p53 Down-regulates Human Matrix Metalloproteinase-1
(Collagenase-1) Gene Expression*
Yubo
Sun
,
Yi
Sun§,
Leonor
Wenger,
Joni L.
Rutter¶,
Constance E.
Brinckerhoff
, and
Herman S.
Cheung
**
From the
Geriatric Research, Education, and Clinical
Center, Veterans Administration Medical Center, Miami, Florida
33101, the ** Department of Medicine, University of Miami
School of Medicine, Miami, Florida 33101, § Department of
Molecular Biology, Parke-Davis Pharmaceutical Research, Warner-Lambert
Company, Ann Arbor, Michigan 48105, and the Departments of
¶ Pharmacology/Toxicology and
Medicine, Dartmouth Medical
School, Hanover, New Hampshire 03755
 |
ABSTRACT |
Recent studies show that the p53
tumor suppressor protein is overexpressed in rheumatoid arthritis (RA)
synovium and that somatic mutations previously identified in human
tumors are present in RA synovium (Firestein, G. S., Echeverri,
F., Yeo, M., Zvaifler, N. J., and Green, D. R. (1997)
Proc. Natl. Acad. Sci. U. S. A. 94, 10895-10900;
Firestein, G. S., Nguyen, K., Aupperle, K. R., Yeo, M., Boyle, D. L.,
and Zvaifler, N. J. (1996) Am. J. Pathol. 149, 2143-2151;
Reme, T., Travaglio, A., Gueydon, E., Adla, L., Jorgensen, C., and
Sany, J. (1998) Clin. Exp. Immunol. 111, 353-3581). We
hypothesize that the abnormality of p53 seen in RA synovium may
contribute to joint degeneration through the regulation of human matrix
metalloproteinase-1 (hMMP-1, collagenase-1) gene expression.
Transcription assays were performed with luciferase reporters driven by
the promoter of the hMMP-1 gene or by a minimal promoter containing
tandem repeats of the consensus binding sequence for activator
protein-1, cotransfected with p53-expressing plasmids. The results
revealed that (i) wild-type (wt) p53 down-regulated the promoter
activity of hMMP-1 in a dose-dependent fashion; (ii) four
of six p53 mutants (commonly found in human cancers) lost this
repression activity; and (iii) this p53 repression activity was
mediated at least in part by the activator protein-1 sites found in the
hMMP-1 promoter. These findings were further confirmed by Northern
analysis. The down-regulation of hMMP-1 gene expression by endogenous
wt-p53 was shown by treatment of U2-OS cells, a wt-p53-containing
osteogenic sarcoma line, and Saos-2 cells, a p53-negative osteogenic
sarcoma line, with etoposide, a potent inducer of p53 expression. p53,
activated by etoposide, appears to block hMMP-1 promoter activity
induced by etoposide in U2-OS cells. In summary, we have shown for the
first time that the hMMP-1 gene is a p53 target gene, subject to p53
repression. Because MMP-1 is principally responsible for the
irreversible destruction of collagen in articular tissue in RA,
abnormality of p53 may contribute to joint degeneration through the
regulation of MMP-1 expression.
 |
INTRODUCTION |
Rheumatoid arthritis
(RA)1 is marked by
destruction of the extracellular matrix and it is believed that, among
other factors, matrix metalloproteinases (MMPs) play an important role
in mediating the degradation of connective tissue matrix components
such as collagens and proteoglycans (4, 5). Collagenase-1 (MMP-1), stromelysin (MMP-3), gelatinase A and B (MMP-2 and MMP-9), and collagenase-3 (MMP-13) are all present at significantly elevated levels
in cartilage, synovial membranes, and synovial fluid of patients with
RA (6-8). The synovium produces substantial amounts of MMP-1, the
major matrix metalloproteinase involved in the degradation of
interstitial collagens, specifically, types I-III. MMP-1 expression has been shown to be stimulated by native collagen type I and collagen
fragments, phorbol esters, growth factors, and cytokines such as
interleukin 1
(IL-1
) and tumor necrosis factor-
(9-12). The
activity of MMP-1 is stringently regulated at three levels: the
promoter, the activation of proenzyme, and the inhibition of active
enzyme. The activator protein-1 (AP-1) binding sites found in the
promoters of human collagenase have been shown to be critical to the
expression of human collagenase (13-16).
The protein product of the p53 tumor suppressor gene plays a
very important role in cell growth control, DNA repair, and apoptosis (17). It has been proposed that p53 acts as an "emergency brake" inducing G1 arrest and apoptosis after DNA damage, either by halting cell division until the damage is fully repaired or by eliminating the
cells with DNA that is irreparably damaged (18-20). Mutational inactivation of p53 is the most frequent genetic alteration in human
cancers, indicating that p53 plays an important role in human
carcinogenesis. p53 mutants often lose wild-type p53 (wt-p53) activity,
and of these, some gain oncogenic activity to promote cellular
transformation (21-23).
p53 is a transcription factor that recognizes a specific consensus DNA
sequence consisting of two copies of a 10-bp motif, 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3', separated by 0-13 bp. Wild-type p53
(but not mutants) efficiently binds to this sequence and transactivates expression of the target genes (24-28). p53 can also repress a wide
variety of cellular and viral promoters that lack p53 binding sites,
including c-fos, bcl-2 and insulin-like growth
factor I receptor (29-34). Furthermore, p53 can bind to the
TATA-binding protein and repress promoter activity (35, 36). p53 has
also been demonstrated to interact with other transcription factors: Sp1 (37), CCAAT-binding factor (38), cAMP response element-binding protein (39), and glucocorticoid receptors (40). Taken together these
observations strongly imply that p53 interacts directly with the
transcription machinery to modulate gene expression. Although wt-p53
can exert its repressive activity on many different genes, most mutants
of p53 have lost this repression activity. For example, the mutant
p53-143A fails to repress the promoter activities of c-fos
(32), Rb (41), and proliferating cell nuclear antigen (33). Mutant
p53-175H is unable to repress the promoter activity of hsp 70 (38) and
fails to bind to the TATA-binding protein to repress transcription from
a minimal promoter (36).
In an earlier study, Firestein et al. (2) reported that the
p53 tumor suppressor protein is overexpressed in RA synovium and
fibroblast-like synoviocytes. Subsequent studies showed that mutant p53
transcripts previously identified in human tumors are present in RA
synovium and fibroblast-like synoviocytes (1, 3). We, therefore,
examined whether p53 inactivation could play a role in the
overexpression of hMMP-1 seen in RA by testing the hypothesis that the
gene transcription of hMMP-1 is subject to wt-p53 regulation. We report
here that, indeed, wt-p53 inhibited hMMP-1 gene expression in a
dose-dependent manner, and that four of six p53 mutants
(commonly found in human cancers) lost most of this repressive
activity. These data provide evidence that p53 is involved in
regulating hMMP-1 gene transcription and that extracellular matrix
degradation attributable to high-level MMP-1 in RA and in certain human
tumors may be, at least partially, related to p53 inactivation.
 |
MATERIALS AND METHODS |
Recombinant human IL-1
was obtained from R & D Systems
(Minneapolis, MN). Phorbol 12-myristate 13-acetate (PMA) was from Calbiochem (San Diego, CA). Dulbecco's modified Eagle's medium (DMEM), OPTIMEM I medium, McCoy's 5A medium, fetal bovine serum (FBS),
stock antibiotic-antimycotic mixture (10,000 units/ml penicillin base,
10,000 µg/ml streptomycin base, and 25 µg/ml Fungizone) were
products of Life Technologies, Inc. Etoposide was purchased from Sigma.
All other chemicals were from Sigma.
DNA Plasmids and Probes--
The hMMP1 promoter/luciferase
reporter plasmids (
4327hMMP1luci,
3400hMMP1luci,
2900hMMP1luci,
1600hMMP1luci, and
512hMMP1luci) used in this study contain the
firefly luciferase gene under the transcriptional control of the hMMP1
promoter and have been described previously (42). The
4327hMMP1SEAP2
repeater plasmid was constructed by subcloning the
4327hMMP1 promoter
into the pSEAP2-Basic Vector (CLONTECH). wt-p53 and
mutant p53-280T (Arg to Thr at amino acid 280) constructs were
generated by reverse transcription polymerase chain reaction (43).
Other p53 mutant constructs encoding five p53 mutants commonly seen in
human cancers were obtained from Dr. A. J. Levine (Princeton
University, Princeton, NJ). They are p53-143A (Val to Ala at amino acid
143), p53-175H (Arg to His at amino acid 175), p53-248W (Arg to Trp at
amino acid 248), p53-273H (Arg to His at amino acid 273), and p53-281G
(Asp to Gly at amino acid 281). The reporter plasmids pAP1-luci,
pSRF-luci, and p53-luci, which contain the luciferase reporter gene
driven by a basic promoter element (TATA box) plus tandem repeats of
the consensus binding sequence for corresponding transcriptional
factors, were obtained from Stratagene (La Jolla, CA). The hMMP-1
(collagenase-1) probe was a 2.05-kb HindIII-SmaI
insert of pCllase 1 plasmid from the American Type Culture Collection
(Rockville, MD). Glyceraldehyde-3-phosphate dehydrogenase was an 800-bp
HindIII insert from a pBS-glyceraldehyde-3-phosphate dehydrogenase plasmid that contains a sequence encoding part of the
mouse glyceraldehyde-3-phosphate dehydrogenase cDNA.
Cell Culture--
Human foreskin fibroblast (HFF) cultures were
established from explants and transferred as described previously (44).
They were grown and maintained in DMEM supplemented with 10% FBS
containing 1% penicillin (50 µg/ml) and streptomycin (50 µg/ml).
All cultures used were third or fourth passage cells. Saos-2 and U2-OS
(osteogenic sarcoma line) cells were propagated in McCoy's medium
supplemented with 15% FBS.
DNA Transient Transfection and Luciferase/SEAP
Assay--
Transfections were performed using the LipofectAMINE
reagent (Life Technologies) following the manufacturer's instructions. Exponentially growing cells were plated at a density of 5 × 105/well in six-well cluster plates (Costar, Cambridge, MA)
in 2 ml of DMEM and 10%FBS (HFF) or in 2 ml of McCoy's and 15%FBS
(Saos-2 and U2-OS) and grown until 80% confluent. (24-36 h). The
cells were then washed, placed in fresh OPTIMEM I medium, and
cotransfected with varying amounts of p53-expressing plasmid and 1.5 µg of hMMP1luci reporter plasmid/or 0.8 µg
4327hMMP1SEAP2
reporter plasmid using 5-7 µl of LipofectAMINE reagent/well. The
parent vector (pCMV) without the p53 coding sequence was used as
control and to maintain the total DNA transfected constant. After
18 h, the cells were gently washed with DMEM, subsequently
incubated with fresh DMEM containing 0.5% serum with or without 5 ng/ml IL-1
(HFF), fresh McCoy's medium with or without PMA at a
final concentration of 200 nM (Saos-2), or fresh McCoy's
medium containing 8% serum with or without 10 µM
etoposide (Saos-2 and U2-OS). The cells were harvested 24 h later
by lysis with reporter lysis buffer (Promega, Madison, WI), and
luciferase activity was determined on an ML 2250 microtiter plate
luminometer and reported as relative light units. For some experiments,
the medium was withdrawn and assayed for SEAP activity following the
manufacturer's instructions (CLONTECH chemiluminescent assay), cells were harvested, and extracts were assayed for luciferase activity. Three transfections, each run in
triplicate, were performed. The luciferase activities were normalized
relative to the amount of protein in the lysates as determined by using
a protein assay kit from Pierce. We were unable to normalize to
-galactosidase activities, because p53 represses the traditional
viral promoters that drive expression of these reporters. However, in
some experiments 0.3-0.5 µg of pCMV-
-galactosidase (Stratagene)
was also included and stained with 1 mg/ml 5-bromo-4-chloro-3-indoyl
-D-galactoside, 2 mM MgCl2, 5 mM K3Fe(CN)6, and 5 mM
K4Fe(CN)6 in phosphate-buffered saline.
Routinely, 5-10% of cells took up enough of the
pCMV-
-galactosidase plasmid to stain an intense blue (when the
amount of pCMV-
-galactosidase plasmid cotransfected increased, the
percentage of cells stained blue increased). We did not normalize
reporter activities against the average percentage of cells stained
blue (it gives results similar to normalizing to total protein; data
not shown). Instead, we presented data as the average of the reporter
activities normalized to total protein and the reporter activities
corrected with the staining results, because the differences in the
staining results were, at least partially, attributable to the
detection limit of the 5-bromo-4-chloro-3-indoyl
-D-galactoside direct staining method. The
5-bromo-4-chloro-3-indoyl
-D-galactoside staining
results may not be a true transfection efficiency indicator in this
specific situation although a better one than expression activity. We
have seen that the percentage of cells stained blue when cotransfected with wild-type p53 expression plasmid (10-200 ng) was ~10-30% less
than that cotransfected with parent vector, whereas under the same
conditions the
4327hMMP1 promoter activity was repressed ~70-90%.
Data were expressed as means ± S.E., and the repression of the
promoter activity of hMMP-1 by wt-p53 or mutant p53 was calculated
based on the maximum level of promoter activity of hMMP-1 in the
presence of pCMV.
RNA Isolation and Northern Analysis--
Saos-2 cells in 100-mm
plates were transiently transfected with 24 µg of wt-p53- or mutant
p53-175H-expressing plasmid. After 18 h, fresh McCoy's medium
containing 0.5% serum or 8% serum was added. The cells were harvested
24 h later, and total cellular RNA was isolated using RNAzol
reagent (Tel-Test, Inc., Friendswood, TX) and quantified by optical
density. The total RNA samples (25 µg) were subjected to Northern
blot analysis. After electrophoresis in 4-morpholinepropanesulfonic
acid electrophoresis buffer, the RNA was transferred in 10 × SSC
buffer by capillary action using a sponge. The RNA was fixed to the
nylon membrane by ultraviolet light exposure with a Stratagene UV
linker. The membrane was prehybridized 2-4 h in 5 × SSPE, 5 × Denhard's reagent, 0.5% SDS, 100 µg/ml denatured salmon sperm
carrier DNA (Life Technologies), and 50% formamide. The filter was
hybridized with random-primed 32P-labeled probes (Life
Technologies). After overnight hybridization at 42 °C in 6 × SSPE, 0.5% SDS, 100 µg/ml denatured salmon sperm carrier DNA, and
50% formamide, the membrane was washed twice in 1 × SSC with
0.1% SDS at room temperature and then washed twice at 55 °C using
0.1 × SSC and 0.1% SDS. The filters were autoradiographed with
Kodak X-OMAT-AR film for 24-72 h at
70 °C. The signal obtained from the Northern blots was normalized to the signal for
glyceraldehyde-3-phosphate dehydrogenase to correct for slight
differences in RNA loading.
 |
RESULTS |
Suppression of hMMP-1 Transcription by the wt-p53 Protein--
To
analyze the effect of wt-p53 protein on the hMMP-1 gene expression, HFF
cells harboring endogenous wt-p53 were transfected with
4327hMMP1luci
reporter plasmid (1.5 µg), a luciferase reporter driven by a 4327-bp
fragment of the hMMP-1 promoter, together with varying amounts of
pCMV-p53. The total amount of DNA transfected was kept constant with
pCMV. As shown in Fig. 1, expression of wt-p53 down-regulated the hMMP-1 promoter activity in a
dose-dependent manner (blank bars). To rule out
the possibility that this transcriptional regulation was resulting from
the interference of the p53-expressing plasmid by endogenous p53, we
also used the human osteosarcoma cell line Saos-2 in the transient
transfection assays. Saos-2 cells have no endogenous p53 (45).
Transcription from the
4327hMMP1 promoter was repressed by wt-p53 in
Saos-2 cells in the same manner as seen in the HFF cells (Fig. 1). We
observed up to a 20-fold reduction in SEAP activity and up to an 8-fold
increase in luciferase activity (p53-luci) when cells were
cotransfected with 0.2 µg of wt-p53 expression plasmid (Fig. 1,
filled bars).

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Fig. 1.
Dose-dependent repression of
hMMP-1 promoter activity by wild-type p53. HFF cells (triplicate)
were cotransfected with varying amounts of the wild-type p53 expression
plasmid pCMVp53 and 1.5 µg of the 4327hMMP1luci reporter plasmid.
Saos-2 cells (six wells) were cotransfected with varying amounts of the
wild-type p53 expression plasmid pCMVp53 together with 4327hMMP1SEAP2
(0.8 µg), p53-luci (0.4 µg), and pCMV- -galactosidase plasmid
using LipofectAMINE in OPTIMEM I medium for 18 h. Cells were then
washed with PBS, and fresh DMEM (for HFF) or OPTIMEM I (for Saos-2)
medium containing 4% serum was added. After 24 h, HFF cells were
harvested and luciferase activity was assayed. For Saos-2 cells, the
medium was withdrawn and assayed for SEAP activity; half of the cells
(triplicate) were then harvested, and protein extracts were assayed for
luciferase activity, and the other half of the cells were subjected to
direct staining as detailed under "Materials and Methods." The
parent vector without p53 coding insert was used to keep total plasmid
transfected constant. It could be seen that with the increase of p53
expression plasmid transfected, reporter activity of p53-luci
increased, but promoter activity of 4327hMMP1 decreased in a
dose-dependent manner. The average percentage of cells
stained blue stayed within the range of 6.6-9%. RLU,
relative light unit.
|
|
wt-p53 Reduced the Activities of hMMP-1 Promoter Induced by PMA and
IL-1
--
PMA and IL-1
are known to induce hMMP-1 gene
expression (16, 46). To examine whether p53 can inhibit the PMA- or
IL-1
-induced hMMP-1 gene transcription, Saos-2 or HFF cells were
transfected with
4327pMMP1luci (1.5 µg) together with 100 ng of
pCMVp53 or pCMV. As shown in Fig. 2, p53
is a strong repressor of the hMMP1 gene at both basal and induced
levels. Co-transfection of the wt-p53 expression plasmid abolished a
7-fold PMA-induced promoter activity in Saos-2 cells (Fig. 2,
top) and a 3-fold IL-1
-induced promoter activity in HFF
cells (Fig. 2, bottom).

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Fig. 2.
Repression of both basal and induced hMMP1
promoter activity by wt-p53. Saos-2 (top) or HFF
(bottom) cells were transfected with 4372phMMP1luci (1.5 µg) together with 100 ng of pCMVp53 or pCMV as control. Half of the
cells were subsequently either stimulated with PMA at a final
concentration of 200 nM (top) or treated with
IL-1 at a concentration of 5 ng/ml (bottom) for 24 h; the other half were incubated in fresh medium without serum as
control. Cells were harvested, and protein extracts were assayed for
luciferase activity. Three independent transfections, each run in
triplicate, were performed, and the results are expressed as the
means ± S.E.M. RLU, relative light unit.
|
|
p53 Mutants either Lost Most or Only Retained Part of This
Repression Activity on the hMMP-1 Promoter--
We next examined
whether p53 mutants (commonly found in human cancer) have lost their
ability to repress the luciferase reporter activity driven by the hMMP1
promoter. As shown in Fig. 3, wt-p53 (100 ng) induced ~20-fold repression of luciferase activity. In contrast,
cotransfection of
4327phMMP1luci with the mutant p53 constructs (100 ng) resulted in either minor repression or reduced repression activity.
Of the six mutants tested, four mutants, p53-143A, p53-175H, p53-280T,
and p53-281G, lost this repression activity almost completely, and the
mutant p53-273H lost approximately one-third of this repression
activity compared with wt-p53. The remaining mutant, p53-248W, retained
most of this repression activity.

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Fig. 3.
Loss of hMMP1 repression by some p53 mutants
commonly found in human cancers. One set of Saos-2 cells was
cotransfected with 4372phMMP1luci (1.2 µg) and
pCMV- -galactosidase (0.3 µg) together with 100 ng of wt-p53- or
p53 mutant-expressing plasmids. The other set was cotransfected with
4372phMMP1luci (1.2 µg) and pCMV- -galactosidase (0.3 µg)
together with 100 ng of pCMV as control. Transfected cells were
subsequently incubated with fresh medium containing 8% serum for
24 h. Half of the cells were lysed, and protein extracts were
assayed for luciferase activity (triplicate), and the other half were
stained for -galactosidase as detailed under "Materials and
Methods." The average percentage of cells stained blue was within the
range of 5-6.1% for all transfections. Relative promoter activity was
calculated by arbitrarily setting the activity of the control
(cotransfected with the parent vector) as 100.
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|
Repression of Endogenous hMMP-1 Messenger by wt-p53--
We
examined the hMMP-1 mRNA levels after transfection of the Saos-2
cells with p53-expressing plasmid. Northern blot (Fig. 4) showed that the hMMP-1 message was
detectable at 0.5% serum (lane 1) and induced by 8% serum
(lane 2). Significantly, serum-induced hMMP-1 expression was
completely suppressed by wt-p53 (Fig. 4, lane 3) but not by
p53 mutant pCMV-175H (Fig. 4, lane 4).

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Fig. 4.
Repression of endogenous hMMP-1 messenger by
wt-p53. Total RNA was isolated from Saos-2 cells after p53
transfection and subjected to Northern analysis. Northern blot showed
that hMMP-1 messenger was repressed by wt-p53. Lane 1,
Saos-2 cells were transfected with pCMV (the parent vector plasmid) and
incubated in McCoy's medium containing 0.5% serum. Lane 2,
pCMV-transfected cells were incubated in McCoy's medium containing 8%
serum. Lane 3, wt-p53-expressing plasmid-transfected cells
incubated in McCoy's medium containing 8% serum. Lane 4,
mutant p53-175H-transfected cells incubated in McCoy's medium
containing 8% serum.
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|
Endogenous wt-p53 Inhibited the Promoter Activity of
hMMP-1--
The repression of hMMP-1 promoter activity by ectopic
expression of wt-p53 suggests that one of the functions of endogenous p53 is to inhibit or modulate hMMP-1 promoter activity. To test whether
the p53 repression of hMMP-1 is a physiologically relevant response,
the ability of induced endogenous p53 to alter the hMMP-1 promoter
activity was examined by the treatment of cells with etoposide, a
topoisomerase II inhibitor and a known potent p53 inducer (47). We
reasoned that if hMMP-1 is a p53 target gene for repression, the hMMP-1
promoter activity should be reduced after p53 induction in cells
containing wild-type p53 but not in p53-negative cells. The optimal
time of induction of endogenous p53 in U2-OS cells by etoposide has
been shown to be at 24 h (47). As shown in Fig.
5, etoposide induced a 5-fold induction
of the luciferase activity of
512hMMP1luci reporter construct in
Saos-2 cells but not in U2-OS cells. The results suggested that
etoposide can induce hMMP-1 expression via signal pathway(s)
independent of p53, and this induction appeared to be repressed by the
endogenous wt-p53 harboring in the U2-OS cells.

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Fig. 5.
Blockage of etoposide-induced activation of
the hMMP-1 promoter by endogenous wt-p53. U2-OS cells, a human
osteogenic sarcoma line harboring endogenous wt-p53, and Saos-2 cells,
a human p53-negative osteogenic sarcoma line, were transiently
transfected with 512phMMP1luci. After the transfection, half of the
cells were treated with etoposide (10 µM), an anticancer
drug and a known p53 inducer, in McCoy's medium containing 8% serum
for 24 h; the untreated other half (in the same medium without
etoposide) were used as control. The means ± S.E.M. were derived
from three independent transfections and assays, each run in
triplicate. Relative promoter activity was calculated by arbitrarily
setting the activity of the control as 100.
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The
512/+67 Fragment Is Sufficient to Mediate p53 Repression
Activity--
To map the site(s) responsible for p53-mediated
repression, various 5' deletions of hMMP1luci plasmids were transiently
cotransfected into Saos-2 cells with wt-p53 expression plasmid. The
hMMP-1 promoter activity of all deletion fragments was repressed
dramatically by wt-p53, resulting in ~90% repression compared with
the control (Fig. 6). The results
indicated that at least one of the major cis-acting elements
involved in the down-regulation of hMMP-1 gene expression resides
within the
512/+67 of the 5' flanking region of the hMMP-1 gene.

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Fig. 6.
Mapping of the minimal promoter sequence
required for p53 repression. Saos-2 cells were transiently
transfected with a series of 5' deletion hMMP1/luciferase reporter
plasmids ( 4372phMMP1luci, 3400phMMP1luci, 2900phMMP1luci,
1600phMMP1luci, and 512phMMP1luci) together with 100 ng of
pCMV-wt-p53 plasmid or pCMV as control. Transfected cells were
subsequently incubated with McCoy's medium containing 8% serum for
24 h. Cells were harvested, and protein extract was assayed for
luciferase activity. The means ± S.E.M. were derived from three
independent transfections and assays, each run in triplicate.
RLU, relative light unit.
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AP-1 Binding Site Is Responsible, at Least in Part, for p53
Repression--
The AP-1 sites (
77 and
181) within the
512/+57
fragment in the hMMP-1 promoter are crucial to both the basal and PMA-
or IL-1
-induced promoter activity of hMMP-1 (13, 15, 16). To test
whether the p53 repression activity could be mediated through AP-1
sites, Saos-2 cells were cotransfected with pAP1-luci or pSRF-luci
together with 100 ng of wt-p53 expression plasmid or pCMV as control.
As shown in Fig. 7, the luciferase
activity of pAP1-luci reporter, but not pSRF-luci reporter, was
repressed substantially by wt-p53. This result indicated that the p53
repression activity on hMMP-1 promoter could be mediated through the
AP-1 sites found in the hMMP-1 promoter and suggested a potential
interaction or cross-talk between AP-1 and p53 on the regulation of
hMMP-1 expression.

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Fig. 7.
AP-1 as a mediator of p53 repression
activity. Saos-2 cells were cotransfected with pAP1-luci or
pSRF-luci together with 100 ng of wt-p53 expression plasmid or pCMV as
control. The cells were harvested 24 h later, and protein extracts
were assayed for luciferase activity. The means ± S.E.M. means
were derived from two independent transfections and assays, each run in
triplicate. RLU, relative light unit.
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|
 |
DISCUSSION |
Overexpression of MMPs has been implicated in a number of
diseases, including arthritis (11, 46, 48) and tumor invasion and
metastasis (49, 50). In RA, a large quantity of MMP-1 produced by the
synovium is mainly responsible for the irreversible degradation of
collagen in the articular joint tissue (6, 12). Similarly, destruction
of the interstitial collagen is also an integral part of tumor invasion
and metastasis (49, 50). Despite the extensive studies on the
regulation of hMMP-1 gene expression, little is known about negative
regulators, which may, when inactivated, contribute to the elevated
level of hMMP-1 gene expression in RA or cancer cells. Here we have
tested the hypothesis that abnormality of p53 in RA or cancers may
contribute to the joint degeneration seen in RA or tumor invasion and
metastasis through the modulation of hMMP-1 expression. Our results
strongly suggest that hMMP-1 is a p53 target gene subject to p53
repression. Overexpression of human wt-p53 can exert a strong
inhibitory effect on human hMMP-1 gene expression, but mutation of p53
at codons 143, 175, 280, and 281 abrogates this inhibition substantially.
It is interesting to observe that p53 repression of the hMMP-1 promoter
was mediated at least in part by AP-1. Because the AP-1 sites at
72
and
181 of the hMMP-1 promoter play a prominent role for the basal
and the PMA- and IL-1
-induced promoter activity of hMMP-1 (11, 15,
16, 51), and because wt-p53 can repress c-fos gene
expression (32), it is possible that the repression activity of wt-p53
on the hMMP-1 promoter could be mediated partially through the AP-1
sites found in hMMP-1 promoter (the
72 and
181 AP-1 sites). wt-p53
protein could decrease the binding of the Fos and Jun families of
transcription factors to the
72 and
181 AP-1 sites by repression of
c-fos expression, and in consequence, suppress the promoter
activity of hMMP-1 gene. The fact that the hMMP-1 promoter lacks the
p53 binding site indicates that p53-induced hMMP-1 repression is
mediated by a p53 binding site-independent mechanism, consistent with
the findings in other known p53-repressed genes (29-34). It will be of
great interest to elucidate the mechanism of potential cross-talk
between p53 (negative) and Ap-1 (positive) in the regulation of hMMP-1 expression.
Two lines of evidence suggest that p53 repression of hMMP-1 is a
physiologically relevant response. First of all, serum-stimulated expression of endogenous hMMP-1 is completely repressed by wt but not
mutant p53. Second, hMMP1luci reporter activities were stimulated by
etoposide significantly only in p53-negative Saos-2 cells but not in
p53-positive U2-OS cells (Fig. 5). Because etoposide induces both AP-1
and NF
b (52), two positive regulators of hMMP-1 expression, it is
not surprising to see hMMP-1 induction by etoposide in Saos-2 cells.
Lack of hMMP-1 induction by etoposide in U2-OS cells, however, implies
that etoposide-activated p53 executes a suppressive activity, thus
diminishing the Ap-1/NF
b effect (11, 13, 15, 16).
During the preparation of this manuscript, Aupperle et al.
(53) reported that the growth rate, invasiveness, and resistance to
apoptosis induced by reactive oxygen species in rheumatoid and normal
fibroblast-like synoviacyte increased significantly after endogenous
p53 protein was inactivated with virus 18 E6 protein. However, no
effect on collagenase mRNA accumulation was observed. One possible
explanation for the discrepancy between these findings and our data
could be attributable to the fact that the wt-p53 protein level is too
low in nontransformed fibroblast-like synoviocytes to induce a
detectable change in collagenase mRNA level. In addition, although
E6 protein can bind to p53 protein to interfere with the
transcriptional function of wt-p53, E6 could possess other pertinent
activities. For instance, staurosporine-mediated apoptosis was reduced
by E6 in p53 knockout mice (54), suggesting that some effects of E6 are
independent of p53 status.
Although the biological significance in vivo of the
suppression of hMMP1 transcription by p53 is not clear at the present time, a strong association of overexpression of MMP-1 and p53 inactivation in RA warrants extensive further investigation. The finding that hMMP-1 is subject to p53 repression, as reported here,
provides the first linkage between p53, a powerful tumor suppressor,
and MMP-1, a matrix-degrading enzyme known to promote joint
degeneration in RA and tumor invasion and metastasis. Inactivation of
p53 found in RA and cancers, therefore, could be causally related to
the genesis of these two diseases.
 |
ACKNOWLEDGEMENTS |
We thank Dr. A. J. Levine (Princeton
University) for the generous gifts of the tumor-derived p53
mutant-expressing constructs. We also thank Drs. D. S. Howell,
L. M. Ryan, and J. F. Woessner, Jr., for critical reading of
the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant AR38421 and a Veterans Administration merit review grant
(to H. S. C.), National Institutes of Health Grant AR08662-33 (to D. S. Howell), and National Institutes of Health Grant AR26599 and a grant from the RGK Foundation (to C. E. B.).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: Miami VA Medical
Center, GRECC, Rm. NHCU 207G, 1201 Northwest 16th St., Miami, FL 33125. Tel.: 305-243-5735; Fax: 305-243-5655; E-mail: hcheung{at}mednet.med.miami.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
RA, rheumatoid
arthritis;
MMPs, matrix metalloproteinases;
IL-1
, interleukin 1
;
PMA, phorbol 12-myristate 13-acetate;
hMMP1luci, human matrix
metalloproteinase-1 promoter/luciferase reporter plasmid;
hMMP1SEAP2, human MMP1 promoter/secreted form of human placental alkaline
phosphatase reporter plasmid;
HFF, human foreskin fibroblast;
AP-1, activator protein-1;
wt, wild-type;
DMEM, Dulbecco's modified Eagle's
medium;
FBS, fetal bovine serum;
CMV, cytomegalovirus.
 |
REFERENCES |
-
Firestein, G. S.,
Echeverri, F.,
Yeo, M.,
Zvaifler, N. J.,
and Green, D. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10895-10900[Abstract/Free Full Text]
-
Firestein, G. S.,
Nguyen, K.,
Aupperle, K. R.,
Yeo, M.,
Boyle, D. L.,
and Zvaifler, N. J.
(1996)
Am. J. Pathol.
149,
2143-2151[Abstract]
-
Reme, T.,
Travaglio, A.,
Gueydon, E.,
Adla, L.,
Jorgensen, C.,
and Sany, J.
(1998)
Clin. Exp. Immunol.
111,
353-358[CrossRef][Medline]
[Order article via Infotrieve]
-
Martel-Pelletier, J.,
McCollum, R.,
Fujimoto, N.,
Obata, K.,
Cloutier, J.-M.,
and Pelletier, J. P.
(1994)
Lab. Invest.
70,
807-815[Medline]
[Order article via Infotrieve]
-
Woessner, J. F., Jr.
(1991)
FASEB J.
5,
2145-2154[Abstract/Free Full Text]
-
Harris, D. E., Jr.
(1993)
in
Textbook of Rheumatology (Kelly, W. N., Harris, E. D., Jr., Ruddy, S., and Sledge, C. B., eds), pp. 833-873, W. B. Saunders, Philadelphia
-
Lindy, O.,
Konttinen, Y. T.,
Sorsa, T.,
Ding, Y.,
Santavirta, S.,
Ceponis, A.,
and Lopez-Otin, C.
(1997)
Arthritis Rheum.
40,
1391-1399[Medline]
[Order article via Infotrieve]
-
Wernicke, D.,
Seyfert, C.,
Hinzmann, B.,
and Gromnica-Ihle, E.
(1996)
J. Rheumatol.
23,
590-595[Medline]
[Order article via Infotrieve]
-
Sudbeck, B. D.,
Pilcher, B. K.,
Welgus, H. G.,
and Parks, W. C.
(1997)
J. Biol. Chem.
272,
22103-22110[Abstract/Free Full Text]
-
Borden, P.,
and Heller, R. A.
(1997)
Crit. Rev. Eukaryotic Gene Expr.
7,
159-178[Medline]
[Order article via Infotrieve]
-
Vincenti, M. P.,
White, L. A.,
Schroen, D. J.,
Benbow, U.,
and Brinckerhoff, C. E.
(1996)
Crit. Rev. Eukaryotic Gene Expr.
6,
391-411[Medline]
[Order article via Infotrieve]
-
Harris, E. D., Jr.
(1990)
N. Engl. J. Med.
322,
1277-1289[Medline]
[Order article via Infotrieve]
-
Aho, S.,
Rouda, S.,
Kennedy, S. H.,
Qin, H.,
and Tan, E. M. L.
(1997)
Eur. J. Biochem.
247,
503-510[Abstract]
-
Pendas, A. M.,
Balbin, M.,
Liano, E.,
Jimenez, M. G.,
and Lopez-Otin, C.
(1997)
Genomics
40,
222-233[CrossRef][Medline]
[Order article via Infotrieve]
-
Mauviel, A.
(1993)
J. Cell. Bichem.
53,
288-295
-
Gutman, A.,
and Wasylk, B.
(1990)
EMBO J.
9,
2241-2246[Abstract]
-
Ko, L. J.,
and Prives, C.
(1996)
Genes Dev.
10,
1054-1072[CrossRef][Medline]
[Order article via Infotrieve]
-
Kastan, M. B.,
Canman, C. E.,
and Leonard, C. J.
(1995)
Cancer Metastasis Rev.
14,
3-15[Medline]
[Order article via Infotrieve]
-
Livingstone, L. R.,
White, A.,
Sprouse, J.,
Livanos, E.,
Jacks, T.,
and Tlsty, T. D.
(1992)
Cell
70,
923-935[Medline]
[Order article via Infotrieve]
-
Yin, Y.,
Tainsky, M. A.,
Bischoff, F. Z.,
Strong, L. C.,
and Wahl, G. M.
(1992)
Cell
70,
937-994[Medline]
[Order article via Infotrieve]
-
Caron de Fromentel, C.,
and Soussi, T.
(1992)
Genes Chromosomes Cancer
4,
1-15[Medline]
[Order article via Infotrieve]
-
Levine, A. J.,
Momand, J.,
and Finlay, C. A.
(1991)
Nature
351,
453-456[CrossRef][Medline]
[Order article via Infotrieve]
-
Hollstein, M.,
Sidransky, D.,
Vogelstein, B.,
and Harris, C. C.
(1991)
Science
253,
49-53[Medline]
[Order article via Infotrieve]
-
Miyashita, T.,
and Reed, J. C.
(1995)
Cell
80,
293-299[Medline]
[Order article via Infotrieve]
-
Okamoto, K.,
and Beach, D.
(1994)
EMBO J.
13,
4816-4822[Abstract]
-
el-Deiry, W. S.,
Tokino, T.,
Veculescu, V. E.,
Levy, D. B.,
Parsons, R.,
Trent, J.,
Lin, D.,
Mercer, W. E.,
Kinzler, K. W.,
and Vogelster, B.
(1993)
Cell
75,
817-825[Medline]
[Order article via Infotrieve]
-
El-Deiry, W. S.,
Kern, S.,
Pietenpol, J. A.,
Kinzler, K. W.,
and Vogelster, B.
(1992)
Nat. Genet.
1,
45-51[Medline]
[Order article via Infotrieve]
-
Kastan, M. B.,
Zhan, Q.,
el-Deiry, W. S.,
Carrier, F.,
Jacks, T.,
Walsh, W. V.,
Plunkett, B. S.,
Vogelstein, B.,
and Fornace, A. J., Jr.
(1992)
Cell
71,
587-597[Medline]
[Order article via Infotrieve]
-
Werner, H.,
Karnieli, E.,
Rauscher, F., III.,
and LeRoith, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8318-8323[Abstract/Free Full Text]
-
Miyashita, T.,
Harigai, M.,
Hanada, M.,
and Reed, J. C.
(1994)
Cancer Res.
54,
3131-3135[Abstract]
-
Donehower, L. A.,
and Bradley, A.
(1993)
Biochim. Biophys. Acta
1155,
181-205[CrossRef][Medline]
[Order article via Infotrieve]
-
Kley, N.,
Chung, R. Y.,
Fay, S.,
Loeffler, J. P.,
and Seizinger, B. R.
(1992)
Nucleic Acids Res.
20,
4083-4087[Abstract]
-
Subler, M. A.,
Martin, D. W.,
and Deb, S.
(1992)
J. Virol.
66,
4754-4762
-
Santhanam, U.,
Ray, A.,
and Sehgal, P. B.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
7605-7609[Abstract]
-
Liu, X.,
Miller, C. W.,
Koeffler, P. H.,
and Berk, A. J.
(1993)
Mol. Cell. Biol.
13,
3291-3300[Abstract]
-
Seto, E.,
Usheva, A.,
Zambetti, G. P.,
Momand, J.,
Hokoshi, N.,
Weinmann, R.,
Levine, A. J.,
and Shenk, T.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
12028-12032[Abstract]
-
Borellini, F.,
and Glazer, R. I.
(1993)
J. Biol. Chem.
268,
7923-7928[Abstract/Free Full Text]
-
Agoff, S. N.,
Hou, J.,
Linzer, D. I. H.,
and Wu, B.
(1993)
Science
259,
84-87[Medline]
[Order article via Infotrieve]
-
Desdouets, C.,
Ory, C.,
Matesic, G.,
Soussi, C. B.,
and Sobczak-Thepot, J.
(1996)
FEBS Lett.
385,
34-38[CrossRef][Medline]
[Order article via Infotrieve]
-
Maiyar, A. C.,
Phu, P. T.,
Huang, A. J.,
and Firestone, G. L.
(1997)
Mol. Endocrinol.
11,
312-328[Abstract/Free Full Text]
-
Shiio, Y.,
Yamamoto, T.,
and Yamaguchi, N.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
5206-5210[Abstract]
-
Rutter, J. L.,
Benbow, U.,
Coon, C. I.,
and Brinckerhoff, C. E.
(1997)
J. Cell. Biochem.
66,
1-15[CrossRef][Medline]
[Order article via Infotrieve]
-
Sun, Y.,
Dong, Z.,
Nakamura, K.,
and Colburn, N. H.
(1993)
FASEB J.
7,
944-950[Abstract/Free Full Text]
-
Cheung, H. S.
(1980)
J. Tissue Cult. Methods
6,
39-40
-
Masuda, H.,
Miller, C.,
Koeffler, H. P.,
Battifora, H.,
and Cline, M. J.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
7716-7719[Abstract]
-
Arend, W. P.,
and Dayer, J.-M.
(1993)
in
Textbook of Rheumatology (Kelly, W. N., Harris, E. D., Jr., Ruddy, S., and Sledge, C. B., eds), pp. 227-247, W. B. Saunders, Philadelphia
-
Bian, J.,
and Sun, Y. I.
(1997)
Mol. Cell. Biol.
17,
6330-6338[Abstract]
-
Brinckerhoff, C. E.
(1992)
Crit. Rev. Eukaryotic Gene Expr.
2,
145-164[Medline]
[Order article via Infotrieve]
-
MacDougall, J. R.,
and Matrisian, L. M.
(1995)
Cancer Metastasis Rev.
14,
351-362[Medline]
[Order article via Infotrieve]
-
Crawford, H. C.,
and Matrisian, L. M.
(1996)
Enzyme Protein
49,
20-37[Medline]
[Order article via Infotrieve]
-
Auble, D. T.,
and Brinckerhoff, C. E.
(1991)
Biochemistry
30,
4629-4635[Medline]
[Order article via Infotrieve]
-
Garcia-Bermejo, L.,
Perez, C.,
Vilaboa, N. E.,
Blas, E. D.,
and Aller, P.
(1998)
J. Cell Sci.
111,
637-644[Abstract/Free Full Text]
-
Aupperle, K. R.,
Boyle, M. H.,
Seftor, E. A.,
Zvaifler, N. J.,
Barbosa, M.,
and Firestein, G. S.
(1998)
Am. J. Pathol.
152,
1091-1097[Abstract]
-
Steller, M. A.,
Zou, Z.,
Shiller, J. T.,
and Baserga, R.
(1996)
Cancer Res.
56,
5087-5097[Abstract]
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