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INTRODUCTION |
Helicobacter pylori is a human pathogen that
infects the gastric mucosa and causes an inflammatory process leading
to gastritis, gastric ulceration, duodenal ulceration,
mucosa-associated lymphoid tissue lymphoma, and gastric cancer
(1). The pathogenesis of gastroduodenal diseases caused by this
bacterium is not well understood. Since the whole genome of
H. pylori was sequenced in 1997, several putative
virulence factors, including VacA (2), IceA, OipA (3), HrgA (4),
lipopolysaccharide, and the neutrophil-activating protein (5), have
been elucidated. The cag pathogenicity island (cagPAI),1 a complex of genes
coding ~30 proteins, has been reported to be a major virulence factor
of H. pylori. The cagPAI is acquired by horizontal transfer
and is found in about 50-70% of H. pylori isolates in
Western countries and in more than 90% of H. pylori isolates in Asian countries, including Japan (6, 7). This lesion codes
for the type IV secretion machinery system forming a cylinder-like
structure connected to epithelial cells (8). Many virulence gene
products or other interactive proteins might be transferred into the
host cells via this system. Peptic ulceration and gastric cancer occur
in some people with H. pylori infection, but the majority
remain asymptomatic. Although differences among the degrees of gastric
mucosal damage caused by different strains should be an important
factor for development of various clinical outcomes, these strain
differences do not provide a complete explanation for individual
differences in H. pylori infection-induced gastric mucosal injury. Therefore, it is presumed that host responses also play
an important role in the outcome of H. pylori infection, interacting with virulence factors and environmental factors. Recent
studies have shown that H. pylori induced various cellular responses, proliferation, apoptosis (9), cytoskeletal rearrangement (10), modification of intracellular signaling molecules (11), vacuolation (12), and cytokine secretion (13). In this study, we
investigated the altered gene expression of host cells infected with
cagPAI-positive or cagPAI-negative H. pylori strain and the association between the altered gene expression and the cellular responses.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Cell Lines--
Biopsy specimens were
obtained from Japanese patients in Hokkaido University Hospital and
were cultured on H. pylori-selective agar plates (Eiken
Chemical Co., Ltd., Tokyo, Japan) under microaerophilic conditions (5%
O2, 10% CO2, 85% N2, at 37 °C; Aaero Pack
Systems, Mitsubishi Gas Chemical, Osaka, Japan) for up to 5 days.
Biopsies were obtained with informed consent from all patients under
protocols approved by our ethics committee. The organisms
were identified as H. pylori by spiral morphology and
positive oxidase, urease, and catalase reactions. One colony on the
agar was collected and cultured again under the same microaerophilic
conditions in brain heart infusion broth (Nissui, Osaka, Japan)
containing 5% (v/v) horse serum for up to 3 days. Aliquots were stored
at
80 °C in 10% phosphate-buffered saline containing 20% (v/v)
glycerol. After thawing of aliquots of the frozen culture, bacterial
suspensions were cultured at 37 °C in brain heart infusion broth
containing 10% fetal calf serum (Invitrogen) under microaerophilic
conditions as described above on a gyratory shaker at 170 rpm for
24-36 h to the plateau phase. The human gastric cell lines AGS,
KATOIII, MKN 28, MKN 45 were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and were maintained in a complete medium
consisting of RPMI 1640 medium supplemented with 10% fetal calf serum,
2 mM L-glutamine, 0.2 mg of ampicillin/ml, and
100 µg of kanamycin/ml.
Co-culture of Epithelial Cells with H. pylori--
Human gastric
epithelial cell lines were cultured in RPMI 1640 containing 10% fetal
calf serum without antibiotics and used at a final concentration of
5 × 105/ml. Bacterial suspensions were cultured at
37 °C in brain heart infusion broth containing 10% fetal calf serum
under microaerophilic conditions as described above on a gyratory
shaker at 170 rpm for 24-36 h to the plateau phase. The bacteria were
then suspended in sterile phosphate-buffered saline. After
centrifugation, the bacteria were resuspended at a final concentration
of 1×107 colony-forming units/ml in RPMI 1640 supplemented
with 10% fetal calf serum and used immediately. Gastric epithelial
cells alone or cells with bacteria were cultured in tissue culture
dishes (Falcon; Becton Dickinson) at 37 °C in a humidified incubator in an atmosphere of 95% air and 5% CO2. The cells were
washed with phosphate-buffered saline three times after 4, 8, 12, and 24 h. Total cellular RNA was extracted from the cells by using Isogen reagent (Nippon Gene, Tokyo, Japan) according to the
manufacturer's instructions, and the amount was measured by absorbance
at 260 nm.
cDNA Microarray Procedure--
Poly(A) RNA was isolated from
total cellular RNA (100 µg) using an MagExtractor (Toyobo, Tsuruga,
Japan) according to the manufacturer's instructions. Total cellular
RNA was incubated with oligo(dT) magnetic beads (in the kit), and then
nonspecific substance was removed by washing. 2 µg of mRNA was
reverse transcribed into cDNA by reverse transcriptase, ReverTraAce
(Toyobo), in the presence of a cDNA synthesis primer.
Biotin-labeled probes were generated by binding of
biotin-16-deoxyuridine triphosphate during synthesis of cDNA. The
human cDNA expression filters, human cancer filters (Toyobo) were
prehybridized at 62 °C for 30 min in 20 ml of PerfectHyb solution
(Toyobo). After denaturalization, cDNA probes were hybridized to
the filters overnight at 62 °C. The membranes were washed three
times with solution 1 (2× SSC and 0.1% SDS) and three times with
solution 2 (0.1× SSC and 0.1% SDS) for 5 min at 62 °C. Specific
signals on the filters were detected by using a chemiluminescence
detection kit, Imaging High (Toyobo), according to the manufacturer's
instructions. CDP-Star was used as the chemiluminescent substrate.
Images and quantitative data of gene expression levels were obtained
using a Fluor-S Multiimager system (Nippon Bio-Rad Laboratories, Tokyo,
Japan) and quantified into intensity of signals by using ImaGene
(BioDiscovery, Inc., Los Angeles, CA).
Northern Blot Analysis--
20 µg of total RNA was
electrophoresed on a 1% agarose gel containing 6.5% formaldehyde and
then transferred onto a nylon membrane. A Smad5 probe was made from
human Smad5 cDNA that corresponded to its whole coding region. Each
probe was labeled with biotin using Biotin-16-dUTP (Roche Diagnostics,
Tokyo, Japan). A human
-actin probe labeled with biotin was used as
a positive control. The membrane was hybridized with the labeled probe
for 20 h at 62 °C in PerfectHyb (Toyobo). After hybridization,
it was washed three times with 2× SSC with 0.1% SDS for 10 min and
washed three times with 0.1× SSC with 0.1% SDS for 10 min at
62 °C. Positive bands were detected by using chemiluminescence
detection kit (Imaging High; Toyobo), and CDP-Star was used as the
chemiluminescent substrate according to the manufacturer's instructions.
mRNA Expression by RT-PCR--
First strand cDNA
templates were synthesized from 2 µg of total RNA using ReverTraAce
and a random primer (Toyobo) according to the manufacturer's
instructions. An aliquot (0.1 µl) of Taq DNA polymerase
and deoxynucleoside triphosphates (Takara Shuzou Co., Ltd., Shiga,
Japan) was mixed with 0.5 µl of a first strand cDNA sample and
each primer. The primers used were Smad5F (5'-CAACACAGCCTTCTGGTTCA-3') and Smad5R (5'-TTGACAACAAACCCAAGCAG-3') for Smad5 amplification. PCR
was performed using a thermal cycler (Takara Shuzou) under the
following conditions: an initial denaturation for 5 min at 94 °C;
30 s at 94 °C, 30 s at 55 °C, and 30 s at
72 °C; and a final extension at 72 °C for 5 min with the number
of cycles at which the band intensity increased linearly with the
amount of mRNA used. The PCR product was then run on 1.5% agarose gel.
Analysis of Apoptosis--
We used two methods to detect
apoptosis of epithelial cells induced by H. pylori
infection. After co-culture of AGS cells with H. pylori for
72 h, DNA was extracted from the control and treated cells using
an apoptosis ladder detection kit (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan). Each DNA (5 µg) was electrophoresed in 2% agarose
gels. The gels were photographed under ultraviolet light, and DNA
ladder formation was observed. Next, we carried out quantitative
analysis of apoptosis. AGS cells were cultured with H. pylori for 72 h in 96-well plates (2×104
cells/well). After centrifugation at 1500 rpm for 5 min, the supernatant was removed, and the pellets were frozen at
80 °C for
15 min. Then the terminal deoxynucleotidyl triphosphate-mediated deoxyuridine triphosphate nick end labeling assay was performed using a
apoptosis screening kit (Wako Pure Chemical Industries, Ltd.) according
to the manufacturer's instructions. The degree of apoptosis was
evaluated numerically by measuring the absorbance (490 nm).
Interference of Smad5 mRNA--
Two 29-mer DNA
oligonucleotides (siRNA oligonucleotide templates) with 21 nucleotides
encoding the siRNA and 8 nucleotides complementary to the T7 promoter
primer were chemically synthesized, desalted, and purified by reverse
phase high pressure liquid chromatography. These sequences were
subjected to a BLAST search (NCBI data base) to ensure that only one
gene was targeted. Two 21-mer oligonucleotides (sense,
5'-AATTACATCCTGCCGGTGATA-3' and antisense, 5'-AATATCACCGGCAGGATGTAA-3') encoding Smad5 had no homology to those of Smad1, 2, 3, 4, and 8 in a
BLAST search. The two siRNA oligonucleotide templates were hybridized
to a T7 promotor primer and were extended by the Klenow DNA polymerase.
The sense and antisense siRNA templates were transcribed by T7 RNA
polymerase and were hybridized to create double-stranded siRNA using a
Silencer siRNA construction kit (Ambion). The control and H. pylori-treated cells were grown in 96-well plates, and cationic
lipid-mediated transient transfections were carried out with 50 ng of
siRNA/well using GeneSilencer siRNA transfection reagent (Gene Therapy
Systems, San Diego, CA). After incubation at 37 °C for 24 h,
Northern blot analysis was performed to assess the effectiveness of RNA
interference, and quantitative analysis of apoptosis was carried out as
described above.
Statistics--
The data are presented as the means ± S.D.
The differences were examined by analysis of variance, and p
values < 0.01 were considered significant.
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RESULTS |
Smad5 Up-regulation in Gastric Epithelial Cell Lines--
We first
examined changes in gastric cellular mRNA expression in response to
co-culture with H. pylori (cagPAI-positive, ATCC 43504 strain) at 8 and 24 h by cDNA microarrays in AGS cells. Eleven
housekeeping genes were used as internal controls to correct the
mRNA abundance. Although the majority of genes indicated only small
differences, the expression level of Smad5 mRNA increased dramatically (Fig. 1), with the relative
fold changes in density to housekeeping genes being 0.4, 22.4, and 21.9 at 0, 8, and 24 h, respectively. The expression of the other Smad
family (Smad1, 2, 3, 4, and 8) mRNA including R-Smads were
increased 0.6-1.3-fold after 24 h co-culture and were not
significant. Northern blot analysis was carried out to confirm the
overexpression of Smad5 mRNA. Total RNA was extracted from AGS
cells treated with H. pylori and untreated AGS cells at 4, 8, 12, and 24 h. Northern blot analysis showed that H. pylori infection up-regulated Smad5 mRNA expression of AGS
cells after 4 h of co-culture (Fig.
2). We examined several other gastric
epithelial cell lines (KATOIII, MKN28, and MKN45) to confirm Smad5
mRNA expression after co-culture with H. pylori by
gene-specific RT-PCR. Smad5 mRNA was highly expressed after co-culture with H. pylori in all tested gastric epithelial
cell lines (Fig. 3a).

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Fig. 1.
H. pylori up-regulated Smad5
expression in AGS cells. cDNA microarray filters were
hybridized with probes from AGS cells co-cultured with H. pylori (cagPAI-positive, ATCC 43504 strain) for 8 or 24 h or
probes from AGS cells alone. The arrows indicate a
differentially expressed Smad5 gene. The left three lanes
indicate housekeeping genes.
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Fig. 2.
H. pylori up-regulated Smad5
expression in AGS cells by Northern blot analysis. H. pylori-induced Smad5 mRNA expression of AGS cells was detected
by Northern blot analysis. Total RNA was extracted from the cells
co-cultured with H. pylori (ATCC43504 strain) for the
indicated time intervals. H. pylori infection up-regulated
Smad5 mRNA expression after 8 h of culture. The fold change of
density was indicated. The -actin probe was hybridized as a
control.
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Fig. 3.
H. pylori up-regulated Smad5
expression in other human gastric epithelial cell lines and in native
gastric mucosa. a, total RNA was extracted from human
gastric epithelial cell lines (KATOIII cells, MKN28 cells, and MKN45
cells) co-cultured with live H. pylori for the
indicated time intervals, and the expression of Smad5 mRNA was
analyzed by RT-PCR using the specific primers. Smad5 mRNA
expressions were up-regulated in all of the tested cells. The fold
change of density was indicated. -Actin was amplified as a control
in parallel. b, total RNA was extracted from the five native
gastric biopsy specimens from five patients infected with
cagPAI-positive strains or the five native gastric biopsy specimens
from five uninfected patients, and the expression of Smad5 mRNA was
analyzed by RT-PCR using the specific primers. Smad5 mRNA were
highly expressed in cagPAI-infected gastric mucosa. The fold change of
density is indicated.
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Smad5 Up-regulation in Native Gastric Mucosa--
We tested
whether Smad5 mRNA was expressed in the 10 native gastric biopsy
specimens by RT-PCR. Smad5 mRNA was highly expressed in the native
gastric specimens from five patients infected with cagPAI-positive
strains, however faintly expressed in the five uninfected gastric
specimens (Fig. 3b).
Effects of cagPAI-positive and cagPAI-negative Strains--
To
assess the role of cagPAI in Smad5 mRNA expression, we tested
cagPAI-positive strains (strains 192, ATCC 43504, 912, 904, and 878)
and cagPAI-negative strains (strains 42 and 273) (14, 15) by Northern
blot analysis. Strains 42 and 273 failed to up-regulate Smad5 mRNA
expression, whereas cagPAI-positive strains clearly enhanced its
expression (Fig. 4a).

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Fig. 4.
cagPAI-positive H. pylori
up-regulated Smad5 expression of AGS cells by Northern blot
analysis. Smad5 mRNA expression of AGS cells after co-cultured
with cagPAI-negative (strains 42 and 273) and cagPAI-positive (strains
192, ATCC 43504, 912, 904, and 878) H. pylori strains at
30 h. a, cagPAI-positive H. pylori strains
induced up-regulation of Smad5 significantly, compared with
cagPAI-negative strains. The fold change of density was indicated.
b, after RNA interference, the up-regulated Smad5 mRNA
expressions induced by H. pylori infection were suppressed
in cagPAI-positive strains. The -actin probe was hybridized as a
control.
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Induction of Apoptosis--
DNA fragmentation was induced in AGS
cells 72 h after having been co-cultured with cagPAI-positive
H. pylori strains (strains 192 and ATCC 43504), whereas no
DNA fragmentation was observed with cagPAI-negative strains (strains 42 and 273) (Fig. 5a).
Quantitative analysis of cellular apoptosis showed that cagPAI-positive
H. pylori strains (strains 192, ATCC 43504, 912, 904, and 878) induced significantly greater levels of apoptosis in AGS cells
than did cagPAI-negative strains and AGS cells alone (Fig.
5b). The difference between the results obtained using
cagPAI-positive strains and cagPAI-negative strains or AGS cells alone
was statistically significant.

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Fig. 5.
H. pylori-induced apoptosis
depended on the presence of cagPAI. a, DNA ladder
formation. DNA fragmentation in AGS cells was induced by only
cagPAI-positive H. pylori strains (strains 192 and ATCC
43504), and not by cagPAI-negative H. pylori strains
(strains 42 and 273). b, quantitative analysis of apoptosis
induced by cagPAI-positive and -negative H. pylori strains
using a terminal deoxynucleotidyl triphosphate-mediated deoxyuridine
triphosphate nick end labeling assay. Only cagPAI-positive H. pylori strains (strains 192, ATCC 43504, 912, 904, and 878)
significantly induced apoptosis in AGS cells. The results are expressed
as the absorbance (490 nm). The bars indicate the means of
four independent experiments. *, p < 0.01 versus AGS alone. **, not significant versus AGS
alone.
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Interference of Smad5 Up-regulation and Apoptosis--
The
up-regulated Smad5 mRNA expression was inhibited by Smad5-specific
RNA interference in all cagPAI-positive strain-infected AGS cells (Fig.
4b). With the suppression of Smad5 mRNA expression, the
induction of apoptosis was completely inhibited in the quantitative apoptosis assay (Fig. 6).

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Fig. 6.
H. pylori-induced apoptosis was
reduced after specific suppression of Smad5 mRNA expression by RNA
interference. Quantitative analysis of apoptosis induced by
H. pylori strains after RNA interference. Interference with
Smad5 mRNA expression suppressed the apoptosis of AGS cells
co-cultured with cagPAI-positive strains (strains 192, ATCC 43504, 912, 904, and 878). The results are expressed as the absorbance (490 nm).
The bars indicate the means of four independent experiments.
**, not significant versus AGS alone.
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DISCUSSION |
The gastric pathogen H. pylori activates
epithelial cell signaling pathways after infection. However, the exact
signaling pathways are still unknown. The host immune response to
H. pylori infection might be of importance with regard to
the various clinical outcomes of infection by this organism. We now
report that H. pylori can up-regulate the Smad5 expression
of gastric epithelial cells and that the Smad5 up-regulation is
involved in H. pylori-induced apoptosis of gastric
epithelial cells. In addition, it was found that the presence of intact
cagPAI is essential for Smad5-mediated apoptosis of epithelial cells.
We speculated that a paracrine or autocrine system of TGF-
and bone
morphogenetic proteins (BMP) from infected H. pylori or AGS
cells are involved in the up-regulation of Smad5 expression. In human
gastric epithelial cells, AGS had TGF-
receptors (TGF-
RII) and
BMP receptors, and up-regulation of Smad5 mRNA
expression was observed after exogenous stimulation of TGF-
1 and BMP
by Northern blot analysis (data not shown). Although TGF-
1 and BMP in co-cultured supernatants from H. pylori-infected and
uninfected AGS cells were measured by enzyme-linked immunosorbent
assay, significant differences were not found (data not shown).
Additionally, TGF-
1 mRNA and BMP mRNA were not up-regulated
after co-culture with H. pylori in cDNA array
experiments. Furthermore, because H. pylori itself did not
possess TGF-
1 or BMP-like genes, which was examined by BLAST search
(NCBI data base), it is unlikely that a paracrine or autocrine pathway
of TGF-
or BMP from AGS infected with H. pylori or direct
production of TGF-
or BMP from H. pylori is involved in
up-regulation of Smad5 expression.
The cagPAI region encodes a novel H. pylori
secretion system, type IV machinery (16), and this apparatus is
essential for the induction of interleukin-8 via an
NF-
B-dependent transcriptional process in human gastric
cells (17, 18). It has recently been shown that CagA is injected
from the attached H. pylori into host cells via the
type IV machinery and that it forms a physical complex with SHP-2, the
Src homology 2 domain-containing tyrosine phosphatase, in a
phosphorylation-dependent manner and stimulates the
phosphatase activity (11, 19). These findings suggest that protein or gene injection through the type IV machinery is a key mechanism for
host-bacterial interaction induced by H. pylori infection. Consequently, it is not surprising that the transcriptional response of
gastric epithelial cells is dependent on the presence of cagPAI. We
therefore examined the Smad5 expression of AGS cells using cagPAI-positive and cagPAI-negative strains and that of the native gastric mucosa infected with H. pylori. Our results
indicated that cagPAI-positive H. pylori strains were able
to activate Smad5 mRNA expression and to induce apoptosis of
the infected epithelial cells but that cagPAI-negative strains were not
able to activate Smad5 mRNA expression or induce apoptosis.
Although CagA is the only H. pylori protein known to
translocate from the bacterium into the cell via the type IV secretion
system, it can be assumed that transfer of unknown genes or gene
products through the type IV machinery might be necessary for
up-regulation of the Smad5 gene in host cells.
It has been reported that mutations in Smad4 played a significant role
in the progression of colorectal tumors (20) and that a subset of
families with juvenile polyposis had germ line mutations in the Smad4
gene and were at increased risk of developing gastrointestinal cancers
(21). However, because there has been no report on Smad5 expression in
gastrointestinal tract, the role of Smad5 in physiological or
pathological status is not known.
Smad family proteins have molecular masses of about 42-65 kDa.
Eight different Smads have been identified in mammals and can be
classified into three subclasses, receptor-regulated Smads (R-Smads),
common mediator Smads (Co-Smads), and inhibitory Smads (I-Smads) (22).
Each member of the Smad family plays a different role in signaling
pathways. R-Smads can be further subdivided into two subtypes, those
phosphorylated after stimulation by TGF-
and BMP. Smad5 belongs to
the latter group (23). Smad5 was isolated as dwarfin-C and was
genetically implicated in TGF-
-like signaling pathways in
Drosophila and Caenorhabditis elegans (24).
Suzuki et al. (25) proposed that Smad5 directs the formation
of the ventral mesoderm and epidermis in Xenopus embryos. In
an antisense oligonucleotide study, Smad5 was shown to mediate the
growth inhibitory effect in hematopoietic cells (26), and Yamamoto
et al. (27) suggested that Smad5 inhibited myogenic
differentiation. Furthermore, BMP actively mediated apoptosis in the
embryonic limb (28), and BMP-2 also induced apoptosis in human myeloma
cell lines, probably via up-regulation of R-Smads (Smads1, 5, and 8)
(29). Many studies have demonstrated that H. pylori induced
apoptosis of gastric epithelial cells (30), suggesting that the
up-regulated Smad5 mRNA expression might be involved in the
apoptosis of gastric epithelial cells induced by H. pylori infection.
We also confirmed that only cagPAI-positive H. pylori
strains were capable of inducing up-regulation of Smad5 mRNA as
well as having apoptotic effects in human gastric cells. Although
virulence factors, VacA, and lipopolysaccharide have been investigated
as possible apoptosis-inducing factors (31), the precise intracellular signaling mechanism of apoptosis induced by H. pylori is
still unknown. Our results indicated that Smad5 up-regulation might be
related to the apotosis induced by cagPAI-positive H. pylori infection as one of the intracellular signaling molecules. We therefore
compared the levels of H. pylori-induced apoptosis before and after suppression of Smad5 mRNA expression by RNA interference, and it was found that the induction of apoptosis was reduced to the
background level after the interference. These observations suggest
that Smad5 up-regulation is a key factor for H. pylori-induced apoptosis. In conclusion, H. pylori
up-regulates Smad5 expression through the presence of cagPAI encoding
type IV secretion machinery, and up-regulated Smad5 induces apoptotic
responses in infected gastric epithelial cells.