From the Departments of Surgery and
§ Physiology and Biophysics, University of Texas Medical
Branch, Galveston, Texas 77555
Received for publication, February 27, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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Colorectal carcinogenesis is a complex, multistep
process involving genetic alterations and progressive changes in
signaling pathways regulating intestinal epithelial cell proliferation, differentiation, and apoptosis. Although cyclooxygenase-2 (COX-2), gastrin-releasing peptide (GRP), and its receptor, GRP-R, are not
normally expressed by the epithelial cells lining the human colon, the
levels of all three proteins are aberrantly overexpressed in
premalignant adenomatous polyps and colorectal carcinomas of humans.
Overexpression of these proteins is associated with altered epithelial
cell growth, adhesion, and tumor cell invasiveness, both in
vitro and in vivo; however, a mechanistic link
between GRP-R-mediated signaling pathways and increased COX-2
overexpression has not been established. We report that
bombesin, a homolog of GRP, potently stimulates the expression
of COX-2 mRNA and protein as well as the release of prostaglandin
E2 from a rat intestinal epithelial cell line engineered to
express GRP-R. Bombesin stimulation of COX-2 expression requires an
increase in [Ca2+]i, activation of extracellular
signal-regulated kinase (ERK)-1 and -2 and p38MAPK, and
increased activation and expression of the transcription factors Elk-1,
ATF-2, c-Fos, and c-Jun. These data suggest that the expression of
GRP-R in intestinal epithelial cells may play a role in carcinogenesis
by stimulating COX-2 overexpression through an activator
protein-1-dependent pathway.
Colorectal cancers are the third leading cause of cancer deaths in
the United States (1). One in 20 Americans is at risk of developing
this disease during their lifetime. Considerable experimental data have
accumulated indicating an important role for cyclooxygenase-2
(COX-2)1 in colorectal
carcinogenesis. COX-2 is a key enzyme in the biosynthesis of
prostaglandins from arachidonic acid and is overexpressed in 85-90%
of human colon cancers and 40-50% of premalignant adenomas (2).
Several large epidemiological studies have shown that mortality from
colorectal cancers decreases (40-50%) in persons who regularly take
aspirin or other nonsteroidal antiinflammatory drugs (3), which inhibit
COX activity. Additionally, experiments with adenomatous polyposis coli
(APC) gene-deficient mice (Min mice) revealed that inhibition of COX
activity with nonsteroidal antiinflammatory drugs resulted in a
reduction in the number and multiplicity of spontaneously formed tumors
(4-6), and APC Like COX-2, the mammalian homologue of bombesin (BBS),
gastrin-releasing peptide (GRP) and its cognate G-protein-coupled
receptor, GRP receptor (GRP-R), are aberrantly overexpressed in
premalignant adenomatous polyps and colorectal cancers. Preston
et al. (8) showed that 24% of colorectal cancers, but not
the adjacent nonmalignant mucosa, exhibited high affinity binding sites
for GRP. Immunohistological analysis of archival tissue specimens from
colonic polyps and colon cancers revealed that 42% (n = 5) of high grade dysplastic polyps and 62% (n = 50)
of colon cancers stained positively for both GRP and GRP-R protein (9).
We have found that 42% (n = 5) of freshly resected
adenomatous polyps and 67% (n = 12) of colorectal
cancers contain cytokeratin-positive cells that exhibit an increase in
the concentration of free intracellular Ca2+
([Ca2+]i) in response to BBS stimulation,
indicating the presence of functional BBS
receptor.2
Although the precise role of BBS-like peptides and GRP-R in colorectal
carcinogenesis has not been defined, recent observations that aspirin
inhibits BBS-induced DNA synthesis in Swiss 3T3 fibroblasts (10) and
GRP stimulates expression of COX-2 in the same cell line (11) have
raised the possibility that GRP-R-mediated signaling pathways may
contribute to the up-regulation of COX-2 expression during colorectal
carcinogenesis. The aims of our study were to examine whether the
expression of GRP-R leads to BBS-dependent up-regulation of
COX-2 in the rat intestinal epithelial cell line, RIE-1, and if so, to
determine the molecular signaling pathways linking GRP-R to the
regulation of COX-2 expression.
We selected the RIE-1 cell line for these studies for several reasons.
1) It is a nontumorigenic intestinal epithelial cell line, which, like
the normal human colonic epithelium, does not express endogenous GRP-R
or other BBS receptor subtypes. 2) Unlike many epithelioid cell lines
derived from cancer cells, the endogenous level of COX-2 expression,
under normal culture conditions, is very low. 3) Constitutive
overexpression of COX-2 in RIE-1 cells increases their tumorigenic
potential (12). 4) The aberrant overexpression of GRP-R and
COX-2 in premalignant adenomatous polyps suggests that these proteins
may play a role in the early stages of colon carcinogenesis.
To evaluate the potential role of GRP-R-mediated signaling pathways in
COX-2 gene expression, we developed RIE-1 cell lines expressing
recombinant GRP-R called RIE/GRPR. We found that the GRP-R agonist,
BBS, markedly stimulates COX-2 mRNA and protein expression as well
as the release of prostaglandin E2 (PGE2) from these cells. The increase in COX-2 expression is largely due to BBS-enhanced transcription of the COX-2 gene and is dependent on an
agonist-stimulated increase in [Ca2+]i,
activation of MAP kinase-dependent pathways, and the
increased expression and activation of activator protein-1 (AP-1)
transcription factor. These findings partially identify the signaling
pathways coupling GRP-R to the up-regulation of COX-2 expression and
identify the regulation of COX-2 gene expression as a potential
mechanism by which aberrantly expressed GRP-R plays a role in
colorectal carcinogenesis.
Plasmids--
The mouse GRP-R expression vector was a gift from
Dr. James F. Battey (National Institutes of Health, Bethesda, MD). The
mouse COX-2 promoter constructs, TIS10L-luc, TIS10-80-luc, and
TIS10-40-luc, as well as the mouse prostaglandin synthase-2
(COX-2) cDNA probe were kindly provided by Dr. Harvey R. Herschman
(UCLA, Los Angeles, CA). The reporter constructs
Gal4-Elk-(307-428), Gal-4-Sap-(268-431), and Gal4-luc were
gifts from Dr. Ralf Janknecht (The Salk Institute, La Jolla, CA).
c-fos-luc and 3XTRE-luc were provided by Dr. Johannes L. Bos
(University of Utrecht, Utrecht, The Netherlands) and Dr. Joan
Massagué (Howard Hughes Medical Institute, New York, NY), respectively. Mouse c-Fos and c-Jun cDNA probes were purchased from
the American Type Culture Collection (Manassas, VA).
Antibodies--
The anti-COX-2 antibodies were obtained from
Cayman Chemical (Ann Arbor, MI). The anti-active ERK-1 and -2 antibody
(pTEpY) was purchased from Promega (Madison, WI).
Anti-phospho-p38MAPK, anti-phospho-ELK-1, and
antiphospho-ATF-2 antibodies were obtained from New England Biolabs,
Inc. (Beverly, MA).
RIE/GRPR Cell Lines--
RIE-1 cells were a gift from Dr.
Kenneth D. Brown (Cambridge Research Station, Babraham, Cambridge, UK).
RIE-1 cells were transfected with mouse GRP receptor using
LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's recommendations. G418-resistant colonies were selected
as described previously (13). The number of binding sites
(Bmax) and their binding affinities (Kd) were determined using [125I]BBS
binding assays as described (14). Agonist-induced changes in
[Ca2+]i were detected using the
Ca2+-sensitive dye Fura-2/AM as previously described (15).
Cells were cultured at 37 °C in a humidified atmosphere of 95% air
and 5% CO2 in Dulbecco's modified Eagle's medium
supplemented with 5% heat-inactivated fetal bovine serum
(Hyclone, Logan, UT) and Geneticin (G418; 400 µg/ml; Life Technologies).
RNA Isolation and Northern Blot Analysis--
Total
cellular RNA was extracted by the method of Chomczynski and Sacchi
(16). RNA samples (30 µg/lane) were separated on 1.2%
agarose-formaldehyde gels and blotted onto Nytran plus filters (Schleicher and Shuell). The blots were hybridized with cDNA probes labeled with [ Western Blot Analysis--
Immunoblot analysis was performed as
described previously (13). The cells were lysed for 30 min in a
solution consisting of 1× PBS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, and 1 mM sodium
orthovanadate. Cellular proteins were denatured by heating, resolved by
SDS-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose membranes and probed with the indicated antibodies and
then with a peroxide-coupled second antibody (Promega). Proteins were
detected using the enhanced chemiluminescence system (ECL; Amersham
Pharmacia Biotech).
PGE2 Assay--
RIE/GRPR cells were plated in
24-well plates. Thirty-six hours later, the medium was replaced with
serum-free Dulbecco's modified Eagle's medium overnight. The cells
were then incubated with or without BBS for the indicated times. Media
were collected from each well and analyzed for PGE2 by
enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI).
Immunofluorescence Microscopy--
RIE/GRPR cells were cultured
in Lab-TekII chamber slides (Nalge Nunc International, Naperville, IL).
Prior to immunostaining with anti-COX-2 antiserum, the cells were
incubated with or without BBS (100 nM) for 6 h at
37 °C, fixed with 4% paraformaldehyde (15 min), permeabilized with
0.3% Triton X-100 (10 min), and incubated in blocking solution (1%
bovine serum albumin in phosphate-buffered saline, 20 min). After
incubating the cells with anti-COX-2 antiserum (1:400) for 90 min at
room temperature, the cells were washed three times with
phosphate-buffered saline and incubated with a goat anti-rabbit IgG
antibody labeled with Alexa 488 (Molecular Probes, Inc., Eugene, OR)
(1:2000, 30 min). Specific immunostaining was visualized with a Nikon
Eclipse fluorescence microscope.
Luciferase Assay--
RIE/GRPR cells were seeded in six-well
plates at a density of 2 × 105 cells/well. After
overnight adhesion, cells were transfected with 2 µg of
promoter/luciferase reporter gene DNA using Fugene 6 (Roche Molecular
Biochemicals). Prior to assaying for luciferase activity, cells were
incubated in Dulbecco's modified Eagle's medium without serum (24 h)
and treated with BBS for 6 h. Luciferase activity in 20 µl of
cell extract was assayed using the Luciferase Assay System (Promega,
Madison WI). Transfection efficiency was assessed using a
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared as previously described (18). An oligonucleotide (Stratagene,
La Jolla, CA) whose sequence corresponded to the AP-1 binding site
consensus sequence was end-labeled with [ Statistical Analysis--
All experiments were
repeated on at least two separate occasions. Results from Northern and
Western blots were quantified by densitometry. Values are expressed as
mean ± S.E. Differences between means were compared using the
analysis of variance test and were considered significantly different
at the level of p < 0.05.
RIE/GRPR Cells--
The RIE-1 cell line, isolated originally from
the rat small intestine, exhibits epithelioid morphology and the normal
rat diploid number of chromosomes, and the cells do not form
colonies in soft agar (19). These properties have made them one of the preferred cell models for examining nontumorigenic intestinal epithelial cell physiology and biochemistry in vitro.
Constitutive overexpression of recombinant COX-2 in RIE-1 cells is
associated with altered cell adhesion to extracellular matrix,
decreased expression of E-cadherin, increased expression of the
antiapoptotic gene product, BCL-2, and decreased apoptosis (12). To
determine whether BBS and GRP-R could regulate endogenous COX-2
expression in RIE-1 cells, cells were stably transfected with an
expression plasmid containing a mouse GRP-R cDNA downstream
of the constitutively active CMV promoter. After selection with G418,
surviving cell clones were evaluated for the level of receptor
expression and activity using radiolabeled ligand binding and
Ca2+-imaging with the calcium indicator dye Fura-2/AM,
respectively. Five clones were isolated with
Bmax values ranging from ~3000 to 8900 binding sites/cell. The calculated affinity constants exhibited a range
of values from 0.33 to 1.3 nM. For the remainder of the
studies, we used the RIE/GRPR cell line with an affinity constant for
BBS of 0.54 nM and Bmax values of
approximately 3000 receptors/cell. Fura-2 imaging experiments revealed
that greater than 99% of these cells exhibited an increase in
[Ca2+]i upon stimulation with BBS (1 nM).
BBS Stimulates COX-2 Expression and Activity--
BBS stimulated
time- and dose-dependent increases in the expression of
COX-2 mRNA and protein in RIE/GRPR cells. Compared with untreated
cells, the level of COX-2 mRNA increased in cells treated with BBS
(100 nM) by 3-, 20-, 15-, 8-, and 0.5-fold at 1, 2, 4, 8, and 24 h, respectively (Fig.
1A). The increase in COX-2
mRNA at 2 h was dependent on the concentration of BBS used to
stimulate the cells. Maximum increases in COX-2 mRNA levels were
detected in cells stimulated with 10, 100, and 1000 nM BBS (Fig. 1B). BBS treatment also stimulated a
time-dependent increase in the level of COX-2 protein.
Western blots revealed an increase in COX-2 protein by 1 h
following BBS (100 nM) stimulation and a peak in expression
at 6 h (Fig. 1C). Untreated RIE/GRPR cells showed no
detectable expression of COX-2 protein. Consistent with the Western
blot data, immunofluorescence staining showed an increase in COX-2
immunoreactivity in RIE/GRPR cells following stimulation with BBS (Fig.
1D, BBS), whereas untreated cells did not exhibit COX-2 immunoreactivity (Fig. 1D, Control).
COX converts arachidonic acid, released from phospholipid stores by the
action of phospholipase A2, to prostaglandin
H2, the common precursor of all prostaglandins. To assess
whether increased COX-2 expression was associated with an increased
prostaglandin synthesis, the levels of PGE2 released from
RIE/GRPR cells were measured using an enzyme-linked immunosorbent
assay. Compared with untreated control cultures, PGE2
levels in the media of RIE/GRPR cells treated with BBS increased
by 6.8-fold at 1 h and continued to increase to 45-fold at 24 h (Fig. 1E).
Increases in [Ca2+]i and
Mitogen-activated Protein Kinase (MAPK) Activity Mediate BBS Regulation
of COX-2 Expression--
Agonist binding to GRP-R initiates the
activation of intracellular signaling pathways (20, 21) involving
specific heterotrimeric G-proteins (22, 23); generation of the second
messengers inositol 1,4,5-trisphosphate and diacylglycerol; release of
Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores
(15); and activation of various protein kinases including
protein kinase C (15), protein kinase D (24), the Src family of
nonreceptor tyrosine kinases (25), and the MAPK cascades (26).
BBS stimulation of RIE/GRPR cells induced a rapid increase in
[Ca2+]i (Fig.
2A). To assess the role of
[Ca2+]i in BBS regulation of COX-2 mRNA
expression, cells were pretreated for 1 h with the
membrane-permeable chelating agent BAPTA-AM (30 µM).
Treatment with the chelator was sufficient to inhibit the BBS-induced
increase in [Ca2+]i (Fig. 2A) and
COX-2 mRNA expression (Fig. 2B) but did not affect
either cell viability, measured by trypan blue exclusion assay
(percentage of blue cells was as follows: vehicle (0.1%
Me2SO), 3.56 ± 0.79% versus BAPTA-AM,
3.23 ± 0.67%), or alter the expression of 18 S ribosomal RNA.
Together, these data indicate that an agonist-induced increase in
[Ca2+]i is required for BBS-stimulated increases
in COX-2 mRNA levels in RIE/GRPR cells.
Mitogen-activated protein kinase pathways mediate the regulation of
COX-2 expression to a variety of extracellular stimuli (27-29). Three
related MAPK cascades have been described (30, 31); they are referred
to as the ERK pathway, the c-Jun N-terminal kinase (JNK) pathway, and
the p38MAPK pathway. The activities of MAPKs are regulated
by upstream dual specificity MAPK kinases (MEKs). MEKs activate MAPKs,
such as ERKs, JNK, and p38MAPK, by phosphorylation on both
threonine and tyrosine residues. To determine whether BBS activated
MAPK pathways in RIE/GRPR cells, the levels of phosphorylated ERK,
p38MAPK, and JNK proteins were determined by immunoblotting.
Bombesin treatment stimulated the activation of the two ERK isozymes,
ERK-1 and -2, as well as p38MAPK (Fig. 2, C and
D) but did not activate JNK (data not shown). Western blots
of RIE/GRPR cell extracts, probed with antibodies selective for the
phosphorylated (activated) forms of ERK-1 and -2, showed that BBS
induced a time-dependent increase in phosphorylated ERK-1
and -2 (Fig. 2C). ERK-1 and -2 phosphorylation was increased 1 min after BBS treatment and reached a peak at 10 min before returning
to base line at 15 and 30 min. A second, smaller increase in ERK-1 and
-2 activation was detected at 60 min after BBS stimulation (Fig.
2C). The active status of ERK-1 and -2 was confirmed by in vitro phosphorylation experiments using
immunoprecipitated ERK-1 and -2 and the substrate, myolin basic
protein. A 5-fold increase in myolin basic protein phosphorylation was
observed when using immunoprecipitated proteins from RIE/GRPR cells
treated for 10 min with BBS versus immunoprecipitated
proteins from untreated cultures (data not shown). In addition to ERK
activation, BBS stimulated a time-dependent activation of
p38MAPK. An increase in the phosphorylated form of
p38MAPK was detected 5 min following BBS treatment (Fig.
2D). In contrast to the transient activation of ERK-1 and
-2, p38MAPK phosphorylation reached a maximum by 5 min and
remained elevated up to 60 min after agonist stimulation (Fig.
2D).
To determine whether MAPK activation was required for BBS-induced
increases in COX-2 mRNA levels, cells were pretreated with selective inhibitors of MEK (PD98059) and p38MAPK
(SB203580). PD98059 (10 µM) and SB203580 (5-20
µM) significantly, but incompletely, inhibited the
BBS-stimulated increases in COX-2 mRNA levels (Fig. 2, E
and F). Although the indicated concentrations of PD98059 and
SB203580 were sufficient to completely inhibit agonist-dependent kinase activation, the inhibition by
either compound alone was insufficient to completely block the
BBS-stimulated increases in COX-2 mRNA levels, suggesting that both
MEK/ERK and p38MAPK-dependent pathways are
partially involved in GRP-R-mediated regulation of COX-2 mRNA levels.
BBS-stimulated COX-2 Promoter Activity--
COX-2 expression is
regulated by both transcriptional and posttranscriptional mechanisms
(32-35). To determine whether BBS regulated COX-2 promoter activity,
RIE/GRPR cells were transfected with different size fragments of the
mouse COX-2 promoter coupled to a luciferase reporter gene. BBS (100 nM) induced a 3.6-fold increase in luciferase activity in
cells transiently expressing TIS10L-luc (positions BBS Activates AP-1 Transcription Factor Regulating COX-2
Expression--
The COX-2 promoter contains multiple potential
cis-activating regulator elements. To date, CRE, E-box,
NF-IL6 (C/EBP
Expression of the c-fos and c-jun genes is
regulated, in part, by ternary complex factors and ATF-2, respectively.
Ternary complex factors belong to the ets domain family of
DNA-binding proteins, which includes Elk-1, Sap1, and Sap2.
Phosphorylation of Elk-1 by MAPKs increases its ability to form
complexes with serum response factor and results in serum response
element-dependent activation of the c-fos
promoter (47, 48). The phosphorylation of ATF-2 by p38MAPK
increases TPA response element-dependent transcriptional
activity of c-jun (48). To determine whether these
transcriptional factors were involved in BBS signaling, we examined the
effect of BBS on their phosphorylation state using antibodies directed
against the phosphorylated (activated) forms of Elk-1 and ATF-2. An
increase in phosphorylated Elk-1 and ATF-2 was detected 5 and 15 min
after BBS treatment and continued for 60 and 30 min, respectively (Fig. 4, A and B).
Additionally, BBS increased the promoter activities of Elk-1 and Sap-1
by 12- and 9-fold, respectively (Fig. 4C).
To assess the role of AP-1 activation in agonist-stimulated COX-2
expression, we treated RIE/GRPR cells with diferulolymethane (curcumin). Curcumin is an inhibitor of AP-1 binding (49-51). RIE/GRPR cells were preincubated with or without curcumin for 1 h and then stimulated with BBS (100 nM) for an additional 2 h.
BBS-induced increases in COX-2 mRNA levels were inhibited in a
dose-dependent manner by curcumin (Fig.
5A), with complete inhibition
at 10 µM. In addition, we found that 10 µM
curcumin completely inhibited BBS-stimulated increases in AP-1 binding
activity (Fig. 5B). Together, these data demonstrate that
BBS stimulation of AP-1 binding is an important intermediate in its
regulation of COX-2 gene expression in the intestinal epithelial cell
line, RIE/GRPR.
The aberrant overexpression of COX-2, BBS-like peptides, and GRP-R
has been demonstrated in various carcinomas, including lung,
pancreatic, gastric, breast, prostate, and colorectal carcinomas (2,
36, 37, 52-61). While a growing body of experimental evidence suggests
that COX-2 plays an important role in the development of colorectal
carcinogenesis, little is known about the molecular mechanisms leading
to its up-regulation. Recent data from mouse Swiss 3T3 fibroblasts
showing that GRP-R activation results in increased COX-2 (11) and
aspirin inhibits BBS-stimulated DNA synthesis (10) expression suggest
that the aberrant overexpression of GRP-R and COX-2 in some adenomatous
polyps and colorectal cancers may be more than coincidental. To
evaluate potential mechanistic links between GRP-R-mediated signaling
pathways and the regulation of COX-2 expression in an intestinal
epithelial cell line, we developed the RIE/GRPR cell lines. In this
cell model, we found that the GRP-R agonist, BBS, is a potent
stimulator of COX-2 expression. The data presented in this report allow
us to partially define the temporal sequence of molecular events
involved in BBS stimulation of COX-2 expression in RIE/GRPR cells (Fig.
6A). Agonist binding to GRP-R
stimulates a rapid and transient increase in
[Ca2+]i, followed by the slower, transient
activation of the MAPKs: ERK-1 and -2 and p38MAPK. The
activation of both ERKs and p38MAPK occurred within 1 min
of BBS stimulation. The levels of phosphorylated ERK-1 and -2 returned
to base line by 15 min, whereas p38MAPK remained activated
for up to 60 min. A second smaller increase in ERK activity was
observed at 60 min. Subsequent to ERK activation, but within the period
of elevated p38MAPK, the levels of c-Fos and c-Jun mRNA
increased. The increases in c-Fos and c-Jun mRNA were preceded by
increased activation (phosphorylation) of the transcription factors
Elk-1 and ATF-2, regulators of the serum response element and TPA
response element transcriptional elements, respectively. The observed
temporal sequence of changing gene expression and protein activation,
coupled with the effects of various selective inhibitors, suggests the model of GRP-R-mediated regulation of COX-2 gene expression depicted in
Fig. 6B.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
716/COX-2 double-knockout
mice showed reduction in both the neoplastic growth and number of
intestinal tumors (7). Although mounting evidence supports an important
role for COX-2 in colorectal carcinogenesis, the molecular mechanisms
leading to COX-2 overexpression in intestinal epithelial cells are not
completely understood.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP by random primer extension.
Specific hybridization was visualized by autoradiography. To ensure RNA
integrity and to confirm equal loading between lanes, the filters were
stripped and rehybridized with a probe for 18 S rRNA.
-galactosidase expression plasmid as described previously (17).
-32P]ATP and
T4 polynucleotide kinase. Electrophoretic mobility shift assay reaction
mixtures contained 50,000 cpm of 32P-end-labeled
oligonucleotide, 20 µg of nuclear protein extract, and 1.0 µg of
poly(dL·dC) (Amersham Pharmacia Biotech) in a final volume of 20 µl. Reaction mixtures were resolved on 4% nondenaturing polyacrylamide gel electrophoresis at 200 V for 2 h. Gels were dried and visualized by autoradiography (17).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BBS stimulated an increase in COX-2
expression and activity in RIE/GRPR cells. A, time
course of COX-2 mRNA expression. Subconfluent, serum-starved
RIE/GRPR cells were treated with BBS (+) (100 nM) for the
indicated time, and the steady-state levels of COX-2 mRNA
expression were determined by Northern blot. B, dose effect.
RIE/GRPR cells were treated with the indicated concentration of BBS for
2 h, and the steady-state levels of COX-2 mRNA were assessed
by Northern blot. C, time course of COX-2 protein
expression. RIE/GRPR cells were incubated with BBS (100 nM), lysed in RIPA buffer at the indicated times, and
analyzed by Western blot as described under "Experimental
Procedures." D, immunofluorescence staining for COX-2.
RIE/GRPR cells were cultured on glass-covered slides. After serum
starving for 24 h, cells were stimulated with BBS
(lower panel, BBS) for 6 h,
fixed, and immunofluorescence-stained as described under
"Experimental Procedures." Untreated cultures are shown in the
upper panel (Control). E,
BBS stimulated the release of PGE2 from RIE/GRPR cells. The
cells were plated in 24-well plates for 36 h, serum-starved for
24 h, and incubated with or without BBS (100 nM) for
1-24 h. Medium in each well was collected and analyzed for
PGE2 levels by enzyme-linked immunosorbent assay.
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Fig. 2.
Bombesin stimulation of COX-2 expression
requires an increase in [Ca2+]i and the
activation of mitogen-activated protein kinases. A, BBS
(100 nM) stimulated an increase in
[Ca2+]i, which was blocked by a 1-h pretreatment
of the RIE/GRPR cells with the chelating agent BAPTA-AM (30 µM). B, effects of BAPTA-AM treatment on
BBS-stimulated COX-2 mRNA abundance. C, BBS stimulated a
time-dependent increase in the levels of activated
(phosphorylated) ERK-1 (pp-ERK-1) and ERK-2
(pp-ERK-2) proteins. D, BBS stimulated a
time-dependent increase in the level of activated
p38MAPK (pp-p38MAPK) protein.
E, inhibition of MEK with the selective inhibitor, PD988059
(10 µM), blocked the BBS-stimulated accumulation of COX-2
mRNA. F, inhibition of the BBS-induced increases in
COX-2 mRNA by the selective p38MAPK inhibitor, SB203580
(5-20 µM).
963 to +30)
compared with untreated control cultures. A 2.3-fold induction was
observed when using a shorter fragment of the COX-2 promoter
(TIS10-80luc,
80 to +30). BBS did not stimulate an increase in
luciferase activity in cells containing the shortest COX-2 promoter
construct (TIS10-40luc (positions
40 to +30)) (Fig.
3A). We also assessed the
effects of BBS treatment on RIE/GRPR cells transfected with rat and
human COX-2 promoter/luciferase reporter constructs, because
differences exist in the sequences of mouse, rat, and human COX-2
promoters. Similar to the mouse promoter, BBS-induced increases in
luciferase activity were detected in cells expressing both the rat and
human COX-2 reporter constructs (data not shown). Together, these data demonstrated that GRP-R-mediated signaling pathways are linked to
regulation of COX-2 promoter activity in RIE/GRPR cells.
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Fig. 3.
BBS increased COX-2 gene transcriptional
activity. A, BBS stimulated the COX-2 promoter
activity. Left, the cis-acting transcriptional
response elements of mouse COX-2 promoter and the constructs of three
COX-2 promoter plasmids; right, the effect of BBS on the
induction of COX-2 promoter coupled to a luciferase reporter gene when
transiently transfected to RIE/GRPR cells. Luciferase activity
(mean ± S.D.) from four independent experiments is expressed
relative to control after normalizing for differences in transfection
efficiency by the -galactosidase plasmid (CMV-
-Gal).
B, BBS (100 nM) stimulated AP-1 activity in
RIE/GRPR cells by electrophoretic mobility shift assay.
C, BBS elicited the mRNA abundance of c-fos
and c-jun by Northern blot analyses, and both were blocked
by the intracellular Ca2+ chelator BAPTA-AM.
D, BBS increased promoter activities of c-fos and
c-jun measured by luciferase assays. *, p < 0.05 versus control, n = 12.
), and NF-
B transcriptional elements have been
identified as being involved in receptor-mediated COX-2 expression (32,
34, 36-40). Additionally, numerous potential cis-activating
consensus sequences have been identified within the COX-2 promoter,
including AP-1, AP-2, SP-1, MEF-2, STAT1, and STAT3 sites (35, 41, 42).
The identities of the cis-elements regulated by BBS- and
GRP-R-mediated signaling pathways are unknown. In several cell models,
BBS is a potent stimulator of the AP-1 transcription factor
complex (43-45). The AP-1 complex is composed of hetero- and
homodimers of the Jun and Fos families of transcription factors, which
bind to a specific DNA consensus sequence (TGA(C/G)TCA) (46).
Electrophoretic mobility shift assay, using an end-labeled
oligonucleotide probe containing the AP-1 consensus binding sequence,
showed an increase in AP-1 binding activity in nuclear protein extracts
of RIE/GRPR cells following treatment with BBS (Fig. 3B).
The BBS-stimulated increases in AP-1 binding activity were detected by
2 h, reached a maximum at 4 h, and decreased thereafter (Fig.
3B, lanes 2-4). Preceding the
increase in AP-1 binding activity was a BBS-stimulated increase in both
c-fos and c-jun mRNA expression. BBS induces
a transient (time- and Ca2+-dependent) increase
in c-Fos and c-Jun mRNA levels (Fig. 3C). Steady-state
mRNA levels of both transcription factors were increased by
0.5 h, peaked by 1 h, and then returned to near base-line
levels by 2 h. Like BBS regulation of COX-2 mRNA levels, the
agonist-stimulated increase in c-Fos and c-Jun mRNA were inhibited
by cells pretreated with BAPTA-AM (30 µM) (Fig.
3C). Additionally, RIE/GRPR cells transfected with either
5'-promoter sequences of c-fos or c-jun coupled
to the luciferase reporter gene showed a 3.3- and 3.5-fold increase in
luciferase activity compared with untreated control cells, respectively
(Fig. 3D). Together, these data indicate that GRP-R
activation stimulates Fos and Jun expression in RIE/GRPR cells through
the activation of their respective promoters.
View larger version (25K):
[in a new window]
Fig. 4.
BBS stimulated phosphorylation of Elk-1
(A) and ATF-2 (B) in RIE/GRPR cells,
as measured by immunoblotting of cell lysates. C, BBS
increased promoter activities of Elk-1 and Sap-1 detected by luciferase
assays. *, p < 0.05 versus control,
n = 12.
View larger version (44K):
[in a new window]
Fig. 5.
Effect of curcumin on COX-2 mRNA
expression and AP-1 activity in response to BBS. A,
curcumin, an AP-1 binding inhibitor, suppressed the BBS-evoked COX-2
mRNA abundance in a dose-dependent fashion.
B, curcumin (10 µM) completely inhibited
BBS-stimulated AP-1 binding activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 6.
A summary of intracellular signaling pathways
required for BBS-stimulated COX-2 expression. A, time
sequence of BBS induction of [Ca2+]i,
ERKs, p38MAPK, Elk-1, ATF-2, c-fos,
c-jun, COX-2 mRNA, and COX-2 protein in RIE/GRPR cells.
B, intracellular transduction pathways for bombesin-evoked
COX-2 expression in RIE/GRPR cells. Activation of GRP-R by BBS results
in increased phosphorylation of mitogen-activated protein kinases (ERKs
and p38MAPK) and the transcriptional factors, ternary
complex factor (includes ELK-1 and Sap-1) and ATF-2; both of the latter
then increase the expression of c-fos and c-jun,
respectively, and the binding activity of AP-1. The activation of AP-1
further stimulates COX-2 promoter and increases the expression of COX-2
mRNA and protein, as well as the release of PGE2.
Inhibitors of MEK (PD98057), p38MAPK (SB203580), and AP-1
(curcumin) all suppress BBS-stimulated COX-2 expression. In addition,
BBS also increases [Ca2+]i, which may be required
for BBS-evoked COX-2 expression.
Mitogen-activated protein kinase pathways mediate the stimulatory
effects of different extracellular stimuli on COX-2 expression in a
stimulus- and cell type-specific manner. We have shown that BBS
stimulation of COX-2 expression in RIE/GRPR cells involves both ERK and
p38MAPK pathways but not the JNK pathway. Similarly, ERK
and p38MAPK pathways mediate the induction of COX-2
expression by transforming growth factor- and interferon-
in
human epidermal keratinocytes and squamous carcinoma cells (62).
Whereas JNK and p38MAPK pathways regulate
interleukin-1
-stimulated COX-2 expression in renal mesangial cells
(63), all three MAPK cascades (ERK, JNK, and p38MAPK) are
involved in the induction of COX-2 expression by physiologic hypertonicity in renal medullary collecting duct cells (29). Regulation
of COX-2 by platelet-derived growth factor is mediated through ERK and
JNK pathways in NIH 3T3 cells (42), but ERK-2 is required for
oxytocin-stimulated PGE2 synthesis in uterine endometrial
and amnion cells (64). Together, these studies demonstrate the central
role MAPK cascades play in regulation of COX-2 expression to a variety
of extracellular stimuli.
We have found that the increase of the steady-state level of COX-2 by
BBS is regulated by a transcriptional mechanism (Fig. 3A).
Furthermore, BBS-mediated COX-2 expression occurs predominantly through
the Ca2+/MAPK/AP-1-dependent signaling
pathways. Activated ERKs can directly phosphorylate transcription
factors, such as Elk-1 and Sap-1, which then bind to the
serum-responsive element of the c-fos promoter (47, 48), and
p38MAPK phosphorylates ATF-2, which binds to the TPA
response element within the c-Jun promoter. We show that BBS stimulated
an increase in the phosphorylation of Elk-1 and ATF-2, which precedes
an increase in the steady-state levels of c-Fos and c-Jun mRNA and
AP-1 binding activity. Inhibition of BBS-induced increases in
[Ca2+]i blocked increases in both COX-2 mRNA
levels and levels of c-Fos and c-Jun mRNA (Figs. 2B and
3C), as well as AP-1 binding (data not shown). Furthermore,
inhibition of ERK and p38MAPK activation suppressed AP-1
binding and COX-2 expression. Finally, inhibition of AP-1 binding with
curcumin blocked BBS-stimulated increases in COX-2 mRNA levels.
These findings indicate that the stimulation of COX-2 expression by BBS
is mediated, in large part, by an AP-1 transcription factor. Our
findings are supported by studies showing that AP-1 activation mediates
the induction of COX-2 in response to stimulation with superoxide,
lipopolysaccharide, interleukin-1, and nitric oxide in RAW264.7
mouse macrophages and human pulmonary type II A549 epithelial cells and
in response to platelet-activating factor and interleukin-1 in human
epidermal keratinocytes (65-68).
Posttranscriptional mechanisms can also play a role in the regulation
of COX-2 expression (34, 35). The stability of COX-2 mRNA is
mediated by sequences within its 3'-untranslated regions (41). In RIE-1
cells, transforming growth factor-1 enhanced Ha-ras-induced COX-2 expression via stabilization of COX-2
3'-untranslated regions (35). In human monocytes, the increase of COX-2
induction by lipopolysaccharide was due to p38MAPK
stabilizing COX-2 mRNA (34). Lasa et al. (41) recently
reported that only 123 nucleotides immediately 3' to the translation
termination condon are required for the regulation of mRNA
stability by p38MAPK. Although the mechanism of
p38MAPK-mediated COX-2 mRNA stabilization is unclear,
the p38MAPK inhibitor (SB203580) has been shown to decrease
the cellular half-life of COX-2 mRNA (34, 41). We found in RIE/GRPR
cells that BBS induced a significant increase in p38MAPK
activation; however, SB203580 treatment did not decrease the half-life
of the agonist-induced increases in COX-2 mRNA levels (data not
shown), suggesting that BBS-induced p38MAPK activation
regulates COX-2 transcription rather than mRNA stability. Which
pathway is favored, transcription versus message stability, may be dependent on the precise kinetics of the p38MAPK
activation. The extent and duration of p38MAPK activation
in response to a specific stimuli may dictate whether pathways involved
in the transcriptional regulation of COX-2 expression are favored over
pathways regulating the stability of COX-2 mRNA.
In summary, our results demonstrated that BBS is a potent inducer of
COX-2 expression in intestinal epithelial cells, and this action is
mediated, at least in part, by
Ca2+/MAPK/AP-1-dependent signaling pathways.
These data suggest that regulation of COX-2 gene expression has a
potential mechanism by which aberrantly expressed GRP-R plays a role in
colorectal carcinogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Kenneth D. Brown for the gift of the RIE-1 cell line, and Drs. James F. Battey, Harvey R. Herschman, Ralf Janknecht, Johannes L. Bos, and Joan Massagué for providing plasmid constructs. We also thank Jell H. Hsieh and Kirk L. Ives for technical support and Eileen Figueroa, Karen Martin, and Steve Schuenke for the preparation of this manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants PO1 DK35608 and R01 DK48345.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: Dept. of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0527. Tel.: 409-772-1285; Fax: 409-772-5611; E-mail: ctownsen@utmb.edu.
Published, JBC Papers in Press, April 5, 2001, DOI 10.1074/jbc.M101801200
2 M. R. Hellmich and C. M. Townsend, Jr., unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: COX-2, cyclooxygenase-2; AP-1, activator protein-1; BBS, bombesin; ERK, extracellular signal-regulated kinase; GRP, gastrin-releasing peptide; GRP-R, GRP receptor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PGE2, prostaglandin E2; TPA, 12-O-tetradecanoylphorbol-13-acetate; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester.
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