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Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, oncostatin M,
or IL-6. IL-4 induction of TIMP-2 expression was dependent upon
transcription. The p38 mitogen-activated protein kinase (MAPK)
inhibitors SB202190 and SB203580 suppressed IL-4-induced TIMP-2
expression, suggesting the involvement of p38 MAP kinase in the
signaling of IL-4 leading to TIMP-2 expression. Immunoblotting analysis
using a specific Ab against phosphorylated p38 MAP kinase
(Thr180/Tyr182) showed that IL-4 induced
phosphorylation of p38 MAP kinase in human dermal fibroblasts.
Furthermore, the p38 MAP kinase assay showed that IL-4 induces p38 MAPK
activation in human dermal fibroblasts. The expression of the
dominant-negative mutant p38 MAPK represses the IL-4-induced TIMP-2
expression in human dermal fibroblasts. Thus, IL-4 can potentially
alter the dermal matrix metabolism by regulating
TIMP-2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -,
platelet-derived growth factor (PDGF), epidermal growth factor (EGF),
IL-1 and -, TNF- and -, IL-4, IL-6, and oncostatin M (OSM),
regulate dermal fibroblast proliferation and ECM deposition
(2, 3, 4, 5, 6, 7, 8, 9). Dermal fibroblasts derived from patients with
systemic sclerosis or keloid appear to be persistently activated and
produce elevated levels of ECM components in tissue cultures. It is
postulated that this persistent fibroblast activation occurs as a
result of chronic exposure to various cytokines, such as TGF-, and
PDGF (10, 11, 12). Matrix metalloproteinases (MMPs) are important breakdown enzymes of ECM components such as collagen. These enzymes consist of collagenases, stromelysins, gelatinases, and membrane-type MMPs, which degrade various components of the ECM (13). A family of tissue inhibitors of metalloproteinases (TIMPs), comprising TIMP-1, -2, -3, and -4 (14, 15, 16, 17), is now recognized as a member of a family of intrinsic common MMP inhibitors. Pathologic turnover of ECM is believed to be a consequence of a TIMP-MMP imbalance (18).
TIMP-1 and TIMP-2 are produced in soluble form, but TIMP-3 is not
soluble and is associated with ECM. Their expressions are regulated
differently. The production of TIMP-1 is enhanced by cytokines, such as
TGF- and OSM (19, 20), and the expression of TIMP-3 is
induced by mitogenic stimuli, such as serum, EGF, and TGF-
(21, 22). The expression of TIMP-2 mRNA was shown to be
down-regulated in response to TGF-, but is unchanged in response to
phorbol ester treatment in human tumor cells (23). TIMP-2
biosynthesis was unaffected by IL-1, TNF-, PDGF, or phorbol ester in
human alveolar macrophages (24).
In this study we investigated the regulation of TIMP-2 expression by
various cytokines believed to be involved in dermal fibrosis using
cultured human dermal fibroblasts. We examined the effects of TGF-,
OSM, IL-6, and IL-4. The results suggest that IL-4 stimulates the
expression of TIMP-2 in dermal fibroblasts. Further analyses suggested
that IL-4 induces TIMP-2 synthesis via the p38 mitogen-activated
protein kinase (MAPK)-dependent pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IL-4, IL-6, OSM, polyclonal rabbit
anti-human IL-4, and polyclonal rabbit anti-human IL-6 were
obtained from Genzyme (Cambridge, MA). Recombinant human TGF- was
purchased from R&D Systems (Minneapolis, MN). SB203580, SB202190,
PD98059, wortmannin, and LY294002 (Calbiochem, La Jolla, CA) were
dissolved in DMSO. Controls were incubated with an equal concentration
of DMSO. The p38 MAPK rabbit polyclonal Ab was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). The phospho-p38 MAP kinase
(Thr180/Tyr182) rabbit
polyclonal Ab and activation transcription factor 2 (ATF-2)
fusion protein were obtained from New England Biolabs (Beverly, MA).
Anti--actin mAb was purchased from Sigma-Aldrich (St. Louis,
MO).
Fibroblast cultures
Human adult skin fibroblasts were grown from explants of forearm skin biopsies from six healthy donors, with institutional approval and informed consent. Cells were cultured in MEM supplemented with 10% FCS. Fibroblast cultures independently isolated from different individuals were maintained as monolayers at 37°C in an atmosphere of 5% CO2 and 95% air. Fibroblasts under the fifth subpassage were used for the experiments. Cell viabilities were determined by trypan blue stain.
Measurement of TIMP-2 levels in the conditioned medium
The TIMP-2 concentration was determined with a specific sandwich ELISA kit (Amersham, Arlington Heights, IL) that used two kinds of mAbs against human TIMP-2, as described previously (25). Normal human dermal fibroblasts (1 x 105 cells) were seeded in six-well plates in MEM with 10% FCS and grown to confluence. Cells were then placed in 0.5 ml of MEM and 0.1% BSA for 24 h before the cytokine treatment. After incubation with various cytokines for the indicated periods, the conditioned medium was collected and clarified by centrifugation. The cells remaining in the dishes were treated with trypsin and counted electronically. The TIMP-2 concentration in the wells was determined as described above and evaluated according to cell numbers per well.
Immunoblotting
For the preparation of medium collection from fibroblasts untreated and treated with various cytokines, cells were placed in MEM and 0.1% BSA for 24 h before cytokine treatment. After incubation with various cytokines for the indicated periods, the conditioned medium was collected and clarified by centrifugation, and the cells remaining in the dishes were treated with trypsin and counted electronically. The samples were normalized for cell number, subjected to electrophoresis on 15% SDS-PAGE slab gels, and then electrotransferred from the gels onto nitrocellulose sheets. The nitrocellulose sheets were then incubated overnight with a rabbit mAb against human TIMP-2 (Fuji Chemical, Tokyo, Japan). Bound Abs were detected with HRP-conjugated anti-rabbit IgG, and immunoreactive bands were visualized with ECL (Amersham) as described previously (26).
RNA preparation and Northern blot analysis
Fibroblasts were grown to confluence in MEM with 10% FCS and then incubated for 24 h in serum-free medium (MEM plus 0.1% BSA) before addition of the cytokine (27). Total RNA was extracted and analyzed by Northern blotting as described previously (27, 28, 29). The full-length 791-bp TIMP-2 cDNA was obtained from American Type Culture Collection (Manassas, VA) and used as the probe (23). Filters were sequentially hybridized with probes for TIMP-2 and GAPDH. Autoradiography was performed, and the densities of the bands were measured using a densitometer.
Assay of p38 MAPK activation
The activation of p38 MAPK was determined by immunoblotting using Abs specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182; New England Biolabs) and with a p38 MAPK assay kit (New England Biolabs) (30).
In both experiments fibroblasts were serum-starved for 24 h and treated with cytokines for the indicated time. Then, the conditioned medium was removed, and the cells were washed with ice-cold PBS. The cells were lysed by scraping into solubilization buffer (50 mM of Tris-Cl (pH 8), 150 mM of NaCl, 2 mM of EDTA, 1% Triton X-100, 1 mM of sodium orthovanadate, 0.2 mM of PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The lysate was incubated at 4°C for 30 min and then centrifuged for 5 min at 4°C. Protein concentrations of lysates were determined using a Bio-Rad (Hercules, CA) protein assay as recommended by the manufacturer.
Immunoblotting was performed as described previously (26). Briefly, cell lysates (30 µg) obtained from fibroblasts were subjected to electrophoresis on 10/20% gradient SDS-PAGE slab gels, and then electrotransferred from the gels onto nitrocellulose sheets. The nitrocellulose sheets were incubated overnight with mAbs specific for phosphorylated, activated forms of p38 MAPK (Thr180/Tyr182; 1/1000 dilution). Bound Abs were detected with HRP-conjugated anti-rabbit IgG, and immunoreactive bands were visualized with ECL (Amersham, Arlington Heights, IL) as described previously (26). Abs against p38 MAPK (Santa Cruz Biotechnology) were also used to confirm that the protein concentrations of p38 MAPK were maintained.
In the p38 MAPK assay (30), 200 µg lysates were incubated with an immobilized phospho-p38 MAPK (Thr180/Tyr182) mAb overnight at 4°C for immunoprecipitation. For kinase assays the beads were incubated with 200 µM of ATP and 2 µg of ATF-2 fusion protein as a substrate for p38 MAPK at 30°C for 30 min. The reaction was terminated with 25 µl of SDS sample buffer. The samples were then boiled for 5 min, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were incubated overnight at 4°C with a phospho-ATF-2 (Thr71) Ab (New England Biolabs). The membranes were washed, then incubated with HRP-conjugated anti-rabbit IgG, and immunoreactive bands were visualized with ECL.
Transfections and constructs
Transient transfections were performed as described previously
(26, 27, 28, 29). Fibroblasts were transfected by the lipofection
technique (FuGene 6 transfection reagent; Roche, Indianapolis, IN) with
various amounts of constructs. Dominant-negative mutants (TGY-AGF) of
p38 (dn p38 ) and p38 (dn p38) MAPK were provided by Dr. J.
Hans (The Scripps Research Institute, La Jolla, CA) (31).
The plasmid used encodes the ERK2 (p42 MAPK) cDNA in which
Thr183 and Tyr185, which
are required to be phosphorylated for activity, were replaced with
either glutamic acid or alanine and phenylalanine, thus rendering the
protein active (32, 33, 34). Plasmids used in transient
transfection assays were twice purified on CsCl gradients. At least two
different plasmid preparation were used for each experiment.
pSV--galactosidase control vector (Promega, Madison, WI) was
transfected to visualize for transfection efficiency. After incubation
overnight, the medium was replaced with serum-free MEM and 0.1% BSA.
The cells were stimulated with cytokines for 24 h in the absence
of serum, and the conditioned medium was collected and clarified by
centrifugation. Then, the cells remaining in the dishes were treated
with trypsin and counted electronically.
Statistical analysis
Statistical analysis was conducted with the Mann-Whitney test for comparison of means. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the regulation of TIMP-2 expression in dermal
fibroblasts, we studied the effects of fibrogenic cytokines, TGF-,
OSM, IL-6, and IL-4, on the expression of TIMP-2 protein in normal
dermal fibroblasts. Ten nanograms per milliliter of each cytokine was
added for 24 h, and TIMP-2 protein levels in conditioned medium
were determined using a specific TIMP-2 ELISA. As shown in Fig. 1
A, TIMP-2 expression was not
significantly affected by TGF-, OSM, or IL-6. Furthermore, TIMP-2
expression was not significantly affected by higher concentrations (25
or 50 ng/ml) of TGF-, OSM, and IL-6. On the other hand, IL-4 induced
secretion of TIMP-2 into conditioned medium approximately 6-fold (Fig. 1A).
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B, IL-4 stimulated TIMP-2 synthesis in
dermal fibroblasts in a dose-dependent manner. TIMP-2 secretion induced
by IL-4 reached the maximum level when the concentration of IL-4 was 25
ng/ml. Furthermore, IL-4 stimulated TIMP-2 synthesis in dermal
fibroblasts in a time-dependent manner. TIMP-2 secretion induced by
IL-4 reached a maximum level 24–48 h after stimulation with IL-4, and
this increase diminished, but was sustained from 48–72 h (Fig. 1C).
Anti-IL-4 Ab (10 µg/ml) completely abolished the IL-4-mediated
induction of TIMP-2 expression (Fig. 1D). This amount of
anti-IL-4 Ab was shown to neutralize the biological activity of
IL-4 (data not shown). However, anti-IL-4 Ab had little effect on
basal TIMP-2 expression. As a control, anti-IL-6 Ab was also used.
As shown in Fig. 1D, anti-IL-6 Ab (10 µg/ml, this
amount of anti-IL-6 Ab was shown to neutralize the biological
activity of IL-6) had little effect on either IL-4-induced TIMP-2
expression or basal TIMP-2 expression.
To further confirm the increase in TIMP-2 secretion after IL-4 stimulation, immunoblotting was performed using a mAb against human TIMP-2. Consistent with the results obtained using a specific TIMP-2 ELISA, IL-4 induced TIMP-2 synthesis in dermal fibroblasts in a dose-dependent manner (data not shown). Furthermore, IL-4 up-regulated TIMP-2 synthesis in a time-dependent manner (data not shown).
To determine whether the IL-4-mediated induction of TIMP-2 expression
was associated with the corresponding mRNA level, human dermal
fibroblasts were incubated in the absence or the presence of 10 ng/ml
IL-4 under the same conditions, and the expression of TIMP-2 mRNA was
analyzed by Northern blotting. The expression of TIMP-2 mRNA was
markedly elevated (
6-fold) after 6 h in comparison with
untreated fibroblasts and gradually decreased after 12–24 h (Fig. 2, A and B). Thus,
the effect of IL-4 on the expression of TIMP-2 mRNA paralleled its
effect on the corresponding protein synthesis.
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To determine whether up-regulation of TIMP-2 synthesis is due to
increased RNA synthesis, the effect of IL-4 on TIMP-2 expression was
examined by treating human dermal fibroblasts with actinomycin D.
Dermal fibroblasts were serum-starved for 24 h and incubated in
serum-free medium for 12 h in the presence or the absence of
actinomycin D (400 ng/ml). Cell viability was determined with trypan
blue stain, which demonstrated that actinomycin D did not cause cell
death. Actinomycin D completely abolished the IL-4-mediated increase in
TIMP-2 mRNA levels (Fig. 3). Thus,
IL-4-induced up-regulation of TIMP-2 synthesis is mediated at the level
of transcription.
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To further understand the mechanism of IL-4-mediated induction of
TIMP-2 synthesis, we investigated the roles of MAPKs using selective
MAPKs inhibitors. First, to block the ERK1/2 pathway, we added PD98059,
a specific inhibitor of the MAPK kinase 1 (MEK1) and MEK2 activation
(35, 36), to dermal fibroblasts 1 h before treatment
with IL-4. Treatment with PD98059 (30 µM) did not change basal TIMP-2
expression or IL-4-mediated induction of TIMP-2 expression (Fig. 4). Next, to block the
phosphatidylinositol 3-kinase (PI3 kinase) pathway, we added wortmannin
or LY 294002, specific inhibitors of PI3 kinase activation (37, 38), to dermal fibroblasts. Neither wortmannin (100 nM) nor LY
294002 (10 nM) changed basal or IL-4-induced TIMP-2 expression (Fig. 4A). In contrast, addition of the selective p38 MAPK
inhibitor SB203580 (10 µM) or SB202190 (10 µM) (39, 40) entirely abolished the IL-4-mediated induction of TIMP-2
expression in a dose-dependent manner (Fig. 4). Cell viabilities were
determined by trypan blue stain, which demonstrated that the addition
of these concentrations of the inhibitors tested did not have a
cytotoxic effect. Furthermore, the synthesis of -actin was not
inhibited by SB203580 or SB202190 (Fig. 4B). These results
suggest that p38 MAPK is involved in IL-4-induced up-regulation of
TIMP-2 secretion in dermal fibroblasts.
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Since inhibition of p38 MAPK significantly decreased IL-4-induced
TIMP-2 expression, we determined the activation of p38 MAPK by
immunoblotting using Abs specific for phosphorylated, activated forms
of p38 MAPK
(Thr180/Tyr182).
Immunoblotting of whole cell extracts revealed that p38 MAPK
Thr180/Tyr182
phosphorylation gradually occurred after treatment with 10 ng/ml IL-4
(Fig. 5). IL-4-induced phosphorylation of
p38 MAPK reached the maximum level after 3 h and diminished after
6 h. Abs against p38 MAPK were also used to confirm that the
protein concentrations of MAPKs were maintained with or without IL-4
stimulation.
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A). In addition, SB203580 (10
µM) and SB202190 (10 µM) entirely abolished the IL-4-induced
activation of ATF-2, whereas PD98059 (30 µM) did not change
IL-4-mediated induction of phosphorylated ATF-2 (Fig. 6B).
These results suggest that treatment with IL-4 results in the
activation of p38 MAPK in human dermal fibroblasts.
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To further confirm the role of p38 MAPK in IL-4-induced TIMP-2
expression in dermal fibroblasts, transient transfection of the
dominant-negative mutant p38 MAPK into dermal fibroblasts was
performed. As shown in Fig. 7, transient
transfection of the dominant-negative mutant p38 MAPK or p38 MAPK
abolished IL-4-induced TIMP-2 expression in dermal fibroblasts in a
dose-dependent manner. Cell viabilities were determined by trypan blue
stain, which demonstrated that the transient transfection of these
amounts of the plasmids did not have a cytotoxic effect. Furthermore,
the synthesis of -actin was not changed by the transient
transfection of these plasmids. On the other hand, transient
transfection of the dominant-negative mutant ERK2 in fibroblasts did
not abolish IL-4-induced TIMP-2 expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expressions of TIMPs are known to be regulated differently. For
example, the production of TIMP-1 is enhanced by cytokines, such as
TGF- or OSM (19, 20), and the expression of TIMP-3 is
induced by mitogenic stimuli, such as serum, EGF, or TGF- (21, 22). Consistent with the previous reports (23, 24),
we found no significant effect of TGF-, OSM, or IL-6 on TIMP-2
expression in human fibroblasts. To our knowledge this is the first
report that IL-4 stimulates the synthesis of TIMP-2 in human dermal
fibroblasts.
IL-4 is known to use the Janus kinase-STAT signaling pathway (46, 47) and the PI3 kinase pathway (48). Recent studies
demonstrated that IL-4 induces p38 MAPKs (49). This is the
first report demonstrating IL-4-induced p38 MAPK activation in
fibroblasts. In this study immunoblotting analyses using
phospho-specific p38 MAPK Abs that detect only the
Thr180/Tyr182-phosphorylated
forms of p38 MAPK demonstrated that IL-4-induced phosphorylation of p38
MAPK in dermal fibroblasts (Fig. 5). Furthermore, p38 MAPK activation
was confirmed using a p38 MAPK assay. In the p38 MAPK assay, IL-4
stimulation increased phosphorylation of ATF-2 (Fig. 6A).
These results suggest that treatment with IL-4 results in the
activation of p38 MAPK in human dermal fibroblasts.
The present study showed that IL-4 up-regulates the synthesis of TIMP-2
in dermal fibroblasts via the p38 MAPK-dependent pathway. We determined
whether the p38 MAPK pathway was involved in the IL-4-induced TIMP-2
synthesis in dermal fibroblasts, using approaches to block the
signaling pathways. First, we used an MEK1-specific inhibitor, PD98059,
which blocks MEK1 activation by Raf, thus preventing downstream
activation of p42/p44 MAPKs. However, it does not inhibit c-Jun
N-terminal kinase or p38 MAPK (35, 36). In our study
pretreatment of fibroblasts with PD98059 did not abolish
IL-4-stimulated TIMP-2 synthesis. Furthermore, neither wortmannin nor
LY294002, specific inhibitors of activation of PI3 kinase (37, 38), affected IL-4-induced TIMP-2 synthesis. In contrast,
addition of selective p38 MAPK inhibitors, SB203580 and SB202190
(39, 40), entirely abolished IL-4-mediated induction of
TIMP-2 expression in a dose-dependent manner (Fig. 4). Recent studies
showed that collagen-dependent induction of MMP-13 requires p38 MAPK
activity (50). These results suggest that p38 MAPK is
involved in the modulation of ECM in dermal fibroblasts. However, the
phosphorylation and activation of p38 MAPK were gradual (Figs. 5 and 6). These slow kinetics suggest that the effect may be indirect.
MAPK modules are involved in the signal transduction of a wide variety
of signals in all eukaryotic organisms. In mammalian cells, three
well-characterized modules coexist: p44/p42 MAPKs, p38 MAPK, and JNK
cascades (51). p38 MAPK (also known as CSBP, RK, and HOG1)
is a recently identified protein kinase that shares sequence similarity
with other MAPKs. p38 MAPK is activated by the dual-specific kinase MKK
3/6, following exposure to products of microbial pathogens,
environmental stress and proinflammatory cytokines, such as TNF- or
IL-1 (52, 53). It will be of great interest to determine
whether similar cytokine-induced p38 MAPK-dependent signaling pathways
operate in vivo to promote ECM modulation.
IL-4 is known to be a fibrogenic cytokine that promotes biogenesis of ECM proteins in normal wound healing and in pathological fibrosis (7, 54). The present findings potentially have important clinical implications in dermal wound healing and many fibrotic diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hironobu Ihn, Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail: in-der{at}h.u-tokyo.ac.jp
3 Abbreviations used in this paper: ECM, extracellular matrix; ATF, activation transcription factor; EGF, epidermal growth factor; ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MMP, matrix metalloproteinase; OSM, oncostatin M; PDGF, platelet-derived growth factor; PI3 kinase, phosphatidylinositol 3-kinase; TIMP, tissue inhibitor of metalloproteinase.
Received for publication August 15, 2001. Accepted for publication November 30, 2001.
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References |
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