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AP-1 TRANSCRIPTION FACTOR M EDIATES BOMBESIN-STIMULATED CYCLOOXYGENASE-2

EXPRESSION IN INTESTINAL EPITHELIAL CELLS

Yan-Shi Guo∗ , Mark R. Hellmich∗ Ψ, Xiao Dong Wen∗ , and Courtney M. Townsend, Jr.∗

Departments of Surgery* and Physiology and BiophysicsΨ

The University of Texas Medical Branch

Galveston, Texas 77555

RUNNING TITLE: Mechanism of BBS-stimulated COX-2 expression

Correspondence to: Courtney M. Townsend, Jr., M.D.

Department of Surgery

The University of Texas Medical Branch

301 University Boulevard

Galveston, Texas 77555-0527

ph: (409) 772-1285

fax: (409) 772-5611

e-mail: ctownsen@utmb.edu

Supported by grants from the National Institutes of Health (PO1 DK35608, R01 DK48345)

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 5, 2001 as Manuscript M101801200 by guest on M

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ABBREVIATIONS

AP-1 activator protein-1

BBS bombesin

COX-2 cyclooxygenase-2

EMSA electrophoretic mobility shift assay

ERK extracellular signal-regulated kinase

GRP-R gastrin-releasing peptide receptor

IP immunoprecipitate

JNK c-Jun N-terminal kinase

LPS lipopolysaccharide

MAPK mitogen-activated protein kinase

MEK MAPK kinase

PG prostaglandin

SRE serum response element

TCF ternary complex factor

TRE TPA response element

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SUMMARY

Colorectal carcinogenesis is a complex, multi-step process involving genetic alterations

and progressive changes in signaling pathways regulating intestinal epithelial cell proliferation,

differentiation and apoptosis. Although cyclooxgenase-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 (BBS), a

homolog of GRP, potently stimulates the expression of COX-2 mRNA and protein as well as the

release of PGE2 from a rat intestinal epithelial cell line engineered to express GRP-R. BBS

stimulation of COX-2 expression requires an increase in [Ca2+]i, activation of extracellular

signal-regulated kinase (ERK)-1 and -2 as well as 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 AP-1-dependent pathway.

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Colorectal cancers are the third leading cause of cancer deaths in the United States (1).

One in twenty 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)

in colorectal carcinogenesis. COX-2 is a key enzyme in the biosynthesis of prostaglandins (PGs)

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 (NSAIDs) (3), which inhibit COX activity. Additionally,

experiments with adenomatous polyposis coli (APC) gene-deficient mice (Min mice) revealed

that inhibition of COX activity with NSAIDs resulted in a reduction in the number and

multiplicity of spontaneously formed tumors (4-6) and, APC∆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.

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 non-malignant 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

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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 (unpublished data).

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

upregulation 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 upregulation 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) They are a non-

tumorigenic intestinal epithelial cell line which, like the normal human colonic epithelium, do

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 GRR-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 PGE2 from these cells. The increase in COX-2 expression is

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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 AP-1 transcription factor. These findings partially identify the

signaling pathways coupling GRP-R to the upregulation 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.

EXPERIMENTAL PROCEDURES

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 mouse PGs-2 (COX-2) cDNA probe were kindly

provided by Dr. Harvey R. Herschman (Los Angeles, CA). The reporter constructs Gal4-

Elk(307-408), Gal-4-Sap(268-431), Gal4-luc were gifts from Dr. Ralf Janknecht (La Jolla, CA).

c-fos-luc and 3XTRE-luc were provided by Dr. Johannes L. Bos (Utrecht, The Netherlands) and

Dr. Joan Massagué (New York, NY), respectively. Mouse c-Fos and c-Jun cDNA probes were

purchased from the American Type Culture Collection (Rockville, MD).

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).

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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 (Gibco/BRL, Grand Island, NY) 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 AM-fura-2 as previously described (15). Cells were cultured at 37oC in a

humidified atmosphere of 95% air and 5% CO2 in DMEM medium supplemented with 5%

heated-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and Geneticin (G418,

400 µg/ml, GIBCO/BRL).

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, Inc., Keene, NH). The blots were hybridized with

cDNA probes labeled with [α32P]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 18S rRNA.

Western blot analysis

Immunoblot analysis was performed as described previously (13). The cells were lysed

for 30 min in a solution consisting of 1 X PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate,

0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 1 mM sodium

orthovanidate. Cellular proteins were denatured by heating, resolved by SDS-polyacrylamide

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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 DMEM 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 ELISA

(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 37oC, fixed with a 4% paraformaldehyde (15 min),

permeabilized with 0.3% Triton X-100 (10 min) and incubated in blocking solution (1% BSA in

PBS, 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 PBS and incubated with a goat anti-rabbit

IgG antibody labeled with Alexa 488 (Molecular Probes, Eugene, OR) (1:2000, 30 min).

Specific immunostaining was visualized with a Nikon Eclipse fluorescence microscope.

Luciferase assay

RIE/GRPR cells were seeded in 6-well plates at a density of 2x105 cells/well. After

overnight adhesion, cells were transfected with 2 µg of promoter/luciferase reporter gene DNA

using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN). Prior to assaying for

luciferase activity, cells were incubated in DMEM 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

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System (Promega, Madison WI). Transfection efficiency was assessed using a β-galactosidase

expression plasmid as described previously (17).

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 [γ-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; Pharmacia Biotech, Inc.) in a final

volume of 20 µl. Reaction mixtures were resolved on 4% non-denaturing polyacrylamide gel

electrophoresis at 200 V for 2 h. Gels were dried and visualized by autoradiography (17).

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 ± SEM.

Differences between means were compared using the analysis of variance (ANOVA) test and

were considered significantly different at the level of p <0.05.

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RESULTS

RIE/GRPR cells

The RIE-1 cell line, isolated originally from the rat small intestine, exhibits epithelioid

morphology, the normal rat diploid number of chromosomes and does not form colonies in soft

agar (19). These properties have made them one of the preferred cell models for examining non-

tumorigenic 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 anti-

apoptotic 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 AM-fura-2, respectively. Five clones were isolated

with Bmax values ranging from approximately 3000 to 8900 binding sites per cell. The calculated

affinity constants exhibited a range of values from 0.33 nM 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 approximate 3000 receptors per 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 to 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

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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 Prostaglandin E2 (PGE2) released from RIE/GRPR cells were measured using an

ELISA 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 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 (IP3) and diacylglycerol (DAG), release of Ca2+ from

IP3-sensitive stores (15) and activation of various protein kinases including protein kinase C

(PKC) (15), protein kinase D (PKD) (24), the Src-family of non-receptor tyrosine kinases (25)

and the mitogen-activated protein kinase (MAPK) cascades (26).

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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, AM-BAPTA (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 (% blue cells; vehicle [0.1% DMSO] 3.56 ± 0.79 vs. AM-BAPTA 3.23 ± 0.67),

or alter the expression of 18S 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 extracellular signal-regulated protein kinase (ERK) pathway,

the c-Jun N-terminal kinase (JNK) pathway and the p38 MAP kinase (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 resides. 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. 2C, 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 baseline at 15 and 30 min. A second,

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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 (IP) ERK-1 and -2 and the substrate, myolin basic protein (MBP). A

5-fold increase in MBP phosphorylation was observed when using IP proteins from RIE/GRPR

cells treated for 10 min with BBS vs. IP 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. 2E, 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

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coupled to a luciferase reporter gene. BBS (100 nM) induced a 3.6-fold increase in luciferase

activity in cells transiently expressing TIS10L-luc (-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 [-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.

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/EBPβ) and NF-κB 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 activator protein-1 (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 (EMSA), using an end-labeled

oligonucleotide probe containing the AP-1 consensus binding sequence, showed an increase in

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AP-1 binding activity in nuclear proteins 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 baseline 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 AM-BAPTA (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-fold and 3.5-fold increase in luciferase activity compared to 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.

Expression of the c-fos and c-jun genes are regulated, in part, by ternary complex factors

(TCFs) and ATF-2, respectively. TCFs 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 (SRF) and results in serum response

element (SRE)-dependent activation of the c-fos promoter (47,48). The phosphorylation of

ATF-2 by p38MAPK increases TPA-response element (TRE)-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 min and 15 min after BBS treatment and continued for 60 min and 30

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min, respectively (Fig. 4A, B). Additionally, BBS increased the promoter activities of Elk-1 and

Sap-1 by 12-fold 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.

DISCUSSION

The aberrant overexpression of COX-2, BBS-like peptides and GRP-R have 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

COX-2 plays an important role in the development of colorectal carcinogenesis, little is known

about the molecular mechanisms leading to its upregulation. Recent data from mouse Swiss 3T3

fibroblasts showing 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

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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, -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 baseline 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 SRE

and TRE 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 Figure

6B.

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 (TGF)-α and

interferon-γ in human epidermal keratinocytes and squamous carcinoma cells (62). Whereas

JNK and p38MAPK pathways regulate interleukin (IL)-1β-stimulated COX-2 expression in renal

mesangial cells (63), all three MAPK cascades (ERK, JNK and p38MAPK) are involved in the

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induction of COX-2 expression by physiologic hypertonicity in renal medullary collecting duct

cells (29). Regulation of COX-2 by PDGF 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 (SRE) of c-fos promoter (47,48), and p38MAPK phosphorylates

ATF-2, which binds to TRE 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, 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 AP-1

activation mediates the induction of COX-2 in response to stimulation with superoxide,

lipopolysaccharide (LPS), IL-1β and nitric oxide in RAW264.7 mouse macrophages and human

pulmonary type II A549 epithelial cells, and to platelet-activating factor and IL-1 in human

epidermal keratinocytes (65-68).

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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 (3′-UTR) (41). In RIE-1 cells, TGF-β1 enhanced Ha-ras-induced COX-2

expression via stabilization of COX-2 3′-UTR (35). In human monocytes, the increase of

COX-2 induction by LPS 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 vs. 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 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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ACKNOWLEDGMENTS

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 wish to thank Jell H. Hsieh and Kirk L. Ives for their

technical support and Eileen Figueroa, Karen Martin, and Steve Schuenke for the preparation of

this manuscript.

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FIGURE LEGENDS

Fig. 1 Bombesin (BBS) stimulated an increase in COX-2 expression and activity in

RIE/GRPR cells. (A) Time course of COX-2 mRNA expression. Sub-confluent,

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 in

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 h to 24 h. Medium in each well was collected and analyzed for PGE2

levels by ELISA.

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

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cells with the chelating agent AM-BAPTA (30 µM). (B) Effects of AM-BAPTA

treatment on BBS-stimulated COX-2 mRNA abundance. (C) BBS stimulated a

time-dependent increase in the levels of activated (phosphorylated) ERK-1

(ppERK-1) and ERK-2 (ppERK-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).

Fig. 3 BBS increased COX-2 gene transcriptional activity. (A) BBS stimulated the

COX-2 promoter activity. The left panel shows the cis-acting transcriptional

response elements of mouse COX-2 promoter and the constructs of three COX-2

promoter plasmids. The right panel shows 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 ± SD) from four

independent experiments is expressed relative to Control after normalizing for

differences in transfection efficiency by the β-galactosidase plasmid (CMV-β-

Gal). (B) BBS (100 nM) stimulated AP-1 activity in RIE-GRP-R 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 intracellular

Ca2+ cheletos BAPTA. (D) BBS increased promoter activities of c-fos and c-jun

measured by luciferase assays. *=p<0.05 vs. control, n=12.

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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

vs. control, n=12.

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.

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 receptor (GRP-R) by

BBS results in increased phosphorylation of mitogen-activated protein kinases

(ERKs and p38MAPK) and transcriptional factors TCF (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.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Yan-Shi Guo, Mark R. Hellmich, Xiao Dong Wen and Courtney M. Townsend, Jrin intestinal epithelial cells

AP-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression

published online April 5, 2001J. Biol. Chem. 

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