From the § MediCity Research Laboratory and the
Departments of Medical Biochemistry and ¶ Periodontology,
University of Turku, FIN-20520 Turku, Finland, the
Department of Dermatology, Turku University Central
Hospital, FIN-20520 Turku, Finland, and the
Turku Centre for
Biotechnology, University of Turku and Åbo Akademi University,
FIN-20520 Turku, Finland
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ABSTRACT |
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Inflammatory cytokines tumor necrosis factor-
and interleukin-1 trigger the ceramide signaling pathway, initiated by
neutral sphingomyelinase-elicited hydrolysis of cell membrane
phospholipid sphingomyelin to ceramide, a new lipid second messenger.
Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1
(matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts.
C2-ceramide activates three distinct mitogen-activated
protein kinases (MAPKs) in dermal fibroblasts, i.e.
extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on
the presence of a functional AP-1 cis-element and is
entirely inhibited by overexpression of MAPK inhibitor, dual
specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1
promoter by C2-ceramide is also effectively inhibited by
kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1
and ERK2, SAPK/JNK activator SEK1, or SAPK
. In addition,
ceramide-dependent induction of MMP-1 expression is
potently prevented by PD 98059, a selective inhibitor of MEK1
activation, and by specific p38 inhibitor SB 203580. These results show
that triggering the ceramide signaling pathway activates MMP-1 gene
expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide
signaling pathway may offer a novel therapeutic approach for inhibiting
collagenolytic activity, e.g. in inflammatory
disorders.
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INTRODUCTION |
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Matrix metalloproteinases (MMPs)1 are a family of zinc-dependent metalloendopeptidases collectively capable of degrading essentially all extracellular matrix components (1, 2). MMPs play an important role in tissue remodeling during fetal development, angiogenesis, and tissue repair, and they are also responsible for excessive breakdown of connective tissue in inflammatory disorders, e.g. rheumatoid arthritis, osteoarthritis, autoimmune blistering disorders of skin, dermal photoaging, and periodontitis (1, 2). In addition, degradation of basement membrane and extracellular matrix by MMPs is crucial for invasion and metastasis of tumor cells. To date, the MMP gene family consists of 16 members, which according to structure and substrate specificity can be divided into subgroups of collagenases, gelatinases, stromelysins, and membrane-type MMPs (2). Collagenase-1 (MMP-1) is the principal fibroblast-derived secreted neutral proteinase capable of degrading native fibrillar collagens of types I, II, III, and V, and it apparently plays an important role in the remodeling of collagenous connective tissues in various physiological and pathological situations. The expression of MMP-1 by fibroblasts is potently up-regulated by cytokines, growth factors, and tumor promoters (see Refs. 1-3).
Tumor necrosis factor- (TNF-
) is a proinflammatory cytokine,
which potently inhibits accumulation of connective tissue components. TNF-
stimulates degradation of extracellular matrix by inducing the
expression of MMP-1 and stromelysin-1 (MMP-3) by fibroblasts (4-6). In
addition, TNF-
inhibits type I collagen gene expression by
fibroblasts in culture (6-9) and in vivo (10),
down-regulates elastin (11) and decorin (12) gene expression at the
transcriptional level, and is able to abrogate the activation of type I
collagen and elastin gene expression by transforming growth factor-
(9, 11). The effects of TNF-
on extracellular matrix formation partially overlap with those of interleukin-1 (IL-1), which also induces expression of MMP-1 and MMP-3 in fibroblasts (see Refs. 1-3
and 5). The cellular effects of TNF-
are mediated by two distinct
cell surface receptors: TNF-RI (TNF-R55) and TNF-RII (TNF-R75), both of
which are expressed by fibroblastic cells (see Ref. 13). We have
recently shown that the effects of TNF-
on the expression of MMP-1,
MMP-3, and type I collagen in dermal fibroblasts are primarily mediated
by TNF-R55 (6). It has been shown that binding of TNF-
to TNF-R55
activates neutral sphingomyelinase, a cell membrane-associated
phospholipase, which hydrolyzes cell membrane structural phospholipid
sphingomyelin to phosphocholine and ceramide, a novel lipid second
messenger (see Refs. 14 and 15). The role of the ceramide pathway in
TNF-
-induced apoptosis in various cells has been recently elucidated
(15). In addition, it has been shown that ceramides activate the
expression of cyclooxygenase, stimulate synthesis of prostaglandin
E2 (16), and enhance production of IL-6 by cultured
fibroblasts (17), indicating a role for this signaling pathway in
mediating the inflammatory effects of TNF-
on fibroblasts. However,
the role of the ceramide pathway as a mediator of the effects of
TNF-
and IL-1 on the synthesis and degradation of extracellular
matrix is not known.
In this study, we show for the first time that triggering the ceramide
pathway with neutral sphingomyelinase or cell-permeable ceramides in
human skin fibroblasts results in marked stimulation of MMP-1
expression and that this effect is dependent on the presence of a
functional AP-1 cis-element in the MMP-1 promoter region as
well as on the activity of extracellular signal-regulated kinase 1/2
(ERK1/2), stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), and p38 mitogen-activated protein kinases (MAPKs). These
results show that the effects of TNF- and IL-1 on MMP-1 gene
expression can be mimicked by activating the ceramide pathway in dermal
fibroblasts, suggesting that targeted modulation of this pathway may
offer a novel approach for therapeutic inhibition of matrix
degradation, e.g. in inflammatory disorders.
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EXPERIMENTAL PROCEDURES |
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Materials--
Neutral sphingomyelinase (from
Staphylococcus aureus) and cycloheximide were obtained from
Sigma. C2- and C6-ceramide,
C2-dihydroceramide, and PD 98059 were obtained from
Calbiochem. SB 203580 was provided by SmithKline Beecham (King of
Prussia, PA). Human recombinant interleukin-1 was obtained from
Boehringer Mannheim (Mannheim, Germany). Human TNF-R55-specific TNF-
(double mutant R32W/S86T) (18) were kindly provided by Dr. Walter Fiers
(University of Gent, Belgium).
Cell Cultures--
Normal human skin fibroblast cultures were
established from punch biopsy obtained from a voluntary healthy male
donor (age 23) and cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM
glutamine, 100 IU/ml penicillin G, and 100 µg/ml streptomycin. Murine
NIH-3T3 fibroblasts were obtained from ATCC (Rockville, MD) and
cultured in similar medium supplemented with 10% calf serum (CS). For
experiments, fibroblast cultures were maintained in culture medium
supplemented with 0.5% FCS for 18 h. Thereafter,
sphingomyelinase, ceramides, TNF-R55-specific human TNF-, or IL-1
was added in concentrations and combinations indicated, and incubations
were continued for 24 h. In experiments involving MAPK inhibitors
or cycloheximide, these were added to the cultures 1 h prior to
the addition of ceramides. To estimate the viability of cells after a
24-h incubation with ceramides and sphingomyelinase, cells were washed
with PBS and stained with 0.4% trypan blue in phosphate-buffered
saline (PBS) for 10 min. Cells were then washed with PBS and fixed with 10% formaldehyde, and the number of stained cells was counted.
RNA Analysis--
Total cellular RNA was isolated from cell
cultures using the single step method (19). Aliquots of total RNA were
fractionated on 0.8% agarose gels containing 2.2 M
formaldehyde, transferred to Zeta Probe filters (Bio-Rad) by vacuum
transfer (VacuGene XL, LKB, Bromma, Sweden), and immobilized by heating
at 80 °C for 30 min. The filters were prehybridized for 2 h and
subsequently hybridized for 20 h with cDNAs labeled with
[-32P]dCTP using random priming. The filters were then
washed, the final stringency being 0.1 × SSC, 0.1% SDS at
60 °C (20). The following cDNAs were used for hybridizations: a
2.0-kb human cDNA for collagenase-1 (MMP-1) (21); a 1.5-kb human
cDNA for stromelysin-1 (MMP-3) (22); a 0.7-kb human cDNA for
TIMP-1 (23); a 1.3-kb rat cDNA for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (24); a human 0.4-kb cDNA for
c-jun (25); a human 1.2-kb cDNA for junB
(26); and a human 3.1-kb genomic fragment for c-fos (obtained from Amersham Corp.). The
[32P]cDNA-mRNA hybrids were visualized by
autoradiography, and the mRNA levels were quantitated by scanning
densitometry of the autoradiographs using MCID software (Imaging
Research Inc., St. Catharines, Ontario, Canada). MMP-1 and MMP-3
mRNA levels were corrected for the levels of GAPDH mRNA in the
same samples.
Assay of MMP-1 Production--
The cells were maintained in
serum-free DMEM for 18 h, after which sphingomyelinase (100 mU/ml)
or C2-ceramide (10 µM) was added either alone
or in combination with TNF-R55-specific TNF- (20 ng/ml) or IL-1
(5 units/ml), and the incubations were continued for 24 h. Equal
aliquots of the conditioned media, relative to cell number (27) were
analyzed for the amount of MMP-1 by Western blotting, as described
previously (6) using a polyclonal rabbit antiserum against human MMP-1
(kindly provided by Dr. Henning Birkedal-Hansen, NIDR, National
Institutes of Health, Bethesda, MD), in a 1:2000 dilution, and the
enhanced chemiluminescence detection system (Amersham). The levels of
immunoreactive MMP-1 were quantitated by densitometric scanning of the
x-ray films.
Transient Transfections and CAT Assays--
Confluent NIH-3T3
fibroblast cultures were transiently transfected either with 4 µg of
the construct p2278CLCAT, which contains 2.278 kb of the human MMP-1
promoter linked to the CAT reporter gene, or with a similar construct
with a mutated AP-1 element (28) (both kindly provided by Dr. William
C. Parks (Washington University, St. Louis, MO)). In co-transfection
experiments, the cells were transiently transfected with the MMP-1
promoter/CAT construct pCLCAT3 (2 µg), which contains 3.8 kb of the
5'-flanking region of human MMP-1 gene linked to the CAT gene (29)
(kindly provided by Dr. Steven Frisch (La Jolla Cancer Research
Foundation, La Jolla, CA)), together with 10 µg of the following
expression plasmids: RSV/AS-c-jun (30), a c-jun
antisense expression construct (kindly provided by Dr. Alain Mauviel
(Thomas Jefferson University, Philadelphia, PA)); SG5-CL100 (31) for
MAPK inhibitor; dual specificity phosphatase CL100 (MAPK phosphatase-1)
(32) (kindly provided by Dr. Steven Keyse (Ninewells Hospital, Dundee,
Scotland)); RSV-Raf-C4 (33) for kinase-deficient Raf-1 and
SAPKKK
RR (34) for kinase-deficient SAPK
(both kindly provided
by Dr. Ulf Rapp (University of Würzburg, Germany));
pEBG-SEK1-K
R (35), specific for kinase-deficient SEK1 (kindly
provided by Dr. John Kyriakis (Harvard University, Boston, MA)); and
CEp4LK71RERK1 or CEp4LK52RERK2 (36), specific for kinase-deficient ERK1
and ERK2, respectively (kindly provided by Dr. Melanie Cobb,
Southwestern Medical Center, Dallas, TX). Control cultures were
co-transfected in parallel with the respective empty expression
vectors.
MAPK Activity Assays--
For assay of ERK1/2 and SAPK/JNK
activity, confluent cultures of human skin fibroblasts were incubated
for 18 h in DMEM containing 0.5% FCS. Thereafter, ceramide was
added, and the incubations were continued for different periods of
time. Cells (2 × 106/sample) were lysed in 400 µl
of lysis buffer (PBS, pH 7.4; 1% Nonidet P-40; 0.5% sodium
deoxycholate; 1 mM Na3VO4; 0.1%
SDS; 1 mM EDTA; 1 mM EGTA; 20 mM
NaF; 1 mM PMSF; and 1 µg/ml aprotinin, leupeptin, and
pepstatin). For immunoprecipitation of ERK1/2, cell lysates were
centrifuged (3000 × g for 15 min), and the supernatant was incubated with an antibody generated against ERK2 (p42 MAPK; Transduction Laboratories, Lexington, KY), coupled to protein A-Sepharose (Sigma). This antibody also cross-reacts with ERK1. Immunoprecipitates were washed three times in lysis buffer and three
times in kinase assay buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.5 mM dithiothreitol). The kinase reaction was carried out by
adding to the immunoprecipitate 20 µl of kinase assay buffer,
including 25 µM ATP, 2.5 µCi of
[-32P]ATP (Amersham), and 1 µg/µl myelin basic
protein (Sigma) as substrate. The reaction was carried out for 15 min
at 37 °C and stopped by adding 3 × Laemmli sample buffer. The
samples were resolved on 12.5% SDS-polyacrylamide gel electrophoresis,
and myelin basic protein phosphorylation was quantified with a phosphor imager (Bio-Rad).
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RESULTS |
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Sphingomyelinase and Ceramides Enhance MMP-1 Expression in Dermal Fibroblasts-- To elucidate the role of ceramide pathway in the regulation of fibroblast collagenase (collagenase-1, MMP-1) gene expression, we first activated this signaling pathway by treatment of human skin fibroblasts with S. aureus neutral sphingomyelinase for 24 h and assayed MMP-1 mRNA levels with Northern blot hybridizations. As shown in Fig. 1A, sphingomyelinase treatment of cells with 1 mU/ml resulted in a marked enhancement (7-fold) in MMP-1 mRNA expression, and an even more potent increase (14-fold) was noted with a concentration of 100 mU/ml after correction of the MMP-1 mRNA abundance for the level of GAPDH mRNA in the same samples (Fig. 1A).
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Sphingomyelinase and C2-ceramide Augment Stimulation of
MMP-1 Production by TNF- and IL-1--
To examine the effect of
sphingomyelinase and C2-ceramide on the production of MMP-1
by dermal fibroblasts, we assayed the amount of immunoreactive MMP-1 in
the conditioned media of cells treated with sphingomyelinase (100 mU/ml) and C2-ceramide (10 µM) using Western
blot analysis. As shown in Fig.
2A, both sphingomyelinase and
C2-ceramide stimulated production of MMP-1 by fibroblasts by 5-fold.
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C2-ceramide Activates the MMP-1 Promoter via AP-1-- Stimulation of MMP-1 gene transcription by various stimuli involves induction of dimeric AP-1 trans-activating factor complex (Jun plus Fos), which binds to the corresponding cis-element in the MMP-1 promoter (see Refs. 1-3). We therefore examined the effect of C2-ceramide on the expression of members of Jun and Fos families in dermal fibroblasts. Treatment of cells with C2-ceramide (100 µM) resulted in rapid stimulation of c-jun mRNA expression, first noted at 1 h of incubation with further increase up to 6 h (Fig. 3A). The levels of junB mRNA were also enhanced by C2-ceramide, the peak induction noted at 2 and 6 h (Fig. 3A). In addition, a rapid induction of c-fos mRNA was detected maximally after 1- and 2-h incubations (Fig. 3A). Interestingly, the levels of c-jun, junB, and c-fos mRNAs were still enhanced after 24 h (Fig. 3A). In the same experiment, the expression of MMP-1 mRNA was first induced after a 6-h exposure to C2-ceramide (Fig. 3A). In parallel experiments, C6-ceramide (100 µM) and sphingomyelinase (100 mU/ml) also induced expression of MMP-1 mRNA with similar kinetics (not shown). As shown in Fig. 3B, enhancement of MMP-1 expression by C2-ceramide was abrogated by co-treatment of cells with cycloheximide (10 µg/ml). Similarly, activation of MMP-1 expression by sphingomyelinase was inhibited by cycloheximide (not shown). These observations show that ceramide-elicited activation of MMP-1 gene expression is dependent on synthesis of new regulatory proteins.
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Ceramide-dependent Activation of the MMP-1 Promoter Is
Mediated by ERK1/2 and SAPK/JNK Pathways--
Induction of the
expression of AP-1 components c-Fos and c-Jun by various stimuli,
e.g. growth factors, cytokines, and tumor promoters, is
mediated by activation of three distinct MAPKs, specifically ERK1/2,
SAPK/JNK, and p38 (see Ref. 37). Of these, the ERK1/2 cascade
(Raf-1/MEK1,2/ERK1,2) is activated by Ras and protein kinase C at
the level of Raf-1 (38). In addition, Raf-1 is activated by
ceramide-activated protein kinase (39) and directly by ceramide (40).
The SAPK/JNK pathway (MEK kinase 1-3/SEK1/JNK) is also activated by
Ras (see Ref. 37). In this context, we wanted to elucidate the role of
the ERK1/2 and SAPK/JNK cascades in mediating the effects of ceramide
on MMP-1 expression in fibroblasts. For this, we first examined the
effects of C2-ceramide on ERK1/2 and SAPK/JNK activity in
dermal fibroblasts. Treatment of cells with C2-ceramide
(100 µM) resulted in a maximal increase (4-fold) in
ERK1/2 activity after 1 h of incubation (Fig.
4A). In parallel cultures,
incubation with C2-ceramide (100 µM) resulted
in maximal stimulation (2-fold) of SAPK/JNK activity at time points of
1 and 2 h (Fig. 4A). These results show that treatment
of dermal fibroblasts with ceramide activates both the ERK1/2 and
SAPK/JNK pathways.
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Ceramide-dependent Activation of MMP-1 Expression Is Abrogated by MEK1 and p38 Inhibitors-- To further elucidate the role of specific MAPK cascades in the ceramide-elicited induction of the expression of the endogenous MMP-1 gene, we utilized two specific chemical inhibitors to distinctly block the ERK1/2 or p38 MAPK pathways. First, we treated dermal fibroblasts with C2-ceramide in combination with PD 98059, a specific inhibitor of MEK1 activation, which prevents activation of ERK1 and ERK2 (43). As demonstrated in Fig. 5A, PD 98059 potently and dose-dependently, but not entirely, inhibited the induction of MMP-1 mRNA levels by C2-ceramide, corroborating the role of the ERK1/2 pathway in ceramide-dependent activation of MMP-1 expression.
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DISCUSSION |
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In this study, we demonstrate for the first time that
triggering the ceramide signaling pathway in human skin fibroblasts by
neutral sphingomyelinase and cell-permeable ceramides induces the
expression of fibroblast collagenase-1 (MMP-1), as determined at
promoter, mRNA, and protein levels. In addition, sphingomyelinase and ceramides activate the expression of stromelysin-1 (MMP-3), a
potent activator of latent MMP-1 (1, 2). Sphingomyelinase also augments
the maximal enhancement of MMP-1 expression by TNF-R55-specific TNF-
and IL-1
, indicating that exogenous sphingomyelinase may further
potentiate the signaling pathways triggered by these inflammatory cytokines. Based on these observations, it is possible that
bacteria-derived sphingomyelinase may play a role in extracellular
matrix degradation by stimulating MMP-1 expression, either alone or in
combination with inflammatory cytokines. In this context, it should be
noted that lipopolysaccharide, a constituent of the cell wall of
Gram-negative bacteria, shows structural similarity to ceramides (46).
However, although lipopolysaccharide induces expression of MMP-1 by
monocytes, it has no effect on the expression of MMP-1 by fibroblasts
(28), indicating that its biological effects are not identical with those of ceramides.
The transient transfection experiments show that C2-ceramide potently activates a 2.3-kb MMP-1 promoter/CAT construct and that activation of a similar MMP-1 promoter construct lacking a functional AP-1 binding element is clearly less potent. These results provide evidence that the maximal stimulation of MMP-1 transcription by ceramide is dependent on the binding of the AP-1 trans-acting factor complex to the corresponding cis-element in the MMP-1 promoter. This notion is also supported by results of co-transfection experiments, in which co-expression of c-jun antisense mRNA clearly, but not entirely, inhibited ceramide-dependent activation of MMP-1 promoter. However, since the low basal activity of the AP-1-deficient MMP-1 promoter construct was also somewhat up-regulated by ceramide, it is possible that the effect of ceramide on MMP-1 promoter also involves activation of other trans-acting factors. In addition to inducing expression of mRNAs for c-Jun and c-Fos, the components of the classical AP-1 dimer, C2-ceramide also induces rapid and transient expression of junB mRNA. Although JunB has been shown to counteract trans-activation of the AP-1-responsive promoter by c-Jun (26), we have recently demonstrated that JunB can also directly activate the MMP-1 promoter (47) and mediate its stimulation by tumor promoter okadaic acid (20). Therefore, we cannot exclude the possibility that JunB-containing AP-1 complexes may be involved in ceramide-elicited activation of the MMP-1 promoter in fibroblasts.
In the present study, triggering the ceramide pathway in dermal
fibroblasts with exogenous ceramide activates both ERK1/2 and SAPK/JNK
classes of MAPKs, and the ceramide-elicited induction of the endogenous
MMP-1 gene is potently inhibited by PD 98059, a specific inhibitor of
activation of MEK1, the MAPK kinase, which activates ERK1/2. This is in
contrast to observations on U937 and bovine aortic endothelial cells in
which ceramide treatment activates SAPK/JNK but has minimal effect on
ERK1/2 activity (48). However, ceramide-treatment of promyelocytic
HL-60 cells also activates the ERK1/2 class of MAPKs (49). In our
transient transfection experiments, overexpression of MAPK inhibitor
CL100, a dual specificity phosphatase, which inactivates ERK1/2,
SAPK/JNKs, and p38 MAPK, entirely inhibited the activation of the MMP-1
promoter by C2-ceramide in fibroblasts. However,
overexpression of kinase-deficient forms of Raf-1, ERK1 and ERK2, SEK1,
or SAPK clearly, but not entirely, inhibited
ceramide-dependent activation of the MMP-1 promoter in
NIH-3T3 fibroblasts. Together, these results provide evidence that the
effect of ceramides on MMP-1 gene expression is mediated via both
ERK1/2 and SAPK/JNK pathways and that activation of both is required
for maximal activation of the MMP-1 promoter by ceramide. It is likely
that ERK1/2 and SAPK/JNK cascades are distinctly activated by ceramide
at the level of MAPK kinase kinases, since Raf-1 does not activate the
SAPK/JNK pathway and SEK1 does not activate ERK1/2 (44, 50). However,
ceramide can also activate MEK1 and ERK1/2 independently of Raf-1 via
protein kinase C
(51) and MEK kinase 1-3 of the SAPK/JNK pathway
can also activate MEK1 and MEK2 (see Ref. 37), providing a putative
Raf-independent pathway for activation of ERK1/2.
As mentioned above, the role of the SAPK/JNK pathway in mediating the
stimulatory effect of ceramide on MMP-1 expression is supported by the
observation that overexpression of kinase-deficient SEK1 or SAPK
clearly, although not entirely, inhibited the up-regulatory effect of
ceramide on the MMP-1 promoter. However, SEK1 may also activate p38
MAPK, which induces expression of c-Jun via phosphorylation and
activation of ATF-2 (37, 44). Our observation that ceramide activates
p38 MAPK and that the up-regulatory effect of C2-ceramide on endogenous MMP-1 expression is partially prevented by the selective p38 inhibitor SB 203580 also provides evidence for the role of p38 MAPK
in the ceramide-mediated activation of MMP-1 expression in dermal
fibroblasts. This is in contrast to endothelial cells, in which
ceramide does not activate p38 (52), indicating cell specificity in the
ceramide-dependent activation of MAPK pathways. p38 MAPK is
also activated independently of the SAPK/JNK cascade by its specific
MAPK kinases, MEK3 and MEK6, in response to environmental stress and
TNF-
(53). It is therefore possible that in fibroblasts ceramides
activate p38 independently of SEK1 via activation of MEK3 or MEK6 by
TAK1 (transforming growth factor-
-activated protein kinase) (54).
Nevertheless, the effect of C2-ceramide on the expression
of the endogenous MMP-1 gene was not entirely inhibited by SB 203580, providing further evidence that the activity of three distinct MAPKs,
i.e. ERK1/2, SAPK/JNK, and p38 is required for maximal
activation of MMP-1 expression by ceramide.
In promyelocytic HL-60 cells and in human monocytic leukemia cell line U937, the induction of apoptosis by C2-ceramide is suppressed by sphingosine-1-phosphate, which inhibits activation of SAPK/JNK and activates ERK1/2 (48). In addition, the initiation of programmed cell death by ceramide in U937 and bovine aortic endothelial cells appears to be entirely mediated via the SAPK/JNK pathway and can be inhibited by overexpression of dominant negative c-Jun (55). In the present study, activation of the ceramide pathway in human skin fibroblasts by treatment with C2-ceramide (100 µM) somewhat affected the viability of cells. However, induction of MMP-1, as well as activation of c-jun expression was also noted with the lower (10 µM) C2-ceramide concentration, with C6-ceramide, and with sphingomyelinase, all of which had no effect on the viability of these cells. Together, these observations indicate that ceramide-dependent stimulation of MMP-1 expression is not associated with altered cell viability and suggest that, in dermal fibroblasts, triggering the sphingomyelin pathway is not alone sufficient to induce programmed cell death.
In conclusion, the results of this study show that the activation of
the ceramide-dependent signaling pathway either by
endogenous ceramides generated by neutral sphingomyelinase or by
exogenously added ceramide analogs potently activates the expression of
collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) in fibroblastic cells.
We also show that in these cells ceramide treatment activates ERK1/2
and SAPK/JNK classes of MAPKs, both of which mediate the
ceramide-elicited activation of MMP-1 gene expression. In addition,
activity of p38 MAPK is required for maximal activation of MMP-1 gene
expression by ceramides. However, our results indicate that inhibition
of any one of these MAPK pathways alone is not sufficient to inhibit ceramide-dependent activation of MMP-1 expression. Based on
the results of this study, it can be suggested that the ceramide
signaling pathway plays an important role in mediating the effects of
TNF- and IL-1 on the expression of MMP-1 and also MMP-3.
Inflammatory cell-derived TNF-
and IL-1 play an important role in
the induction of MMP-1 expression in conditions characterized by
increased collagenolytic activity, such as rheumatoid arthritis,
osteoarthritis, autoimmune blistering disorders of skin, and
periodontitis. It is likely that targeted inhibition of the ceramide
pathway and three distinct MAPKs implicated in this study may be
feasible for inhibiting induction of MMP-1 expression by TNF-
and
IL-1, thus offering novel possibilities for therapeutic intervention of
extracellular matrix degradation.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Eeva Virtanen, Emma Metsämäki, Marita Potila, and Leila Saarinen is gratefully acknowledged. We also thank Drs. E. Bauer, M. Kurkinen, D. Carmichael, P. Fort, M. Karin, J. Minna, U. Rapp, J. Kyriakis, S. Keyse, M. Cobb, A. Mauviel, W. C. Parks, and S. Frisch for plasmids.
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FOOTNOTES |
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* This work was supported by grants from the Academy of Finland, the Sigrid Jusélius Foundation, the Cancer Research Foundation of Finland, and Turku University Central Hospital and by personal grants from the Duodecim Society and Culture Fund of Southwestern Finland (to J. 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.
** To whom correspondence should be addressed: MediCity Research Laboratory, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland. Fax: 358-2-3337000; Tel.: 358-2-3337025; E-mail: velkah{at}utu.fi.
1 The abbreviations used are: MMP, matrix metalloproteinase; TNF, tumor necrosis factor; IL, interleukin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK/JNK, stress-activated protein kinase/Jun N-terminal kinase; MEK, MAPK/ERK kinase; SEK1, SAPK/ERK kinase-1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; CS, calf serum; kb, kilobase pair; TIMP, tissue inhibitor of metalloproteinases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; MOPS, 4-morpholinepropanesulfonic acid; mU, milliunit(s).
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REFERENCES |
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