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Disruption of tumor necrosis factor receptor p55 impairs collagen turnover in experimentally induced sclerodermic skin fibroblasts.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 4, April 2003, pp 1117–1125
DOI 10.1002/art.10896
© 2003, American College of Rheumatology
Disruption of Tumor Necrosis Factor Receptor p55
Impairs Collagen Turnover in Experimentally
Induced Sclerodermic Skin Fibroblasts
Hiroyuki Murota, Youichiro Hamasaki, Tomoki Nakashima, Kazuo Yamamoto,
Ichiro Katayama, and Toshifumi Matsuyama
Objective. To determine the role of tumor necrosis
factor receptor p55 (TNFRp55)–mediated signaling in
the pathogenesis of scleroderma.
Methods. A murine model of scleroderma that
closely resembles systemic sclerosis in humans was
used. Wild-type and TNFRp55-deficient (TNFRp55ⴚ/ⴚ)
mice received a subcutaneous injection of bleomycin
each day. The extent of skin fibrosis was determined by
measurements of the dermal thickness, as well as histologic examinations. Expression levels of fibrogenic
cytokines, procollagen ␣1, and matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-9 messenger RNA
(mRNA) were analyzed, both in vivo and in vitro, by
reverse transcriptase–polymerase chain reaction assay
or Western blotting.
Results. TNFRp55ⴚ/ⴚ mice began to develop severe sclerotic changes of the dermis on day 3 of the
subcutaneous injections of bleomycin, while wild-type
mice did not. The expression levels of fibrogenic cytokines, procollagen ␣1, and MMP-2 and MMP-9 mRNA
were unaffected in the skin of both wild-type and
TNFRp55ⴚ/ⴚ mice, with or without bleomycin treatment. Induction of MMP-1 expression was significantly
inhibited in the skin from bleomycin-treated
TNFRp55ⴚ/ⴚ mice, and this phenomenon was also
observed in vitro.
Conclusion. These results indicated that signaling mediated by TNFRp55 plays an essential role in
MMP-1 expression and a key role in the collagen
degradation process in this murine model. This study
might provide a basis for understanding the pathogenesis of scleroderma and formulating therapeutic intervention.
Scleroderma is an autoimmune disease that is
characterized by progressive fibrosis of the skin and
internal organs, including the lungs and gastrointestinal
tract. Skin fibrosis is caused by massive production of
fibrous connective tissue in the dermis. Studies of
growth factors (e.g., transforming growth factor ␤
[TGF␤]) have suggested that they also have a role in
the development of fibrosis. Later in the course of
the response, TGF␤ is primarily associated with extracellular matrix production and up-regulation of
platelet-derived growth factor (PDGF) receptors in the
scleroderma fibroblast (1). Interleukin-4 (IL-4) is also
secreted by T cells and increases the synthesis of
collagen by the fibroblast (2), and elevated levels of IL-4
are found in the blood (3,4), bronchoalveolar lavage
cells (5), and skin (6) of scleroderma patients. These
cytokines might be involved in mechanisms that
exacerbate the symptoms of this disease, but not in its
onset.
Tumor necrosis factor (TNF) affects the growth,
differentiation, and function of a multitude of cell types
that are mediators of inflammation and cellular immune
responses. Expression of TNF is detectable during the
very early stages of scleroderma (7), and the serologic
level of TNF increases with the clinical severity and
biologic activity of the disease (8). Additionally, genetic
Supported in part by grants-in-aid for scientific research from
the Ministry of Education, Science, Sports, and Culture, Japan, and by
a grant from NDR Corporation, Gifu, Japan.
Hiroyuki Murota, MD, Youichiro Hamasaki, MD, PhD,
Tomoki Nakashima, PhD, Kazuo Yamamoto, PhD, Ichiro Katayama,
MD, PhD, Toshifumi Matsuyama, MD, PhD: Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki, Japan.
Address correspondence and reprint requests to Toshifumi
Matsuyama, MD, PhD, Department of Molecular Microbiology and
Immunology, Nagasaki University Graduate School of Biomedical
Sciences, Nagasaki 852-8523, Japan. E-mail: tosim@net.nagasakiu.ac.jp.
Submitted for publication April 30, 2002; accepted in revised
form January 8, 2003.
1117
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MUROTA ET AL
analyses have revealed that microsatellite polymorphisms of the TNF and lymphotoxin ␣ (LT-␣) alleles
are associated with scleroderma (9,10). The serum concentration of the soluble TNF receptor p55 (TNFRp55)
correlates with the severity of the disease (11–13).
Because the soluble form of TNFRp55 neutralizes TNF
in cytotoxic assays, and is functionally active as a TNF
antagonist (12), alteration of the TNFRp55 signaling
pathway might be a key factor in the pathogenesis of
scleroderma. To evaluate the role of TNFRp55 signaling
in the onset of skin sclerosis, TNFRp55-deficient
(TNFRp55⫺/⫺) mice were used in a bleomycin-induced
dermal fibrosis model. It has been shown that daily
subcutaneous injections of bleomycin to mice gradually
increase dermal sclerosis after 3–4 weeks (14). Surprisingly, in the present study the TNFRp55⫺/⫺ mice exhibited severe sclerotic skin after only 3–5 days of daily
subcutaneous injections of bleomycin. Alteration of the
TNFRp55 signaling pathway might exacerbate this disease.
MATERIALS AND METHODS
Animals and cells. Mice deficient for the TNFRp55
gene were obtained as previously described (15). Animal care
was in accordance with the institutional guidelines of Nagasaki
University. TNFRp55⫺/⫺ mice were backcrossed to B6
background 5 times. Wild-type and TNFRp55⫺/⫺ mouse embryo
fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s
medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal
bovine serum (BioWhittaker, Walkersville, MD) and streptomycin at 37°C in a 5% CO2 atmosphere.
Histologic analysis. The mice were killed and back skin
was removed on the day after the final injection. The skin
pieces were fixed with 10% formalin, embedded in paraffin,
and sectioned using a microtome. They were then stained with
hematoxylin and eosin (H&E).
Analysis of glycosaminoglycans. Glycosaminoglycans,
which were obtained by ␤-elimination and pronase digestion
from proteoglycans, were treated with chondroitinase ABC,
chondroitinase AC, Streptomyces hyaluronidase, or NaNO3, as
previously described (16,17). The resultant reaction mixtures
were analyzed by electrophoresis on cellulose acetate membranes.
Collagen analysis of the sclerotic skin. Six-millimeter
circles of biopsied skin were homogenized in acetic acid at 4°C
to extract collagen. One milligram of pepsin was added to each
homogenized sample, which was incubated at 4°C for 24 hours
with shaking. The pepsin-solubilized material was collected
after removal of the insoluble residue by centrifugation at
35,000g for 60 minutes at 4°C. The extracted collagen was
analyzed using 5% polyacrylamide gel, and the gels were
stained with Coomassie brilliant blue to identify the pepsinresistant collagen.
Reverse transcriptase–polymerase chain reaction (RTPCR). Total RNA was extracted using an RNeasy mini kit
(Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer. First-strand complementary DNA
(cDNA) was synthesized using an RT-PCR kit (Stratagene, La
Jolla, CA) with oligo dT primers. Thereafter, the cDNA was
amplified for 25 cycles for fibrogenic cytokines, procollagen ␣1
(pro␣1), and matrix metalloproteinases (MMPs). As an internal control, ␤-actin was amplified under the same conditions.
The oligonucleotide primers used for RT-PCR were as follows:
for TNF sense 5⬘-GCGACGTGGAACTGGCAGAAG-3⬘, antisense 5⬘-GGTACAACCCATCGGCTGGA-3⬘; for TGF␤ sense
5⬘-GCTAATGGTGGACCGCAACAACG-3⬘, antisense 5⬘-CTTGCTGTACTGTGTGTCCAGGC-3⬘; for PDGF sense 5⬘CACATCGGCCAACTTCCT-3⬘, antisense 5⬘-TCACACGCCACGCACATC-3⬘; for IL-4 sense 5⬘CGAAGAACACCACAGAGAGTGAGCT-3⬘, antisense 5⬘-GACTCATTCATGGTGCAGCTTATCG-3⬘; for pro␣1 sense 5⬘-AGAGCGGAGAGTACTGGGATCGAC-3⬘, antisense 5⬘-TCTGAGCTCGATCTCGTTGGAT-3⬘; for MMP-2 sense 5⬘-TGCAACCACAACCAACTACG-3⬘, antisense 5⬘-TCTGCGATGAGCTTAGGGAA-3⬘; for MMP-9 sense 5⬘-CTCTGAATAAAGACGACATAGACGGC-3⬘, antisense 5⬘-TCTGAGCTCGATCTCGTTGGAT-3⬘, for LT-␣ sense 5⬘-TGGCTGGGAACAGGGGAAGGTTGAC-3⬘, antisense 5⬘-CGTGCTTTCTTCTAGAACCCCTTGG-3⬘, for ␤-actin sense 5⬘-GTGGGCCGCTCTAGGCACCAA-3⬘, antisense 5⬘-CTCTTTGATGTCACGCACGATTTC-3⬘.
Western blot analysis. Skin samples were frozen in
liquid nitrogen and were homogenized in 0.1M NaCl, 0.01M
Tris HCl (pH 7.6), 1 mM EDTA (pH 8.0), and 0.02 mg/ml
complete protease inhibitor (Roche Diagnostics, Mannheim,
Germany). For cultured cell sampling, ⬃5 ⫻ 105 cells were
solubilized at 4°C in a lysis buffer (0.5% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 100
␮g/ml phenylmethylsulfonyl fluoride, and 1 mM sodium
orthovanadate) for 30 minutes at 4°C. The protein extracts
(20 ␮g of each) were analyzed with an anti–MMP-1 antibody (Sigma, St. Louis, MO), an anti–MMP-2 antibody (Fuji
Yakuhin, Saitama, Japan), an anti-phosphorylated c-Jun
N-terminal kinase/stress-activated protein kinase (JNK/SAPK)
antibody (New England Biolabs, Beverly, MA), and an antiJNK/SAPK antibody (New England Biolabs), as previously
described (18). An antiactin antibody (Chemicon, Temecula,
CA) was used as a control.
Detection of TNFRp55 on the cell surface by flow
cytometric analysis. The expression of TNFRp55 on wild-type
MEFs was analyzed by flow cytometry with fluorescein
isothiocyanate–labeled TNFRp55 monoclonal antibody, as
previously described (35).
RESULTS
Severe fibrosis in skin from TNFRp55ⴚ/ⴚ mice
following bleomycin treatment. After a daily subcutaneous injection of 100 ␮g of bleomycin, wild-type mice
began to exhibit sclerosis on day 14 of the injections. In
contrast, on day 3 of bleomycin treatment, the skin of the
TNFRp55⫺/⫺ mice exhibited severe sclerosis. Skin sections stained with H&E from bleomycin-injected
TNFRp55⫺/⫺ mice were histologically characterized by
thickened and homogeneous collagen bundles and inflammatory infiltrates, whereas the skin from bleomycin-
TNFRp55-MEDIATED SIGNALING IN PATHOGENESIS OF SCLERODERMA
1119
treated TNFRp55⫺/⫺ mice underwent a time-dependent
increase and exhibited a significant difference as compared with that of the skin of bleomycin-treated wildtype mice during days 3–7 of injections (Figure 2). On
day 14, when the skin of wild-type mice started to
thicken, the skin of TNFRp55⫺/⫺ mice instead started
to become atrophic. These results indicate that disruption of the TNFRp55 gene enhanced the effect of
bleomycin to cause the development of thickened
skin.
Collagen accumulation in the skin of bleomycintreated TNFRp55ⴚ/ⴚ mice. To investigate the etiology of
bleomycin-induced thickened skin in TNFRp55⫺/⫺
mice, the contents of the extracellular matrix in skin of
wild-type and TNFRp55⫺/⫺ mice with or without bleomycin treatment were studied (Figures 3A–C). An azanstained section of the skin of TNFRp55⫺/⫺ mice obtained after 3 days of bleomycin treatment exhibited
thickened collagen bundles, while that of the skin of
wild-type mice did not (Figure 3A). In the sclerotic skin
of TNFRp55⫺/⫺ mice after 1–5 days of bleomycin injections, the content of both the ␣1 and ␣2 collagen chains
was significantly increased as compared with that in
normal, untreated mice and the skin of bleomycintreated wild-type mice (Figure 3B). The collagen content at the site of bleomycin injection was almost 3-fold
that in the skin of the untreated mice and the bleomycintreated wild-type mice. In contrast, no sclerotic skin or
accumulation of collagen was detected at sites far from
the bleomycin injection site (Figure 3B). This clearly
indicates that sclerotic skin and collagen accumulation
Figure 1. Histopathologic evaluation of dermal sclerosis in tumor
necrosis factor receptor p55–deficient (TNFRp55⫺/⫺) mice. A, Wild-type
and TNFRp55⫺/⫺ mice received a subcutaneous injection of 100 ␮g of
bleomycin/day for 2 weeks. Skin sections from days 3, 5, 7, and 14 of
bleomycin treatment were stained with hematoxylin and eosin. Skin
sections from day 7 of saline treatment (100 ␮l/day) were used as a
control. B, Inflammatory cell infiltration in bleomycin-treated skin on day
3. (Original magnification ⫻ 10 in A; ⫻ 40 in B.)
injected wild-type mice developed severe inflammatory
infiltrates (Figure 1B), but neither sclerosis nor fibrosis
(Figure 1A). The thickness of the skin of bleomycin-
Figure 2. Dermal thickness in wild-type and tumor necrosis factor
receptor p55–deficient (TNFRp55⫺/⫺) mice treated with bleomycin.
Wild-type and TNFRp55⫺/⫺ mice received a subcutaneous injection of
100 ␮g of bleomycin/day (n ⫽ 5). In TNFRp55⫺/⫺ mice, dermal
thickness increased in a time-dependent manner related to dosage,
whereas wild-type mice did not exhibit an increase in dermal thickness.
Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05.
1120
Figure 3. Biochemical analysis of bleomycin-induced sclerotic dermis
in wild-type and tumor necrosis factor receptor p55–deficient
(TNFRp55⫺/⫺) mice. A, Azan-stained sections of skin from wild-type
and TNFRp55⫺/⫺ mice, obtained on day 3 of bleomycin treatment
(original magnification ⫻ 50). B, Type I collagen content of skin tissue.
Bleomycin was injected at 100 ␮g/day, and total protein was extracted
from the lesional skin and skin located 3 cm from the site of injection,
on days 0, 1, 2, 3, and 5. All lanes were subjected to densitometry
scanning using GeneTools software (Syngene, Cambridge, UK). Experiments were performed on 2 separate occasions with similar results.
C, Glycosaminoglycan content of skin tissue. HA ⫽ hyaluronic acid;
DS ⫽ dermatan sulfate.
were induced only at bleomycin injection sites. Furthermore, the expression level of another component of the
extracellular matrix, proteoglycan, was also analyzed
(Figure 3C). Hyaluronic acid and dermatan sulfate were
evenly expressed in both genotypes. Taken together,
MUROTA ET AL
these results might suggest that bleomycin-induced
thickened skin is caused by an accumulation of collagen.
Lack of effect on collagen synthesis in the skin of
bleomycin-treated TNFRp55ⴚ/ⴚ mice. Because collagen
turnover is regulated by 2 reciprocal pathways, collagen
synthesis and collagen degradation, the induction of
expression of some fibrogenic cytokines in the skin of
bleomycin-treated mice was examined using RT-PCR
analysis (Figure 4). The expression levels of IL-4 and
PDGF were not influenced by the bleomycin treatment. TGF␤ messenger RNA (mRNA) was reduced,
but TNF mRNA was induced in a time- and dosedependent manner related to the bleomycin injections.
There were no significant changes between phenotypes
in the expression levels of these cytokines. The LT-␣
transcript peaked 5 days after bleomycin treatment in
wild-type mice, whereas it peaked at 12 hours in
TNFRp55⫺/⫺ mice. However, at the protein level, there
was no difference between the 2 genotypes (data not
shown). Although we currently do not know the reason,
it seems that LT-␣ is not involved in this pathology. The
expression level of pro␣1 mRNA was not altered in the
skin of TNFRp55⫺/⫺ mice as compared with the skin of
wild-type mice (Figures 5A and B). These results indicate that the collagen synthesis pathway was not affected
in the skin of TNFRp55⫺/⫺ mice.
Inhibition of MMP-1 expression in the skin of
TNFRp55ⴚ/ⴚ mice following bleomycin treatment. To
determine whether the collagen degradation pathway
was affected, the expression levels of MMPs in the skin
of bleomycin-treated mice (Figures 5 and 6) were
examined. First, the expression of MMP-2 and MMP-9,
which are representative of gelatinases, were analyzed
by RT-PCR (Figures 5A and B). Their expressions
were not altered in the skin of bleomycin-treated
TNFRp55 ⫺/⫺ mice. Although the expression of
MMP-1 (collagenase) was increased in the skin of
wild-type mice following bleomycin treatment, it was
significantly reduced in the skin of TNFRp55⫺/⫺
mice (Figure 6). A discrepancy was found in the low
MMP-2 transcript measured 3 and 5 days after bleomycin treatment and the high protein concentration
of MMP-2 detected at the protein level. It has been
reported that bleomycin can induce MMP-2 expression in alveolar epithelial cells (19,20), and some
shedded MMP-2 was stored in endosomal vesicles for
recycling (21). Thus, it is possible that we detected
not only newly produced MMP-2 but also endosomestored MMP-2 at the protein level. These results raised
the hypothesis that aberrant MMP-1 expression in
TNFRp55⫺/⫺ mice might cause severe accumulation of
TNFRp55-MEDIATED SIGNALING IN PATHOGENESIS OF SCLERODERMA
1121
Figure 4. Polymerase chain reaction analysis of cytokine gene expression in bleomycin-treated wild-type and tumor necrosis factor receptor
p55–deficient (TNFRp55⫺/⫺) mice. A, Mice received a subcutaneous
injection of 100 ␮g of bleomycin/day, and total RNA was isolated from
the lesional skin at 12, 24, and 72 hours, and 5 days. The cDNA was
prepared for the detection of TNF, lymphotoxin ␣ (LT-␣), transforming growth factor ␤1 (TGF␤1), platelet-derived growth factor (PDGF),
and interleukin-4 (IL-4) mRNA. Representative results are shown. B,
Results of quantitative analysis of the increase in TNF, LT-␣, TGF␤1,
PDGF, and IL-4 mRNA levels normalized to ␤-actin, from 3 independent experiments. Lanes 1–5, 0 hours, 12 hours, 24 hours, 72 hours,
and 5 days, respectively, after bleomycin treatment in wild-type mice.
Lanes 6–10, 0 hours, 12 hours, 24 hours, 72 hours, and 5 days,
respectively, after bleomycin treatment in TNFRp55⫺/⫺ mice. Values
are the mean and SEM.
collagen in bleomycin-treated skin, and that TNFRp55
might play an important role in the regulation of
MMP-1 expression.
Inhibition of MMP-1 expression in TNFRp55ⴚ/ⴚ
MEFs following TNF treatment. To confirm the above
hypothesis, wild-type and TNFRp55⫺/⫺ MEFs were
treated with TNF in vitro (Figure 7). The induction of
MMP-1 expression was impaired in TNFRp55⫺/⫺
MEFs, while wild-type MEFs were able to induce
MMP-1 expression following TNF treatment. Because
TNFRp55⫺/⫺ MEFs express functional TNFRp75, this
indicated that signaling mediated by TNFRp55, but not
TNFRp75, is essential for the induction of MMP-1
expression. Next, we examined whether the nuclear
factor ␬B (NF-␬B) pathway in TNFRp55⫺/⫺ MEFs was
functional by using another activator of NF-␬B, IL-1␣.
IL-1␣ induced the translocation of p65 to the nucleus
(data not shown), and MMP-1 was induced in a manner
comparable with that in wild-type MEFs. Because TNF
also activates activator protein 1 (AP-1), and the induction of MMP-1 was demonstrated by the binding of AP-1
to TPA responsive element (TRE) in MMP-1 promoter
(22), JNK activities in TNF-treated wild-type and
TNFRp55⫺/⫺ MEFs were examined (Figure 7B). The
phosphorylation of JNK was detected in TNFRp55⫺/⫺
MEFs as in wild-type MEFs, although its appearance
was delayed until 60 minutes after treatment. The
delayed kinetics of JNK activation in TNFRp55⫺/⫺
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MUROTA ET AL
pathway in vivo. Alternatively, some negative regulatory
pathway(s) in MMP-1 induction may have been aberrantly dominated in the absence of TNF/TNFRp55
signaling.
Up-regulation of TNFRp55 in bleomycin-treated
wild-type MEFs. To examine the effect of bleomycin on
the expression of TNFRp55, the cell surface expression
of the TNFRp55 protein was analyzed by flow cytometry
(Figure 8). The up-regulation of TNFRp55 protein on
bleomycin-treated wild-type MEFs was noted. Taken
together, the data indicated that bleomycin induces TNF
and TNFRp55 expression in fibroblasts, and then
TNFRp55-mediated signaling activated JNK and NF␬B. The subsequent production of MMP-1 protein led to
the degradation of collagen. The absence of this cascade
sequence might lead to the development of the severe
sclerotic skin in TNFRp55⫺/⫺ mice following bleomycin
treatment.
DISCUSSION
While there has been much research into the
effects of the TNF/TNFR system on scleroderma, little is
known about its effects on the development of skin
sclerosis. The experimental data presented here show
that TNFRp55 is a key regulator in the induction of
sclerotic skin. TNFRp55⫺/⫺ mice developed severe skin
sclerosis and accumulation of collagen on day 3 of
bleomycin treatment, whereas wild-type mice did not.
As described above, there is a possibility that the mutual
antagonism of TNF and soluble TNFRp55 is involved in
the mechanism of scleroderma pathogenesis. This murine model shows that impaired TNFRp55 signaling
Figure 5. Polymerase chain reaction analysis of procollagen ␣1, matrix metalloproteinase 2 (MMP-2), and MMP-9 gene expression in
bleomycin-treated wild-type and tumor necrosis factor receptor p55–
deficient (TNFRp55⫺/⫺) mice. A, Mice received a subcutaneous
injection of 100 ␮g of bleomycin/day. Total RNA was isolated from the
lesional skin at 0, 3, and 5 days. The cDNA was prepared for the
detection of procollagen ␣1, MMP-2, and MMP-9 mRNA. Representative data are shown. B, Results of quantitative analysis of the
increase in procollagen ␣1, MMP-2, and MMP-9 mRNA levels normalized to ␤-actin, from 3 independent experiments. Values are the
mean and SEM levels in wild-type mice (open bars) and TNFRp55⫺/⫺
mice (solid bars).
MEFs was not seen when IL-1␣ was used (Figure 7B).
Although the activation pathways for NF-␬B and AP-1
were functional with the stimulation of IL-1␣, MMP-1
induction was largely dependent on the TNF/TNFRp55
Figure 6. Western blot analysis of MMP-1 and MMP-2 protein expression in bleomycin-treated wild-type and TNFRp55⫺/⫺ mice. Mice
received a subcutaneous injection of 100 ␮g of bleomycin/day. Total
protein was isolated from the lesional skin at 0, 1, 3, and 5 days. All
lanes were subjected to densitometry scanning. Experiments were
performed on 2 independent occasions with similar results. Representative data are shown. See Figure 5 for definitions.
TNFRp55-MEDIATED SIGNALING IN PATHOGENESIS OF SCLERODERMA
Figure 7. Expression of MMP-1 protein in wild-type and
TNFRp55⫺/⫺ mouse embryo fibroblasts (MEFs) in the presence of 20
ng/ml of TNF or interleukin-1␣ (IL-1␣). A, Whole cell lysates were
prepared from wild-type and TNFRp55⫺/⫺ MEFs treated with TNF
for 0, 24, 48, or 72 hours. The blots were reprobed with an anti-actin
antibody. B, Phosphorylation of c-Jun N-terminal kinase/stressactivated protein kinase (JNK/SAPK) in wild-type and TNFRp55⫺/⫺
MEFs in the presence of 20 ng/ml of TNF or 10 ng/ml of IL-1␣ was
examined to show the level of JNK/SAPK activation. Whole cell lysates
were prepared from wild-type and TNFRp55⫺/⫺ MEFs treated with
TNF or IL-1␣ for 0, 30, 60, or 120 minutes. The blots were reprobed
with an anti-JNK/SAPK antibody. Representative data from 2 independent experiments are shown. See Figure 5 for other definitions.
might also occur in scleroderma patients. Until now, it
was believed that the increase in the serum concentration of TNF reflected the inflammatory stages and also
the extent of internal involvement (23,24). The results of
this study suggest that it may in fact function to improve
the clinical condition.
In the course of scleroderma, a gradual increase
of soluble TNFRp55 in serum occurs and may lead to
the exacerbation of disease symptoms. However, an
increase of soluble TNFR in serum has been described
in patients with other autoimmune diseases, e.g., systemic lupus erythematosus, mixed connective tissue dis-
1123
ease, and rheumatoid arthritis (RA) (12,25,26). Perhaps
not only TNFRp55 signaling, but also other abnormalities, are required for exhibiting specific symptoms of
these diseases.
In this study, a bleomycin-induced dermal fibrosis
model was used. One of the characteristic histologic
features of bleomycin-induced sclerosis is the presence
of infiltrating mononuclear cells in the dermis in its early
stages. However, it has been suggested that participation
of T cells or B cells, as well as mast cells, is not essential
for the development of bleomycin-induced dermal sclerosis, since similar pathology was induced in both SCID
mice and mast cell–deficient WBB6F1-W/Wv mice (27).
It was shown that bleomycin up-regulates type I collagen
and fibronectin mRNA in cultured normal skin fibroblasts (28). Exposure of rat lung fibroblast cultures to
bleomycin results in elevated TGF␤ mRNA synthesis
and TGF␤ protein, which is followed by increased
procollagen gene transcription (29). A recent study
showed that TGF␤ is a mediator of the fibrotic effect of
bleomycin at the transcriptional level and that the TGF␤
response element is required for bleomycin stimulation
of the pro␣1(I) collagen promoter (30). These previously published results (27–30) indicate that bleomycin
increases extracellular matrix production even without
the involvement of immune cells.
We show here that TNFRp55-mediated signaling
is essential for bleomycin-induced MMP-1 expression.
Figure 8. Up-regulation of tumor necrosis factor receptor p55
(TNFRp55) by wild-type mouse embryo fibroblasts (MEFs) following
bleomycin treatment. Wild-type MEFs were treated with 100 ␮M of
bleomycin for 24 hours. Flow cytometric analyses of wild-type MEFs
without bleomycin treatment (dark gray), or with 100 ␮M of bleomycin
treatment for 24 hours (black) stained with TNFRp55. Antibodystained wild-type MEFs with bleomycin treatment were used as a
control (light gray). Representative data from 3 independent experiments are shown FL ⫽ fluorescence.
1124
The role of TNF in extracellular matrix regulation is not
limited to MMP-1. Indeed, a TNFRp55-specific form of
mutant TNF markedly induces collagenase and stromelysin 1 gene expression in dermal fibroblasts (31). In
addition, TNFRp55-specific TNF suppresses type I collagen mRNA levels as potently as wild-type TNF (31).
Therefore, the abnormality in TNF/TNFRp55-mediated
signaling might be involved in the aberrant regulation of
fibrosis seen in scleroderma patients. However, an intrinsic abnormality was noticed in scleroderma fibroblasts. It was reported that the connective tissue growth
factor (CTGF) protein, which promotes collagen synthesis, was constitutively expressed in scleroderma fibroblasts, but not in the normal counterpart (32). The
CTGF protein is induced by TGF␤ exclusively in connective tissue cells (33–35). Although TNF was able to
repress TGF␤-induced CTGF and collagen synthesis in
skin fibroblasts, the basal level of CTGF expression in
scleroderma fibroblasts was unaffected (32). Therefore,
we propose that at least 2 mechanisms are operative in
progressive fibrosis: the inhibition of TNF/TNFRp55mediated signaling, and the TNFRp55-independent, intrinsically aberrant regulation of fibrogenic molecules,
such as CTGF.
It is interesting that MMP-1 is not so much
induced in control mice as it is suppressed in
TNFRp55⫺/⫺ mice after bleomycin treatment. Therefore, the suppression, but not the inability of induction,
of MMP-1 seems to cause sclerodermic skin. Unexpectedly, bleomycin stimulation of TNFRp55⫺/⫺ MEFs did
not cause the suppression of MMP-1 mRNA transcription (data not shown). In the absence of TNF/TNFRp55
signaling, it is possible that some cytokines aberrantly
regulate the expression of MMP-1 in vivo. TGF␤ and
TNF are known to antagonize each other’s function. A
molecular mechanism of this reciprocal inhibition is at
least partly attributable to the competition between
TGF␤-activated Smad-3 and TNF-activated AP-1 for
limiting the amount of the transcriptional coactivator
p300 (36–38). Because TGF␤ expression in the lesional
skin is similar between the 2 genotypes and MMP-1 is
under the control of TGF␤, it is reasonable to speculate
that the inhibition of TGF␤ in the expression of MMP-1
is exaggerated without TNF/TNFRp55 signaling.
Regarding MMP-2, the expression level was
maintained much longer in TNFRp55⫺/⫺ mice (Figure
6). A recent study showed that synthesis and/or secretion
of MMP-2 was increased in cultured fibroblasts from a
scleroderma patient (39), and MMP-2 was proposed to
be involved in the pathophysiology of the disease by
initiating microvascular damage, and leading to fibro-
MUROTA ET AL
blast activation. Interestingly, TGF␤ increases the expression of MMP-2 in cultured fibroblasts (40). From
this point of view, TGF␤ might exert its positive regulatory activity in MMP-2 expression in the absence of
TNF/TNFRp55 signaling. Studies to clarify of the role of
TGF␤ signaling in TNFRp55⫺/⫺ mice are now under
way.
In recent clinical trials on RA, TNF has been the
target of immunotherapy using anti-human TNF chimeric monoclonal antibodies (41,42). It has also been
reported that serum MMP-1 and MMP-3 were reduced
in RA patients following anti-TNF therapy (43). Based
on the present results, it should be kept in mind that
these patients may be at risk for the side effect of
scleroderma-like symptoms.
ACKNOWLEDGMENTS
The authors thank Drs. A. Koda, H. Ichinose, and M.
Miyazaki for encouragement, and Ms Fumiyo Tujita for secretarial work.
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disruption, turnover, induced, experimentale, p55, skin, necrosis, factors, receptov, scleroderma, impair, collagen, tumors, fibroblasts
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