From the Department of Cell Biology and Anatomy,
Weill Medical College of Cornell University, New York, New York 10021, the § Strang Cancer Research Laboratory at the Rockefeller
University, New York, New York 10021, the
Department of Cell
Biology, Vanderbilt University School of Medicine, Nashville, Tennessee
37232, the
Department of Medicine, Weill
Medical College of Cornell University, New York, New York 10021, and
the §§ Institute for Molecular Biology and
Biotechnology, McMaster University, Hamilton,
Ontario L86 4K1, Canada
Received for publication, November 27, 2000, and in revised form, March 16, 2001
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ABSTRACT |
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The inducible prostaglandin synthase
cyclooxygenase-2 (COX-2) is aberrantly expressed in intestinal tumors
resulting from APC mutation, and is also transcriptionally
up-regulated in mouse mammary epithelial cells in response to
Wnt1 expression. Wnt1 is a mammary oncogene that encodes a secreted
signaling factor. Targeted expression of Wnt1 in murine
mammary glands results in epithelial hyperplasia with subsequent
carcinoma formation (1). Wnt1 signaling leads to stabilization of
a cytosolic pool of Cox-2, the inducible isoform of prostaglandin synthase, is aberrantly
expressed in human colorectal cancers, and also in tumors from mouse
colorectal cancer models carrying germline Apc mutations (14-18). Additionally, COX-2 overexpression has now been
detected in multiple human cancers including those of the skin, head
and neck, lung, breast, and stomach (19-24). Strikingly,
COX-2 overexpression in murine mammary gland is sufficient
to induce tumorigenesis (25). Thus, considerable interest is focused on
COX-2 as a potential therapeutic target for the prevention or treatment
of cancer. Both genetic ablation and pharmacological inhibition of
COX-2 have resulted in reduced tumorigenesis in several animal cancer models (26-30), and selective COX-2 inhibitors have also proved effective in reducing the number of colorectal polyps in familial adenomatous polyposis patients (31). Several mechanisms have been
proposed to account for the role of COX-2 in tumorigenesis. Cox-2 overexpression in epithelial cells is associated with
enhanced invasiveness and suppression of apoptosis (25, 32, 33). Prostaglandin overproduction is likely to have pleiotropic consequences including stimulation of proliferation and local immunosuppressive effects that could facilitate tumorigenesis. Recent data also demonstrate a role for Cox-2 in angiogenesis (34-39).
Modulation of COX-2 protein levels can be achieved via multiple
mechanisms, including transcriptional activation, mRNA
stabilization, and altered COX-2 protein stability. We have previously
demonstrated transcriptional up-regulation of Cox-2 in
response to Wnt1 expression in mouse mammary epithelial
cells (13). The goal of the current study was to elucidate the
mechanism(s) underlying Wnt1-mediated induction of Cox-2.
Given that COX-2 is also up-regulated in intestinal tumors
resulting from APC mutation, we initially hypothesized that
Cell Culture--
Generation, characterization, and
culture of the control and Wnt1-expressing mouse mammary
epithelial cell lines have been previously described (13). C57/MV7 and
RAC/MV7 are control populations infected with MV7 retrovirus, while
C57/Wnt-1 and RAC/Wnt-1 are Wnt1-expressing populations, and
RAC/Wnt-1 #9 is a clonal subline selected for high level
Wnt1 expression. Identical culture conditions were used for
the parental cell line C57MG. 293 human embryonic kidney cells were
grown in Dulbecco's modified Eagle's medium (4.5 g/liter
D-glucose) containing 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin.
Cell Transfection and Luciferase Assays--
293 cells were
transfected using LipofectAMINE (Life Technologies), according to the
manufacturer's instructions. Briefly, cells were seeded in 24-well
plates at 1 × 105 cells/well. 20 h after cell
seeding, 6 wells were transfected for 4 h using a transfection
mixture consisting of 1.2 ml of serum-free Dulbecco's modified
Eagle's medium, 3.6 µl of LipofectAMINE, and 2.2 µg of total
plasmid DNA (including 0.2 µg of pRL-TK). Where necessary "empty"
vectors were included to maintain constant amounts of DNA. Lysates were
prepared 48 h after transfection, and Firefly and Renilla
luciferase activities were measured using a Dual Luciferase Reagent kit
(Promega) and a Monolight 2010 luminometer (Analytical Luminescence
Laboratory). Firefly activity was normalized to the Renilla activity,
and results were expressed as percentage of control activity. To
transfect 293 cells for protein analysis or RNA preparation, cells were
plated at 5 × 106 cells per 10-cm plate, and
transfections scaled-up proportionally to surface area. Similar
conditions were used to transfect C57MG cells, except that cells were
plated at 2.5 × 104 cells/well, and 5.4 µl of
LipofectAMINE was used for transfection.
Mammary Tissues and Tumors--
A breeding colony of
Wnt1 transgenic mice (1) was maintained by crossing
Wnt1 transgenic B6/SJL males (obtained from the Jackson
Laboratory) with strain-matched females. Mice were genotyped by
polymerase chain reaction analysis of tail-tip DNA. The primers used were: 5'-CCAGAACACAGCATGGCTTCCAACG-3' and
5'-ACTCCACACAGGCATAGAGTGTCTGC-3'. These primers amplify a
425-base pair fragment present in the transgene, but not in the
endogenous Wnt1 gene. Wnt1 transgenic animals
were sacrificed when tumors were 1 cm in diameter, and wild type
littermates were simultaneously sacrificed. Tumors and mammary glands
were snap-frozen in liquid nitrogen and stored at Protein Analysis--
Transfected 293 cells were lysed, and
lysates analyzed for expression of Myc-epitope-tagged proteins and
ERK2, using 9E10 monoclonal and 122 polyclonal antibodies,
respectively, as previously described (40). Cox-2 protein in mammary
glands and tumors was assayed using a coupled
immunoprecipitation/immunoblotting assay. 10 mg of mammary gland or
tumor tissue was sonicated in 1 ml of RIPA buffer, and the sonicate was
centrifuged at 10,000 × g for 10 min at 4 °C. The
resulting supernatant was precleared by incubation at 4 °C with goat
IgG, rabbit IgG, and Protein-G-PLUS agarose (Santa Cruz Biotechnology
Inc.). Cox-2 protein was immunoprecipitated by incubation at 4 °C
for 1 h with 10 µl each of rabbit polyclonal and goat polyclonal
anti-Cox-2 antibodies (Oxford Biomedical Research, Inc. and Santa Cruz
Biotechnology Inc., respectively), followed by an additional 16-h
incubation after addition of 20 µl of Protein A-agarose. Beads were
recovered by centrifugation (5 min, 3,000 × g,
4 °C), and washed 4 times with RIPA buffer prior to resuspension in
Laemmli sample buffer. Cox-2 protein was detected by Western blotting
after running the immunoprecipitates on SDS-polyacrylamide gels as
described previously (13).
RNA Preparation and Northern Blotting--
RNA was prepared from
confluent cells and from mammary glands and tumors using RNAzol B
(Tel-Test, Inc.) according to the manufacturer's instructions. Frozen
tissues were finely minced prior to homogenization in RNAzol B. Northern blot analysis was performed as described previously using
MOPS/formaldehyde gels (13).
Plasmids--
Northern probes used were as follows: human COX-2
(S. M. Prescott, University of Utah, Salt Lake City, UT),
murine PEA3 (41), murine ERM (42), murine ER81 (42), and GAPDH (13).
The COX-2 promoter reporter construct COX-2-LUC contained
nucleotides Transient transfections were performed to determine whether the
COX-2 promoter was susceptible to regulation by -Catenin stabilization is a consequence
of both APC mutation and Wnt signaling. We have previously
observed coordinate regulation of the matrilysin promoter by
-catenin and Ets family transcription factors of the PEA3 subfamily.
Here we show that while
-catenin only weakly activates the
COX-2 promoter, PEA3 family transcription factors are
potent activators of COX-2 transcription. Consistent with this, PEA3 is up-regulated in Wnt1-expressing
mouse mammary epithelial cells, and PEA3 factors are highly expressed
in tumors from Wnt1 transgenic mice, in which
Cox-2 is also up-regulated. Promoter mapping experiments
suggest that the NF-IL6 site in the COX-2 promoter
is important for mediating PEA3 responsiveness. The NF-IL6 site is also
important for COX-2 transcription in some colorectal cancer
lines (Shao, J., Sheng, H., Inoue, H., Morrow, J. D., and DuBois,
R. N. (2000) J. Biol. Chem. 275, 33951-33956),
and PEA3 factors are highly expressed in colorectal cancer cell lines. Therefore, we speculate that PEA3 factors may contribute to the up-regulation of COX-2 expression resulting from both
APC mutation and Wnt1 expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, and consequently
transcriptional activation by
-catenin·TCF1 complexes
(2, 3). Additionally,
-catenin may induce TCF-independent transcription (4-6). Inappropriate activation of the Wnt signaling pathway has been detected in numerous tumors, arising as a consequence of Wnt gene misexpression, APC mutation, or
mutation of other components of the pathway such as axin and
-catenin itself (7). Multiple transcriptional targets of Wnt
signaling have now been identified, some of which are likely to
contribute to tumorigenesis. Of these, several have been demonstrated
to be directly activated by
-catenin, including cyclin D1,
c-myc, matrilysin, and peroxisome proliferator
activated receptor
(8-12). In addition, we have shown
transcriptional up-regulation of Cox-2 in
Wnt1-expressing mouse mammary epithelial cells (13), but did
not determine whether this was due to direct regulation of the
Cox-2 promoter by
-catenin.
-catenin might regulate the COX-2 promoter. Here we
examine the effect of both
-catenin and Ets family transcription
factors of the PEA3 subfamily on COX-2 promoter activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C prior to use.
1432/+59 of the human COX-2 promoter linked to
luciferase (43). In addition, the following truncated COX-2
promoter constructs were used:
327/+59,
220/+59,
124/+59, and
52/+59 (43). Constructs were also utilized in which mutations had
been introduced into the
327/+59 backbone. KBM, ILM and CRM have
mutagenized NF-
B (
223/
214), NF-IL6 (
132/
124), and CRE
(
59/
53) sites, respectively (43). The stromelysin-1 promoter
construct p754TR-Luc (42) was used to compare activation by Ets factors
with that of the COX-2 promoter. p754TR-Luc was co-transfected with
expression vectors encoding Ets factors plus c-Jun, since Ets
factors alone were insufficient to induce stromelysin-1 promoter
activity. The TOPFLASH vector, an artificial
-catenin·TCF-responsive promoter reporter (44), was used to
confirm that overexpressed
-catenin could drive transcription. The
following expression vectors were used: pMT23
-catenin (myc-tagged;
45), pCANmycPEA3 (42), pcDNA-ER81 (42), pCANmycERM (42), Ets-1 (S. Hiebert, Vanderbilt University, Nashville, TN), pSG5-Ets-2 (D. Watson, Medical University of South Carolina, Charleston, SC),
pCMX-c-jun (R. Wisdom, Vanderbilt University), and pCMV-LIP, encoding
dominant negative C/EBP
(46, 47). In addition, expression vectors encoding C/EBP
, C/EBP
, and C/EBP
were obtained from S. McKnight (University of Texas, Southwestern, TX).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin. The 293 human embryonic kidney cell line was selected for these experiments based on ease of transfection (4). 293 cells were co-transfected with a
-catenin expression vector and with COX-2-LUC, a luciferase reporter construct containing 1.4 kilobase pairs of the
human COX-2 promoter (43). In addition, we tested the effect
of the Ets family transcription factor PEA3 since we had previously
observed synergistic activation of another target gene, matrilysin, by
PEA3 and
-catenin (42). Overexpression of
-catenin stimulated the
activity of TOPFLASH, an artificial
-catenin/TCF-responsive promoter
reporter (44), by 5-10-fold (data not shown). However, we observed
only very weak activation of the COX-2 promoter construct COX-2-LUC by
-catenin (Fig.
1A). The mean increase
observed in six experiments in response to
-catenin was 28%
(p < 0.03). In contrast, PEA3 activated COX-2-LUC up
to 20-fold (Fig. 1, A and B). Together PEA3 and
-catenin elicited a greater than additive response in some
experiments (Fig. 1A), although the observed trend was not
statistically significant.
View larger version (10K):
[in a new window]
Fig. 1.
Effects of -catenin
and PEA3 on COX-2 promoter regulation.
A, effect of PEA3 and
-catenin on COX-2
promoter activity. 293 cells were transfected with combinations of
expression vectors encoding
-catenin and PEA3, together with a
COX-2 promoter luciferase reporter construct (COX-2-LUC),
and with pRL-TK as an internal control. Luciferase activities were
measured as described under "Experimental Procedures." Results
shown are the mean + S.D. of six replicates from a
representative experiment, expressed relative to activity in control
cells transfected with empty expression vectors. B, PEA3
dose dependence of COX-2 promoter activation. 293 cells were
transfected with increasing amounts of PEA3 expression vector, plus
COX-2-LUC and pRL-TK, and luciferase assays were performed as described
above. Results shown are the mean ± S.D. of 6 replicates.
One potential explanation for the contrasting potencies of PEA3 and
-catenin in activating the COX-2 promoter could be
different expression levels of the two proteins. To directly compare
expressions levels, lysates were prepared from 293 cells transfected
with expression vectors for PEA3 or
-catenin (both of which were Myc epitope-tagged) and analyzed by Western blotting with anti-Myc epitope
antibody 9E10. On this basis, PEA3 was expressed at 2-4-fold higher
levels than
-catenin (Fig. 2).
However, transfection of 50-fold less PEA3 than used in this experiment
was sufficient to potently activate the COX-2 promoter (Fig.
1B), and it therefore seems unlikely that this small
difference in expression levels is sufficient to explain the
differential responsiveness of the COX-2 promoter to
-catenin and PEA3.
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We also examined the response of the COX-2 promoter to the
Ets factors Ets-1 and Ets-2, in comparison with the PEA3 subfamily members PEA3, ER81, and ERM. Of these, PEA3 was the most potent activator of the COX-2 promoter (Fig.
3). ER81 and ERM were also capable of
inducing significant activation, in contrast with the weaker responses
elicited by Ets-1 and Ets-2. It was not possible to compare expression
levels of the various factors directly, since the cDNAs were not
uniformly epitope-tagged. However, since Ets-2 caused much stronger
activation of the stromelysin-1 promoter than did PEA3 in the same
experiment, we conclude that the COX-2 promoter is
preferentially responsive to PEA3 family members (Fig. 3). Strikingly,
PEA3 alone was also sufficient to activate transcription of the
endogenous COX-2 gene. Transient transfection of PEA3 into 293 cells caused accumulation of COX-2 transcript (Fig.
4). In contrast, -catenin alone did
not stimulate significant transcription of the endogenous
COX-2 gene, nor did it enhance the response to PEA3,
consistent with the data obtained using the COX-2 promoter reporter construct (Fig. 1A).
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Since we had previously observed Cox-2 up-regulation in
Wnt1-expressing cell lines (13), we were interested to
determine whether PEA3 factors could play a role in this up-regulation. In particular, we had observed significantly increased Cox-2
transcript in response to Wnt1 in C57MG cells, a mouse mammary
epithelial cell line which undergoes Wnt1-induced morphological
transformation (48). Therefore PEA3 transcripts were
measured in C57MG-derived cell lines, and also in mammary tumors from
Wnt1 transgenic mice. We observed up-regulated
PEA3 expression in C57MG cells in response to
Wnt1 expression (Fig.
5A). This observation is
consistent with the Cox-2 up-regulation observed in these
cells, and suggests a role for PEA3 in Cox-2 promoter
regulation in these cells. To test this, the effects of PEA3 and
-catenin on COX-2 promoter activity in C57MG cells were
compared. PEA3 overexpression increased COX-2
promoter activity to 205% (p < 0.03; Fig.
6). A smaller degree of activation was
observed in response to
-catenin in several experiments, but was not
statistically significant. These experiments were limited by the poor
transfection efficiency associated with C57MG cells (4). Nevertheless,
our data suggest that, both in 293 and C57MG cells (Figs. 1 and 6),
PEA3 is a more potent activator of the COX-2 promoter than
-catenin.
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In addition to being up-regulated in Wnt1-expressing C57MG cells, PEA3 was highly expressed in all tumors examined from Wnt1 transgenic mice (Figs. 5, B and C), contrasting with the very low expression level in normal virgin mammary gland (41) (Fig. 5B). ERM was also highly expressed in all Wnt1 tumors tested, while no expression was detected in wild type mammary gland (Fig. 5D; data not shown). Comparison of all three factors revealed that, in contrast to PEA3 and ERM, ER81 was expressed at a relatively low level in the tumors (Fig. 5E, data not shown), albeit at higher levels than in virgin mammary gland (Fig. 5F).
Our observation of high level expression of PEA3 factors in tumors from
Wnt1 transgenic mice, coupled with the demonstration that
PEA3 activates the COX-2 promoter, prompted us to test
whether Cox-2 was also up-regulated in
Wnt1-expressing mammary tumors. Levels of Cox-2 protein were
compared in normal mammary glands from wild type mice and in tumors
from Wnt1 transgenic mice. We observed a significant
increase in Cox-2 protein in the three tumors tested compared with
virtually undetectable basal levels in wild type mammary gland (Fig.
7). These data suggest that increased expression of PEA3 factors may contribute to Cox-2
up-regulation in these tumors.
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In order to map the PEA3-responsive elements in the COX-2
promoter, a range of promoter reporter constructs were used in which deletions or site-specific mutations had been introduced (43). Progressive truncation of the promoter sequentially deletes binding sites for NF-B, NF-IL6, and finally the CRE (Fig.
8A). No significant diminution
in PEA3 responsiveness was observed until residues
220 to
125 were
deleted (Fig. 8B). The
124/+59 construct was virtually
unresponsive to PEA3, suggesting that the PEA3 response element(s) lie
between
220 and
125. Since an NF-IL6-binding site is present in
this region of the promoter, we next tested the responsiveness to PEA3
of various site-specific promoter mutants, including the ILM construct
in which the NF-IL6 site is mutagenized. This experiment was primarily
intended to rule out involvement of this site in the PEA3 response.
However, to our surprise, we found that mutation of the NF-IL6 site
completely and specifically abolished both basal and PEA3-stimulated
COX-2 promoter activity (Fig.
9). In contrast, mutation of the NF-
B
site had no effect on PEA3 responsiveness. Mutation of the CRE site
reduced both basal and PEA-3-stimulated activity, such that the index
of stimulation exhibited by the CRM construct was not reduced relative
to the wild type construct. Collectively, these data strongly implicate the NF-IL6 site in mediating PEA3 responsiveness. Since we have previously found mutation of the NF-IL6 site to have little effect on
COX-2 promoter responsiveness to the lipid ceramide (49), we
believe these data to reflect a specific involvement of the NF-IL6 site
in mediating PEA3 responsiveness.
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The NF-IL6 site is a consensus binding site for transcription factors
of the C/EBP family. Several C/EBP factors have been identified (50).
C/EBP is generally associated with differentiation, while C/EBP
and C/EBP
are primarily implicated in mediating gene activation
during inflammation and cell proliferation. Our data demonstrating the
importance of the NF-IL6 site for PEA3 responsiveness suggested that
C/EBP factors might be involved in PEA3-mediated activation of the
COX-2 promoter. To test this hypothesis we used several
C/EBP expression constructs, including a dominant negative variant, LIP
(liver-enriched inhibitory protein). Consistent with previous
observations in other cell types (43, 51), both C/EBP
and C/EBP
stimulated COX-2 promoter activity, while C/EBP
had
little effect on basal promoter activity (Fig. 10A). None of the C/EBP
isoforms tested enhanced the response to PEA3 (data not shown).
However, LIP, which functions as a dominant negative C/EBP due to the
absence of a transactivation domain (47), caused
dose-dependent inhibition of the PEA3 response (Fig.
10B). This inhibitory effect of LIP suggests that C/EBP
factors may be involved in mediating COX-2 promoter
responsiveness to PEA3 (see "Discussion").
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DISCUSSION |
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Transcriptional up-regulation of COX-2 has previously
been observed under conditions of nuclear -catenin accumulation,
such as in intestinal tumors and Wnt1-expressing cell lines.
The ability of
-catenin to activate transcription in complex with
other transcription factors has led to speculation that
-catenin
might directly regulate the COX-2 promoter. As an initial
test of this hypothesis, we examined the regulation of COX-2
promoter activity by overexpressed
-catenin. We also tested the
potential role of PEA3 factors, since we had previously observed
coordinate regulation by
-catenin and PEA3 of the matrilysin
promoter (42). We observed that
-catenin caused only very weak
activation of a COX-2 promoter reporter construct (Figs.
1A and 6). Although it remains possible that there are
-catenin-responsive elements lying upstream of the 1.4-kilobase pair
promoter sequence used, our data are consistent with the recent report
by Haertel-Wiesmann et al. (52), who found Wnt-3A-mediated
Cox-2 induction to be resistant to antisense
-catenin
oligonucleotides. Furthermore, expression of
-catenin in 293 cells
was insufficient to activate transcription from the endogenous
COX-2 gene (Fig. 4). These data suggest that
COX-2 is not a direct target of
-catenin, and raise the
possibility that
-catenin may activate COX-2 via
up-regulation of an intermediary transcription factor.
In contrast to the weak response to -catenin, PEA3 and the related
factors ER81 and ERM potently activated the COX-2 promoter (Figs. 1 and 3). The degree of activation was such that activation of
the endogenous COX-2 gene could be achieved simply by
PEA3 overexpression (Fig. 4). To our knowledge, this is the
first report of COX-2 promoter activation in response to any
Ets family transcription factor. Previously, multiple transcription
factor binding motifs have been implicated in regulation of the
COX-2 promoter. Thus, both NF-IL6 and CRE sites are
important for Cox-2 induction in mouse mast cells, murine
macrophages, and bovine endothelial cells in response to IgE receptor
aggregation, lipopolysaccharide, and phorbol ester/lipopolysaccharide,
respectively (43, 53, 54). In contrast, tumor necrosis factor-
stimulation of a mouse osteoblastic cell line activates
Cox-2 via the NF-IL6 and NF-
B sites (55), while the CRE
site appears critical for Cox-2 transcription in response to
v-Src, platelet-derived growth factor, serum, and ceramide (49, 56,
57). COX-2 can also be regulated post-transcriptionally by mRNA
stabilization (58-60).
In this study, we found the NF-IL6 site to be essential for
PEA3-mediated COX-2 promoter induction (Figs. 8 and 9).
C/EBP and C/EBP
, but not C/EBP
, caused modest stimulation of
the COX-2 promoter (Fig. 10A), as has previously
been reported for other cell types (43, 51), but overexpression of wild
type C/EBPs did not enhance PEA3 responsiveness. In contrast, a
dominant negative C/EBP diminished both basal and PEA3-stimulated
promoter activity (Fig. 10B). Several models can be proposed
to explain these data. PEA3 might bind directly to the NF-IL6 site, in
which case the truncated dominant negative C/EBP protein could be
antagonistic via competition with PEA3 for the NF-IL6 site. However,
the NF-IL6 site does not resemble a consensus PEA3 site
(5'-CCGGA(A/T)GC-3') (61). Alternatively, simultaneous binding of a
C/EBP factor to the NF-IL6 site and of PEA3 to a cognate binding site
might be required for activation. Several potential Ets-binding sites are present in the minimal PEA3-responsive fragment of the
COX-2 promoter (Fig. 11),
although none corresponds exactly to a consensus PEA3 site. Precedents
for synergistic transcriptional activation by Ets factors and C/EBP
factors have previously been described (62, 63). Thus it seems likely
that binding of C/EBP and PEA3 to proximal sites may be required for
induction of COX-2 transcription in our system. However, we
have not excluded the possibility that PEA3 might bind to C/EBP
proteins at the NF-IL6 site without itself binding to DNA.
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As discussed above, NF-IL6 sites in the COX-2 promoter have previously been implicated in the response to multiple agents (43, 53-55). Interestingly, NF-IL6 sites are also implicated in COX-2 up-regulation in mouse skin tumors (51), and mutagenesis of the NF-IL6 site reduces COX-2 promoter activity in the human colon cancer cell lines HCA7 and LS-174 (64). This latter result is particularly intriguing since we have previously detected high level PEA3 expression in intestinal tumors and colorectal cancer-derived cell lines (42), and have also observed PEA3-induced COX-2 promoter activation in colorectal cancer cell lines.2 Thus PEA3-mediated regulation of the COX-2 promoter via the NF-IL6 element may explain the importance of this site in human colorectal cancer lines (64). The correlation between PEA3 expression and Cox-2 expression both in intestinal tumors and in Wnt1-expressing cells and tumors (Figs. 5 and 7) suggests that PEA3 contributes to COX-2 up-regulation in response to both Wnt1 expression and APC mutation.
The coincidence of PEA3 expression in intestinal tumors
resulting from APC mutation and in mammary tumors caused by
Wnt1 expression suggests that PEA3 up-regulation is a common
consequence of activation of the Wnt/-catenin signaling pathway.
This raises the intriguing possibility that PEA3 itself may be a target
of
-catenin. To address this directly, the effect of
-catenin on
a PEA3 promoter reporter construct has been assayed.
Preliminary data indicate that
-catenin overexpression in Cos cells
can stimulate PEA3 promoter activity up to
5-fold.3 If PEA3
transcription can be regulated by
-catenin, it is unclear why
ectopic expression of
-catenin was insufficient to induce COX-2 transcription in 293 cells, when PEA3 was sufficient
(Figs. 1 and 4). One potential explanation could be differing PEA3
expression levels achieved by transfection with
-catenin and PEA3.
Since PEA3 is known to positively regulate its own transcription (65), ectopic PEA3 expression may lead to much greater levels of PEA3 expression than can be achieved in the same time frame by expression of
-catenin. Interestingly, the PEA3 promoters of human, mouse, and
chicken all contain a TCF-binding site, conserved in both sequence and
position relative to the transcriptional start site,3
suggesting that PEA3 is most likely a direct target of
-catenin.
PEA3 family members have recently been shown to regulate the promoter
of another gene, matrilysin, in synergy with -catenin (42). Aberrant
matrilysin expression is detected in intestinal tumors, and matrilysin
is also up-regulated in Wnt1-expressing mammary cell lines
and tumors.4 Thus both
COX-2 and matrilysin are responsive to Wnt
signaling and are regulatable by PEA3 factors. In addition, we have
recently observed synergistic activation by
-catenin and PEA3 of the
murine Twist
gene.5 Finally, we note that
the promoters of several genes known to be
-catenin-responsive also
contain consensus Ets-binding sites. For example, the
Drosophila gene Even-skipped is coordinately regulated via TCF and Ets-binding sites (66), and the cyclin D1 promoter can be activated cooperatively by PEA3,
-catenin, and c-Jun.6 Together these
data lead us to speculate that PEA3 factors may contribute to
regulation of several target genes of the Wnt/
-catenin pathway.
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ACKNOWLEDGEMENTS |
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We thank John Timmer for helpful discussions and Jay Patel for technical assistance. We thank L. Sealy, S. M. Prescott, T. Tanabe, A. Ashworth, R. Kypta, S. Hiebert, R. Wisdom, S. McKnight, H. Clevers, and D. Watson for gifts of plasmids.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants CA47207 (to A. M. C. B.) and CA89578 (to A. J. D.), United States Department of the Army Grant DAMD17-98-1-8057 (to A. J. D.), and a grant from the Irving Weinstein Foundation (to L. R. H.).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: Strang Cancer Research Laboratory, Rockefeller University, Box 231, 1230 York Ave., New York, NY 10021. Tel.: 212-734-0567; Fax: 212-472-9471; E-mail: lrhowe@med.cornell.edu.
** Supported by American Cancer Society Pilot Project Grant IRG-58-009-41.
¶¶ Supported by the Canadian Institutes of Health Research and the Canadian Breast Cancer Research Initiative.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M010692200
2 H. C. Crawford and L. R. Howe, unpublished data.
3 C. Messier and J. A. Hassell, unpublished data.
4 L. R. Howe, O. Watanabe, J. Leonard, and A. M. C. Brown, unpublished data.
5 L. R. Howe and H. C. Crawford, manuscript in preparation.
6 C. Messier, H. C. Crawford, and J. A. Hassell, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
TCF, T cell factor;
C/EBP, CCAAT/enhancer-binding protein;
COX, cyclooxygenase;
CRE, cyclic
AMP response element;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
LIP, liver-enriched inhibitory protein;
MG, mammary glands;
NF-IL6, nuclear factor interleukin-6;
NF-B, nuclear factor-
B;
MOPS, 4-morpholinopropanesulfonic acid.
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