From the Centers for Immunology and Microbial Disease
and ** Cell Biology and Cancer Research, Albany Medical College, Albany,
New York 12208, the ¶ Dartmouth Medical School, Hanover, New
Hampshire 03755-3833, and the
Department of Pediatrics,
University of California, San Francisco, California 94143-0748
Received for publication, January 10, 2001
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ABSTRACT |
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Manganese-superoxide dismutase (Sod2)
removes mitochondrially derived superoxide (O Reactive oxygen species are constantly generated in aerobic
organisms during normal metabolism as well as in response to both internal and external stimuli. Although low levels of reactive oxygen
species are indispensable in several physiological processes ranging
from cell proliferation to apoptosis, high levels of reactive oxygen
species and their inefficient removal result in oxidative stress that
can be potentially toxic to the cells. An imbalance in reactive oxygen
species has been hypothesized to play a causative role in many disease
pathologies, including cancer (1), ischemia/reperfusion injury (2),
degenerative diseases such as photoaging (3), atherosclerosis (4),
osteoarthritis and neurodegeneration (5). A feature often associated
with these diseases is a malfunctioning of the connective tissue
remodeling process due to increased activity of extracellular
matrix-degrading metalloproteinases
(MMPs).1 The matrix
metalloproteinase family is composed of at least 20 zinc-dependent extracellular endopeptidases that are
primarily expressed as inactive precursors or zymogens (6). MMP
expression is low in normal tissues and is induced when extracellular
matrix remodeling is required; in all the above-mentioned disease
pathologies and during senescence, there is a coincident increase in
the production of reactive oxygen species and MMPs.
The mitochondrial manganese-containing superoxide dismutase
(Sod2) is one of the numerous enzymatic and non-enzymatic
defenses that exist to combat oxidant injury. Sod2 catalyzes
the diffusion-limited removal of superoxide to
H2O2 at its site of production. It has recently
been shown that the Sod2-dependent production of
H2O2 leads to the expression of MMP-1 (7). In
addition, Sod2 (CD1Sod2tm1Cje)
heterozygous knockout mice develop premature interstitial fibrosis and
accumulate collagen when compared with wild-type age-matched controls
(8). Whether the accumulation of collagen in the
Sod2 This study demonstrates that the Sod2-dependent
H2O2 production leads to the activation of the
ERK1/2 signaling cascade and subsequent downstream transcriptional
increases in MMP-1 expression. Furthermore, a specific single
nucleotide polymorphism sensitizes the MMP-1 promoter to
Sod2-dependent activation. Thus, the cell has
evolved to utilize the mitochondria as an important source of the
signaling molecule H2O2 via Sod2 and
provides a mechanistic rationale for the increased expression of
Sod2 and MMP in numerous disease pathologies.
Cell Culture and Reagents--
HT-1080 human fibrosarcoma cells
were cultured in MEM supplemented with 10% fetal calf serum, 1000 units/ml penicillin, 500 µg/ml streptomycin, and 1 mg/ml neomycin in
a 37 °C humidified incubator containing 5% CO2.
Constructions of recombinant Mn-SOD and catalase plasmids and
transfections were previously described in detail (9, 10). Mouse
embryonic fibroblasts (MEFs) derived from Sod2-deficient
mice (CD1Sod2tm1Cje) were kindly provided by
Drs. C. J. Epstein and T. T. Huang (University of
California, San Francisco, CA). The fibroblasts were cultured individually in 50% Dulbeccos' modified Eagle's medium and
50% F-12 supplemented with 15 mM HEPES and
L-glutamine (Fisher) and 10% fetal calf serum in
25-cm2 flasks in 3% oxygen. They were split 1:4 and
passaged every 5 days. Cells were treated with recombinant human TNF
(R&D Systems, Minneapolis, MN), phorbol 12-myristate 13-acetate, or
mouse IL-1 (R&D Systems).
Construction and Transfection of Full-length Human IL-1 RNA Isolation and Northern Blot Analysis--
Cell lines were
grown to confluence in 25-cm2 culture flasks, and
treatments were performed as described in the figure legends. Total
cytoplasmic RNA was extracted with TRIzol reagent (Life Technologies,
Inc.) according to the manufacturer's instructions and quantified
spectrophotometrically. Northern blot analysis was performed as
previously described (11). To normalize for RNA loading and transfer,
the membrane was hybridized to a GAPDH or actin probe.
cDNA Probes--
The human MMP-1, a probe
1.3-kilobase pair fragment, was isolated from plasmid was kindly
provided by Dr. G. I. Goldberg (Washington University School of
Medicine, St. Louis, MO). All other Northern probes were made as
described in detail by Dumin et al. (12).
Reverse Transcription-PCR (RT-PCR) mRNA
Analysis--
RT-PCR analyses of MMP-1 and Sod2 were
carried out as described by Melendez and Davies (9) with slight
modifications. GAPDH was co-amplified in the linear amplification range
as a loading control. The primers used for the PCR amplification were
designed by utilizing OLIGO primer analysis software (National
Biosciences, Inc.). The primers were as follows: human 5'-MMP-1,
5'-GGAGGAAATCTTGCTCAT-3'; human 3'-MMP-1, 5' CTCAGAAAGAGCAGCATC-3';
5'-GAPDH, 5' CATCATCCCTGCCTCTACTGG-3'; 3'-GAPDH, 5'-TCTCTTCCTCTT
GTGCTCTTG-3'; 5'-SOD2, 5'-TCCCCGACCTGCCCTACGAC-3'; and 3'-SOD2,
5'-CATTCTC CCAG- TTGATTACAT-3'.
Transient Transfections of Human MMP-1 Promoter
Constructs--
Cell lines were transiently cotransfected with the
pGL3-MMP-1(1G) or pGL3-MMP-1(2G) construct (13) and pCMV.SPORT Dichlorofluorescein Fluorescence--
Construction of the
expression vectors, characterization of the cell lines, and FACScan
analysis of DCF fluorescence were performed as described in detail by
Rodriguez et al. (14).
Detection of ERK1/2 Phosphorylation by
Immunoblotting--
Control (CMV) cells at 80-90% confluence in
100-mm dishes were made quiescent by incubation in serum-free MEM for
24 h. The cells were then stimulated with 20% fetal calf serum
for 15 min, washed three times with ice-cold phosphate-buffered saline,
and placed on ice. The cells were lysed by incubation for 10 min on ice
with 400 µl of hypotonic buffer (20 mM HEPES (pH 7.9),
0.1 mM EDTA, and 10 mM KCl), 0.2% Nonidet
P-40, protease inhibitors (3 µg/ml aprotinin and 1 mM
phenylmethylsulfonyl fluoride), phosphatase inhibitors (0.1 mM sodium orthovanadate and 20 mM sodium
fluoride), 10% glycerol, and 1 mM dithiothreitol. The
solubilized proteins were centrifuged at 14,000 × g in
a microcentrifuge (4 °C) for 1 min, and the supernatants were stored
at Gelatin Zymography--
Serum-free conditioned medium from cells
at 80-90% confluence was prepared by washing two times with
phosphate-buffered saline, followed by incubation with serum-free MEM.
MMPs were concentrated from the serum-free conditioned medium by
incubation with 250 µl of gelatin-agarose beads (Sigma) overnight at
4 °C. The beads were then centrifuged at 10,000 × g
and eluted in electrophoresis sample buffer (final concentration of
2.25% SDS, 9% glycerol, and 45 mM Tris (pH 6.8), and
bromphenol blue) by incubation for 30 min at room temperature, followed
by centrifugation at 14,000 × g for 2 min. Equal
volumes of the eluate were then resolved on a 10% nondenaturing
SDS-polyacrylamide gel impregnated with 1 mg/ml gelatin (Sigma). MMP-2
and MMP-9 gelatinolytic activities were measured as described elsewhere
(15).
Sod2-dependent Regulation of MMP-1 Expression--
To
establish whether the expression of MMP-1 is regulated by
Sod2, we first measured the levels of MMP-1 in HT-1080
fibrosarcoma cell lines designed to overexpress Sod2
utilizing RT-PCR (10). Overexpression of Sod2 by 15-fold
resulted in a dramatic increase in both the basal expression of the
MMP-1 mRNA and the steady-state concentrations of
H2O2, both of which were reversed upon
coexpression of catalase in either the cytosolic or mitochondrial
compartment (Fig. 1, A and
B). The addition of O Regulation of MMP-1 by Mn-SOD Is IL-1 MMP Expression is Sod2-dependent--
To further
establish the linkage between Sod2 and the expression of
MMP-1, MEFs derived from Sod2-deficient mice
(CD1Sod2tm1Cje) were analyzed for the expression of
MMP-13 mRNA (a mouse functional analog of human MMP-1) in response
to numerous stimuli (20). As analyzed by Northern blotting, the
wild-type MEFs responded to the induction of MMP-13 by TNF, whereas the
heterozygotes did not. This response was also shown to be specific for
MMP-13 since the induction of IL-6 and IL-1 by TNF was unaffected by
the loss of Sod2 activity (Fig.
3C). This suggests that Mn-SOD
may be required for the TNF-mediated induction of MMP-13. To determine
if Mn-SOD was also involved in signaling the expression of MMP-13 by
other stimuli, Sod2+/+ and
Sod2 Sod2 Enhances MMP-1 Promoter Activity--
Control at the level of
transcription is a major factor determining MMP-1 expression, and a
single nucleotide polymorphism (1G/2G) at base pair Sod2 Signals MMP-1 Expression by Activating ERK1/2--
Both AP-1
and Ets transcription factors are regulated by MAPK signaling pathways
(21). The three most well characterized MAPK pathways include the
ERK1/2, JNK/SAPK, and p38 pathways. In general, ERK1/2 is activated by
mitogens and phorbol esters, whereas JNK/SAPK and p38 are mainly
stimulated by environmental stress and inflammatory cytokines. All
three pathways have been implicated in the expression of MMPs, and the
specific MAPK pathway that regulates MMP expression can vary depending
on the inducing agent and cell line. To test which of the MAPK pathways
are involved in the induction of MMP-1 by Sod2, control and
Sod2-overexpressing cells were treated with the specific
inhibitors of ERK1/2 (PD98059) and p38 (SB203580) (Fig.
4B, inset).
Northern blotting demonstrated that the ERK1/2 inhibitor blocked basal
MMP-1 expression and dramatically decreased MMP-1 mRNA levels in
the HT15 cell lines. The p38 inhibitor increased MMP-1 expression in
both control and HT15 cell lines. The ERK1/2 inhibitor also blocked the
induction of MMP-1 by TNF. The effect of the MAPK inhibitors on MMP-1
expression was also observed with the MMP-1 promoter constructs (Fig.
4B). Furthermore, the inductive properties of SB203580 could
be blocked by PD98059. It was thought possible that the ability of
PD98059 to inhibit MMP-1 expression may be a property of its ring
structure and potential antioxidant properties. To test this
hypothesis, an alternative ERK1/2 inhibitor structurally unrelated to
PD98059 was tested on MMP-1 promoter activity. The specific ERK1/2
inhibitor U0126 blocked the Sod2-dependent
increase in both 1G and 2G promoter activities, whereas its
non-inhibitory analog (U0124) had no comparable effect (Fig.
4C). The active forms of both ERK1/2 and p38 can be
distinguished from their inactive forms by phospho-specific antibodies.
Immunoblot analysis of extracts from control and HT15 cells showed an
increase in the activated forms of ERK1/2 in the Sod2-overexpressing cells that was reversed upon
coexpression of catalase (Fig.
5A). The constitutive
activation of ERK1/2 in the HT15 cell lines was also blocked by PD98059
(Fig. 5B). Interestingly, SB203580 activated ERK1/2 in both
the control and HT15 cell lines, which may explain its ability to
induce MMP-1 expression. The ability of SB203580 (20-50
µM) to activate ERK1/2 has also recently been reported in
erythroleukemic cell lines and was not observed when lower
concentrations of the drug were used (22). However, in the present
study, both low and high doses of SB203580 were capable of activating
MMP-1 promoter activity (Fig. 4D). The above findings
indicate that the Sod2-dependent generation of
H2O2 leads to the activation of ERK1/2 and are
in agreement with studies demonstrating that reactive oxygen species
can activate MAPK pathways (23).
The proximal AP-1 element located near position Under normal physiological conditions, the steady-state
concentrations of H2O2 are well within the
buffering capacity of the mitochondrial glutathione redox system.
However, when Sod2 levels increase, the
glutathione-buffering capacity of the mitochondria may be overwhelmed
by H2O2. We propose that it is this excess mitochondrial H2O2 that signals the downstream
events that lead to the expression of MMP-1 and possibly other MMP
family members. The expression of catalase in either the cytosolic or
mitochondrial compartment can block
Sod2-dependent H2O2
production and decrease MMP-1 expression (Fig. 1, A and
B). The inability of the endogenous catalase to effectively
detoxify the mitochondrially generated H2O2 may
be due to its restricted presence in the peroxisomal compartment. An
increase in H2O2 production leads to the
phosphorylation of ERK1/2 by a yet unknown mechanism, as well as the
subsequent activation of the MMP-1 promoter. This signaling cascade may
be utilized under a variety of conditions in which Sod2
levels are elevated. Sod2, IL-1, and MMP-1 expression are
elevated in aging systems both in vitro and in
vivo. The increased expression of collagenase may result from an
elevation in Sod2 levels in response to increasing levels of
IL-1 or other inflammatory cytokines. Increases in the levels of
H2O2 that result from the enhanced dismutase
activity in aged cells may explain the ability of antioxidant strategies to reduce the severity of UV-induced premature skin aging
(3) and to extend life span in experimental models of aging (24).
Reactive oxygen species have also long been implicated in mitogenic
signaling and cancer. Recently, the ability of estrogen derivatives to
selectively kill human leukemia cells has been attributed to the
specific inhibition of SOD activity (25). Several independent groups
have also reported that the elevated levels of Sod2 may
correlate with aggressiveness in gastric and colorectal adenocarcinomas
and are reflective of a poor overall survival of these patients
(26-28). The finding that Sod2 overexpression can
dramatically enhance MMP-1 promoter activity may explain the above
observations, as excessive MMP-1 production is a major contributor to
the stromal degradation involved in tumor invasion.
H2O2 has been implicated as one of the critical
signaling molecules leading to the expression of MMP-1 in dermal
fibroblasts (29). H2O2 activates AP-1, an
essential transcription factor required for the induction of MMP-1 (17,
29). The expression of MMP-1 is also effectively blocked by
antioxidants such as N-acetylcysteine, a precursor of
glutathione (7, 30), or by treatment with catalase (17). Agents that
decrease the H2O2-detoxifying capacity of the
cell such as buthionine sulfoximine (a glutathione synthesis inhibitor)
and aminotriazole (a catalase inhibitor) enhance basal MMP-1 production
(7). The retinoic acid analog tretinoin, which effectively blocks the
increase in expression of MMPs following low-dose UV irradiation (3),
also blocks AP-1 activation (31). The ability of tretinoin to
antagonize the process of photoinduced skin aging has been attributed
to its antioxidant properties. Recent studies in cultured renal
mesangial cells demonstrated that tretinoin protects from
H2O2-induced cytotoxicity by increasing both
cellular reduced glutathione and catalase levels (32). Thus,
H2O2 has been shown to be important in the
signaling events that lead to the expression of MMP-1. We (14) and
others (7, 33, 34) have shown that overexpression of Mn-SOD can
increase the levels of intracellular peroxides in a number of cell
lines. In addition, highly metastatic MCF-7 adriamycin-resistant breast cancer cells and gastric tumors both show high levels of MMP-1 (35, 36)
and Sod2 (37, 38) compared with their non-metastatic counterparts. Furthermore, many of the agents that induce the expression of MMP-1 also increase Mn-SOD levels. These agents include
TNF (39, 40), IL-1 (40, 41), UV irradiation (42, 43), phorbol esters
(44), and in vitro replicative senescence (45).
Sod2 is the only antioxidant enzyme that is modulated in
response to pathogenic stimuli, including lipopolysaccharides, growth factors, and cytokines (40); ionizing radiation (42, 43); phorbol
esters (44); and redox-cycling drugs and in vitro
replicative senescence (45). These findings indicate that cells have
evolved to utilize the mitochondria as a major source of the potent
intracellular signaling molecule H2O2 via
Sod2. The MMPs may represent just one family of genes whose
expression is tightly regulated by Sod2-derived H2O2.
1607 that creates an
Ets site adjacent to an AP-1 site at base pair
1602 and has
been shown to dramatically enhance transcription of the MMP-1 promoter.
Luciferase promoter constructs containing either the 1G or 2G variation
were 25- or 1000-fold more active when transiently transfected into
Sod2-overexpressing cell lines, respectively. The levels of
MMP-2, -3, and -7 were also increased in the
Sod2-overexpressing cell lines, suggesting that
Sod2 may function as a "global" redox regulator of MMP
expression. In addition, Sod2
/+
mouse embryonic fibroblasts failed to respond to the cytokine-mediated induction of the murine functional analog of MMP-1, MMP-13. This study
provides evidence that the modulation of Sod2 activity by a
wide array of pathogenic and inflammatory stimuli may be utilized by
the cell as a primary signaling mechanism leading to matrix metalloproteinase expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/+ mice is due to increased
collagen synthesis or degradation is not known. These observations have
led us to evaluate the importance of Sod2 in regulating the
expression of the collagen-degrading enzyme MMP-1.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Expression Vector--
A full-length 2.4-kilobase pair IL-1
sequence was PCR-amplified with NotI-XbaI ends
from an HT-1080 fibrosarcoma cell cDNA library. The resulting
product was inserted into the pCR2.1 TA cloning vector (Invitrogen). A
2.4-kilobase pair fragment containing the full-length IL-1
sequence
was then removed from the resulting plasmid by digestion with
NotI and XbaI. This fragment was ligated into
pRCMV, precut with NotI and XbaI. The
pRCMV/IL-1
construct was sequenced bidirectionally using the
Taq DyeDeoxy terminator cycle sequencing kit and an ABI
Model 310 DNA sequencer (Applied Biosystems, Inc.). Cells were
transfected with 1 µg of the plasmids and LipofectAMINE Plus reagent
(Life Technologies, Inc.) according to the instructions provided by the
manufacturer. HT-1080 cells were washed, and 0.8 ml of serum-free MEM
was added per well. The DNA-LipofectAMINE Plus complexes were added to
the medium and incubated at 37 °C. After 3 h, 0.8 ml of MEM
with 20% fetal calf serum was added, and the cells were grown
overnight. The next day, the cells were diluted 1:4 and transferred to
a 75-cm2 flask. After incubation overnight, selective
medium was added containing 10% fetal bovine serum, 1000 units/ml
penicillin, 500 µg/ml streptomycin, and 1 mg/ml neomycin; 10 days
later, resistant cells were harvested, and ~100 cells were plated in
a 75-cm2 flask. The cells were grown until visible colonies
were observed. Single colonies were transferred to 24-well
plates for further characterization. IL-1
immunoblot analysis was
performed using a rabbit anti-human IL-1
polyclonal antibody (R&D
Systems) according to the manufacturer's specifications.
-gal
using LipofectAMINE Plus reagent according to manufacturer's
instructions. The cells were lysed 18 h post-transfection, and the
luciferase reporter activity was determined using the Promega assay system.
80 °C. Extracted proteins were then quantified using the BCA
protein reagent (Pierce). 40 µg of denatured protein in 5%
2-mercaptoethanol was separated on a 10% polyacrylamide gel by
SDS-polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membranes (Bio-Rad) at 100 V for 1 h. The membranes
were then blocked for 2 h at room temperature in Tris-buffered
saline containing 0.1% Tween 20 and 5% nonfat dry milk. The blots
were then incubated overnight at 4 °C with primary antibody (mouse
anti-phospho-ERK1/2 monoclonal antibody, Santa Cruz Biotechnology) at
1:1000 in Tris-buffered saline containing 0.01% Tween 20. This was
followed by incubation with secondary antibody (biotinylated anti-mouse
IgG) at 1:50,000 for 2 h at room temperature. Detection of the
phosphorylated forms of the protein was performed by the addition of
the Vectastain EliteABC reagent, followed by Pierce
Supersignal chemiluminescent substrate and exposure to Kodak X-Omat
radiographic film. The immunoblot was then reprobed by incubation with
standard stripping buffer for 15 min at 60 °C, washed twice,
blocked, and then reprobed using rabbit polyclonal antibody (Promega)
that recognizes ERK1/2 regardless of its phosphorylation state at a
1:1000 dilution. The blot was incubated with biotinylated anti-rabbit
antibody (1:50,000 dilution), and the signal was detected as described above.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Redox modulation of MMP-1.
A, RT-PCR analysis of MMP-1 in control transfectants
(CMV and CMVmCAT), Sod2 overexpressors (HT15 = 15-fold
increase in Sod2 levels), and cells overexpressing catalase
in the cytosolic (CAT) or mitochondrial (mCAT)
compartment and Sod2. GAPDH was co-amplified in the linear
amplification range as a loading control. bp, base pair.
B, left panel, a representative FACScan analysis
of DCF fluorescence in untreated CMV, HT15, and HT15mCAT cell lines
(stippled trace) or DCF-treated CMV (gray trace),
HT15 (black trace), and HT15mCAT (shaded area)
cell lines; right panel, flow cytometric analysis of
intracellular peroxides using DCF fluorescence in CMV, HT15, and
HT15mCAT cell lines. The mean fluorescence of DCF was analyzed in a
FACScan. Values are in arbitrary fluorescence units and represent the
mean ± S.E. of four independent experiments for untreated and
DCF-treated cell lines. * and , p < 0.005 compared
with control and HT15, respectively.
-independent--
Previous
studies have demonstrated that IL-1
is a potent inducer of MMP-1 in
many biological systems (16) and is modulated by reactive oxygen
species (9, 17). Analysis of IL-1
levels in
Sod2-overexpressing cells showed a 3-fold increase in basal IL-1
levels that was reversed when catalase was coexpressed in these
cell lines (Fig. 2A). To
examine whether IL-1
was responsible for the
Sod2-dependent changes in MMP-1 gene expression,
Sod2-overexpressing cells were treated with a concentration
of IL-1 receptor antagonist (IL-1RA) that has previously been shown to
block the IL-1-mediated induction of MMP-1 (18). Neither acute nor
chronic treatment of the HT15 cell line with IL-1RA was effective in
decreasing the expression of MMP-1 (Fig. 2B). Furthermore,
in contrast to many other mesenchymal cells, HT-1080 fibrosarcoma cells
did not respond to IL-1
by increasing the expression of MMP-1 (Fig.
2C, upper panel). It is nevertheless possible
that intracellular IL-1
modulates the expression of MMP-1 since
intracellular pro-IL-1
is constitutively active (19). To address
this question, HT-1080 fibrosarcoma cells were transfected with an
IL-1
expression vector. Cloned transfectants with levels of IL-1
mRNA and protein that were equivalent to those observed upon
Sod2 overexpression showed no increase in basal MMP-1
expression (Fig. 2C, middle and lower panels). Thus, Sod2 overexpression alone is sufficient
to signal MMP-1 expression.
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Fig. 2.
Redox-dependent regulation of
MMP-1 is IL-1 -independent. A,
analysis of IL-1
levels using enzyme-linked immunosorbent assays as
previously described (9). Results are expressed as the
mean ± S.E. of two independent experiments. *,
p < 0.05 when compared with CMV and HT15,
respectively; ***, p < 0.001 when compared with CMV.
B, effect of IL-1RA on the spontaneous production of MMP-1
by Sod2 overexpressors. Cells were cultured in the presence
(+) or absence (
) of IL-1RA at the indicated doses for 24 or 72 h. The cells were then harvested for total RNA isolation, and Northern
analysis was performed using aliquots of 15 µg of total RNA in each
lane. MMP-1 mRNA levels was assessed by hybridization with a
full-length 32P-labeled MMP-1 probe. C, Northern
blot analysis of MMP-1 mRNA levels in control transfectants treated
with IL-1
(10 ng/ml) in either the presence or absence of IL-1RA
(500 ng/ml) (upper panel); parental (HT-1080) and
IL-1
-overexpressing cells (HTIL-1
) treated with TNF (10 ng/ml)
for 24 h and analysis of mRNA levels of IL-1
, MMP-1, and
GAPDH by Northern blotting (middle panel); IL-1
immunoblot analysis of control and Sod2- and
IL-1
-overexpressing cell lines (lower panel).
/+ MEFs were treated
with either IL-1 or phorbol 12-myristate 13-acetate. The induction of
MMP-13 by IL-1
and phorbol myristate acetate was severely impaired
in the Sod2
/+ fibroblasts compared
with the Sod2+/+ fibroblasts (Fig. 3,
A and B). To validate the genetic background of
the MEFs, RT-PCR analysis was performed to monitor Mn-SOD mRNA content (Fig. 3D). RT-PCR analysis produced the expected
size product in the Sod2+/+ MEFs, whereas the
Sod2
/+ MEFs produced two
products. The lower size band reflects the targeted disruption of exon
3 in one of the Mn-SOD alleles. Mn-SOD activity levels also reflect the
genetic background of each of the cell lines (data not shown).
Furthermore, Mn-SOD content had no effect on the induction of IL-6 by
both TNF and IL-1
or on the levels of the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (Fig. 3D). Our data
indicate that Mn-SOD plays an obligatory role in the induction of
interstitial collagenase and that this is specific for MMP-13, as the
induction of IL-1 and IL-6 in response to TNF was not altered by the
levels of Mn-SOD. Furthermore, MEFs respond to IL-1
by signaling
MMP-13 expression and, in such a setting, require Sod2.
Taken together, these findings indicate that Sod2 is
important for the IL-1-, TNF-, and phorbol 12-myristate
13-acetate-mediated induction of MMP-13.
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Fig. 3.
Sod2 is required for expression of
the mouse functional analog of human MMP-1, MMP-13. A,
confluent cultures of passage 6 cells derived from
Sod2 /+ and
Sod2+/+ mouse fibroblasts were treated with
IL-1
(10 ng/ml), TNF-
(30 ng/ml), or phorbol 12-myristate
13-acetate (PMA; 100 nM). Cells were lysed
24 h post-treatment, and total RNA was isolated and subjected to
Northern analysis. MMP-13 mRNA levels were assessed by
hybridization with a 32P-labeled MMP-13 probe for rat
collagenase. After autoradiography, the same membrane was stripped and
hybridized with a 32P-labeled actin probe. B,
quantitative evaluation of data from the Northern blot in A
after actin normalization was performed using a STORMTM
PhosphorImager (Molecular Dynamics). C, Northern
blot analysis of MMP-1, IL-6, IL-1
, and GAPDH from passage 4 Sod2-deficient fibroblasts treated for 18 h with or
without TNF (10 ng/ml) The values were obtained after normalizing to
-actin. D, RT-PCR analysis of Sod2 mRNA
levels in mouse embryonic fibroblasts. Sod2 and GAPDH
mRNA levels were measured in control and IL-1 (10 ng/ml)- and TNF
(30 ng/ml)-treated cells.
1607 that creates
an Ets site adjacent to an AP-1 site at base pair
1602 has been shown
to dramatically enhance transcription (13). Accordingly, luciferase
promoter constructs containing either the 1G or 2G variation were
transiently transfected in control and HT15 cell lines, and the effect
on basal transcription was analyzed. The activities of the 1G and 2G
promoter constructs in the HT15 cell line were increased ~20- and
1000-fold, respectively, compared with the control cell line (Fig.
4A). The increase in MMP-1 promoter activity of both
constructs by Sod2 overexpression was reversed by
coexpression of catalase, indicating an
H2O2-dependent regulation. An
increase in the 2G frequency in tumor cell lines has been postulated to
increase their invasive behavior. In such a setting, the level of
Sod2 may well be a determinant of the metastatic potential
of tumor cell lines by regulating the expression of MMP-1.
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Fig. 4.
Sod2 regulates MMP-1
transcriptional activity. A, transcriptional regulation of
MMP-1 by Sod2 overexpression. The indicated cell lines were
transiently transfected with the pGL3- MMP-1(1G) (inset)
or pGL3-MMP-1(2G) construct (13). The cells were lysed 18 h
post-transfection, and the luciferase reporter activity was determined
using the Promega assay system. B, effect of ERK1/2 MAPK and
p38 MAPK inhibitors on MMP-1 transcription.
pGL3-MMP-1(1G/2G)-transfected cells were treated with the ERK1/2
inhibitor PD98059 (PD; 50 µM) or the p38
inhibitor SB203580 (SB; 20 µM) (Calbiochem) or
both for 18 h. The cell lysates were then assayed for luciferase
activity. Inset, effect of PD98059 and SB203580 on MMP-1
mRNA expression. Cells were treated with PD98059 (50 µM) or SB203580 (20 µM) in either the
presence or absence of TNF (10 ng/ml). 15 µg of total RNA was then
analyzed by Northern blot hybridization. Blots were sequentially
hybridized with 32P-labeled cDNA probes encoding human
MMP-1 and GAPDH. C, the dual ERK1/2 inhibitor blocks the
Sod2-dependent increases in MMP-1 promoter
activity. Cell lines were transfected with the indicated
pGL3-MMP-1(1G/2G)-reporter constructs as described for
A and treated overnight with 20 µM U0126 or
U0124 (the inactive analog). D, SB203580 enhances MMP-1
promoter activity in a dose-dependent manner. CMV and HT15
cell lines were transfected as described for A and
treated with the indicated concentrations of SB203580. Luciferase
activity is expressed in cpm. Unless otherwise indicated, the values
represent -fold induction compared with induction of CMV
pGL3-MMP-1(1G)-transfected cells and represent the mean results from
3-12 individual experiments (n = 2-3 for each
individual experiment).
View larger version (54K):
[in a new window]
Fig. 5.
Sod2 regulates MMP-1 transcription
by activating ERK1/2. A, increased
phosphorylation of ERK1/2 (p42/p44) by Sod2 overexpression.
Cell lysates from confluent cells grown in complete medium (CMV, HT15,
and HT15mCAT), quiescent CMV cells in serum-free medium (CMV
sfm), and quiescent cells stimulated with fetal bovine serum
(20%) for 15 min (CMV+FBS) were analyzed for ERK1/2 by
Western blotting. B, spontaneous up-regulation of ERK1/2
(p42/p44) phosphorylation by SB203580. Control and
Sod2-overexpressing cells were treated for 24 h with
either PD98059 (PD; 50 µM) or SB203580
(SB; 20 µM). Cells were lysed and analyzed for
phosphorylated ERK1/2 as described under "Materials and Methods."
C, enhanced levels of MMP-2, -3, and -7 in Sod2
overexpressors. Left panel, gelatin zymography; right
panel, RT-PCR. D, Sod2 is activated by a
variety of stimuli, leading to an increase in the steady-state
production of H2O2.
H2O2 activates ERK1/2, leading to the
transcriptional activation of the MMP-1 promoter and subsequent
extracellular matrix (ECM) degradation.
70 plays a major role
in the transcriptional regulation of MMP-1 gene expression and is found
near this position in each of the inducible human MMP promoters (21).
Because of the conservation of the promoter regions between MMPs, it is
possible that other MMPs respond to Sod2 overexpression. In
support of this hypothesis, the gelatinolytic activity of MMP-2 and the
mRNA levels of MMP-3 and MMP-7 were increased in the
Sod2 overexpressors compared with control cell lines (Fig.
5C, left panel). The observed decline in
MMP-9 activity in the HT15 cell line may represent a negative
regulatory aspect of Sod2 in MMP expression. Thus,
Sod2 may regulate a broad spectrum of MMPs and function as a
"global" redox regulator of metalloproteinases.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Center for
Immunology and Microbial Disease, MC151, Albany Medical College, 47 New
Scotland Ave., Albany, NY 12208. Tel.: 518-262-8791; Fax: 518-262-6161;
E-mail: melenda@mail.amc.edu.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M100199200
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ABBREVIATIONS |
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The abbreviations used are: MMPs, matrix metalloproteinases; ERK, extracellular signal-regulated kinase; MEM, modified Eagle's medium; Mn-SOD, manganese-superoxide dismutase; MEFs, mouse embryonic fibroblasts; TNF, tumor necrosis factor; IL-1, interleukin-1; IL-1RA, interleukin-1 receptor antagonist; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DCF, dichlorofluorescein; CMV, cytomegalovirus; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase.
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