Tài liệu Báo cáo khoa học: Fra-1 targets the AP-1 site/2G single nucleotide polymorphism (ETS site) in the MMP-1 promoter docx

Fra-1 targets the AP-1 site/2G single nucleotide polymorphism
(ETS site) in the MMP-1 promoter
Grant B. Tower
1,
*, Charles I. Coon
2
, Karine Belguise
3
, Dany Chalbos
3
and Constance E. Brinckerhoff
1,2
Department of
1
Biochemistry and
2
Medicine at Dartmouth Medical School, Hanover, NH, USA;
3
Institut National
de la Sante
´
et de la Recherche
´
Me
´
dicale, Endocrinologie Moleculaire et Cellulaire des Cancers, Montpellier, France
The matrix metalloproteinase (MMP) family degrades the
extracellular matrix. One member of this family, MMP-1,
initiates the breakdown of interstitial collagens. The
expression of MMP-1 is controlled by the mitogen activated
protein kinase (MAPK) pathway(s) via the activity of acti-

vator protein-1 (AP-1) and polyoma enhancing activity-3/
E26 virus (PEA3/ETS) transcription factors through con-
sensus binding sites present in the promoter. Another ETS
site in the MMP-1 promoter is created at )1607 bp by a
single nucleotide polymorphism (SNP), which contains two
guanines (5¢-GGAT-3¢; Ô2G SNPÕ), rather one guanine
(5¢-GAT-3¢; Ô1G SNPÕ), adjacent to an AP-1 binding site at
)1602 bp. The 2G SNP displays greater transcriptional
activity than the 1G SNP, and AP-1 and Ets families of
transcription factors cooperate to increase transcription. The
2G SNP has been linked to the incidence and the progression
of several cancers and is also associated with non-neoplastic
diseases; although the underlying mechanism(s) has yet to be
elucidated. In this study we demonstrate that the expression
of Fos-like region antigen (Fra-1), an AP-1 transcription
factor component that also correlates strongly with neo-
plastic disease, is necessary for MMP-1 transcription in
A2058 melanoma cells. The inhibition of Fra-1 expression
preferentially downregulates transcription from the MMP-1
promoter DNA containing the 2G SNP, compared to DNA
containing the 1G SNP. This study provides evidence that,
in cooperation with the 2G DNA polymorphism, the AP-1
family member, Fra-1, contributes to the high constitutive
expression of MMP-1 in melanoma cells.
Keywords: antisense; MAPK; matrix metalloproteinase;
melanoma and metastasis; signal transduction.
The extracellular matrix (ECM) acts as a structural support
network within tissues and as a barrier to cell migration.
Additionally, the destruction of the ECM can regulate
disease progression and severity in a variety of pathological

situations [1–3]. The matrix metalloproteinase (MMP)
family is responsible for degradation of the ECM, and the
MMP subfamily of collagenases specifically cleaves the
interstitial collagens (types I, II and III). MMP-1 is a
collagenase that is expressed at very low levels in normal
physiological situations, and expression can be increased
transiently when remodeling of the ECM is required (e.g.
wound healing, uterine resorption and development).
However, in many pathological states, MMPs are expressed
at high levels. This is due to continuous induction by
external stimuli, as seen in rheumatoid arthritis and
athlerosclerosis, or to constitutive expression because of
an activating mutation in a regulatory pathway [4–7]. High
constitutive levels of MMP-1 correlate with a poor prog-
nosis in many types of cancer [8–10].
The mitogen activated protein kinase (MAPK) signaling
pathway is composed of four main cascades, three of which
are known to control the expression of MMP-1. They are
the extracellular response kinase (ERK 1/2), the p38 and the
Jun N-terminal kinase, and they activate factors that target
multiple activator protein-1 (AP-1) and polyoma enhancing
activity-3/E26 virus (PEA3/ETS) factor binding sites within
the MMP-1 promoter [11–16]. These families of transcrip-
tion factors act synergistically when AP-1 and ETS
consensus sites are located in proximity to one another
[17,18], such as the AP-1/ETS site located at )73/)88 bp in
the MMP-1 promoter [19].
We have described a single nucleotide polymorphism
(SNP) at )1607 bp that generates an additional ETS site
when two guanines (5¢-GGAT-3¢; Ô2G SNPÕ) are present

instead of one guanine (5¢-GAT-3¢; Ô1G SNPÕ) (Fig. 1), and
this SNP results in increased transcription from the
2G-containing promoter relative to the 1G-containing
promoter [20,21]. We have verified this ETS site by
demonstrating that it binds recombinant Ets-1 protein,
and that the adjacent AP-1 site at )1602 bp binds recom-
binant c-Jun. Further, we carried out gel shift analyses
with nuclear extracts from A2058 melanoma cells and
Correspondence to C. E. Brinckerhoff, Department of Biochemistry,
505 Vail Building, Hanover, NH 03755, USA.
Fax: + 1 603 650 1128, Tel.: + 1 603 650 1609,
E-mail: brinckerhoff@dartmouth.edu
Abbreviations:MMP,matrixmetalloproteinase;MAPK,mitogen
activated protein kinase; AP-1, activating protein; PEA3/ETS, poly-
oma enhancing activity-3/E26 virus; FGF, fibroblast growth factor;
SNP, single nucleotide polymorphism; ERK, extracellular response
kinase; Fra, Fos-related antigen; Elf, ETS-like factor; DMEM,
Dulbecco’s Modified Eagles Medium; CMV, cytomegalovirus;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
*Present address: Schering-Plough, 2015 Galloping Hill Rd., K-15,
D-129C Kenilworth, NJ 07033, USA.
(Received 11 June 2003, revised 4 September 2003,
accepted 8 September 2003)
Eur. J. Biochem. 270, 4216–4225 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03821.x
competition experiments with AP-1 and PEA3 oligonucleo-
tides to show specificity of binding [20].
Several studies correlate the presence of the 2G SNP
with invasive cancers, including melanoma [22,23], and
colorectal [24–26], lung [27], endometrial [28] and ovarian
cancers [29,30]. The 2G polymorphism is also associated

with non-neoplastic diseases, such as premature rupture of
fetal membranes [31], increased risk of internal carotid
artery stenosis [32], rate of decline in lung function [33],
lower bone mineral density [34] and increased severity of
chronic periodontitis [35]. These studies support the
hypothesis that the 2G polymorphism may increase
expression from the MMP-1 promoter and influence the
degree of pathology resulting from dysregulated matrix
degradation. However, the mechanisms mediating the
increase in MMP-1 from the 2G promoter have not been
elucidated. We have previously described one mechanism
responsible for the increase in expression from the
2G-containing promoter in A2058 melanoma cells, which
constitutively express copious amounts of MMP-1 [15]. In
this system, we determined by means of transient transfec-
tions with MMP-1 reporter constructs, that the MAPK
pathway, ERK 1/2, targets the 2G SNP at )1607 bp and
theadjacentAP-1siteat)1602 bp. Activation and/or
production of transcription factors binding to these sites
are responsible for the elevated expression of MMP-1 from
the 2G allele compared to that from the 1G allele [15].
However, it is likely that other mechanisms exist, which
may also influence this response.
Our previous study showed that the chemical inhibitors
PD098059 and U0126 blocked the phosphorylation of
ERK1/2 and reduced expression of MMP-1 mRNA [15].
We also determined that continuous synthesis of new
proteins is necessary for MMP-1 expression [15] and that
the levels of these proteins are decreased when ERK 1/2
signaling is disrupted by PD098059. Since the expression

and the mRNA stability of the AP-1 family member Fra-1 is
dependent on ERK activity [36], we hypothesized that Fra-1
may be produced by A2058 cells, that it may be necessary
for MMP-1 gene expression, and that it may be reduced by
PD098059.
Fra-1 has been implicated in the regulation of MMP-1
[37–40], MMP-13 [41,42] and the urokinase plasminogen
activator receptor [43], a receptor in the fibrinolytic cascade
that activates MMPs. Fra-1 is necessary for the induction of
MMP-1 by mitogens [44,45] and it binds to the AP-1
responsive element in the proximal promoter [39,46]. As the
ERK pathway often regulates induction of the mitogen,
fibroblast growth factor (FGF), which induces expression of
MMP-1, Fra-1 may contribute to the transcriptional
regulation of both FGF and MMP-1. Indeed, FGF
responsiveness in the MMP-1 promoter requires both the
AP-1 and ETS sites within a 63-bp region, from )123 bp to
)61 bp, to which Fra-1 binds [39]. These studies have
suggested that Fra-1 interacts with Ets transcription factors
to induce MMP-1 gene expression. The present study
extends these findings to include a role for Fra-1 in
augmenting MMP-1 transcription from the AP-1 site at
)1602 bp and the 2G SNP (ETS site) at )1607 bp.
Materials and methods
Cell culture
Stock cultures of A2058 melanoma cells were grown in
150-mm culture plates in Dulbecco’s Modified Eagles
Medium (DMEM) containing 10% fetal bovine serum,
L
-glutamine, and penicillin/streptomycin (37 °C, 5% CO

2
),
and passaged at confluency [20]. For most experiments,
confluent cultures of cells were washed twice with Hanks
balanced salt solution to remove traces of serum and
cultured in serum-free DMEM and 0.2% lactalbumin
hydrolysate. Cells were treated with increasing concentra-
tions of PD098059 (Calbiochem), an inhibitor of the
ERK 1/2 pathway, solubilized in dimethyl sulfoxide.
Electrophoresis mobility shift assay (EMSA)
Nuclear extracts were prepared from untreated A2058 cells,
or from cells treated with PD098059 [20]. Oligonucleotide
probes containing 35 or 36 bp from regions of the MMP-l
promoter encompassing the lG/2G site at )1607 bp and the
AP-1 site at )1602 bp, from )1621 bp to )1586 bp [20],
were end-labeled with [
32
P]dATP[cP] by T4 Kinase (Gibco/
BRL) in 1 · polynucleotide kinase buffer (Roche) and
2 · 10
6
c.p.m. were incubated with 5 lg of nuclear extract
for 20 min at 4 °C. This amount was needed for probe
excess with the 2G oligonucleotide. Lesser amounts showed
depletion of free probe at the bottom of the gel. Binding
reactions were separated on 7% Tris/glycine polyacrylamide
gels and visualized by autoradiography.
Transient transfection and luciferase assay
One microgram of 4372 bp of the MMP-l promoter DNA
containing either 1G or 2Gs at )1607 bp, linked to the

luciferase reporter, was transiently transfected into cells
using Geneporter (Gene Therapy Systems). To further
elucidate the role of AP-1 sites in the MMP-1 promoter,
reporter constructs containing mutations in selected AP-1
sites were used [15,20]. To alter the expression levels of
Fra-1, expression constructs containing the Fra-1 cDNA
in the sense and antisense orientation driven by the
Fig. 1. Diagram of the MMP-1 promoter region spanning the region
from )1585 bp to )1620 bp. The 1G SNP DNA contains an AP-1
consensus at )1602 bp, and the sequence 5¢-GAA-3¢ at )1607 bp. The
2G SNP DNA has the AP-1 site at )1602 bp and the sequence
5¢-GGAA-3¢ at )1607 bp, conferring a consensus ETS site.
Ó FEBS 2003 Fra-1 and the 2G SNP (Eur. J. Biochem. 270) 4217
cytomegalovirus (CMV) minimal promoter in a pCl vector
[47] as well as empty vector to keep the DNA concentration
constant, were used. Twenty-four hours post-transfection,
cells were washed and incubated with serum free medium,
with or without 5 l
M
PD098059 for 24 h and cell lysates
were then assayed for luciferase activity in a luminometer.
All transfections were carried out in triplicate. Disparities in
relative light unit (RLU) values among different experi-
ments are due to the use of different luminometers.
Northern blot analyses
Total RNA (10 lg) was harvested from cells with Trizol
(Gibco) and separated on a 5% formaldehyde, 1% agarose
gel, transferred to a GeneScreen membrane (NEN Life
Sciences) and probed with a
32

P-labelled cDNA for MMP-1
[47], c-Fos (a generous gift from I. Verma, the Salk Institute,
La Jolla, CA, USA) and Fra-1. The Fra-1 probe was
created by PCR, using primers to amplify a region unique
to the transcript. The forward primer for Fra-1 was
5¢-TCTGGGCTGCAGCGAGAGATTGAGGAG-3¢ and
the reverse primer was 5¢-GGAGGAGACATTGGC
TAGGGTGGCATC-3¢, giving a product of 435 bp. PCR
reactions were separated on a 1.5% agarose gel by
electrophoresis; the band of the correct size was excised
and purified. The purified PCR fragment was labeled with
[
32
P]dCTP[aP] for probing. Levels of glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) mRNA probed
with a
32
P-labelled cDNA controlled for loading.
Western blot analysis
Whole cell lysates were harvested by mechanical scraping
and collected with 2 · SDS buffer [15]. Proteins were
separated on 10% Tris/HCl Ready Gels (Bio-Rad) and
transferred to Immobilon
TM
-P poly(vinylidene difluoride)
membranes (Millipore). All membranes were blocked in 5%
dry milk (Carnation) at room temperature for 1 h. Blots
were probed with antibodies against Fra-1, JunD, c-Fos,
Ets-1, Ets-2, and ETS-like factor (Elf-1; Santa Cruz, Inc.,
Santa Cruz, CA, USA) overnight at 4 °C, as suggested by

manufacturer. Blots were then incubated with an anti-rabbit
horseradish peroxidase-linked secondary antibody (Cell
Signaling, Beverly, MA, USA) at a dilution of 1 : 2000 at
room temperature for 1 h. Bands were visualized by electro-
chemiluminesence. The membranes were stripped as des-
cribed in [48] and reprobed with an antibody against actin
(Oncogene, Boston, MA, USA) for 1 h at room tempera-
ture. Blots were subsequently probed with an anti-goat IgM
secondary antibody (Oncogene) at a dilution of 1 : 2000 for
1 h at room temperature. Bands were visualized by electro-
chemiluminesence.
Results
Increasing concentrations of the ERK specific inhibitor,
PD098059, lead to a decrease in protein binding to the 2G
SNP. Our earlier studies demonstrated that the 2G SNP
functioned as a bone fide ETS site, as measured by
competition experiments and by the specific binding of
recombinant Ets-1 protein [20]. Further, treatment of A2058
melanoma cells with the chemical inhibitor PD098059
blocked the phosphorylation of ERK 1/2 and reduced
MMP-1 mRNA [15]. This reduction in MMP-1 mRNA
occurred at the level of transcription, was greater in the 2G
SNP-containing promoter than in the 1G SNP-containing
promoter, and required the AP-1 site at )1602 bp [15].
Therefore, we hypothesized that this inhibition may be a
consequence of reduced binding of nuclear proteins to the
AP-1 site and SNP at )1602 bp and )1607 bp, respectively.
We tested this hypothesis with an EMSA, using a 35/36 bp
nucleotide probe containing either 1G or 2Gs and nuclear
extracts from untreated cells or from cells treated with

PD098059 for 24 h. Figure 2 illustrates that extracts from
untreated cells bind to both the 1G and 2G probes. In
agreement with previous data [20,49], the binding to the 2G
probe is greater than to the 1G probe, with a 2G specific
band, indicated by a star. Treating cells with increasing
concentrations of PD098059 results in a decrease in the
intensity and/or a complete disappearance of some bands,
indicated by arrows. In contrast, bands that are more
intense in the lanes containing the 1G probe appear
unchanged in the presence of the inhibitor (–). Thus, a
decrease in the expression of MMP-1 in the presence of
PD098059 may be due, at least in part, to a decrease in the
binding of transcription factors to the MMP-1 promoter
Fig. 2. Effects of the MEK/ERK inhibitor PD098059 on transcription
factor binding. EMSA of transcription factors binding to [
32
P]dATP[cP]
end-labeled 35- and 36-mer, 1G and 2G (respectively) oligonucleotides
in the presence of increasing concentrations of PD098059 for 24 h.
Lanes 1 and 2, free probe alone; lanes 3 and 4, extract from cells
incubated in serum-free media alone; lanes 5–8, extract from cells
incubated in the presence of 5 l
M
(5 and 6) and 10 l
M
(7 and 8)
PD098059. *, 2G specific band; fi , inhibitable activities that bind 2G
to a greater extent; –, bands with greater affinity to 1G and not inhibited
by PD098059. EMSA was performed four times for reproducibility.
4218 G. B. Tower et al. (Eur. J. Biochem. 270) Ó FEBS 2003

region containing the AP-1 site at )1602 bp and the 2G
SNP at )1607 bp.
Fra-1 protein is decreased in response to PD098059
Previously, we demonstrated that recovery of MMP-1
mRNA expression after inhibition by PD098059 in A2058
melanoma cells required de novo protein synthesis [15]. This
finding, together with the data in Fig. 2, led us to
hypothesize that inhibition of the MEK/ERK pathway by
PD098059 may decrease the level of a transcription factor(s)
required for MMP-1 expression and may explain the
decrease in protein binding to the AP-1/2G site. Therefore,
to determine which factors in the AP-1 or Ets family might
be downregulated, we compared protein levels of AP-1/Ets
family members in cells left untreated or treated for 24 h
with increasing concentrations of PD098059. Western blot
analysis showed that in A2058 cells, c-Fos, Fra-1, and JunD
of the AP-1 family and Ets-1, Ets-2 and Elf-1 of the Ets
family are expressed constitutively (Fig. 3A, and data not
shown). We could not detect Fra-2. Fra-1 levels were
downregulated in response to PD098059 (Fig. 3A). The
two bands in Fig. 3A represent the phosphorylated
protein (upper band) and the nonphosphorylated form
(lower band) of Fra-1. Both forms of the protein are
decreased by PD098059 treatment, suggesting that levels
of Fra-1 protein and its subsequent phosphorylation are
mediated by the ERK pathway. We found that c-Fos was
also downregulated by PD098059, but its level of expres-
sion was very low (data not shown). Thus, Fra-1 is the
principal Fos family member expressed in these cells.
Further, while c-Fos expression can be regulated by many

pathways [50,51], Fra-1 mRNA synthesis is solely regu-
lated by the ERK 1/2 pathway [36,52–55]. Since MMP-1
expression in these cells is unaffected by other signal
transduction inhibitors [15], it is likely that Fra-1 is the
dominant Fos family member involved in constitutive
MMP-1 expression in these cells. Therefore, we have
focused on the possible role of Fra-1 in the transcription
of MMP-1.
Fra-1 mRNA is reduced in response to PD098059 prior
to inhibition of MMP-1 gene expression
To ascertain whether there is a correlation between Fra-1
and MMP-1 expression, dose–response and time-courses
comparing the inhibition of Fra-1 and MMP-1 mRNAs
were performed. Fra-1 has two mRNA transcripts, with the
1.7-kb transcript being dominantly expressed compared to
the 3.3-kb transcript, which results from an extended 3¢
untranslated region [56], presumably due to an alternative
polyadenylation site. Since both transcripts are presumed to
encode the Fra-1 protein [56], whether the reduction of one
transcript precedes the other or is more completely down-
regulated may be unimportant. The dose–response for
inhibiting Fra-1 and MMP-1 gene expression was similar,
with complete inhibition of MMP-1 mRNA and the 3.3-kb
transcript of Fra-1 at 5–10 l
M
PD098059 and substantial
inhibition of the 1.7-kb Fra-1 transcript with 10 l
M
PD098059 (Fig. 3B).
For the time-course experiment, A2058 cells were either

left untreated or treated with PD098059 (5 l
M
). RNA was
harvested immediately after addition of PD098059 (time
zero), and at subsequent time points for measurement of
Fra-1 and MMP-1 mRNA. The level of the 3.3-kb
transcript of Fra-1 began to decrease within 1 h of
treatment, and was undetectable by 6 h. Reduction of the
1.7-kb transcript lagged behind the 3.3-kb transcript,
beginning at 2 h and reaching maximal inhibition by 4 h.
This reduction of the Fra-1 transcript preceded that of
MMP-1 mRNA, which began at 4 h and was nearly
complete by 10 h (Fig. 4). The increase in MMP-1
mRNA levels in untreated cultures beginning at 4 h has
been observed previously in response to removal of serum-
containing medium [57]. This increase in MMP-1 mRNA
may be a stress response or a consequence of removing
transforming growth factor-b, which is present in serum
and can repress the expression of MMP-1 [58]. These
findings indicate that Fra-1 is downregulated in response
to PD098059 prior to MMP-1, and suggest that Fra-1
may be involved in regulating MMP-1 expression in these
cells.
Fig. 3. Inhibition of Fra-1 protein and mRNA levels by PD098059. (A)
Fra-1 protein levels from confluent cultures of A2058 cells incubated in
serum-free media with or without increasing concentrations of
PD098059. Whole cell extracts were separated by SDS/PAGE, trans-
ferredtopoly(vinylidenedifluoride)membranesandprobedwithFra-1
antibody. The blot was stripped as described [48] and reprobed with an
antibody against a-actin for 1 h. (B) Dose–response of PD098059 on

Fra-1 and MMP-1 mRNA levels in A2058 cells. GAPDH was used as
a loading control. Dose–response experiments were performed at least
three times for reproducibility.
Ó FEBS 2003 Fra-1 and the 2G SNP (Eur. J. Biochem. 270) 4219
Fra-1 expression drives transcription from the MMP-1
promoter in a dose-responsive manner
Evidence thus far has indirectly implicated Fra-1 as one
factor contributing to constitutive expression of MMP-1 in
A2058 melanoma cells: Fra-1 is expressed in these cells,
and its pattern of repression in response to PD098059 is
coordinate with that of MMP-1. To more directly link Fra-1
to MMP-1 expression, we used a fra-1 sense expression
construct under control of the CMV promoter, which has
been described previously [47]. Cells were transiently
cotransfected with MMP-1 promoter DNA containing
either the 1G or 2G SNP, driving luciferase, along with
increasing amounts of the Fra-1 expression construct.
Expression from the 2G SNP was initially 2.7-fold higher
than the 1G SNP, and increasing quantities of Fra-1
preferentially augmented transcription from this promoter
(Fig. 5A). With increasing amounts of Fra-1, expression of
the 1G promoter construct increased 2.9-, 5.2- and 8.8-fold
over the 1G control construct, compared to increases of
4.8-, 10.4- and 12.9-fold for the 2G construct. For each
concentration of Fra-1, the increase in expression from the
2G SNP was approximately twofold greater than the
increase in expression from the 1G SNP. Therefore, Fra-1
can drive transcription from both the 1G and 2G-containing
promoters but has the more dramatic effect on the 2G allele.
Fra-1 antisense reduces MMP-1 transcription

in A2058 cells
To further address the role of Fra-1 in MMP-1 expres-
sion, we used an antisense construct. A2058 cells were
cotransfected with the 1G or 2G MMP-1 promoter
constructs and increasing doses of a construct expressing
Fra-1 antisense cDNA, which has been shown to lower
AP-1 activity in a dose-dependent manner in MCF-7 and
MDA-231 breast cancer cells [47]. We reasoned therefore
that it should antagonize production of endogenous Fra-1.
Unfortunately, however, because the transfection effi-
ciency was too low (< 10%), we could not measure
changes in the level of Fra-1 protein. Nonetheless, Fig. 5B
shows that expression of both the 1G and 2G-containing
constructs was inhibited by fra-1 antisense. Repression of
luciferase activity from the 1G SNP was  50% at all
doses of Fra-1 antisense. In contrast, reporter expression
from the 2G SNP promoter was inhibited in a dose-
dependent fashion (Fig. 5B), with maximal inhibition at
 75%. These results are similar to those seen with
PD098059, where the 1G allele was inhibited by 50% and
the 2G allele by  50–70% [15]. Thus, the effects of
decreasing Fra-1 expression mimic the effects of inhibition
by PD098059, and the reduction in Fra-1 expression by the
addition of 3 lg of antisense construct abolished the
difference in expression between the 1G and 2G alleles.
Therefore, in these melanoma cells, Fra-1 expression is
required for the increase in transcription from the 2G SNP
promoter over the 1G SNP promoter.
The AP-1 site at )1602 bp is necessary for modulation
of MMP-1 expression from the 2G SNP promoter

To identify the sequences in the MMP-1 promoter that are
targeted by Fra-1, 1G/2G MMP-1 constructs with either
wild-type or mutated AP-1 sites at )73 bp or )1602 bp were
Fig. 4. Downregulation of Fra-1 and MMP-1 mRNA levels prior to MMP-1 by PD098059. A2058 cells were grown until confluent, washed with
Hanks balanced salt solution and transferred to serum-free media with or without 5 l
M
PD098959.RNAwasharvestedat0,0.5,1,2,4,6,8,10and
12 h. RNA blots were probed with Fra-1 and MMP-1 and subsequently, with GAPDH probes as a loading control, as described in Materials and
methods.
4220 G. B. Tower et al. (Eur. J. Biochem. 270) Ó FEBS 2003
transfected into A2058 cells. The AP-1 site at )73 bp is
important for basal and growth factor induced expression
(via ERK activation) [19]. The AP-1 site at )1602 bp is
adjacent to the 2G SNP, which is targeted by the ERK 1/2
pathway, and it may also be responsible for growth factor
induced expression of MMP-1 [15]. As seen in Fig. 6,
expression of luciferase from the MMP-1 reporter
constructs with a wild-type AP-1 site at )1602 bp was
equivalent to previous reports [15], in that there is greater
expression from the wild-type 2G construct than from the
1G. However, as noted previously [15], this difference in
expression was absent in the constructs containing a
mutated AP-1 site at )1602 bp (Fig. 6). In this experiment,
some cells were also transfected with the Fra-1 antisense
construct in order to determine whether the absence of
Fra-1 can lower gene expression when the AP-1 sites are
mutated. We found that expression of Fra-1 antisense
abolished the higher level of expression from the 2G
MMP-1 reporter construct so that all constructs, wild-type
or with a mutated AP-1 site at )1602 bp, show equivalent

activity. These findings corroborate our previous results,
which show that when A2058 cells were transfected with the
same wild-type and mutant constructs and subsequently
treated with PD098059, reporter levels were nearly identical
[15], thereby suggesting that the ERK inhibitor and Fra-1
antisense may be working in a similar manner.
We next examined the effect of mutating the AP-1 site
at )73 bp in the MMP-1 promoter (Fig. 6, inset). Com-
pared to constructs containing a wild-type site at )73 bp,
mutating this site substantially decreased expression
( 90%) from both the 1G and 2G SNP promoters
(compare 1G vs. 2G wild-type in Fig. 6 with 1G vs. 2G
inset). While the absolute levels of expression from both
alleles were reduced when the AP-1 site at )73 bp is
mutated, the difference between expression of the 1G vs.
2G alleles remained intact, further demonstrating that the
proximal AP-1 site is not involved in the augmented
expression from the 2G allele. Co-transfection of con-
structs with the mutated AP-1 site at )73 bp and Fra-1
antisense inhibited expression from both the 1G and 2G
constructs but downregulated the 2G construct to a
greater extent (49% repression vs. 77% repression,
respectively). The fact that Fra-1 antisense can inhibit
transcription from the 1G SNP construct with the mutant
AP-1 site at )73 bp indicates that Fra-1 can induce MMP-
1 through AP-1 sites other than that at )73 bp. We
conclude that the )73 bp site, in contrast to the AP-1 site
at )1602 bp, is not involved in the difference between the
1G and 2G SNP.
Fra-1 rescues the 1G/2G response in cells treated

with PD098059
To confirm that the attenuation of Fra-1 by PD098059
results in reduced MMP-1 expression, we attempted to
reverse this repression by ectopic expression of Fra-1. A2058
cells were treated with PD098059 and cotransfected with the
1G or the 2G MMP-1 promoter constructs along with
increasing amounts of the Fra-1 expression vector. Exo-
genous Fra-1, expressed under control of the CMV
promoter, would not be subject to the same regulation as
endogenous Fra-1 gene, and the high level of expression
should allow the exogenous protein to be phosphorylated
even in the presence of 5 l
M
PD098059. Although higher
concentrations of the drug might block the phosphorylation
of all exogenous Fra-1, they may also introduce toxicity and
nonspecific effects. Thus, as expected, in cells transfected
with the empty vector, there is about a threefold increase in
expression of the 2G allele compared to the 1G allele, and
treatment with 5 l
M
PD098059 abolished this difference
(Fig. 7). We reasoned that any increase in expression from
the 2G allele in these treated cells cotransfected with Fra-1
would be due to rescue by Fra-1. Indeed, compared to the
empty vector, we found that cotransfection of increasing
amounts of the Fra-1 expression plasmid augments the level
Fig. 5. Effect of Fra-1 expression on MMP-1 transcription. (A) Fra-1
sense expression. A2058 cells were transfected with 1 lgof1G/2G
MMP-1 promoter constructs driving luciferase and the pCl empty

vector or with increasing concentrations of the Fra-1 overexpressing
vector, as described in Materials and methods. For the empty vector,
the difference in expression between the 1G and 2G constructs is
indicated above the line spanning both the 1G and 2G bars
(mean ± SD). With increasing amounts of Fra-1 expression, (*)
indicates that the value is statistically significant (P £ 0.02), compared
to the 1G or 2G value in cells transfected with the empty vector. (B)
Fra-1 antisense expression. A2058 cells were transfected with 1 lgof
1G/2GMMP-1promoterconstructsdrivingaluciferasereporterand
the pCl empty vector, or with increasing quantities of pCl fra-1 anti-
sense (AS) vector. The percent inhibition compared to the empty
vector control is indicated above the bar. The (*) indicates that the
value is statistically significant (P £ 0.05) compared to the previous
respective 1G or 2G data point. Transfection experiments were per-
formed three times for reproducibility.
Ó FEBS 2003 Fra-1 and the 2G SNP (Eur. J. Biochem. 270) 4221
of transcription from both the 1G and 2G promoter
constructs. The increase is greater in cells transfected with
the 2G construct compared to the 1G construct, increasing
from 1.06-fold for the empty vector to 2.1-fold for cells
transfected with 3.0 lg Fra-1. Increasing the amount of the
Fra-1 not only restored the difference between the 1G and
2G alleles, but also partially abrogated the repression of the
2G allele by PD098059. Therefore, expression of Fra-1 is
reduced by PD098059 and this protein is required for
increased expression from the 2G allele over the 1G allele.
Discussion
The SNP in the MMP-1 promoter at )1607 bp results in an
ETS binding site (the 2G SNP) and is correlated with
increased transcription [20]. Subsequent investigations that

demonstrated an association between the 2G allele and
several cancers have generated interest as to the identity of
factors that activate this site. In this study, we identify
Fra-1 as one of the potential transcription factors driving
MMP-1 transcription and enhancing expression through
the AP-1 site at )1602 bp, but only in combination with
the 2G SNP (ETS site) at )1607 bp. In the presence
of increasing concentrations of the ERK 1/2 inhibitor,
PD098059, the dose–responses of Fra-1 and MMP-1
expression are similar (Fig. 3B). The time-course of inhibi-
tion of Fra-1 and MMP-1 mRNAs in response to
PD098059 treatment (Fig. 4) further supports a probable
role of Fra-1 in MMP-1 transcription. These correlations
between Fra-1 and MMP-1 expression prompted us to find
direct evidence implicating Fra-1 in constitutive MMP-1
production.
AP-1 factors bind DNA as Jun/Jun homodimers or Jun/
Fos heterodimers, which have affinity for AP-1 responsive
elements. A2058 melanoma cells express both JunD (data
not shown) and Fra-1 constitutively and these proteins may
form a transactivating complex with other transcription
factor families, such as members of the Ets family [18].
Furthermore, Fra-1 is a potent transactivator in the
presence of JunD, where either factor may be inhibitory
without the other [42,59]. In addition to the reduction in
phosphorylation and expression of Fra-1, inhibition of the
ERK pathway also decreases the stability of Fra-1 protein
and its ability to bind to DNA [36]. With the exception of
Fra-2, this contrasts with other AP-1 members, which bind
Fig. 6. Mutational analysis of AP-1 sites at )1602 bp and )73 bp. A2058 cells were cotransfected with 1G/2G MMP-1 reporter constructs

containing a wild-type AP-1 site or a mutated AP-1 site at either )1602 bp or (inset) )73 bp. Additionally all cells were transfected with either a Fra-
1 expression, Fra-1 antisense (AS) or control construct. Data points are the mean ± SD and the statistical significance of the 1G compared to the
2G is **P ¼ 0.002. The transfection experiment was performed three times for consistency.
Fig. 7. Partial rescue of MMP-1 transcription in cells treated with
PD098059. A2058 cells were transfected with 1 lg of 1G or 2G MMP-1
promoter constructs driving luciferase and the pCl vector or with
increasing quantities of pCl Fra-1 overexpression vector. After 24 h
cells were incubated in serum-free media with or without 5 l
M
PD098059 for an additional 24 h. The fold increase of 2G expression
over 1G expression from the 2G allele treated with 5 l
M
PD098059
compared to the expression from the untreated 1G allele are charted
below the graph. Data points represent the mean ± SD. **P £ 0.05 is
the statistical significance of expression of the 1G allele compared to
the 2G allele.
4222 G. B. Tower et al. (Eur. J. Biochem. 270) Ó FEBS 2003
constitutively to AP-1 consensus sequences in the presence
or absence of MAPK signaling [50,60]. Additionally, Fra-1
is the factor that determines the DNA-binding activity of
the AP-1 transcription factor complex [61]. Thus, if Fra-1 is
binding to the AP-1 site at )1602 bp, PD098059 should
reduce binding activity since ERK 1/2 is required for
stability/DNA binding [36]. Our EMSA results suggest the
presence of a multi-family association of proteins required
to enhance synthesis of MMP-1 through the 2G SNP/AP-1
site [20,31,49], of which Fra-1 may be a component. Specific
complexes bind to the probe containing the AP-1 site and
2G SNP as seen in Fig. 2, and these complexes are absent in

the lanes containing the probe with the 1G SNP. Although
increasing concentrations of PD098059 reduce the binding
of complexes to the probe containing the 1G SNP, a more
pronounced decrease is observed in the binding of com-
plexes to the 2G SNP probe. Attempts to supershift
complexes with antibodies raised to Fra-1 or other AP-1
factors were unsuccessful (data not shown), potentially due
to the inability of the antibodies to access the epitope.
Experiments with a Fra-1 antibody from another source
(Geneka, Canada) also failed to supershift (data not
shown). The downregulation of Fra-1 may decrease the
total number of complexes formed, thereby diminishing the
cooperative binding to the promoter and, subsequently
decreasing MMP-1 production.
Transfection with increasing amounts of Fra-1 sense and
antisense expression constructs had a greater effect on
transcription from the 2G SNP than from the 1G allele
(Fig. 5). It has been previously reported that Fra-1 protein
binds to the proximal AP-1 site at )73 bp in the MMP-1
promoter [39]. Thus, the increase in expression from the
1G promoter in the presence of increasing amounts of Fra-
1 may be due to the effects of Fra-1 on the proximal AP-1
site. The greater increase in the induction of the 2G
promoter may result from Fra-1 acting not only at the
proximal site but also at the AP-1 site at )1602 bp adjacent
to the 2G SNP. Furthermore, our data implicate Fra-1 in
contributing to the increase in MMP-1 expression from the
2G SNP promoter via a cooperative mechanism between
theAP-1siteat)1602 bp and the 2G SNP at )1607 bp
(Fig. 6). Our previous results show that when A2058 cells

were transfected with wild-type and AP-1 mutant at
)1602 bp constructs and treated with PD098059, reporter
levels were identical from either the 1G or 2G promoter
[15]. Thus, expression of Fra-1 antisense and PD098059
[15] have similar effects on transcription of the MMP-1
promoter. The similarities between the wild-type and AP-1
()1602 bp) mutant constructs in response to Fra-1 anti-
sense and ERK 1/2 inhibition provide additional support
for the concept that inhibition of Fra-1 is one target of
PD098059 [15], with the subsequent inhibition of MMP-1
mRNA.
The ectopic expression of Fra-1 was able to moderately
rescue transcription from the 2G allele in the presence of
PD098059 (Fig. 7), but was unable to fully overcome this
repression. This partial rescue is probably because the
inhibition of the ERK 1/2 pathway greatly reduces phos-
phorylation and stability of downstream targets, including
Fra-1 [62] and because Fra-1 needs to be phosphorylated to
transactivate transcription. Young et al. [62] determined
that the threonine at 231 in Fra-1 is phosphorylated by the
ERK pathway and is essential for the transactivation of
transcription by Fra-1. We attempted to overcome the need
for phosphorylation by the ERK pathway by changing the
threonine at 231 to an aspartic acid residue (T231D), which
may mimic a phosphothreonine. However, the T231D
Fra-1 mutant was unable to transactivate MMP-1 tran-
scription to the same levels as wild-type Fra-1 or rescue
MMP-1 expression in the presence of PD098059 (data not
shown). Two possible reasons why the T231D mutation
did not work are that the mutant protein may have been

unstable or the aspartic acid residue was unable to mimic
the phosphorylation state. Although the A2058 cells
express c-Fos, levels of the protein are low (data not
shown). However, it is possible that c-Fos may play a
minimal role in the residual expression of MMP-1 in the
presence of Fra-1 antisense.
Fra-1 had previously been implicated in MMP-1 expres-
sion through the AP-1/ETS site in the proximal promoter
[39,46], but it is the first transcription factor to be identified
that leads to the increase in expression from the MMP-1 2G
SNP through its cooperation with the adjacent AP-1 site.
Fra-1 expression, like the presence of the 2G polymorphism,
has been associated with the more aggressive and metastatic
forms of breast cancer [63] and melanoma [64], further
supporting the possible connection between Fra-1 expres-
sion and the 2G polymorphism.
Acknowledgements
Supported by grants from the NIH, AR-26599, CA-77267, the
Department of Defense DAMD17-00-1-0221 (CEB), predoctoral
fellowships from the Komen Foundation Austin, Texas (DISS00-
000354), and the Department of Defense DAMD17-01-1-0225 (GBT),
Institut National de la Sante
´
et de la recherche Me
´
dicale, the
Association pour la Recherche sur le Cancer (grant no. 5825) (DC),
the Fondation pour la Recherche Me
´
dicale and the Ligue Nationale

Contre le Cancer (KB).
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