Dig Dis Sci
DOI 10.1007/s10620-006-9661-8
ORIGINAL PAPER
Geranylgeranylacetone Induces Cyclooxygenase-2 Expression
in Cultured Rat Gastric Epithelial Cells Through NF-κB
Tsutomu Nishida · Yuki Yabe · Hai Ying Fu · Yujiro Hayashi · Kayoko Asahi ·
Hiroshi Eguchi · Shingo Tsuji · Masahiko Tsujii · Norio Hayashi · Sunao Kawano
Received: 8 March 2006 / Accepted: 24 October 2006


C Springer Science+Business Media, LLC 2006
Abstract Geranylgeranylacetone (GGA) effectively pro-
tects the gastric mucosa against noxious agents. The precise
mechanisms underlying the gastroprotective actions of GGA
are not known. To elucidate the precise mechanism of GGA,
the effect of GGA treatment on COX-2 expression in rat
gastric epithelial (RGM1) cells was investigated. We used
a prostaglandin E2 (PGE2 ) enzyme-linked immunoassay
kit and Western blot analysis to measure PGE2 production
and COX-2 induction by GGA treatment in serum-starved
RGM1 cells. Gel-shift assay, Western blot analysis, and a
reporter assay were performed to determine which COX-2
promoter was involved in GGA-induced COX-2 expression.
GGA treatment dose dependently increased COX-2 expres-
sion and PGE2 production. The nuclear factor (NF)-κB sites
of the COX-2 gene promoter were critical for GGA-mediated
COX-2 expression. GGA induces COX-2 expression and in-
creases PGE2 production in serum-starved RGM1 cells via
activation of the NF-κB sites of COX-2 gene promoters.
Keywords COX-2 . Geranylgeranylacetone .
Prostaglandin E2 . Gastric epithelial cells . NF-κB .
RGM1 cells
The first two authors contributed equally to this work.
T. Nishida · Y. Yabe · H. Y. Fu · Y. Hayashi · K. Asahi ·
H. Eguchi · S. Kawano
Department of Clinical Laboratory Science, School of Allied
Health Sciences, Faculty of Medicine, Osaka University,
Osaka, Japan
T. Nishida (
) · S. Tsuji · M. Tsujii · N. Hayashi
Department of Gastroenterology and Hepatology,
Clinical Research Building (A8),
Osaka University Graduate School of Medicine,
2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
e-mail: tnishida@gh.med.osaka-u.ac.jp
Introduction
Geranylgeranylacetone (GGA; 3:2 (5E:5Z) geometrical
mixture of [9E, 13E]-6,10-14,18-tetramethyl-5,9,13,17-
nonadecatetraen-2-one], an acyclic polyisoprenoid, is an an-
tiulcer drug that is widely used in Japan for patients with pep-
tic ulcers and gastritis [1]. Various mechanisms have been
proposed for the protective effect of GGA. GGA protects
the gastric mucosa against noxious agents such as aspirin,
aspirin plus HCl [2], and absolute ethanol in rats [3]. GGA
increases the synthesis and secretion of gastric mucin [4] as
well as the components of high molecular weight glycopro-
teins [5] and surface-active phospholipids [6, 7]. It has been
reported that GGA induces heat shock protein (HSP) 70 in
rat gastric mucosa [8] and liver [9]. Elevation of other cyto-
protective mediators, such as prostaglandins (PGs) [10] and
nitric oxide [11], has also been postulated in GGA-induced
gastroprotection, although the precise mechanisms have not
yet been examined.
PGs have an important role in gastric mucosal defense [12,
13]. PG synthesis is governed by PG endoperoxide synthase
or cyclooxygenase (COX), which consists of two isoforms.
The constitutive isoform (COX-1) is dominantly expressed
in platelets, prostate, and stomach. The mitogen-inducible
isoform (COX-2) is minimally expressed in normal stomach
[14, 15]. COX-2 expression is enhanced, however, in gastric
epithelial cells after growth stimulation in vitro and in gastric
epithelium after acid-induced damage in vivo [16, 17]. We
recently demonstrated that COX-2 protein is overexpressed
during the healing of gastric lesions and a COX-2 specific
inhibitor delays ulcer healing in the rat, suggesting an im-
portant role for this isozyme in gastric ulcer healing [18].
In the human COX-2 gene, consensus sequences of nuclear
factor (NF)-κB, NF-interleukin (IL)-6, and cyclic AMP re-
sponse element (CRE) sites are found at nucleotides −223
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to −214, −132 to −124, and −59 to −53, respectively
[19, 20]. It is not known, however, whether GGA stimulates
COX-2 expression and PGE2 production.
In the present study, we investigated whether GGA stimu-
lates endogenous PGE2 production and influences transcrip-
tion modulating factors using the luciferace reporter vector
driven by the human COX-2 promoter region mutated at the
NF-κB, NF-IL-6, and CRE sites that was stably transfected
into rat gastric mucosal (RGM1) cells.
Materials and methods
Cell culture
Nontransformed rat gastric mucosal cells (RGM1) [21], de-
rived from rat gastric mucosa, were cultured in Dulbecco’s
modified Eagle’s medium (DMEM)/F12 Ham (Sigma Chem-
ical Co., St. Louis, MO) supplemented with 10% fetal bovine
serum (FBS; Sigma) and 1% antibiotics-antimycotic (AA;
Invitrogen Corp., Carlsbad, CA) at 37◦ C in a 95% air, 5%
CO2 humidified atmosphere.
Analysis of COX-1 and COX-2 proteins by
Western Blot analysis and PGE2 measurement
RGM1 cells were seeded at 3.8 × 105 /well in six-well
plates and cultured for 24 hr to reach subconfluence. GGA
was provided by Eisai Co. Ltd., Tokyo. After 24 hr of
serum starvation, the medium was replaced with supple-
mented DMEM/F12 Ham–10% FBS with or without GGA
(0, 0.5, 1.0, 2.0, and 4.0 mM) at the start of the experi-
ment. After 6 hr of culture, the supernatant was removed
and PGE2 concentration was determined in the cell culture
medium by enzyme-linked immunosorbent assay (ELISA)
using a PGE2 enzyme immunoassay kit (Cayman Chem-
ical, Ann Arbor, MI) according to the manufacturer’s in-
structions. The cells were subsequently washed three times
with phosphate-buffered saline (PBS), then were lysed in
a RIPA buffer (1 × PBS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) contain-
ing 0.1 mg/ml phenylmethylsulfonyl fluoride and 51 µg/ml
aprotinin, followed by 30-min incubation on ice. Samples
were homogenized and centrifuged at 13,400 × g for 20 min
at 4◦ C. Total protein was quantified using the DC Protein As-
say kit (Bio-Rad Laboratories, Richmond, CA). Cell lysates
(30 µl/sample) were dissolved in 10% SDS-polyacrylamide
gels and transferred to a polyvinylidine difluoride mem-
brane. After blocking with 5% nonfat powdered milk in Tris-
buffered saline (TBS) containing 0.1% Tween 20 (TBS-T)
for 1 hr, the membrane was incubated overnight at 4◦ C with
antihuman COX-1 or COX-2 antibody (Santa Cruz Biotech-
nology Inc., Santa Cruz, CA) followed by incubation with
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Dig Dis Sci
Fig. 1 Schematic representation of reporter vectors containing the
5 -flanking region of the COX-2 gene promoter. The construct of the
long type of reporter vector containing the 5 -flanking region of the
COX-2 gene promoter contains three promoter sites (NF-IL-6, NF-κB,
and CRE) (−327/+59). The short type of reporter vector containing the
5 -flanking region of the COX-2 gene promoter (−52/+59) contained
no promoters. Distances are given as nucleotide positions relative to
the transcriptional start site as +1. Luc, luciferase reporter
appropriate horseradish peroxidase-conjugated bound rabbit
anti-goat antibody (DAKO, Glostrup, Denmark). Signal was
detected using the enhanced chemiluminescence protocol as
described by the manufacturer (Pierce, Rockford, IL). Time-
points of the study were determined according to the results
of our previous studies [16, 17, 22, 23].
Reporter assay for NF-κB, NF-IL-6,
and CRE activity
RGM1 cells were seeded at 7 × 104 /well in six-well plates
and cultured for 24 hr to reach subconfluence. Reporter con-
structs containing different lengths of the human COX-2 pro-
moter, as shown in Fig. 1 [24], were a gift from Dr. H. Inoue
(National Cardiovascular Center Research Institute, Osaka,
Japan). The construction of the reporter vector for the human
COX-gene phPES2 (Triple M) has been described previously
[24]. The long-type construct contained the NF-κB, NF-IL-
6, and CRE (−327/+59) sites, whereas no promoters were
inserted in the short-type construct (−52/+59). Constructs
of COX-2 gene reporters with a mutation in the NF-κB, NF-
IL-6, or CRE region of the COX-2 promoter, which were
used in this study, are shown in Fig. 2.
Fig. 2 Schematic representation of reporter vectors containing the
5 -flanking region of the COX-2 gene promoter with mutations. The
luciferase (Luc) reporter vector driven by the COX-2 promoter region
(−327/+59) mutated at the NF-κB, NF-IL-6, or CRE site. The mutated
sequences are listed as follows: NF-κB site (−233/−214), changed
from GGGACTACCC to cacACTACCC; NF-IL-6 site (−132/−124),
changed from TTACGCAAT to TTggtaccT; and CRE site (−59/−53),
changed from TTCGTCA to TTgagCt. In the reporter assay, the mutated
promoters are combined with the Luc gene, so that their promoter
activities are reflected to photoluminescence of the Luc, not to COX-2
activity or to PGE2 production of the transfected cells
Dig Dis Sci
Reporter constructs (0.2 µg) were mixed with 20 ng/ml
of the control vector pRL-TK in 50 µl of OPTI-MEM (In-
vitrogen Corp., Carlsbad, CA). The solution was mixed with
1 µl of lipofectamine (Invitrogen Corp.) diluted in 150 µl of
OPTI-MEM and incubated at room temperature for 30 min.
The two vectors, diluted in 200 µl solution, were then co-
transfected into RGM1 cells after the cells were washed twice
with OPTI-MEM. The cells were incubated at 37◦ C for 24 hr
in a 5% CO2 atmosphere. The medium was replaced with
DMEM/F12 Ham supplemented with 10% FBS, and GGA
was added to the reaction. After 6 hr of culture, cells were
harvested and assayed with a PicaGene dual-luciferase assay
kit (PG-DUALSP; Tokyo Ink Co. Ltd., Tokyo) according to
the manufacturer’s instructions. Briefly luciferase and sea
pansy luciferase activity were measured with a luminometer
and relative activity was calculated.
Detection of phosphorylated NF-κB
and phosphorylated protein I-κB by Western Blot analysis
and electrophoretic mobility gel shift assay (EMSA)
RGM1 cells were seeded at 3.8 × 105 /well in six-well plates
and cultured for 24 hr to reach subconfluence. After 24 hr
of serum starvation, the cells were cultured in medium sup-
plemented with 1 mM GGA for 15 min. Sample proteins
were collected by the methods described above. Cell lysates
(30 µl/sample) were dissolved in 10% SDS-polyacrylamide
gels and transferred to a polyvinylidine difluoride mem-
brane. After blocking with 5% nonfat powdered milk in
TBS-T for 1 hr, the membrane was incubated overnight
at 4◦ C with phosphorylated NF-κB and phosphorylated in-
hibitory protein I-κB antibody (Cell Signaling, Technology,
Inc., Beverly, MA) followed by incubation with the appro-
priate horseradish peroxidase-conjugated bound secondary
antibody (DAKO). Signal was detected using the enhanced
chemiluminescence protocol as described by the manufac-
turer (Pierce).
An EMSA Gel-Shift Kit (Panomics Inc., Redwood City,
CA) was used to detect the nuclear translocation of NF-κB.
RGM1 cells were seeded at 3.8 × 105 /well in six-well plates
with DMEM/F12 medium. After cells were serum-starved
for 24 hr, culture medium containing 10% FBS and 1 mM
GGA was added for 15 min.
Nuclear extracts were prepared using the Nuclear/Cytosol
Fractionation Kit (Bio Vision) according to the manu-
facturer’s instructions. The nuclear protein was harvested
and NF-κB binding with DNA was detected accord-
ing to the manufacturer’s instructions. The NF-κB se-
quence consensus oligonucleotide used in this study was
5 -AGTTGAGGGGACTTTCCCAGGC-3 . Instead of GGA,
20 ng/ml tumor necrosis factor (TNF)-α was added to the
culture medium as a positive control.
Fig. 3 Effect of GGA on COX-1 and COX-2 expression. RGM1 cells
were cultured as described in the text. After GGA treatment, cells were
harvested and used for Western blot analysis with COX-1 and COX-2
antibodies
Statistical analysis
Results are shown as mean ± SD. Statistical significance
was determined by the two-tailed Student’s t-test to compare
luciferase activity and the Kruskal-Wallis test to examine
the dose-dependent change in PGE2 production. A P value
< 0.05 was considered statistically significant.
Results
Effect of GGA on COX-2 expression
and PGE2 production in RGM1 cells
After 24 hr of serum starvation, RGM1 cells were cultured
for 6 hr in medium containing FBS and GGA (0, 0.5, 1.0, 2.0,
and 4.0 mM). COX-1 and COX-2 expression was examined
by Western blot analysis (Fig. 3). COX-1 expression was
unaltered by GGA. Conversely, GGA induced COX-2 ex-
pression. As PGE2 is a major metabolite of arachidonic acid,
and its synthesis is catalyzed by COX-1 and COX-2, we in-
vestigated the effect of GGA on PGE2 production in RGM1
cells. Figure 4 shows the production of PGE2 in RGM1 cells
after stimulation with GGA at varying concentrations. West-
ern blot analysis indicated that GGA significantly increased
COX-2 production in the culture medium compared with no
GGA treatment.
GGA induced COX-2 expression via
the COX-2 gene promoter NF-κB
The reporter assay indicated that the luciferase activity in the
long-type construct was significantly increased after GGA
treatment (Fig. 5). The luciferase activity in the short-type
construct was lower than that in the long-type construct and
did not change with GGA treatment.
Using the reporter constructs shown in Fig. 2, we investi-
gated the GGA-induced changes in luciferase activity. When
the COX-2 reporter genes with a mutation in the NF-κB
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Fig. 4 Effect of GGA on PGE2 production in RGM1 cells. RGM1 cells
were seeded at 3.8 × 105 /well in six-well plates and cultured for 24 hr
to reach subconfluence. After 24-hr serum starvation, GGA was added
at different concentrations for up to 6 hr. The supernatant was then
collected and PGE2 was measured with a PGE2 enzyme immunoassay
kit. ∗ P < 0.05 vs without GGA treatment; n = 6
region were transfected into RGM1 cells, the luciferase ac-
tivity was not induced by GGA treatment compared with the
long-type construct. On the other hand, GGA-induced lu-
ciferase activity was not changed when the COX-2 reporter
genes with a mutation in the CRE region or NF-IL-6 region
were transfected into RGM1 (Fig. 6).
Fig. 5 Effect of GGA on long-type promoter (L) and short-type pro-
moter (S) activities. Using a reporter assay, COX-2 gene transcription
was measured after GGA stimulation for 6 hr. ∗ P < 0.05 vs long-type
promoter without GGA treatment; n = 6
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Dig Dis Sci
Fig. 6 Effect of GGA on the three promoter activities. COX-2 gene
reporters with a mutation in the NL-IL-6, NF-κB, or CRE region and
the normal COX-2 gene reporter were transfected into RGM1 cells. Lu-
ciferase activity was measured after 6 hr of GGA stimulation. ∗ P < 0.05
vs without GGA treatment; n = 6
To confirm the GGA-induced NF-κB activation, we inves-
tigated NF-κB and I-κB phosphorylation by Western blot-
ting analysis. GGA treatment induced both NF-κB and I-κB
phosphorylation. TNF-α treatment (20 ng/ml) was used as a
positive control (Fig. 7). The EMSA gel shift assay revealed
that GGA-activated NF-κB was bound to DNA, similarly to
TNF-α-activated NF-κB (Fig. 8).
Discussion
GGA is a unique antiulcer drug. GGA exerts protective ac-
tions against a variety of tissues and their injury, includ-
ing neuronal injury [25], endotoxic shock [26], ischemia-
reperfusion [9], and myocardial ischemia [27]. GGA stim-
ulates the synthesis and secretion of mucin in vivo [4] and
in vitro [10] and prevents degradation of the gastric mucosa
when it is exposed to several types of insults and/or stress
Fig. 7 Effect of GGA on NF-κB and I-κB phosphorylation. RGM1
cells were seeded at 3.8 × 105 in six-well plates. After 24-hr serum
starvation, medium containing 10% FBS and 1.0 mM GGA was added
for 15 min. Cells were then harvested and used for Western blot analysis
with phosphorylated NF-κB and phosphorylated I-κB antibodies. TNF-
α was used as a positive control
Dig Dis Sci
Fig. 8 Nuclear translocation of NF-κB was detected by GGA treat-
ment. Cell culture and treatment with GGA were the same as in Fig. 7.
TNF-α was used as a positive control
[1, 28–30]. It has been suggested that GGA stimulates the
synthesis and secretion of mucin by cultured gastric mucus-
secreting cells without increasing their production of PGs.
Nam et al. reported that GGA alone did not induce changes
in COX-2 expression in a short-term in vivo study [11].
The pharmacologic actions of GGA were thought to resem-
ble those of PGs [3, 31]. Furthermore, Terano et al. found
that indomethacin suppressed GGA-induced gastric mucosal
protection against ethanol, suggesting that PGs have impor-
tant roles in GGA-induced gastric mucosal protection [29].
It was, therefore, not known whether GGA affected COX-2
over the long term.
Expression of COX-2 in gastrointestinal epithelia has
been examined extensively in many laboratories including
ours [17, 18, 22, 23, 32]. COX-2 is one of the immediate
early genes whose transcripts are fairly unstable due to the
AU-rich sequences of the 3 untranslated regions. COX-2
protein is an isozyme of PG endoperoxidase, whose major
product is PGE2 . Although phospholipase A2 and microso-
mal PGE synthase are also involved, COX is the rate-limiting
enzyme in PGE2 production. RGM1 cells are known to ex-
press COX-2 in response to various stimuli including fetal
calf serum [32] and TGF-α [17]. Since RGM1 cells also
express COX-1 constitutively, it takes hours to observe sig-
nificant enhancement of PGE2 production due to induction
of COX-2.
The present study demonstrated that GGA increased
PGE2 by upregulating COX-2 in RGM1 cells. Considering
the unstable nature of the COX-2 transcript, the expression
of COX-2 may not clearly depend on the dose of GGA.
Moreover, the present study also examined which COX-2
gene promoter is stimulated by GGA. The COX-2 gene pro-
moter contains a canonical TATA box and various putative
transcriptional regulatory elements, such as NF-κB, NF-IL-
6/C/EBP, PEA3, NFAT, CRE, AP-2, and SP-1 [19]. The
nucleotide sequences of the human [19], mouse [33], and rat
[34] COX-2 genes in their 5 -flanking regions share 60–63%
similarity among the 275-bp nucleotide residues upstream of
the transcriptional start site. It is noteworthy that in all three
genes the sequences homologous to the consensus NF-IL-6
and CRE sites are conserved.
It is noteworthy that NF-κB upregulates not only COX-2,
but also various genes such as inducible nitric oxide synthase
(iNOS). Such redundant upregulation of multiple genes may
protect gastric mucosa from a wide range of insults. In fact,
Nam et al. reported that GGA also protects gastric mucosa
by increasing nitric oxide [11]. Depending on the stimu-
lus and cell type, these transcription factors can modulate
COX-2 expression. To investigate the promoter site involved
in GGA-induced COX-2 expression, we used a series of
reporter vectors for the COX-2 gene and measured their lu-
ciferase activity after the addition of GGA. Mutations at
the NF-κB site, but not the CRE or NF-IL-6 site, decreased
the response to GGA compared with normal controls, in-
dicating that the NF-κB promoter regions were critical in
GGA-induced COX-2 gene expression.
NF-κB has a central role in regulating numerous genes
in response to cellular stress that promote the dissociation
of I-κB through phosphorylation followed by ubiquitina-
tion and degradation. Moreover, recent studies indicate that
phosphorylation of NF-κB subunit p65 modulates NF-κB
transcription activity [35]. Following GGA treatment, NF-
κB and I-κB were phosphorylated to levels similar to those
after treatment with TNF-α, an NF-κB signaling activator.
Furthermore, using an NF-κB gel shift assay, nuclear translo-
cation of NF-κB was detected following treatment with ei-
ther GGA or TNF-α. NF-κB transcribes not only COX-2
but also various inflammatory enzymes such as iNOS. Al-
though the effects of GGA on the induction of nitric oxide
synthase were beyond the scope of the present study, GGA
might have cytoprotective effects depending on nitric oxide
and other factors related to NF-κB.
We reported that COX-2 upregulation induces numerous
vascular growth factors and accelerates angiogenesis [36]. In
addition, PGE2 stimulates vascular endothelial growth factor
expression in endothelial cells [37], and angiogenic factors
such as vascular endothelial growth factor have an impor-
tant role in the healing of gastric ulcers [38]. Moreover, it
was reported that PG directly protects isolated human gastric
gland cells against ethanol and indomethacin injury in vivo
[39]. Therefore, COX-2 upregulation and increased PGE2
production are expected to facilitate ulcer healing. Further
investigation is required to determine whether COX-2 induc-
tion by GGA accelerates ulcer healing via angiogenesis.
HSP70 was found to be involved in the cytoprotective
effect of GGA [8, 40, 41]. Recently Zhang et al. reported
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that upregulation of HSP70 is associated with induction of
COX-2 and synthesis of PGE2 in endothelial cells in vitro
[42]. They demonstrated that in endothelial cells exogenous
HSP70 could activate NF-κB and upregulate the expres-
sion of proinflammatory cytokines and COX-2. Furthermore,
Saika et al. reported that there is a differential temporal and
spatial expression of HPS70 and COX-2 mRNAs in rat stom-
ach following ethanol ingestion [43]. Upregulation of HSP70
mRNAs was observed in the mucosa surrounding the deep
erosions and in the smooth muscle cells of muscularis mu-
cosae, muscularis externa, and blood vessels in response to
intragastric ethanol. On the other hand, COX-2 mRNA was
detected in surface mucous cells. These distinct gene expres-
sions in the mucosa suggest that not only COX-2 and PGs but
also HSPs and other cytoprotective factors are involved in
gastric cytoprotection. Possible interactions between GGA
and various cytoprotective mediators, such as HSP, COX-2-
induced PGE2 , and nitric oxide, remain to be investigated in
future studies.
In conclusion, GGA induces COX-2 expression and PGE2
production in untransformed rat gastric epithelial cells. The
reporter assay and EMSA demonstrate that GGA activates
the binding of NF-κB to the COX-2 gene promoter and tran-
scription of the downstream gene. Thus, GGA upregulates
COX-2 at transcription levels, which may contribute to the
gastroprotective functions of GGA.
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