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J. Biol. Chem., Vol. 276, Issue 50, 47329-47337, December 14, 2001
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From the Department of Molecular Genetics, University of Illinois
at Chicago College of Medicine, Chicago, Illinois 60607-7170
Received for publication, August 10, 2001, and in revised form, September 27, 2001
Cyr61 is a heparin-binding, extracellular
matrix-associated protein of the CCN family, which also includes
connective tissue growth factor, Nov, WISP-1, WISP-2,
and WISP-3. Cyr61 is capable of multiple functions, including induction
of angiogenesis in vivo. Purified Cyr61 mediates cell
adhesion and induces adhesive signaling, stimulates cell migration,
enhances cell proliferation, and promotes cell survival in both
fibroblasts and endothelial cells. In this study, we have used cDNA
array hybridization to identify genes regulated by Cyr61 in primary
human skin fibroblasts. The Cyr61-regulated genes fall into several
groups known to participate in processes important for cutaneous wound
healing, including: 1) angiogenesis and lymphogenesis (VEGF-A and
VEGF-C); 2) inflammation (interleukin-1 Cyr61 is a member of the CCN protein family, which also includes
CTGF, Nov, WISP-1, WISP-2, and WISP-3 (1, 2). These vertebrate-specific
proteins are characterized by a four-domain modular structure that
share sequence similarities to insulin-like growth factor binding
proteins, von Willebrand factor, thrombospondin, and growth factor
cysteine knots (1, 3, 4). The expression of Cyr61 is
associated with the cardiovascular and skeletal systems during
embryonic development (5). Consistent with this observation, Cyr61 can
act as both an angiogenic inducer and a chondrogenesis differentiation
factor. Purified Cyr61 supports endothelial cell adhesion and induces
cell migration through integrin Although a number of activities have been documented for Cyr61 as
described above (7-10, 14), information on its effects on gene
expression is still scant. Cyr61 was shown to elevate type II collagen
expression in mesenchymal cells undergoing chondrogenic differentiation
(12), whereas fibroblasts adhered to Cyr61 express high levels of MMP1
(collagenase-1) and MMP3 (stromelysin-1) (14). To understand its
function fully, we used cDNA microarray hybridization to identify
genes regulated by purified Cyr61 in human dermal fibroblasts.
Interestingly, the Cyr61-regulated genes thus identified encode
proteins that control angiogenesis, inflammation, ECM remodeling, and
cell-ECM interaction, all of which are processes vital for cutaneous
wound healing (15-17). The notion that Cyr61 is important in wound
repair is further supported by the finding that Cyr61 expression is highly induced and tightly regulated in granulation tissue during cutaneous wound healing. Furthermore, Cyr61 regulates gene expression in the presence of other ECM molecules or serum growth
factors and its effect can last for at least 5 days, consistent with
the tissue environment in which healing occurs and the time course of
wound healing in vivo. We propose a model in which the inducible expression of Cyr61 in granulation tissue directly
and indirectly regulates the processes of inflammation, angiogenesis, matrix remodeling, and cell-ECM interaction during wound repair.
Cell Culture, Proteins, Antibodies, and Reagents--
Normal
human fibroblasts (1064SK) derived from skin biopsy of healthy newborn
were obtained from the American Type Culture Collection (CRL-2076). The
cells were maintained in Iscove's modified Dulbecco's medium
(Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Intergen,
Purchase, NY) at 37 °C with 5% CO2 and used before passage 8. Both wild-type and mutant Cyr61 (Cyr61DM) proteins were
produced in a baculovirus expression system using Sf9 cells and
were purified from serum-free insect cell conditioned medium on
Sepharose-S columns as described (8, 9). FN, vitronectin, laminin, and
type I collagen were purchased from Collaborative Biomedical (Bedford,
MA). BSA, heparin (sodium salt, from porcine intestinal mucosa),
cycloheximide, 5,6-dichlorobenzimidazole riboside, X-gal, Harris
hematoxylin, and eosin-Y solutions were from Sigma. PD98059 was
purchased from Calbiochem-Novabiochem Corp. (San Diego, CA), as were
monoclonal antibodies against human MMP1 (clone 41-1ES), MMP2 (clone
42-5D11), MMP3 (clone 55-2A4), uPA (clone 6), PAI-1 (clone 2), and
TIMP1 (clone 7-6C1). Monoclonal antibodies against human IL-1 cDNAs--
Human cDNA clones of IL-1 cDNA Array Hybridization--
The Atlas human cancer
cDNA expression array kit was from CLONTECH
Laboratories, Inc. (Palo Alto, CA). The array contains 588 unique human
cDNAs (listed at
www.CLONTECH.com/atlas/genelists/index.shtml). Human skin fibroblasts grown to near confluence were made quiescent by
serum starvation, achieved by first washing the cells three times with
phosphate-buffered saline to remove traces of serum factors, and then
incubating the cells for 24 h in Iscove's modified Dulbecco's
medium with no serum or protein factors added. The cells were then
stimulated by adding purified recombinant Cyr61 or BSA to 10 µg/ml
for an additional 24 h. Total RNA isolation, 32P-cDNA probe labeling, hybridization, and high
stringency wash were all performed following protocols of the cDNA
array kit provided by the manufacturer. The intensity of hybridization
signals was quantified by PhosphorImager and normalized against
internal controls (GAPDH and actin) on the same blot. The signal of
MMP3 (positive control) normalized to GAPDH is 0.3, falling within the
range of normalized signals detected for other regulated genes, which spans from 0.14 for IL-1 RNA Analysis, Immunoblotting, and p42/p44 MAPKs
Activation--
Total cellular RNA was isolated, resolved on an
agarose-formaldehyde gel, and blotted onto a nylon membrane using
standard protocols (18). Radioactive probes were generated by enzymatic incorporation of [32P]dCTP into indicated human
cDNAs. The blots were washed at high stringency (0.2× SSC; 0.1%
SDS at 65 °C) and analyzed by PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
To examine secreted proteins, conditioned media were collected after
cell incubation for times indicated and were centrifuged to remove
cellular debris. The conditioned media were concentrated using
Centricon YM-10 (molecular mass cut-off, 10 kDa), and 25 µl of the
concentrate (from 0.5 ml of original conditioned medium exposed to
4 × 105 of cells) were electrophoresed on 10%
SDS-polyacrylamide gel and analyzed by immunoblotting with specific
antibodies using standard protocols (19).
For analysis of p42/p44 MAPKs activation, 1064SK fibroblasts were
serum-starved for 24 h and treated with 10 µg/ml of Cyr61 for
various times. Total cell lysates were prepared and applied on
SDS-polyacrylamide gel electrophoresis, and immunoblotting was carried
out using rabbit polyclonal antibodies against the dually
phosphorylated active forms of p42/p44 MAPKs
(pThr183/pTyr185) at 1:5000 dilution as
suggested by the manufacturer (Promega, Madison, WI). To detect total
MAPK protein, anti-p42/p44 MAPKs rabbit polyclonal antibody was used (Promega).
Wound Healing and Histology--
To study Cyr61
promoter activity during the healing process of skin wounds, C57BL/6
mice heterozygous for the Cyr61 locus were used (20). In the
mutated allele, the first exon and the 5' half of the second exon of
Cyr61 were replaced with the bacterial lacZ gene,
thereby placing expression of Identification of Cyr61-regulated Genes in Human Skin
Fibroblasts Using cDNA Arrays--
Primary human skin
fibroblasts were grown to confluence and serum-starved prior to
stimulation with either Cyr61 or BSA (10 µg/ml each) in serum-free
medium for 24 h. Total RNA was isolated and used to prepare
32P-labeled cDNA probes for hybridization to cDNA
arrays (Fig. 1). Negative controls of the
array (blank spots, M13mp18(+) strand, Cyr61 Up-regulates Angiogenic Factors and the Inflammatory Cytokine
IL-1 Expression of Extracellular Proteases and Their Inhibitors--
We
examined the effect of Cyr61 on fibroblast expression of secreted
proteases and their naturally occurring inhibitors. RNA blotting (Fig.
3A) showed that Cyr61 treatment elevated MMP1 and MMP3
mRNA levels 3-5-fold after 12 h and >20-fold after 24 h. Up-regulation of TIMP1, an inhibitor of MMP1 and MMP3, occurred with
slower kinetics and resulted in a 4-fold increase in mRNA level
after 24 h. MMP2 expression, by contrast, was unaffected by Cyr61.
Regulation of uPA by Cyr61 followed an unusual but consistently observed biphasic time course. The uPA mRNA level was initially unaltered (2 h) but became suppressed by 6 h. Longer exposure (12-24 h) to Cyr61 resulted in a 3-5-fold increase in uPA mRNA. Consistent with the observation that PAI-1, a major inhibitor of uPA,
is co-expressed with uPA in fibroblasts (26), Cyr61 also up-regulated
PAI-1 expression by about 15-20-fold in 12-24 h (Fig. 3A).
As expected, all genes tested were expressed at basal levels in control
cells throughout the course of the experiments.
The accumulation of specific proteins in conditioned media of cells
treated with various amounts of Cyr61 was examined by immunoblotting
(Fig. 3B). Whereas the levels of MMP1, MMP3, and TIMP1
proteins were elevated by as little as 0.1 µg/ml Cyr61, up-regulation
of uPA required a higher dose of Cyr61 (1 µg/ml). In each case, 5 µg/ml of Cyr61 elicited maximal accumulation of protein. The level of
PAI-1 protein in conditioned medium was elevated >20-fold after 12-24
h of Cyr61 treatment, whereas the level of MMP2 protein was unaffected
by Cyr61, consistent with their mRNA accumulation profiles (Fig. 3,
B and C). Thus, Cyr61 treatment of human skin
fibroblasts results in a robust induction of MMP1, MMP3, TIMP1, uPA,
and PAI-1 in a time- and dosage-dependent manner at both
the mRNA and protein levels.
Cyr61 Down-regulates Type 1 Collagen and Up-regulates Integrin
Cyr61 Heparin Binding Capacity, p42/p44 MAPK Activities, and de
Novo Protein and RNA Syntheses Are Required for Cyr61-regulated Gene
Expression--
Because the heparin binding activity of Cyr61 is
indispensable for its ability to support fibroblast adhesion (9), we
tested whether this activity is also necessary for Cyr61 to regulate gene expression. The recombinant protein Cyr61DM harbors mutations in
the heparin-binding motifs and fails to bind heparin, is unable to
support fibroblast adhesion, but is still able to mediate endothelial cell adhesion through integrin
Cyr61 treatment elicited a marked and sustained activation of p42/p44,
evident after 12-24 h (Fig.
6A). Interestingly, the kinetics of activation are slow but consistent with the regulation of
gene expression by Cyr61, which also requires 12-24 h (with the
exception of VEGF-A). We therefore tested whether p42/p44 MAPKs
activities are required for regulation of gene expression by Cyr61.
Indeed, up-regulation of most genes by Cyr61 was severely attenuated in
the presence of the extracellular signal-related kinase kinase
inhibitor PD98059 (Fig. 6B). Suppression of Col1
The relatively slow kinetics of gene activation shown above suggested
that Cyr61-mediated gene regulation is indirect and that biosynthesis
of protein effectors may be required. We tested this possibility by
examining the effects of cycloheximide and 5,6-dichlorobenzimidazole
riboside, which inhibit translation and RNA polymerase II activity,
respectively. As shown in Fig. 7, both
induction of MMP1 and suppression of Col1 Expression of Cyr61 during Cutaneous Wound Healing--
The genes
regulated by Cyr61 described above, including those involved in
inflammation, angiogenesis, and matrix remodeling, are known to
participate in cutaneous wound healing (15-17), suggesting that Cyr61
plays a role in regulating wound repair. To investigate this
possibility, we examined Cyr61 expression during skin wound healing using transgenic mice that express the bacterial
lacZ gene encoding Cyr61 Activity in the Presence of ECM Proteins or Serum--
The
expression of Cyr61 in response to wounding implicates its
role in cutaneous wound healing, a process that occurs over several
days and in the context of ECM proteins, growth factors, and cytokines.
To test whether Cyr61-regulated gene expression is modulated by the
presence of ECM components, we allowed fibroblasts to adhere to dishes
precoated with FN, vitronectin, laminin, or type I collagen in
serum-free medium before treatment with Cyr61 or BSA for 24 h. RNA
blot analysis showed that Cyr61 up-regulated MMP1 mRNA by >20-fold
while suppressing Col1
To address the effects of serum growth factors on Cyr61 activity, we
added Cyr61 (5 µg/ml) to cell culture medium containing 10% fetal
bovine serum. Fibroblasts were maintained in such a medium for up to 5 days, with medium renewal on day 3 as needed. Cyr61 is
inducible by TGF-
Thus, even in a growth factor-rich environment (10% serum), Cyr61 can
still exert its effect to up-regulate expression of IL-1 Cyr61-regulated Gene Expression in the Presence of
TGF-
These results suggest that TGF- The principal finding of this study is that Cyr61, an
ECM-associated angiogenic protein, regulates the expression of a
genetic program for wound healing in fibroblasts. This conclusion is
based on two observations: 1) the Cyr61 gene is inducibly
expressed in granulation tissue during wound repair and 2) Cyr61
regulates the expression of genes involved in angiogenesis,
inflammation, matrix remodeling, and cell-matrix interactions. Thus,
the roles of Cyr61 in wound healing extend beyond its activity as an
angiogenic inducer and may coordinate multiple events through
regulation of specific genes.
Purified Cyr61 elicits gene expression changes in human
fibroblasts in serum-free medium in a dose-dependent
manner. Site-specific mutations in the Cyr61 polypeptide abolished its
ability to regulate gene expression, demonstrating that this
activity is an intrinsic property of the Cyr61 polypeptide (Fig.
5A). Cyr61 most likely acts as a matricellular protein
(34) rather than a conventional growth factor or cytokine, given
that it: 1) associates with the ECM upon secretion (35), 2) is
structurally related to other ECM proteins such as von Willebrand
factor and thrombospondin, 3) supports cell adhesion and induces
adhesive signaling, and 4) acts through binding to integrin receptors.
The dosage requirements for Cyr61 to regulate different genes vary,
ranging from 0.1 to 1 µg/ml (~2.5-25 nM) for uPA and
MMPs (Fig. 3B) to 1-5 µg/ml for IL-1 Cyr61 binds heparin with high affinity (35), and the interaction of
Cyr61 with cell surface HSPGs is required for it to support fibroblast
adhesion (9). Two lines of evidence showed that Cyr61 regulation of
gene expression is also dependent on its capacity to bind heparin: 1) a
heparin-binding defective mutant of Cyr61 was unable to regulate gene
expression (Fig. 5A) and 2) soluble heparin, in quantities
sufficient to saturate the heparin-binding site of Cyr61, abrogated its
ability to enhance gene expression (Fig. 5B). Interaction
with HSPGs alone may not be sufficient, however, because numerous
extracellular proteins also can bind heparin but do not have the same
activities. It is likely that another cell surface receptor(s),
together with HSPGs, may be necessary to determine the specific effects
of Cyr61 on gene expression. It is of interest to note that cell
surface HSPGs are important for wound healing. For instance, null
mutation of the syndecan-4 gene results in dramatically delayed skin
wound healing in mice (22).
Although p42/p44 MAPKs were activated by Cyr61 (Fig. 6A),
the unusually slow kinetics (more than 6 h of exposure to Cyr61 before activation occurs) suggest that activation is mediated through
secondary factor(s) induced by Cyr61. This is consistent with the
observation that the gene regulation activity of Cyr61 was abrogated by
inhibitors of transcription or protein synthesis (Fig. 7). Because the
extracellular signal-related kinase kinase inhibitor, PD98059, severely
blunted Cyr61 enhancement of gene expression (Fig. 6B), it
follows that the activities of p42/p44 MAPKs may be required for Cyr61
to regulate gene expression. Functional AP-1 elements have been found
in the promoter regions of genes encoding IL-1 Cyr61 and CTGF are members of the CCN family, and both are encoded by
immediate-early genes that are transcriptionally activated by serum,
platelet-derived growth factor, basic fibroblast growth factor, and
TGF- A number of ECM proteins are capable of regulating MMP expression in
fibroblasts, endothelial cells, or macrophages; these include type 1 collagen (56, 57), FN proteolytic fragments (37, 38, 58), laminin (39),
SPARC (36), tenascin-C (59), and thrombospondin-2 (60). It is of
interest to note that FN fragments are capable of up-regulating
fibroblast expression of MMPs, IL-1 Even though Cyr61 is an immediate-early gene inducible by
TGF- Based on the current study and information available in literature, we
propose a working model for the mechanism of Cyr61 action in cutaneous
wound healing (Fig. 10). After injury,
Cyr61 is produced both by dermal fibroblasts (Fig. 8) and endothelial cells.2 As an ECM-associated
signaling protein (8, 35), Cyr61 interacts with integrin receptors to
induce adhesive signaling (9, 14), cell migration, and enhancement of
growth factor-induced mitogenesis (10). Although Cyr61 interacts
directly with endothelial cells through integrin
We thank Ningyu Chen for kindly providing
Cyr61DM, Tatiana M. Grzeszkiewicz for critical reading of the
manuscript, and members of the laboratory for helpful discussions.
*
This work was supported by National Institutes of Health
Grants CA46565 and CA80080 (to L. F. L.).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.
Published, JBC Papers in Press, October 2, 2001, DOI 10.1074/jbc.M107666200
2
C.-C. Chen, F.-E. Mo, and L. F. Lau,
unpublished results.
The abbreviations used are:
ECM, extracellular
matrix;
BSA, bovine serum albumin;
CTGF, connective tissue growth
factor;
FN, fibronectin;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
HSPGs, heparan sulfate proteoglycans;
IL-1
The Angiogenic Factor Cyr61 Activates a Genetic Program
for Wound Healing in Human Skin Fibroblasts*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
); 3) extracellular matrix
remodeling (MMP1, MMP3, TIMP1, uPA, and PAI-1); and 4) cell-matrix
interactions (Col1
1, Col12, and integrins 3 and
5). Cyr61-mediated gene expression requires heparin
binding activity of Cyr61, cellular de novo transcription, and protein synthesis and is largely dependent on the activation of
p42/p44 MAPKs. Cyr61 regulates gene expression not only in serum-free
medium but also in fibroblasts cultured on various matrix proteins or
in the presence of 10% serum. These effects of Cyr61 can be sustained
for at least 5 days, consistent with the time course of wound healing
in vivo. Interestingly, Cyr61 can interact with
transforming growth factor-1 to regulate expression of specific
genes in an antagonistic, additive, or synergistic manner. Furthermore,
we show that the Cyr61 gene is highly induced in dermal
fibroblasts of granulation tissue during cutaneous wound repair.
Together, these results show that Cyr61 is inducibly
expressed in granulation tissues after wounding and that Cyr61
activates a genetic program for wound repair in skin fibroblasts. We
propose a model in which Cyr61 integrates its activities on endothelial cells, fibroblasts, and macrophages to regulate the processes of
angiogenesis, inflammation, and matrix remodeling in the context of
cutaneous wound healing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
v3,
enhances growth factor-induced mitogenesis, and induces
neovascularization in rat cornea (6-8). As an
ECM1-associated protein,
Cyr61 supports cell adhesion, migration, and proliferation in
fibroblasts (9, 10). Overexpression of Cyr61 in human tumor
cells enhances their tumorigenicity in immunodeficient mice, increasing
tumor size and vascularization (7, 11). In addition, Cyr61 enhances
chondrogenic differentiation of mouse limb bud mesenchymal cells in
micromass cultures, suggesting a role in skeletal development (12).
Cyr61 is a ligand of multiple integrin receptors, which mediate some of
its activities in different cell types (6, 9, 10, 13). In fibroblasts,
Cyr61 induces cell adhesion and adhesive signaling through integrin
61 and heparan sulfate proteoglycans (9,
14), cell migration through integrin v5
and cell proliferation through integrin
v3 (10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(clone
8516.311) and VEGF-A (clone 26503.11) were from R&D Systems Inc.
(Minneapolis, MN). Affinity-purified anti-human VEGF-C polyclonal
antibody was purchased from Zymed Laboratories Inc.
(San Francisco, CA).
, MMP1, MMP2,
uPA, TIMP1, Col11, Col12, FN, integrin 3, integrin
1, and GAPDH were all obtained from the American Type
Culture Collection. Partial human cDNAs corresponding to VEGF-A,
VEGF-C, integrin 5, MMP3, and PAI-1 were generated by
reverse transcription followed by polymerase chain reaction. First
strand cDNA was synthesized from total RNA isolated from human skin
fibroblasts by reverse transcription. Partial cDNAs of human
VEGF-A, VEGF-C, integrin 5, MMP3, and PAI-1 were
amplified from first strand cDNA by polymerase chain reaction using
primer sets that correspond to nucleotides 198-226 and 622-590 of the
human VEGF-A cDNA sequence (GenBankTM accession number
M32977), nucleotides 1165-1191 and 1559-1533 of the human VEGF-C
cDNA (GenBankTM accession number U43142), nucleotides
2094-2117 and 2367-2341 of the human integrin 5
cDNA (GenBankTM accession number X06256), nucleotides
1493-1521 and 1763-1736 of the human MMP3 cDNA
(GenBankTM accession number X05232), and nucleotides
359-381 and 1121-1098 of the human PAI-1 cDNA
(GenBankTM accession number X04429).
to 2.9 for TIMP1.
-galactosidase under the control of
the endogenous Cyr61 promoter. The morphology, growth, and
breeding of these mice are indistinguishable from those of normal
C57BL/6 mice. Wounds were created on the backs of 2-3-month-old female
Cyr61 heterozygous mice according to published protocols
with modifications (21, 22). The mice were anesthetized with a single
intraperitoneal injection of Nembutal, and one full-thickness incision wound of about 5-6 mm in length was created along the mid-line on the back of each mouse by cutting through the skin and
panniculus carnosus. The wounds were left open, and scabs formed over
them within a day. Three animals each were sacrificed at 1, 3, 5, 7, and 14 days, respectively, and their wounds were harvested, fixed in
0.2% paraformaldehyde, and frozen in Tissue-Tek O. C. T. compound, purchased from Sakura Finetechnical Co. (Tokyo). Histological
sections (16 µm) of the wounds were collected serially and stained
with X-gal solution to reveal -galactosidase activity (23). For
comparison, adjacent tissue sections were stained with hematoxylin and eosin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
DNA, and pUC18) yielded no
signal. MMP3 cDNA was represented on the array and was used as a
positive control, because we have previously shown the Cyr61
up-regulates MMP3 as a cell adhesive substrate (14). cDNA array
hybridization showed up-regulation of MMP3 by Cyr61, with a fold
induction consistent with results obtained by RNA blotting (see Fig.
3A). Approximately 15% of the DNA on the arrays hybridized
to cDNA probes, and ~40 genes were found to be differentially
expressed (>2-fold difference in signal). Of these, 12 have been
confirmed by further analyses described below.
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Fig. 1.
cDNA array hybridization. 1064SK
fibroblasts were serum-starved for 24 h and treated with Cyr61 or
BSA (10 µg/ml each) for 24 h. Total RNA was isolated and reverse
transcribed with [32P]dATP to prepare cDNA probes,
which were then hybridized to the cDNA arrays. Arrows
point to examples of differential hybridization signals.
--
Although purified Cyr61 is pro-angiogenic in
vitro and induces angiogenesis in vivo (7, 8), there
has been no previous report regarding its ability to regulate the
expression of angiogenic factors or cytokines. It is thus noteworthy
that cDNA array analysis showed Cyr61 regulation of the potent
angiogenic inducer VEGF-A, the angiogenic/lymphogenic factor VEGF-C
(24, 25), and the inflammatory cytokine IL-1. To confirm these
results, human skin fibroblasts were treated with Cyr61 or BSA for
2-24 h, and gene expression was analyzed by RNA blotting. Cyr61
elevated the VEGF-A mRNA level >2-fold after 6 h and >5-fold
after 24 h (Fig. 2A). VEGF-C and IL-1 mRNAs were up-regulated with slower kinetics (12-24 h), resulting in a 3-5-fold and 5-20-fold enhancement, respectively. Because VEGF-A, VEGF-C, and IL-1 are secreted
proteins, their accumulation in conditioned medium was examined by
immunoblotting. Significant levels of these proteins were secreted by
cells treated with Cyr61 but not by BSA-treated cells (Fig.
2B). In contrast, the MMP2 (gelatinase A) level was
unaffected by Cyr61 (Fig. 3). These data
show, for the first time, that VEGF-A, VEGF-C, and IL-1 expression
are up-regulated by Cyr61 in fibroblasts at both the mRNA and
protein levels.
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Fig. 2.
Time course of IL-1 ,
VEGF-A, and VEGF-C expression upon Cyr61 treatment. Serum-starved
skin fibroblasts were treated with 10 µg/ml of either Cyr61 or BSA
for various durations. A, 15 µg of total RNA isolated at
indicated times was electrophoresed on a 1% agarose gel, followed by
RNA blotting and hybridization with indicated cDNA probes. GAPDH
mRNA expression serves as the control for sample loading.
B, conditioned media of fibroblasts treated with either
Cyr61 or BSA (10 µg/ml) for 24 h were harvested. Protein was
subjected to 10% SDS-polyacrylamide gel electrophoresis, followed by
immunoblotting with specific monoclonal antibodies against human
IL-1, VEGF-A, VEGF-C, and MMP-2, respectively. The data on both
panels are representative of at least three experiments.
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Fig. 3.
Time course and dose response of Cyr61
induction of extracellular proteases and their inhibitors.
A, skin fibroblasts were treated with 10 µg/ml of Cyr61 or
BSA in serum-free medium for various durations. Total RNA was isolated
at the times indicated and analyzed by RNA blotting using indicated
cDNA probes. GAPDH was monitored as a loading control.
B, fibroblasts were treated with various concentrations of
Cyr61 from 0 to 10 µg/ml (as indicated at the top) in
serum-free medium for 24 h. The conditioned media were collected,
and proteins were analyzed by immunoblotting with specific monoclonal
antibodies against human MMP1, MMP2, MMP3, TIMP1, and uPA as indicated.
C, cells were treated with either 10 µg/ml of Cyr61 or BSA
as a control for various times. Protein in conditioned medium was
analyzed by immunoblotting with monoclonal antibodies against human
MMP2 and PAI-1. The data shown for all panels are representative of at
least three experiments.
3 and 5 Subunits Expression--
CTGF, a
related protein with sequence homology to Cyr61, has been reported to
mediate up-regulation of type I collagen by TGF- (27, 28). It is
thus surprising that Cyr61 down-regulates the expression of
1 and 2 subunits of human type 1 collagen (Col1A1 and Col1A2) in a
dosage-dependent manner (Fig.
4A). The major cell surface
receptors for matrix proteins such as collagen and FN are integrins,
and expression of the integrin 3 and 5 subunits was found to be induced by Cyr61 via cDNA array analysis. Both integrin 3 and 5 subunits can
heterodimerize with 1 subunits in fibroblasts to form
receptors that bind denatured collagen and FN, respectively. Expression
of integrin 5 was elevated 5-fold in 12 h and of
both 3 and 5 was increased >10-fold in
24 h by Cyr61 (Fig. 4B). The level of integrin
1 mRNA, however, was unaffected by Cyr61.
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Fig. 4.
Cyr61 down-regulates expression of
Col1 1 and Col12 and
up-regulates expression of integrin
3 and
5. A, skin fibroblasts
were treated with Cyr61 at various concentrations from 0 to 10 µg/ml
(as indicated at the top) for 24 h. Total RNA was
analyzed by RNA blotting and hybridization with cDNA probes
specific for human Col11, Col12, and FN. GAPDH was monitored as a
loading control. B, fibroblasts were treated with Cyr61 (10 µg/ml) for various times as indicated. RNA blotting and hybridization
were performed with cDNA probes specific for human integrin
3, 5, and 1 subunits. The
data shown for both panels are representative of three
experiments.
v3 (9) and
to stimulate fibroblast migration through integrin
v5 (10). As shown in Fig.
5A, Cyr61DM was unable to
up-regulate MMP3 or integrin 5, whereas wild-type Cyr61
caused a >10-fold mRNA induction of both genes (Fig.
5A). The binding of Cyr61 to cell surface heparan sulfate
may be important, because when soluble heparin was added in the culture
medium to saturate the heparin-binding capacity of Cyr61, up-regulation of MMP1 and VEGF-A by Cyr61 was abrogated (Fig. 5B). These
results show that the heparin binding activity of Cyr61 is
indispensable for its ability to regulate the expression of at least
several genes, although we cannot rule out the possibility that Cyr61 may also regulate the expression of a subset of genes through a heparin
binding-independent mechanism.
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Fig. 5.
Heparin binding activity of Cyr61 is required
for gene regulation. A, skin fibroblasts were treated
with 10 µg/ml of either wild-type (Cyr61WT) or mutant (Cyr61DM) Cyr61
in serum-free medium for 24 h. Total cell RNA was isolated and
analyzed by RNA blotting. B, fibroblasts were treated with
10 µg/ml Cyr61 either in the absence (control) or presence (heparin)
of 1 mg/ml of soluble heparin for 12 h. Total cellular RNA was
isolated and analyzed by RNA blotting. The data shown for both panels
are representative of three experiments. GAPDH was monitored as a
loading control.
1 mRNA, however, appeared unchanged by inhibition of extracellular signal-related kinase kinase/MAPK signaling.
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Fig. 6.
p42/p44 MAPKs are activated by Cyr61 and are
necessary for Cyr61 regulation of gene expression. A,
skin fibroblasts were serum-starved for 24 h before treatment with
10 µg/ml of Cyr61 or BSA for various durations. Total cell lysates
prepared from cells harvested at indicated times were electrophoresed
and immunoblotted with affinity-purified polyclonal antibodies against
dually phosphorylated (pTEpY) forms of p42/p44 MAPKs. The same blots
were stripped and reprobed with antibodies against p42/p44 MAPKs.
B, fibroblasts were treated with 10 µg/ml of Cyr61 or BSA
either in the absence (control) or presence of 20 µM
PD98059 for 24 h. Total RNA was analyzed by RNA blotting and
hybridization with indicated specific cDNA probes. The data shown
for both panels are representative of three experiments.
1 mRNA by Cyr61 were
abrogated by these inhibitors. Taken together, these results show that
Cyr61 regulation of gene expression requires its heparin binding
activity, p42/p44 MAPK activities, de novo transcription, and synthesis of protein mediators.
View larger version (81K):
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Fig. 7.
Requirement of de novo
mRNA and protein synthesis for Cyr61-regulated gene
expression. Serum-starved skin fibroblasts were treated with 10 µg/ml of Cyr61 or BSA in the absence (control) or presence of 10 µg/ml cycloheximide (CHX) or 40 µM of
5,6-dichlorobenzimidazole riboside for 24 h. Total RNA was
isolated and analyzed by RNA blotting and hybridization with indicated
cDNA probes. The data shown are representative of two
experiments.
-galactosidase under the control of
the endogenous Cyr61 promoter (20). The first two exons of
one of the Cyr61 alleles were replaced by the
lacZ gene in these heterozygous mice, which are viable and
fertile and show no apparent signs of abnormality. We created full
thickness incisional skin wounds on the backs of these mice, and skin
samples of the wounded area were collected at various times after
wounding and stained for -galactosidase activity (Fig.
8). Little or no -galactosidase
staining was observed within the first 3 days of wounding, but strong
staining was observed by the fifth day in the granulation tissue, where
large numbers of fibroblasts appeared underneath the migrating and
proliferating keratinocytes (Fig. 8). -Galactosidase activity
remained high 1 week after wounding, when re-epithelialization of the
wound by migrating keratinocytes was complete. At this point the
fibroblasts adopted myofibroblast morphology and aligned themselves in
parallel bundles underneath the keratinocytes at right angles to the
wound, consistent with the process of wound contraction (29). By 2 weeks after wounding, the granulation tissue was resolved, and -galactosidase activity was barely detected (Fig. 8). Thus,
Cyr61 is strongly expressed in dermal fibroblasts of the
granulation tissue during wound healing, coincident with ECM remodeling
and wound contraction.
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Fig. 8.
Cyr61 expression during cutaneous wound
healing. Full thickness skin incision wounds were created on the
backs of female C57/BL mice with one allele of Cyr61 mutated, such that
the endogenous Cyr61 promoter drives expression of
Escherichia coli -galactosidase (lacZ) (see
"Materials and Methods"). The skin wound and surrounding tissues
were collected on the indicated days after wounding. Frozen
sections from the middle of the wounds were stained with
X-gal for -galactosidase activity. Adjacent sections were stained
with hematoxylin and eosin (H&E). Bar, 0.2 mm.
1 mRNA to less than 30% of control level,
irrespective of the ECM protein used as substrate (Fig.
9A). Thus, regulation of gene
expression by Cyr61 was unaffected by the presence of the ECM proteins
tested.
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Fig. 9.
Cyr61 regulation of gene expression in
different culture environments. A, skin fibroblasts
were adhered to culture dishes pre-coated with 10 µg/ml each of FN,
vitronectin (VN), laminin (LN), or type 1 collagen (Col.I) in serum-free medium for 24 h. Cyr61
or BSA was added to medium to 10 µg/ml and incubated for 24 h.
Total RNA was isolated and analyzed by RNA blotting. B,
proliferating fibroblasts were cultured in medium containing 10% FBS,
to which Cyr61 or BSA was added to 5 µg/ml, and TGF- 1 was added to
20 ng/ml. The media were renewed on day 3 with the factors supplemented
at the same level as before. At times indicated, total RNA was isolated
and analyzed by RNA blotting and hybridization with specific cDNA
probes as indicated. C, serum-starved fibroblasts were
treated for 24 h with various concentrations (from 0 to 10 µg/ml) of Cyr61, either in the presence or absence of 20 ng/ml of
TGF-1. Total RNA was isolated and analyzed by RNA blotting. The data
shown for all panels are representative of two experiments.
1 (30), a growth factor with recognized roles in
skin wound repair (15, 31). To compare the effects of TGF-1 and
Cyr61, we also added TGF-1 (20 ng/ml) in parallel cultures. The
results showed that on day 1, in contrast to serum-free cultures (Figs.
2 and 3A), Cyr61 had a minimal effect in enhancing IL-1,
VEGF-A, and MMP1 expression, although Col11 was suppressed to about
50% of control (Fig. 9B). By day 3, when cultures became nearly confluent, Cyr61 strongly enhanced IL-1, VEGF-A, and MMP1 expression, whereas Col11 mRNA was suppressed to a minimal level barely detectable. These effects became even more pronounced after 5 days of culture (Fig. 9B). In contrast, TGF-1 suppressed
IL-1 and MMP1 mRNA expression to below control levels, yet
slightly elevated VEGF-A, Col11, and MMP2 expression (Fig.
9B), and these effects of TGF-1 were sustained throughout
5 days of culture.
, VEGF-A,
and MMP1 and suppress Col11 expression. The effects of Cyr61 on gene
expression lasted for at least 5 days; these kinetics closely mirror
the time course of wound healing in vivo (Fig. 8). Whereas
both Cyr61 and TGF-1 enhance VEGF-A expression, major differences in
their effects on gene expression were observed: 1) Cyr61 enhances but
TGF-1 suppresses the expression of IL-1 and MMP1 (Fig.
9B) and 2) Cyr61 suppresses but TGF-1 induces the
expression of both Col11 and Col12 (Figs. 4 and 9B)
(32, 33).
1--
Although TGF-1 can induce Cyr61 synthesis and both
appear to be involved in wound repair, these factors have different
effects on gene expression (Fig. 9B). Thus, we tested the
effects of the combination of Cyr61 and TGF-1 on fibroblast gene
expression. Various amounts of Cyr61 (0.1-10 µg/ml) were added to
serum-starved cells either alone or with TGF-1 (20 ng/ml) for
24 h. TGF-1 alone caused >2-fold induction of Col11
mRNA, whereas Cyr61 at low concentrations (0.1-1 µg/ml) had no
effect (Fig. 9C). However, when Cyr61 was present at 10 µg/ml, Col11 expression was suppressed to 1/3 of the basal level.
When both Cyr61 (10 µg/ml) and TGF-1 were added together, Col11
expression became the same as basal level, which was 3-fold higher than
when Cyr61 was added alone and yet 50% lower than when TGF-1 was
added alone. Thus, the opposing effects of Cyr61 and TGF-1
neutralized one another, resulting in no change in Col11 expression.
The expression of integrin 5 subunit was up-regulated by
either TGF-1 (~2-fold) or Cyr61 (5-fold). The presence of both
Cyr61 and TGF-1 together did not enhance integrin 5
beyond 5-fold (Fig. 9C). TGF-1 alone suppressed MMP1
expression to below basal level, whereas Cyr61 alone (10 µg/ml)
caused >15-fold elevation of MMP1 mRNA. When Cyr61 and TGF-1
were added together, MMP1 was suppressed to an undetectable level.
Thus, the effect of TGF-1 on MMP1 expression overrode that of Cyr61.
Regulation of PAI-1 expression by TGF-1 and Cyr61 followed yet
another pattern. PAI-1 expression was enhanced by either TGF-1 or
Cyr61 by a similar magnitude (~10-fold). When added together, PAI-1
expression became further elevated, and the effect of TGF-1 and
Cyr61 appeared synergistic (Fig. 9C).
1 and Cyr61 induce different but
interacting signaling mechanisms to regulate gene expression. Interestingly, each of the four genes tested above responded in a
different manner to the mixture of Cyr61 and TGF-1. Thus, the effects of Cyr61 and TGF-1 on Col11 expression were antagonistic; in integrin 5 expression, the effects of Cyr61 and
TGF-1 overlapped; in MMP1 expression, TGF-1 completely suppressed
the strong inducing effect of Cyr61; and in PAI-1 expression, effects
of Cyr61 and TGF-1 were synergistic. These results indicate a
complex interaction between the signaling pathways induced by Cyr61 and
TGF-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, Col11, and
Col12 (Fig. 4A and data not shown). These effective
concentrations are higher than those required of soluble growth factors
and cytokines but consistent with the dosage requirements for other
matricellular and ECM proteins, such as tenascin, SPARC, collagen,
laminin, and the 120-kDa FN fragment, to regulate gene expression
(36-43). Although Cyr61 expression is strongly induced by
mitogenic signals in fibroblasts (1), it should be noted that the
actual local concentration of Cyr61 as an ECM-associated molecule in
tissues has been difficult to assess.
, VEGF-A, and MMP1,
and p42/p44 MAPKs were shown to activate transcription through these
AP-1 elements (44-46). Although p42/p44 MAPKs are also involved in the
down-regulation of type 1 collagen gene expression by basic fibroblast
growth factor, platelet-derived growth factor, and ceramide (47, 48), PD98059 was unable to block Cyr61-suppression of Col11 expression (Fig. 6B). Therefore, alternative pathways for the
down-regulation of type 1 collagen must exist. Interestingly, CTGF, a
protein closely related to Cyr61 and also expressed in granulation
tissue, has been reported to enhance rather than suppress type 1 collagen synthesis in NRK fibroblasts (27). Although CTGF interacts
with the many of the same receptors as Cyr61 (13, 14, 49), it has also
been shown to bind the low density lipoprotein receptor-related protein
(50). Differential utilization of receptors in human skin fibrobasts
and NRK fibroblasts for Cyr61 and CTGF may explain the differences in
gene expression effects.
1 (51-53). Because platelet-derived growth factor, basic
fibroblast growth factor, and TGF-1 are released by activated platelets and damaged tissue upon wounding, the actions of these growth
factors may explain the expression of Cyr61 and
CTGF in wounds (Fig. 8) (54, 55). The kinetics of
Cyr61-regulated gene expression are consistent with the wound healing
process in vivo. Among the first genes up-regulated by Cyr61
is the angiogenic factor VEGF-A (6 h), consistent with the need for
nutrient and oxygen supply to cells of the healing wound. Subsequent
expression of VEGF-C may enhance the angiogenic process and/or promote
lymph vessel formation (24, 25). Although Cyr61 up-regulates both MMPs
and their natural inhibitor TIMP1, expression of the proteases (MMP1
and MMP3) precedes that of the inhibitor (TIMP1) (Fig. 3A). We speculate that elevation of proteases prior to their inhibitors allows ECM degradation to occur in a temporally regulated fashion.
, and IL-1 but do not affect
expression of angiogenic factors such as VEGF-A (38, 58). To our
knowledge, Cyr61 is the only matricellular protein (34) capable of
regulating genes that play roles encompassing several major processes
in wound repair, including angiogenesis, inflammation, tissue
remodeling, and cell-ECM interaction. Furthermore, only Cyr61 has been
shown to have long term effects on gene regulation lasting for at least 5 days (Fig. 9B), consistent with the time course of wound
healing in vivo. Importantly, the effects of Cyr61 on
gene expression were observed even in cells grown in 10% serum (Fig.
9B), showing that Cyr61 actions are not mitigated by the
presence of serum growth factors. Thus, Cyr61 appears to have more
diverse functions in skin wound healing compared with other
ECM-associated proteins examined to date.
1, the effects of Cyr61 and TGF-1 on fibroblast gene
expression are not identical. Where both are present, the effects of
Cyr61 and TGF-1 on fibroblast gene expression can be antagonistic, additive, or synergistic. The effect of TGF-1 on MMP1 expression completely overrides that of Cyr61 (Fig. 9C). During skin
wound healing, the peak of TGF-1 expression correlates with
granulation tissue formation when ECM proteins are being actively
produced and deposited, whereas the peak of Cyr61 expression
occurs about 3 days later, coincident with ECM remodeling and
granulation tissue contraction (Fig. 8) (31). We speculate that
fibroblasts produce more collagen and fewer MMPs during the early phase
of wound healing when the level of TGF-1 is high and that of Cyr61
low, thus favoring a net accumulation of ECM and granulation tissue
formation. Subsequently, when TGF-1 level decreases and Cyr61 is
highly expressed, collagen expression is decreased, whereas MMP
expression is increased, thus facilitating ECM remodeling.
v3 to induce pro-angiogenic activities
(7, 8), it also up-regulates synthesis of VEGF-A and VEGF-C in fibroblasts (Fig. 2), further enhancing the angiogenic potential of the
immediate milieu. In addition, Cyr61 enhances fibroblast synthesis of
IL-1, which may act upon macrophages to enhance the inflammatory
response. It is of interest to note that macrophage is an important
source of growth factors necessary to stimulate fibroplasia and
angiogenesis in granulation tissue (16). In addition, exposure of
fibroblasts to Cyr61 leads to elevated expression of ECM-degrading
proteases and their inhibitors, including MMP1, MMP3, TIMP1, uPA, and
PAI-1, and down-regulation of type I collagen (Figs. 3 and
4A), thereby leading to ECM remodeling. The expression of
provisional matrix integrin subunits 3 and
5 is enhanced by Cyr61 as well (Fig. 4B).
This hypothetical model proposes that in response to wounding, Cyr61 is
induced and subsequently activates genes that play multiple and
coordinated roles in wound healing, including angiogenesis,
inflammation, and ECM remodeling (Fig. 10). The functional roles of
Cyr61 in cutaneous wound healing clearly merit further investigation in
the context of wounding models in whole animals.
View larger version (30K):
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Fig. 10.
A model for Cyr61 function during cutaneous
wound healing. Cyr61 is an ECM-associated protein that can act on
(solid arrows) endothelial cells, fibroblasts, macrophages,
and platelets (8,9,13). Cyr61 is synthesized by both endothelial cells
and fibroblasts (dashed arrows); in fibroblasts Cyr61
synthesis is induced by serum growth factors (51). Cyr61 not only acts
directly on endothelial cells to promote angiogenesis (7) but also
stimulates fibroblasts to produce VEGF to enhance angiogenesis further.
In addition, Cyr61 stimulates fibroblast synthesis of IL-1 (Fig. 3),
which can act on macrophages to promote the inflammation response.
Fibroblast expression of MMPs, TIMP1, uPA, PAI-1, and integrin subunits
3 and 5 is enhanced (
) by Cyr61,
whereas type 1 collagen expression is down-regulated (
),
contributing to ECM remodeling. Finally, through affecting
angiogenesis, ECM remodeling, and inflammation, Cyr61 may coordinate
these processes to facilitate cutaneous wound healing.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Molecular
Genetics, University of Illinois at Chicago College of Medicine, 900 South Ashland Ave., Chicago, IL 60607. Tel.: 312-996-6978; Fax:
312-996-7034; E-mail: lflau@uic.edu.
ABBREVIATIONS
, interleukin-1;
MAPK, mitogen-activated protein kinases;
MMP, matrix
metalloproteinase;
PAI-1, plasminogen activator inhibitor-1;
TIMP1, tissue inhibitor of metalloproteinase-1;
TGF-1, transforming growth
factor-1;
uPA, urokinase-type plasminogen activator;
VEGF, vascular
endothelial growth factor;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactoside.
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