1 Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 565-0871 Osaka; and 2 Department of Molecular Pharmacology, Cancer Research Institute, Kanazawa University, 920-0934 Kanazawa, Japan
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ABSTRACT |
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Although hypergastrinemia is
frequently observed in individuals with a chronic Helicobacter
pylori infection, its pathophysiological significance in gastric
mucosal inflammation is unclear. The present study was designed to
determine if gastrin induces the expression of CXC chemokines in
gastric epithelial cells. Human and rat gastric epithelial cells,
transfected with gastrin receptor, were stimulated with gastrin. The
expression of mRNAs for human interleukin-8 (IL-8) and rat
cytokine-induced neutrophil chemoattractant-1 and release of human IL-8
protein were then determined by Northern blot analysis and ELISA,
respectively. Gastrin not only induced the expression of mRNAs for
these chemokines but also stimulated IL-8 protein release. A luciferase
assay using IL-8 promoter genes showed that nuclear factor (NF)-B is
absolutely required and activator protein-1 (AP-1) is partly
required for the maximum induction of IL-8 by gastrin. An
electrophoretic mobility shift assay revealed that gastrin is capable
of activating both NF-
B and AP-1. In addition, the inhibition of
NF-
B abrogated gastrin-induced chemokine expression. These results
suggest that gastrin is capable of upregulating CXC chemokines in
gastric epithelial cells and therefore may contribute to the
progression of the inflammatory process in the stomach.
mucosa; inflammation; chemokines; transcription factors
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INTRODUCTION |
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GASTRIC EPITHELIAL CELLS PRODUCE chemokines such as interleukin (IL)-8, cytokine-induced neutrophil chemoattractant-1 (CINC-1), and monocyte chemoattractant protein 1 (MCP-1) (1, 19, 30, 32, 40), which are potent chemotactic and activating factors for leukocytes (3). IL-8 and CINC-1 belong to the CXC chemokine family, whereas MCP-1 is a prototype of CC chemokines. IL-8 plays a significant role in several types of human gastric injury through the attraction and activation of neutrophils. Although a rat counterpart of IL-8 has not yet been identified, CINC-1 is thought to play a pivotal role in rat gastric injury.
Presently available evidence suggests that IL-8 may play a significant role in the pathogenesis of gastritis, which is closely associated with Helicobacter pylori infection (1, 19, 30, 32, 40). Several studies have shown that H. pylori is capable of upregulating epithelial cell IL-8 production in gastric epithelial cell lines (42). Furthermore, gastric mucosal levels of IL-8 are increased in parallel with the histological severity of gastritis (2, 32). Prolonged production of IL-8 by gastric epithelial cells could result in the recruitment of leukocytes to gastric tissues (35). Infiltrated leukocytes would produce a number of proinflammatory cytokines, reactive oxygens, and chemical mediators, which would further contribute to the progression of inflammatory processes.
Gastrin is a peptide hormone that stimulates gastric acid secretion and
the growth of fundic mucosa in the stomach (38). Hypergastrinemia is found not only in patients with gastrin-producing endocrine cell tumors or with type A atrophic gastritis
(6) but also in individuals with H. pylori-associated gastritis (24). It has been
reported that proinflammatory cytokines including IL-1, tumor
necrosis factor (TNF)-
, and IL-8 (4, 5) are able to
stimulate gastrin release from G cells. In addition, IL-1
, which can
also act as a potent inhibitor of acid production, may cause
hypochlorhydria, which results in hypergastrinemia (46).
Although the findings of some investigators suggest that
hypergastrinemia may contribute to the progression of chronic atrophic gastritis (8, 39), the pathophysiological significance of gastrin in the inflammatory and immune process has not been well studied. The purpose of the present study was to examine the effect of
gastrin on expression of CXC chemokines in human and rat gastric epithelial cells. Since epithelial cell lines originating from gastric
epithelial cells and expressing gastrin receptor have not been
identified, we decided to produce such cell lines by DNA transfection.
We previously developed a rat gastric epithelial cell line that stably
expresses human gastrin receptor. In the present study, we report on
the transfection of the gastrin receptor cDNA into MKN28 cells, a human
gastric cancer cell line. Using these cell lines, we have shown that
gastrin is capable of upregulating production of CXC chemokines through
the activation of the transcription factors nuclear factor (NF)-B
and activator protein-1 (AP-1). This represents the first report that
demonstrates that gastrin induces chemokine expression in gastric
epithelial cells.
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MATERIALS AND METHODS |
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Reagents.
RPMI-1640 medium, DMEM, FCS, and Genecitin (G418) were obtained from
GIBCO BRL (Grand Island, NY). Gastrin 17, cholecystokinin-8 (CCK-8),
and somatostatin were purchased from the Peptide Institute (Osaka,
Japan). IL-1 was a generous gift from Otsuka Pharmaceutical (Tokushima, Japan). TNF-
, pyrrolidine dithiocarbamate (PDTC), 12-O-tetradecanoylphorbol 13-acetate (TPA), staurosporine,
and PD 98059, a selective mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, were obtained from Sigma (St. Louis, MO). Selective gastrin/CCK-B-receptor antagonist L-740093 (28)
and epidermal growth factor (EGF) receptor-specific tyrphostin AG1478 (17) were donated by Merck Sharp & Dohme Research
Laboratories (Rahway, NJ) and Dr. A. Levitzki (Hebrew University,
Jerusalem, Israel), respectively. Radiochemicals
([
-32P]dCTP and [
-32P] ATP) were
purchased from Dupont NEN (Boston, MA).
Cell lines and transfection. MKN28 cells, a human gastric adenocarcinoma cell line (21), were obtained from the Japanese Cancer Research Cell Bank (no. JCRB0253). These cells were grown in RPMI-1640 medium supplemented with 10% FCS, 100 U/ml of penicillin G, and 100 µg/ml streptomycin. MKN28 cells were transfected according to the liposome-mediated transfection method (LipofectAMINE PLUS; Life Technologies, Rockville, MD) with pSV2-neo alone or pSV2-neo plus pSVhGAS-R, a plasmid containing the full-length human CCK-B/gastrin receptor cDNA, which was kindly provided by Dr. T. Horiuchi (Exploratory Research Laboratories, Daiichi Pharmaceutical, Tokyo, Japan). Cells were selected for their ability to grow in the presence of 1.0 mg/ml G418.
RGaR9 cells were established in our laboratory (20) by transfection of the full-length human CCK-B/gastrin receptor cDNA to RGM1 cells, a rat gastric epithelial cell line, obtained from the Riken Cell Bank (Tsukuba, Japan) (13, 16). The cells were grown in DMEM supplemented with 10% FCS, 100 U/ml penicillin G, and 100 µg/ml streptomycin.Bacterial preparations. We have used the urease-positive Tox+ CagA+ wild-type H. pylori 60190 strain (ATCC 49503). An extract of water-soluble proteins were prepared as described (4). In the experiments with bacterial suspensions, cells were incubated with bacterial preparations at a concentration of 1 × 108 CFU/ml in DMEM supplemented with 5% FCS.
Mitogenic assays. The cells were seeded onto 96-well plates, grown to confluence, and incubated in serum-free medium for 48 h. The cells were subsequently incubated for 18 h with human gastrin 17 with or without the CCK-B/gastrin receptor antagonist L-740093 and were labeled with 1 µCi/ml [3H]thymidine (Amersham, Arlington Heights, IL) 18-22 h later. The cells were washed three times with PBS and harvested by trypsinization, and the incorporated [3H]thymidine was measured by a Beta system (Pharmacia, Uppsala, Sweden).
Northern blot analysis.
The cells, which were grown in 90-mm dishes until they reached
confluence, were serum deprived for 24 h and then stimulated with
108 M human gastrin 17, 10
8 M human CCK-8,
10
8 M human somatostatin, 10 ng/ml IL-1
, or 10 ng/ml
TNF-
in a serum-free medium. Each reagent was dissolved in 10 µl
distilled water. The control cells were treated with a medium
containing 10 µl distilled water alone. After 0, 0.5, 1, 3, 5, 7, 24, or 48 h, total RNA was extracted by the guanidinium thiocyanate
method (7). Approximately 15 µg of each RNA was
electrophoresed onto 1.0% agarose/2.2 M formaldehyde denaturing gels,
transferred to Hybond-N+ membranes (Amersham, Arlington,
IL), and ultraviolet cross-linked (1,200 J/M2).
Hybridization was performed using cDNA probes, which were labeled by
random priming (Multiprime DNA labeling system; Amersham) with [
-32P]dCTP in Rapid-hyb buffer (Amersham). After
hybridization, the membranes were exposed to Kodak XOMAT AR film
(Eastman Kodak, Rochester, NY). In some experiments, the cells were
preincubated with the gastrin/CCK-B receptor antagonist L-740093 (10 nM), the inhibitor of NF-
B, PDTC (~10-50 µM), the selective
MEK inhibitor PD-98059 (50 nM), or the EGF receptor-specific tyrphostin
AG1478 (250 nM). To determine the role of protein kinase C (PKC) in
gastrin-induced chemokine expression, the cells were preincubated with
staurosporine (100 nM) for 1 h or with TPA (500 nM) for 24 h
before gastrin 17 treatment.
Measurement of human IL-8 production by ELISA.
Cells were seeded and grown to confluence in 24-well plates. The cells
were then stimulated with 108 M gastrin 17, 10 ng/ml
IL-1
, 10 ng/ml TNF-
, or H. pylori extract (1 × 107 CFU/ml) in a medium containing 5% FCS. In several
experiments, the cells were preincubated with L-740093 (10 nM) or PDTC
(10~50 µM) 1 h before stimulation. After the cells were
incubated for 24 h, the concentration of human IL-8 in the
conditioned media was evaluated by using a commercially available ELISA
kit (Biosource International, Camarillo, CA) according to the
manufacturer's guidelines.
Luciferase assay.
The 5'-flanking region of the IL-8 gene, spanning from bp 133 to +44
was subcloned into a luciferase expression vector as previously
described, and site-directed mutagenesis of the NF-
B, AP-1,
and NF-IL-6 binding sites was then carried out (14). For the luciferase assay, cells were transfected with 5 µg of each luciferase vector along with 1 µg of pRL-SV40 expression vector as an
internal control by using LipofectAMINE PLUS. After a 24-h incubation
period, the cells were treated with or without 10
8 M
gastrin 17 for 24 h. The cells were then lysed with 1× luciferase lysis buffer (Toyo Ink, Tokyo, Japan). Luciferase activity was measured
using the PicaGene reagent kit (Toyo Ink) in a Lumat LB9501 luminometer
(Berthold, Wildbad, Germany). The enzyme activity was normalized for
efficiency of transfection on the basis of SeaPansy luciferase
activity, and relative values were determined. Transfection experiments
were carried out three times independently, and the average of these
values was calculated.
Electrophoretic mobility shift assay.
Nuclear proteins were extracted from the cells which had been incubated
in the presence or absence of 108 M gastrin for 0.5 h as described by Dignam et al. (9). Protein concentrations were determined using the method involving bicinchoninic acid (BCA protein assay reagent, Pierce, Rockford, IL)
(34). Electrophoretic mobility shift assay (EMSA) was
performed using gel shift assay systems (Promega, Madison, WI)
according to the manufacture's guidelines. Double-stranded
oligonucleotide probes for NF-
B (5'-AGTTGAGGGGACTTTCCCAGGC-3') and
AP-1 (5'-CGCTTGATGAGCAGCCGGA A-3') were labeled with
[
-32P]ATP. Each 10 µg of nuclear proteins were
incubated with a labeled probe (5 × 104 cpm) and 0.5 µg/ml poly(dI-dC) in 10 µl of binding buffer [4% glycerol with
(in mM) 1 MgCl2, 0.5 EDTA, 0.5 dithiothreitol, 50 NaCl, and 10 Tris · HCl, pH 7.5] for 20 min at room
temperature. Samples were loaded onto 4% polyacrylamide gel
(acryoamid/N,N'-methylene bisacryoamide, 30:1)
with 0.5× Tris borate buffer. After electrophoresis, the gels were
dried and exposed to Kodak XOMAT AR film.
Statistical analysis. Data are expressed as means ± SE. Statistical comparisons between groups were performed with the Mann-Whitney U-test. A P value of < 0.05 was considered to be statistically significant.
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RESULTS |
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Expression of gastrin/CCK-B receptor mRNA by transfected MKN28
cells.
The plasmid pSVhGAS-R containing a full-length human CCK-B/gastrin
receptor cDNA was transfected into MKN28 cells. Forty-three G418-resistant clones were picked and screened for the expression of
human CCK-B/gastrin receptor mRNA. We found that one of these clones,
designated MKGR26, strongly expressed the 2.7-kb gastrin receptor mRNA
(Fig. 1A). In contrast, the
parental MKN28 cells did not express CCK-B/gastrin receptor. The
following experiments were performed with MKGR26 cells. When we assayed
the effects of gastrin 17 on cell growth, we found that gastrin dose
dependently increased the incorporation of [3H]thymidine
into MKGR26 cells (Fig. 1B). However, the effects of gastrin
on MKGR26 cells were inhibited by treatment of these cells with the
CCK-B/gastrin receptor antagonist L-740093 (Fig. 1B).
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Effect of gastrin on the expression of chemokine transcripts.
We next examined the issue of whether gastrin 17 alters the expression
of mRNAs for chemokines in MKGR26 cells and RGaR9 cells. IL-8 mRNA was
induced in MKGR26 cells within 1 h, reached a peak level at 5 h, and then gradually decreased after the cells were incubated with
108 M gastrin 17 (Fig.
2A). In contrast, neither
parental MKN28 cells nor cells transfected with pSV2-neo alone showed
any response to gastrin 17 (data not shown). CINC-1 mRNA was induced in
RGaR9 cells within 30 min, reached a peak level at 1 h, and then
decreased after the cells were incubated with 10
8 M
gastrin 17 (Fig. 2B). Gastrin-induced expression of IL-8
mRNA was dose dependent and inhibited by preincubation with the gastrin receptor antagonist L-740093 (Fig. 2C). Gastrin and CCK-8
are equally effective on IL-8 mRNA expression (Fig. 2D).
However, somatostatin did not affect IL-8 mRNA expression (Fig.
2D).
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Effect of gastrin on the expression of IL-8 protein.
We also determined the effect of gastrin on IL-8 levels in the culture
media of MKGR26 cells. Treatment with gastrin dose dependently
increased the concentration of IL-8 in the culture media (Fig.
4A). This effect of gastrin on
IL-8 secretion was abolished by pretreatment with the selective
CCK-B/gastrin receptor antagonist L-740093 (Fig. 4A).
IL-1, TNF-
, and H. pylori extract also significantly
increased IL-8 levels in the culture media (Fig. 4B).
Synergistic interaction was observed between gastrin and IL-1
,
TNF-
, or H. pylori relative to the production of IL-8 (Fig. 4B).
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Transcriptional regulation of the IL-8 gene by gastrin.
It is known that the expression of IL-8 is regulated at the
transcriptional level (22, 23). As a result, the effects
of gastrin on IL-8 gene transcription were examined. Gastrin enhanced luciferase activities in MKGR26 cells that had been transfected with
the luciferase expression vector containing a minimally essential promoter region of the IL-8 gene (bp 133 to + 44) by threefold (Fig. 5). This promoter region contains
three important cis-acting elements for IL-8 gene
transcription, namely the NF-
B, AP-1, and NF-IL-6 binding sites. The
contribution of each element to the gastrin-induced activation of IL-8
gene transcription was next examined. The mutation of the
NF-
B-binding site completely abolished gastrin-induced enhancement
in luciferase activities (Fig. 5). The mutation of the AP-1 binding
site partially inhibited gastrin-induced enhancement, as evidenced by
luciferase activities, whereas the mutation of the NF-IL-6 binding site
had no effect on luciferase activities (Fig. 5). These results indicate
that the NF-
B binding site is absolutely required and the AP-1
binding site is partially required for gastrin-induced IL-8 gene
transcription.
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Gastrin increased NF-B- and AP-1-specific binding activities in
MKGR26 cells.
Because the NF-
B and AP-1 binding sites appeared to be required for
the gastrin-induced transcription of the IL-8 gene, EMSA was performed
to determine whether gastrin is capable of activating NF-
B and AP-1.
Treatment with gastrin activates NF-
B within 30 min, and this effect
was abrogated by pretreatment with L-740093 or PDTC (Fig.
6A). Although AP-1 appeared to
be activated to a considerable extent in this cell line without gastrin
stimulation, the presence of gastrin further increased AP-1 binding
activity significantly. This activation was diminished when the cells
were pretreated with L-740093. Pretreatment with PDTC, however, had no
significant effect on the gastrin-induced increase in AP-1 binding
activity (Fig. 6B).
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Effects of inhibition of NF-B on gastrin-induced chemokine
expression.
To further clarify the involvement of NF-
B on the extent of
chemokine expression, we examined the effect of PDTC on the
gastrin-induced expression of chemokines. Pretreatment with PDTC
inhibited the expression of IL-8 transcripts in MKGR26 cells (Fig.
7A) as well as that of CINC-1
mRNA in RGaR9 cells (Fig. 7B) in a dose-dependent manner.
Pretreatment with PDTC also abolished the release of IL-8 after gastrin
stimulation in MKGR26 cells (Fig. 8).
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Effects of inhibition of protein kinases on gastrin-induced
chemokine expression.
It is known that CCK-B/gastrin receptor-mediated signals involve the
activation of PKC and MAPK (33, 43). In an earlier report,
we indicated that gastrin induces the expression of EGF-like growth
factors, which, in turn, activate EGF receptor (20). As a
result, we examined the effects of the MEK inhibitor PD-98059, the EGF
receptor tyrosine kinase inhibitor AG1478, and PKC inhibitor staurosporine on the gastrin-induced expression of chemokines. As
expected, the gastrin receptor antagonist L-740093 completely inhibited
the effect of gastrin, whereas PD-98059, AG1478, and staurosporine
partially inhibited gastrin-induced expression of IL-8 or CINC-1
transcripts in MKGR26 cells and RGaR9 cells (Fig. 9). Downregulation of PKC by pretreatment
with TPA also partially inhibited gastrin induction of CINC-1 in RGaR9
cells (Fig. 9B).
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DISCUSSION |
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The present study shows that gastrin induces the expression of chemokines, including IL-8 and CINC-1, in gastric epithelial cells that have been transfected with the gastrin receptor. Chemokines represent a recently described family of inflammatory cytokines that have leukocyte chemotactic and activating properties (3). The chemokine superfamily has been divided into two major subgroups: the CXC chemokines and CC chemokines. In general, the CXC chemokines, which include IL-8 and CINC-1, appear to primarily affect neutrophils, whereas CC chemokines, which include MCP-1, functionally act on monocytes and lymphocytes (3). Of these chemokines, IL-8 is thought to be important in the initiating and perpetuating of the inflammatory process of H. pylori-associated gastritis (1, 19, 30, 32, 40). Previous studies have shown that different strains of H. pylori are able to induce the expression of IL-8 in parallel with their pathogenicity in gastric epithelial cell lines (31). In addition, mucosal IL-8 correlates with the infiltration of polymorphonuclear cells in H. pylori-associated gastritis (35). It is generally recognized that hypergastrinemia often occurs early in the course of H. pylori infection (24, 25, 36). The present study also shows that gastrin dramatically enhances the H. pylori-induced release of IL-8. Thus gastrin may modulate the expression of IL-8 and contribute to the progression of inflammatory processes during H. pylori infection.
Prolonged IL-8 expression by gastric epithelial cells could result in
mucosal infiltrates, which produce large amounts of proinflammatory
cytokines such as IL-1 and TNF-
(26, 37, 45). These
cytokines may, in turn, stimulate the expression of epithelial
chemokines and further promote the inflammatory and immune process. The
data herein show that gastrin dramatically potentiates IL-8 expression
in gastric epithelial cells induced by H. pylori, IL-1
,
or TNF-
. Hypergastrinemia associated with H. pylori
infection often is resolved after eradication of the infection
(25, 36) and therefore is recognized to be secondary to
gastric inflammation. In fact, IL-1
and TNF-
are capable of
stimulating the release of gastrin from G cells. In addition, IL-1
,
which can also act as a potent inhibitor of acid production, may cause
hypochlorhydria, which results in hypergastrinemia (46). Our results suggest that hypergastrinemia is not only a result of
inflammation but also may promote gastric inflammation synergistically with the inflammatory cytokines.
The expression of the IL-8 gene is primarily controlled at the
transcriptional level (22, 23). A nucleotide sequence
analysis of the 5'-regulatory region of the IL-8 gene shows the
presence of potential binding sites for several transcription factors, including NF-B, AP-1, and NF-IL-6 (22, 23, 44). It is
known that the transcription of the IL-8 gene requires either a
combination of NF-
B and AP-1 or of NF-
B and NF-IL-6, depending on
the type of cells (1, 19, 44). In our experiments, the
NF-
B binding site was found to be absolutely required, whereas the
AP-1 binding site was partially required for the gastrin-induced
transcription of the IL-8 gene. EMSA showed that gastrin enhances the
binding activities of both NF-
B and AP-1. PDTC, a well-known
inhibitor of I-
B degradation (18), abrogated not only
the gastrin-induced activation of NF-
B but also the expression of
IL-8 mRNA and protein as well. These results suggest that gastrin
induces IL-8 expression via the activation of NF-
B and AP-1 and that
the activation of NF-
B is indispensable for gastrin-induced IL-8
expression. Similarly, NF-
B appears to be required for the
gastrin-induced expression of CINC-1.
The gastrin receptor is identical to the CCK-B receptor, and both CCK-A
and gastrin/CCK-B receptors belong to the family of G-protein-coupled
receptors (33, 43). As expected, the present study shows
that CCK-8 and gastrin are equally effective in inducing the expression
of IL-8 mRNA. The binding of an agonist to CCK-A or CCK-B receptor
activates phospholipase C, which catalyses the hydrolysis of
phosphatidylinositol biphosphate, thus generating inositol
1,4,5-triphosphate and diacylglycerol, which mobilize intracellular
Ca2+ and activate PKC, respectively (33, 43).
It is interesting to note that recent studies indicate that CCK is
capable of activating NF-B and inducing chemokines in pancreatic
acinar cells via the CCK-A receptor (11, 12). In vivo
experiments also showed that supramaximal stimulation of CCK-A
receptors causes acute pancreatitis, suggesting that NF-
B activation
is an important early event (10, 27). Since the activation
of PKC appears to be required for CCK-induced chemokine expression in
pancreatic acinar cells, the effect of the PKC inhibitor staurosporine
on gastrin-induced chemokine expression was tested. Staurosporine was
found to significantly inhibit the gastrin-induced expression of IL-8
and CINC-1, suggesting that this effect is, in part, mediated by a
PKC-dependent pathway.
Gastrin receptor-mediated signals also involve the activation of MAPK
and the upregulation of c-jun and c-fos
(33, 43). We previously showed (20) that
gastrin induces EGF-like growth factors as autocrine/paracrine growth
factors through a MAPK pathway. It has also been reported that EGF can
activate NF-B and induce IL-8 secretion in certain epithelial cell
lines (15, 29). Our present study shows that the selective
EGF receptor kinase inhibitor AG1478 as well as MEK inhibitor PD-98059
significantly inhibits the induction of chemokines by gastrin. Thus
EGF-like growth factors may partly mediate the effect of gastrin on
chemokine expression.
In conclusion, the present study demonstrates for the first time that
gastrin induces IL-8 production in gastric epithelial cells through the
activation of NF-B and AP-1. Our findings suggest a possible role of
gastrin in the development of gastric inflammation. Further studies are
needed to clarify the involvement of gastrin in the inflammatory and
immune process of H. pylori-associated chronic gastritis.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Miyazaki, Dept. of Internal Medicine and Molecular Science, B5, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita Osaka 565-0871, Japan (E-mail: miyazaki{at}imed2.med.osaka-u.ac.jp).
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.
Received 13 December 2000; accepted in final form 2 May 2001.
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