From the Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany
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ABSTRACT |
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Due to its potent effect on fibroblast
proliferation and extracellular matrix deposition, connective tissue
growth factor (CTGF) seems to play an important role in the
pathogenesis of fibrotic disease. Since glucocorticoids are frequently
used for the therapy of these disorders, we determined a potential
effect of these steroids on CTGF expression. In cultured fibroblasts, a
striking induction of CTGF expression was observed after dexamethasone treatment and occurred in a time- and dose-dependent
manner. This effect was obviously not mediated by the CTGF inducer
transforming growth factor-1, since expression of this factor was
down-regulated by the glucocorticoid. Most importantly, CTGF expression
levels also increased substantially in various tissues and organs by systemic glucocorticoid treatment of mice. After cutaneous injury, a
strong induction of CTGF expression was seen in the wounds of nontreated mice. However, no further increase in the levels of CTGF
mRNA occurred in wounded skin compared with unwounded skin of
glucocorticoid-treated animals, suggesting the presence of other
factors in the wound that might compensate for the effect of the
steroids. Tumor necrosis factor-
was identified as a possible mediator of this effect because this factor suppressed CTGF expression in cultured fibroblasts and also blocked the glucocorticoid-induced CTGF production by these cells. These findings indicate that
glucocorticoids stimulate CTGF expression in normal tissues and organs
but not in highly inflamed areas.
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INTRODUCTION |
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Connective tissue growth factor
(CTGF)1 is a potent and
ubiquitously expressed growth factor that has been shown to play a unique role in fibroblast proliferation and in the stimulation of
extracellular matrix production by these cells (1). The 38-kDa protein
was originally identified in conditioned medium from human umbilical
vein endothelial cells (2) and is also secreted by fibroblasts in
vitro. CTGF belongs to a cysteine-rich protein family that
includes serum-induced immediate-early gene products such as
Fisp12/BIGM2, the mouse homologue of CTGF (3), Cyr61, whose expression
correlates with chondrogenesis in mice (4), CEF-10, a v-Src-induced
peptide (5), and the putative avian oncoprotein Nov (6). These proteins
appear to function in a wide variety of biological processes such as
embryonic development and tissue repair. CTGF expression was shown to
be selectively stimulated by transforming growth factor-1 (TGF-
1)
in cultured fibroblasts, whereas other growth factors such as
platelet-derived growth factor, epidermal growth factor, and basic
fibroblast growth factor had no effect on the CTGF mRNA levels in
these cells (7). The identification of a novel TGF-
-responsive
element in the CTGF promotor proved that this induction is a direct
effect on the transcriptional activation of the CTGF gene (8). Due to its mitogenic action on fibroblasts and its ability to induce the
expression of the extracellular matrix molecules collagen type I (
1
chain), fibronectin, and integrin
5 in these cells in
vitro (1), CTGF plays an important role in connective tissue cell
proliferation and extracellular matrix deposition. Apparently, it acts
as a mediator of TGF-
1 in these processes (9). Moreover, it has been
shown to promote cell adhesion (10). In a wound healing model, which
was based on harvesting tissue from subcutaneously implanted steel mesh
chambers, a strong induction of CTGF mRNA was found (7). Because
wound healing requires fibroblast proliferation and connective tissue
deposition by these cells, CTGF might play an important role in the
induction of these processes. In addition to the physiological process
of wound repair, CTGF also seems to be an important player in the
pathogenesis of various fibrotic disorders. Thus it was shown to be
overexpressed in scleroderma, keloids, and other fibrotic skin
disorders (11), as well as in stromal-rich mammary tumors (12) and in
advanced atherosclerotic lesions (13).
Recently, we demonstrated a strong overexpression of CTGF mRNA in affected areas of inflammatory bowel disease patients, whereby a strong correlation between the levels of CTGF mRNA and the production of extracellular matrix molecules was observed.2 Moreover, areas displaying stenosis showed a particularly strong overexpression of CTGF mRNA.
Chronic fibrosis of major organs causes severe medical problems ranging from disfigurement to progressive disability and death. Considering that chronic inflammation may induce some forms of fibrosis, it seems logical to apply anti-inflammatory therapy, whereby glucocorticoids are frequently used. However, the use of these steroids is somewhat controversial in treating fibrosis, because these drugs are frequently ineffective in blocking the progress of the disease. Although they have been shown to be beneficial in the treatment of some cases of liver and lung fibrosis, they seem to aggravate other conditions such as cardiac fibrosis (15, 16).
Since CTGF is highly overexpressed in several fibrotic diseases, we
investigated whether glucocorticoids had any effect on the expression
of CTGF. Here we identified dexamethasone, a common glucocorticoid, as
a novel inducer of CTGF mRNA and protein expression in
vitro and in vivo. This effect was independent of
TGF-1, because we could show that dexamethasone represses its
mRNA expression. Considering that prolonged administration of
anti-inflammatory steroids leads to a delay in wound repair and an
increase in local wound complications, as demonstrated by a series of
experiments and clinical studies (17, 18), we also examined the effect of dexamethasone on the expression of CTGF during the healing of
full-thickness excisional wounds.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
Murine BALB/c 3T3 fibroblasts were cultured in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum and
penicillin/streptomycin (100 units/ml, 100 µg/ml). For experiments,
cells were grown for 3-4 days to 70% confluence. 1 h after the
medium was changed, the cells were treated with dexamethasone or
TNF-. In a combined treatment with dexamethasone and cytokines,
cytokines were added at the same time as dexamethasone. Aliquots of
cells were harvested before and at different time points after the
addition of these reagents. Total RNA was isolated from these cells and
used for RNase protection analysis. Each experiment was repeated at
least twice.
Animal Care-- BALB/c mice were obtained from the animal care facility of the Max Planck Institute of Biochemistry. They were housed and fed according to federal guidelines, and all procedures were approved by the local government of Bavaria.
Glucocorticoid Treatment of Mice--
For glucocorticoid
treatment of mice, two independent experiments were performed. 12 female BALB/c mice (6-12 weeks of age) were injected daily
subcutaneously at 9 a.m. and 6 p.m. with 1 mg (experiment 1)
or 0.5 mg (experiment 2) dexamethasone in phosphate-buffered saline per
kg body weight for either 1 or 3 days. Control animals were injected
with phosphate-buffered saline. After 1 or 3 days of treatment, animals
were sacrificed at 2 p.m. and tissues from each time point were
pooled, frozen in liquid nitrogen, and stored at 80 °C until used
for RNA isolation.
Wounding and Preparation of Wound Tissues--
Three independent
wound healing experiments were performed with female BALB/c mice (8-12
weeks old), as already described (19). For each experiment, 27 animals
were anesthesized by intraperitoneal injection of avertin. The hair on
the animals' backs was cut with fine scissors, and the skin was wiped
with 70% (v/v) ethanol. Six full-thickness wounds were generated on
each animal by excising the skin and the panniculus carnosus. The
wounds were allowed to dry to form a scab. At 12 h, 1, 3, 5, 7, and 14 days after wounding, four animals were sacrificed, and the
wounds were harvested. The complete wound, including 2 mm of the
margins, was excised at each time point. A similar amount of back skin
from three nonwounded animals served as a control. Wounds from animals
at each time point were combined, frozen immediately in liquid nitrogen
and stored at 80 °C until used for RNA isolation.
RNA Isolation and RNase Protection Analysis-- Total RNA isolation was performed as already described (20). RNase protection assays were carried out according to Werner et al. (19). Briefly, antisense transcripts were synthesized in vitro with T3 or T7 RNA-polymerase and [32P]UTP (800 Ci/mmol) using the templates described below. RNA aliquots of 20 µg were hybridized at 42 °C overnight with 100,000 cpm of the labeled antisense probe. As a loading control, 1-µg aliquots of the same batch of RNAs were loaded onto a 1% agarose gel and stained with ethidium bromide. Nonhybridized RNA was digested at 30 °C for 1 h with RNases A and T1, which were subsequently digested by proteinase K at 42 °C for 15 min. Protected fragments were separated on 5% acrylamide, 8 M urea gels and detected by autoradiography.
DNA Templates--
DNA templates for RNase protection analysis
were generated by polymerase chain reaction using primers corresponding
to the published murine sequences. For CTGF, a 170-base pair fragment corresponding to nucleotides 1019-1188 (3) was cloned, and for
TGF-1, the template described previously (21) was used. Furthermore,
a 172-base pair fragment of human CTGF corresponding to nucleotides
1012-1183 (2) was used.
Western Blot Analysis of CTGF Protein-- Cells were grown in 5 ml of Dulbecco's modified Eagle's medium with 10% fetal calf serum and penicillin/streptomycin (100 units/ml, 100 µg/ml) per 10-cm Petri dish with or without dexamethasone. 6 h after addition of the glucocorticoid, conditioned medium from 6 Petri dishes was harvested and centrifuged to remove cell debris. Heparin-binding proteins were precipitated from the supernatant with 120 µl of heparin-Sepharose (1:1 slurry; Amersham Pharmacia Biotech) overnight at 4 °C. Heparin-Sepharose beads were sedimented by centrifugation and washed three times with 20 mM Tris-HCl, pH 7.4, 300 mM NaCl. Heparin-Sepharose-bound proteins were extracted by a 5-min incubation in Laemmli sample buffer at 95 °C, and the amount representing 1 Petri dish was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane, CTGF protein was detected using a rabbit polyclonal antiserum directed against a carboxyl-terminal peptide (NH2-CFESLYYRKMYGDMA-COOH) and an alkaline phosphatase detection system (Promega). Conditioned media from nontreated and solvent-treated cells were used as negative controls.
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RESULTS |
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To determine a potential effect of dexamethasone on the expression
of CTGF, we treated exponentially growing 3T3 fibroblasts with 1 µM dexamethasone for different time periods and analyzed total cellular RNA by RNase protection assay with a CTGF antisense probe (Fig. 1). In comparison to
solvent-treated controls, dexamethasone treatment resulted in a strong
induction of CTGF mRNA expression. Maximal induction was seen as
early as 1 h after addition of the steroid, and increased CTGF
mRNA levels were still observed after 10 h. A similar kinetics
of CTGF induction was seen in three independent experiments. As shown
in Fig. 2, this dexamethasone-mediated
induction was dose-dependent, whereby a concentration of
100 nM dexamethasone was sufficient to elicit a maximal
response (upper panel). This concentration caused a 14-fold
stimulation of CTGF mRNA expression. 10 nM
dexamethasone caused an 8-fold induction, whereas a concentration of 1 nM had no effect. Because these concentrations correspond to the appropriate pharmacological rank of order of potency, the effect
of dexamethasone on CTGF mRNA expression is likely to be mediated by the glucocorticoid receptor. The same induction of CTGF
mRNA expression was obtained in analogous experiments with human
primary fibroblasts, although to a lesser extent (not shown). To
exclude the possibility that the high CTGF mRNA levels are the
result of increased expression of TGF-1, the only known inducer of
CTGF so far, we assayed the same batch of RNAs for TGF-
1 mRNA expression in an RNase protection analysis with a TGF-
1 specific antisense probe (Fig. 2, lower panel). Dexamethasone
concentrations of 10-1000 nM reduced the expression of
TGF-
1 mRNA by approximately 80%, whereas 1 nM had
no effect on TGF-
1 expression levels. This demonstrates that the
induction of CTGF expression by dexamethasone is not mediated by
TGF-
1 but rather is a direct effect of the glucocorticoid.
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Having demonstrated the induction of CTGF by dexamethasone at the mRNA level, we wanted to find out if this could be verified at the protein level. To address this question, we treated exponentially growing 3T3 fibroblasts as before (Fig. 2) with different concentrations of the glucocorticoid and harvested conditioned medium after 6 h. Heparin-binding proteins were enriched by their capacity to bind to heparin-Sepharose and analyzed by Western blotting using a rabbit polyclonal antibody directed against a carboxyl-terminal peptide of CTGF. The Western blot in Fig. 3 shows essentially a similar pattern of induction for the CTGF protein as previously observed for its mRNA, with the exception of a slight decrease of the protein after addition of the highest concentration of dexamethasone.
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To investigate whether the results we obtained in vitro with
cultured 3T3 fibroblasts had any significance in vivo and
thus any relevance for therapeutic applications, BALB/c mice were daily injected with pharmacological doses of dexamethasone. After 1 and 3 days of treatment, we harvested different tissues and isolated total
cellular RNA. RNase protection assays confirmed our in vitro studies; CTGF mRNA expression was significantly up-regulated in heart, kidney, and back skin, all of which are organs frequently affected by severe fibrosis, whereas TGF-1 mRNA expression was down-regulated in these tissues (Fig. 4).
The same results were obtained with lung and liver (not shown).
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Since impaired wound healing by corticosteroids is a well known
phenomenon that may be due to the suppression of the inflammatory phase
of wound healing (22, 23), we tested the influence of dexamethasone on
the CTGF mRNA expression in a full-thickness excisional wound
healing model. As a first step we determined the time course of
expression during wound healing in nontreated mice. For this purpose we
isolated mRNA from skin as well as from full-thickness wounds at
different intervals after injury and performed RNase protection assays
(Fig. 5). CTGF mRNA was maximally expressed between 12 h and 1 day after wounding and reached basal levels after 7 days. Interestingly, we recently observed similar kinetics of induction for the CTGF inducer TGF-1 (21). To study the
influence of glucocorticoids on this process, mice were treated with
dexamethasone for 7 days prior to wounding, and treatment was
continued for the duration of the experiment. Surprisingly, no major
differences in CTGF mRNA expression were observed in wounded skin
of dexamethasone-treated mice compared with control mice. Fig.
6 shows the CTGF mRNA expression in
normal skin and 1 and 5 days after wounding in an RNase protection
analysis. Although in normal skin a significant induction of CTGF
mRNA expression could be observed as already shown in Fig. 4, only
a slight enhancement of CTGF mRNA expression after glucocorticoid
treatment was seen after wounding, whereby the difference was more
pronounced after 5 days.
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To explain this finding, we wondered if there might be any factors
present in an acute wound that could down-regulate the CTGF mRNA
expression and thus were able to compensate for the induction by
dexamethasone. Inflammatory processes always involve the release of
pro-inflammatory cytokines such as tumor necrosis factor (TNF-
)
and interleukin-1
. Therefore, we analyzed the CTGF mRNA
expression in exponentially growing 3T3 fibroblasts after treatment
with TNF-
. TNF-
down-regulated CTGF mRNA levels by 90%
within 8 h and almost completely suppressed CTGF mRNA
expression after 24 h (Fig. 7). In
an additional experiment we determined if this cytokine might have a
modulating effect on the induction of CTGF expression by
glucocorticoids. For this purpose, exponentially growing 3T3
fibroblasts were treated simultaneously with dexamethasone and TNF-
or IL-1
for different time periods (Fig.
8). RNase protection analysis revealed
that TNF-
almost completely abolished the inducing effect of
dexamethasone on the expression of CTGF mRNA, whereas IL-1
had
no effect. These data demonstrate that TNF-
can compensate for the
stimulatory effect of dexamethasone on CTGF expression.
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DISCUSSION |
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Tissue injury initiates a series of tightly regulated processes involving inflammation, the proliferation of fibroblasts, endothelial and epithelial cells, and the deposition of extracellular matrix. If this initially positive process gets out of control, pathological fibrosis can occur in almost any organ or tissue in the body, whereby the most frequently affected sites are liver, kidney, lung, and skin (15, 24). Fibrosis can have severe consequences, ranging from disfigurement to failure of an entire organ and death. Because CTGF is a potent inducer of fibroblast proliferation and deposition of extracellular matrix molecules (1), it seems plausible that aberrant expression of this factor can substantially contribute to the development of fibrotic disease. And indeed, CTGF has been shown to be transiently overexpressed during normal repair processes such as wound healing (Ref. 7, and this study), but permanently overexpressed in various fibrotic conditions (11, 13).2
Although the development of antifibrotic drugs has become a field of significant interest, there is still a lack of effective therapy. Since fibrosis is often the result of acute or chronic inflammation, anti-inflammatory therapy using glucocorticoids is often applied but is only effective in blocking the progress of the disease in some cases.
In addressing the question of whether glucocorticoids influence the
expression of CTGF, we identified dexamethasone as a novel potent
inducer of this growth factor on the mRNA and protein level in vivo and in vitro. This effect is not mediated
by the CTGF stimulator TGF-1, since it was down-regulated by the
glucocorticoid (this study and Ref. 25). Because the concentration
range of dexamethasone corresponds to the pharmacological order of
potency, the effect of dexamethasone on CTGF mRNA expression is
likely to be mediated by binding of the hormone to the glucocorticoid receptor. The most common mechanism of action of such a
ligand-activated steroid receptor is the binding to a glucocorticoid
responsive element in the 3'-untranslated region of a gene. However,
neither the promotor of the murine, nor of the human CTGF gene (for
alignment, see Ref. 8) contains the most common glucocorticoid
responsive element. Nevertheless, the existence of other glucocorticoid
responsive elements in the CTGF promotor cannot be excluded. Another
possibility is the activation of gene expression by protein-protein
interaction of the activated glucocorticoid receptor with another
transcription factor. Such a mechanism has recently been shown for the
transcription of the gene encoding the milk protein
-casein where
the glucocorticoid receptor apparently acts as a
ligand-dependent coactivator of Stat5, independently of its
DNA binding function (26).
The dramatic induction of CTGF expression by dexamethasone could at
least partly explain the phenomenon that steroids can further increase
the fibrotic condition in some patients as observed for the
mineralocorticoid aldosterone in cardiac fibrosis. This hormone uses
the same receptor as glucocorticoids in nonepithelial cells and
therefore exerts the same effects as glucocorticoids in these cells
(16). However, other forms of fibrosis seem to benefit from
glucocorticoid treatment, for example, some forms of liver and lung
fibrosis (15). Furthermore, matrix deposition during wound healing is
significantly reduced by dexamethasone (17), leading to a severe delay
in wound closure and reduced tensile strength. In these cases, the
induction of CTGF expression by dexamethasone might be counteracted by
other factors. We compared CTGF expression during wound healing in
glucocorticoid-treated and nontreated mice. In a full-thickness
excisional wound healing model, peak induction of CTGF mRNA
expression was observed between 12 and 24 h after injury in
nontreated mice. This seems logical, because large amounts of the CTGF
inducer TGF-1 are released from platelets upon hemorrhage (27). In
addition, this kinetics correlates with the time course of induction of
TGF-
1 on the mRNA level in the same wound healing model (21).
However, a very different kinetics of CTGF mRNA expression had been
obtained with a different wound healing model (7), where subcutaneously implanted steel chambers had been used. In this model peak induction of
CTGF mRNA expression was observed after 9 days. The fact that a
different cell population was assayed in this experimental design provides a possible explanation for the delay. For example, the chamber
only contained newly formed granulation tissue, whereas cells situated
at the wound edge did not contribute to the tested RNA pool. Our model,
on the contrary, assays all cells in the wound bed itself and in the
vicinity of the wounds.
Surprisingly, dexamethasone treatment of mice had no significant effect
on the CTGF mRNA levels during wound repair, particularly at early
stages of the repair process. A substantial increase in CTGF mRNA
expression was only observed in nonwounded, but not in wounded skin.
Three reasons might account for the similar levels of CTGF mRNA in
the wounds of glucocorticoid-treated and nontreated mice. First,
dexamethasone could down-regulate the CTGF inducer TGF-1, and the
reduced TGF-
1 levels might in turn compensate for the CTGF mRNA
up-regulation by the glucocorticoid. This hypothesis is supported by
our finding that dexamethasone reduces TGF-
1 mRNA levels during
wound healing (21). However, since platelets are the major source of
TGF-
1 protein in a wound, the reduced expression of this factor
within the wound is unlikely to cause major changes in CTGF expression.
Second, the overexpression of CTGF mRNA during wound healing and
the induction by dexamethasone might not be additive, so no further
enhancement of the expression level could occur. Third, and most
importantly, other factors present in an acute wound, might counteract
the induction of CTGF by glucocorticoids. Because serum growth factors
such as platelet-derived growth factor, epidermal growth factor and
basic fibroblast growth factor, which are also abundantly present in an
acute wound, have no effect on the CTGF mRNA expression (7),
pro-inflammatory cytokines such as IL-1
and TNF-
might be the
most likely candidates. These cytokines are highly expressed during the
inflammatory phase of wound healing (28) and in other inflammatory
conditions (29).
To test this hypothesis, we analyzed CTGF mRNA expression in
fibroblasts after treatment with TNF-. TNF-
not only suppressed the basal expression of CTGF in exponentially growing fibroblasts but
also completely abolished the induction of CTGF mRNA expression by
dexamethasone in these cells. One putative mechanism of this inhibition
might be the interaction of the AP-1-complex and the activated
glucocorticoid receptor, which have been shown to mutually block their
transactivation function (14).
Collectively, these data suggest that dexamethasone up-regulates
CTGF expression in various tissues and organs, although this up-regulation is likely to be counteracted by TNF- in inflamed areas. The actual effect of the dexamethasone-mediated increase in CTGF
expression on the extent of fibrosis is as yet unknown. However,
because elevated levels of CTGF have been shown to correlate with
increased matrix deposition and fibrosis (11, 13)2 our
finding might at least partially explain the phenomenon that glucocorticoid treatment can sometimes even aggravate the course of
fibrotic disease.
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ACKNOWLEDGEMENTS |
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We thank Dr. P. H. Hofschneider for support and A. Stanzel for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by a Grant from the Deutsche Forschungsgemeinschaft (WE 1983/3-1) and a Hermann and Lilly Schilling award (to S. W.).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.
Present address: NOVARTIS Pharma AG, 4002 Basel, Switzerland.
§ To whom correspondence should be addressed. Tel.: 89-8578-2269; Fax: 89-8578-2814; E-mail: werner{at}biochem.mpg.de.
1
CTGF, connective tissue growth factor; IL-1,
interleukin-1
; TGF-
1, transforming growth factor-
1; TNF-
,
tumor necrosis factor
.
2 Dammeier, J., Brauchle, M., Falk, W., Grotendorst, G. R., and Werner, S. (1998) Int. J. Biochem. Cell Biol., in press
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REFERENCES |
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