,
fibroblasts adhered to Cyr61 or CTGF formed integrin
6
1-containing focal complexes localized
to the leading edges of filopodia and lamellipodia (Fig. 4). Similar
patterns of focal complexes can be discerned by immunostaining for
paxillin and talin, consistent with the known association of paxillin
with focal adhesion complexes, and with the demonstrated direct
interaction between talin and the cytoplasmic domains of engaged
integrins (72). Furthermore, both FAK, a tyrosine kinase that plays
central roles in integrin signaling, and its substrate paxillin were
activated by tyrosyl phosphorylation when cells were adhered to Cyr61
or CTGF (Fig. 5). Together, these data show that fibroblast adhesion to
Cyr61 or CTGF is mediated through integrin
6
1 and cell surface HSPGs, leading to
formation of integrin
6
1-containing focal complexes, cytoskeleton reorganization, and cell spreading with the
formation of lamellipodia and filopodia. These morphological changes
are accompanied by signaling events including the activation of FAK,
paxillin, and Rac. These observations provide compelling evidence that
Cyr61 and CTGF function as bona fide adhesive signaling molecules.
The activities of MAPKs are central to a variety of cell
functions, in particular cell proliferation, differentiation, and gene
expression (73). Cells in suspension have no contact with solid surface
and therefore are round in shape, and MAPK activity is down-regulated.
Once reattached, cell spreading occurs and MAPKs are transiently
activated. In the present study, we employed this cell
detachment-reattachment scheme to test if Cyr61 and CTGF will also lead
to MAPK activation. Remarkably, cell adhesion to Cyr61 and CTGF caused
a marked and sustained activation of p42/p44 MAPKs in skin fibroblasts
lasting for at least 9 h, compared with the transient activation
observed when cells were plated on type I collagen, fibronectin, or
laminin (Fig. 7). To our knowledge, this sustained activation of MAPK
by Cyr61 and CTGF is unique among adhesive substrates in fibroblasts.
This prolonged activation of MAPK following adhesion is consistent with
the ability of Cyr61 and CTGF to enhance proliferation through
augmentation of growth factor-induced DNA synthesis (10, 11).
There exist conflicting reports about whether integrin
6
1 can activate MAPK. Cross-linking of
the mAb GoH3 to integrin
6
1 in
fibroblasts kept in suspension did not induce MAPK (74). However,
expression of integrin
6a and
6b subunits
separately in the P388D1 mouse macrophage cell line, which normally
does not express endogenous
6 integrins (75),
showed activation of MAPK only in
6a-expressing cells
but not in
6b-expressing cells when both were plated on
laminin-1 (76). One of the major differences between these studies is
the agents used to activate integrin. While the former study used mAb
that binds to the extracellular domain of integrin
6,
the other employed a natural
6
1 ligand that also binds to cell surface HSPGs. It has been observed that a
heparin-binding peptide derived from thrombospondin-1 can synergize with T-cell receptor to enhance activation of MAPKs (77). It is
possible that integrin
6
1 requires
costimulation of cell surface HSPGs to activate MAPKs and that Cyr61
and CTGF may sustain more prolonged activation of MAPKs by virtue of
their dual affinity for integrin
6
1 and HSPGs.
Aside from Cyr61 and CTGF, natural ligands for integrin
6
1 include laminin, invasin (78), and
fertilin (79). Invasin is a bacterial protein involved in invasion of
mammalian tissues, and fertilin is a sperm surface protein involved in
the binding of sperm to egg. Since neither invasin nor fertilin is an
endogenous substrate, these proteins are not likely to play a role in
wound repair. The presence of laminin is restricted to the basement membrane of epidermis and associated with vascular structures in dermis
(80, 81). Given its confined presence in the basement membrane, laminin
is unlikely to have important roles in the majority of skin fibroblasts
in the granulation tissue. In contrast, both Cyr61 and CTGF are induced
in granulation tissues during cutaneous wound repair and are likely to
be the principal adhesion ligands of integrin
6
1 during wound healing. In addition,
unlike laminin, which interacts with a multitude of integrins, Cyr61
and CTGF appear to utilize only integrin
6
1 in adhesion of dermal fibroblasts, thus providing a unique paradigm for studying integrin
6
1-mediated adhesive signaling in a
natural context, free of interference from other interacting integrins.
As adhesive substrates, proteolytic fragments of fibronectin containing
the cell binding domain have been observed to induce MMP-1 and MMP-3
through integrin
5
1 (82). Other ECM
substrates, such as vitronectin, laminin, type I collagen, and intact
fibronectin, either have no effect on MMP-1 and MMP-3 expression or
induce their expression only transiently (82, 83) (Fig. 8). Fibroblast adhesion to Cyr61 or CTGF, on the other hand, induces a more prolonged activation of MMP-1 and MMP-3 lasting for at
least 24 h, resulting in accumulation of MMP-1 and MMP-3 proteins
in the conditioned media (Fig. 8, B and C). Since
CTGF expression has been associated with fibrosis and subcutaneous
injection of CTGF induces granulation tissue (12), our finding that
both Cyr61 and CTGF can up-regulate MMP gene expression may appear
paradoxical. However, the induction of metalloproteinases is indeed
consistent with a role for these proteins in matrix remodeling of
granulation tissue during wound healing, where the degradation of a
provisional matrix and the synthesis of new matrix must occur
simultaneously (84, 85). Moreover, degradation of the ECM can promote
the migration of endothelial cells in angiogenesis.
Several lines of evidence support the roles of Cyr61 and CTGF in wound
healing: 1) expression of cyr61 and CTGF genes
are minimal in normal dermis and becomes highly induced in dermal fibroblasts in granulation tissue during wound repair; 2) both Cyr61
and CTGF are angiogenic inducers and may recruit new blood vessels to
supply nutrients to sites of wound healing; and 3) Cyr61 and CTGF
promote chemotaxis and proliferation in fibroblasts. Encoded by
immediate early genes, Cyr61 and CTGF are induced
by serum growth factors implicated in wound healing, including bFGF and
TGF-
1 (4, 7). It is interesting to note that in fibroblasts TGF-
1
suppresses the expression of MMP-1 and MMP-3 (86, 87), whereas both are
induced by bFGF, Cyr61, and CTGF (Fig. 8) (86, 88). Thus, although it
has been suggested that CTGF can mediate some of the activities of
TGF-
1 (89, 90), their effects on gene expression are not identical.
With respect to induction of MMP-1 and -3, the activities of Cyr61 and
CTGF closely parallel those of bFGF instead of TGF-
1. The interplay
between the actions of bFGF, TGF-
1, and the induced effectors, Cyr61
and CTGF, is likely to be complex and remains to be investigated.