Helicobacter pylori induces gastric mucosal intestinal metaplasia through the
inhibition of interleukin-4-mediated HMG box protein Sox2 expression
Sho Asonuma, Akira Imatani, Naoki Asano, Tomoyuki Oikawa, Hidetomo Konishi, Katsunori Iijima,
Tomoyuki Koike, Shuichi Ohara, and Tooru Shimosegawa
Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
Submitted 19 November 2007; accepted in final form 4 June 2009
Asonuma S, Imatani A, Asano N, Oikawa T, Konishi H, Iijima
K, Koike T, Ohara S, Shimosegawa T. Helicobacter pylori induces
gastric mucosal intestinal metaplasia through the inhibition of inter-
leukin-4-mediated HMG box protein Sox2 expression. Am J Physiol
Gastrointest Liver Physiol 297: G312–G322, 2009. First published
June 11, 2009; doi:10.1152/ajpgi.00518.2007.—Helicobacter pylori
is a major cause of the transdifferentiation into intestinal metaplasia
that may develop gastric cancer. However, the molecular pathogenesis
of this transdifferentiation is poorly understood. A SRY-related HMG
box protein Sox2 is an essential transcription factor of organ devel-
opment in brain, lung, and stomach. Our aim of this study was to
investigate the mechanism responsible for regulation of Sox2 in host
Th1-dominant response to H. pylori. Sox2 protein was immunohisto-
chemically expressed in both human oxyntic and pyloric glands with
H. pylori infection, but not in intestinal metaplasia. Western immu-
noblotting of gastric epithelial cell lines showed that IL-4, a Th2-
related cytokine, dose dependently enhanced Sox2 expression among
H. pylori infection-mediated cytokines. Small changes of Sox2 ex-
pression were observed after each treatment with IFN-?, IL-1?, or
TNF-?. IL-4-mediated Sox2 induction was suppressed by the inhibi-
tion of STAT6 activation with STAT6 RNA interference, and elec-
trophoretic mobility shift assay indicated that activation of the Sox2
promoter by IL-4 occurred through the action of STAT6. Further-
more, H. pylori and IFN-? inhibited the phosphorylation of STAT6,
resulting in the suppression of IL-4-mediated Sox2 expression. Im-
munohistochemical analyses showed significantly the suppressed
STAT6 activity in H. pylori-infected human gastric mucosa. Addi-
tionally, downregulation of Sox2 by knockdown experiments led to
intestinal phenotype with expressions of Cdx2 and MUC2. These
results suggest that H. pylori and IFN-? interfere with the differenti-
ation into oxyntic and pyloric glands by the downregulation of Sox2
on IL-4/STAT6 signaling, which may contribute to the transdifferen-
tiation into intestinal metaplasia.
transdifferentiation; STAT6 signaling; Cdx2
HELICOBACTER PYLORI (H. pylori) is a gram-negative spiral-
shaped organism that colonizes human gastric mucosa (26).
Although most infected people remain asymptomatic, the bac-
terium causes gastritis, gastroduodenal ulcer, gastric adenocar-
cinoma, and lymphoma (15, 19). Correa (7) has proposed that
H. pylori infection causes chronic active gastritis and then
leads to intestinal metaplasia in both the antrum and body of
stomach, which are thought to be precursor lesions of the
development of well-differentiated adenocarcinoma.
In view of local inflammation, once infected, polymorpho-
nuclear neutrophils and mononuclear cells infiltrate into gastric
mucosa as the host immune response to H. pylori, resulting in
the production of proinflammatory cytokines such as IL-8,
IFN-?, TNF-?, IL-1?, and IL-4 (22, 29, 36, 42). The profile of
these cytokines depends on the balance between type 1 and
type 2 T helper (Th) lymphocytes. In H. pylori infection,
gastric inflammatory response predominantly shifts to Th1
response (29, 42). Furthermore, it has been reported that Th1
immune response might be important in the progression of gastric
atrophy and intestinal metaplastic formation in a concurrent in-
fection model with Helicobacter felis and helminth (13).
Hence, Th1 predominant inflammatory response to H. pylori
may be a key factor of the transdifferentiation to gastric
atrophy and intestinal metaplasia, which are considered pre-
Recent studies have suggested that several differentiation
genes such as Cdx2, Pdx1, Sonic hedgehog, and Oct1 (2, 10)
play an important role in this transdifferentiation. Especially, a
homeobox gene Cdx2 is induced at a late stage of the trans-
differentiation to intestinal metaplasia (3, 40), because it is
thought to be an initiator gene in the differentiation of intestinal
epithelium, controlling many genes such as MUC2 (28), lac-
tase (11), intestinal alkaline phosphatase (1), and furin (14).
However, molecular pathogenesis of this transdifferentiation
by the host immune response to H. pylori is poorly understood.
A sex-determining region Y-box 2 (Sox2), a member of the
high mobility group (HMG) domain proteins, is a crucial
transcription factor for the maintenance of embryonic stem
(ES) cells pluripotency (5, 43), and organ development of
neural tissues (39) and sensory epithelia (24, 46). In addition,
Sox2 also contributes to the development of foregut-derived
organs such as esophagus, lung, and stomach, but not to the
development of hindgut-derived organs of intestine (20, 38). In
the stomach, Sox2 was first reported as a protein expressed in
epithelial cells during the morphogenetic process of embryonic
chick stomach (38). Even in the human adult stomach, the
expression of Sox2 was observed on the RNA level (48).
Moreover, in vitro analyses showed that Sox2 upregulated
stomach-specific expressions of pepsinogen A (45) and
MUC5AC (25) genes. These studies suggest that Sox2 is one of
the candidate genes related with the differentiation in the
The aim of the present study was to analyze the molecular
mechanism responsible for the regulation of Sox2 by the
immune response to H. pylori and to investigate the role of
Sox2 in the transdifferentiation from oxyntic and pyloric
glands to intestinal metaplasia.
MATERIAL AND METHODS
Tissue samples. Specimens of H. pylori-infected human gastric
mucosa were endoscopically obtained from H. pylori-infected patients
with gastritis, gastric cancer, MALToma, gastric ulcer, or duodenal
Address for reprint requests and other correspondence: A. Imatani, Division
of Gastroenterology, Tohoku Univ. Graduate School of Medicine, 2-1 Seiryo-
machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan (e-mail: firstname.lastname@example.org.
Am J Physiol Gastrointest Liver Physiol 297: G312–G322, 2009.
First published June 11, 2009; doi:10.1152/ajpgi.00518.2007.
0193-1857/09 $8.00 Copyright © 2009 the American Physiological Societyhttp://www.ajpgi.orgG312
ulcer at Tohoku University Hospital (Sendai, Japan). Nonneoplastic
gastric mucosa distant from carcinomas was collected. In addition,
biopsy specimens after H. pylori eradication were also obtained from
six patients among them. The presence of H. pylori infection was
confirmed by at least one positive result of three different methods for
determining the H. pylori status: histology, rapid urease test, [13C]urea
breath test. In addition, to confirm H. pylori eradication, H. pylori
negativity was defined by all of these three different tests 2 mo after
eradication. This study was conducted with an approval from the
Ethical Review Board of Tohoku University and examined after
obtaining of informed consent from the subjects.
Cell culture. AGS, Caco-2, and HT29 were obtained from the
American Type Culture Collection (ATCC, Manassas, VA). NUGC4,
KATOIII, GCIY, and THP-1 were obtained from Cell Resource
Center for Biomedical Research, Tohoku University (Sendai, Japan).
MKN45 and MKN28 were purchased from the Health Science Re-
search Resources Bank (Osaka, Japan). AGS was cultured in F-12
(Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated
fetal bovine serum (FBS). The other cell lines were cultured in
RPMI-1640 (Invitrogen) with 10% FBS. All cell lines were incubated
at 37°C in a 5% CO2atmosphere for experiment.
Growth of bacteria and conditions of infection. H. pylori (cagA?
strain 43504; ATCC) was grown on Skirrow plates (Nissui, Tokyo,
Japan) and incubated at 37°C in a microaerophilic condition (10%
CO2, 5% O2, and 85% N2). After an overnight incubation on the
plates, bacteria were cultured in a Brucella broth (Invitrogen) supple-
mented with 10% FBS for 2 days. Bacterial numbers were standardized
by optical density measurement at 600 nm [OD600, 1.5 ? 3.6 ? 108
colony forming units (cfu)/ml] according to the previous report (23).
Immunohistochemistry. Tissue samples were fixed in 10% buffered
formalin and embedded in paraffin. Serial sections cut from paraffin
blocks (3 ?m) were dewaxed. After the inhibition of endogenous
peroxidase activity, antigen retrieval was carried out by microwaving
the sections in 10 mmol/l citrate buffer for 30 min. For first antibodies,
we applied anti-Sox2 1:2,000 (Chemicon, Temecula, CA), anti-Cdx2
1:80 (BioGenex, San Ramon, CA), anti-MUC5AC 1:100 (Novocastra
Laboratories, Newcastle, UK), anti-MUC2 1:100 (Novocastra Labo-
ratories), anti-Ki-67 1:200 (DakoCytomation, Glostrup, Denmark),
and anti-phosphorylated-signal transducers and activator of transcrip-
tion 6 (pSTAT6) 1:300 (Cell Signaling Technology, Beverly, MA)
and incubated sections overnight at 4°C. After the application of
appropriate secondary antibodies for 30 min, bindings were visualized
with a mixture of 0.03% diaminobenzidine and 0.05% hydrogen
peroxide in 0.05 M Tris?HCl buffer. Sections were counterstained
with hematoxylin in the standard fashion.
The pSTAT6-positive ratio was calculated by counting pSTAT6-
positive epithelial cells in the entire gastric pits longitudinally sec-
tioned and representing them as a percentage of the total epithelial
cells in a gastric pit. At least 10 well-oriented gastric pits (more than
500 epithelial cells) were examined for each specimen. The count was
performed in high-power view ( ?100) by two observers (S. Asonuma
and A. Imatani) independently. Only nuclei stained unequivocally
were considered positive.
Stimulation with cytokines and H. pylori. Human gastric epithelial
cells, AGS, and MKN45 were stimulated with IFN-? (R&D Systems,
Minneapolis, MN), IL-1? (Roche, Indianapolis, IN), TNF-? (Roche),
and IL-4 (PeproTech, London, UK), respectively, according to each
procedure. Another set of stimulation with IL-4 (50 ng/ml) for 8 h was
performed in these cell lines after the pretreatment with H. pylori or
IFN-? for 24 h.
Western blot analysis. Proteins were extracted by using a lysis
buffer that consisted of 0.05 M Tris?HCl (pH 7.5), 0.15 M NaCl, 1%
Table 1. Sequences of primers for RT-PCR
cDNA Primer SequenceSize
F, forward; R, reverse.
Fig. 1. Representative immunohistochemical
sections of Sox2, Ki-67, and MUC5AC in
Helicobacter pylori-infected human oxyntic
mucosa. Sox2 is strongly expressed in the
nuclei of proliferative cells, mostly labeled
with Ki-67. In the foveolar and oxyntic glan-
dular cells, it is sporadically expressed. Orig-
DOWNREGULATION OF SOX2 EXPRESSION IN H. PYLORI INFECTION
AJP-Gastrointest Liver Physiol • VOL 297 • AUGUST 2009 • www.ajpgi.org
33. Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F. Oct-3/4 and
Sox2 regulate Oct-3/4 gene in embryonic stem cells. J Biol Chem 280:
34. Orsini B, Ottanelli B, Amedei A, Surrenti E, Capanni M, Del Prete G,
Amorosi A, Milani S, D’Elios MM, Surrenti C. Helicobacter pylori cag
pathogenicity island is associated with reduced expression of interleukin 4
(IL-4) mRNA and modulation of the IL-4?2 mRNA isoform in human
gastric mucosa. Infect Immun 71: 6664–6667, 2003.
35. Otsubo T, Akiyama Y, Yanagihara K, Yuasa Y. SOX2 is frequently
downregulated in gastric cancers and inhibits cell growth through cell-
cycle arrest and apoptosis. Br J Cancer 98: 824–831, 2008.
36. Peek RM Jr. Events at the host-microbial interface of the gastrointestinal
tract IV. The pathogenesis of Helicobacter pylori persistence. Am J
Physiol Gastrointest Liver Physiol 289: G8–G12, 2005.
37. Pellicano ` A, Sebkova L, Monteleone G, Guarnieri G, Imeneo M,
Pallone F, Luzza F. Interleukin-12 drives the Th1 signaling pathway in
Helicobacter pylori-infected human gastric mucosa. Infect Immun 75:
38. Que J, Okubo T, Goldenring JR, Nam KT, Kurotani R, Morrisey EE,
Taranova O, Pevny LH, Hogan BL. Multiple dose-dependent roles for
Sox2 in the patterning and differentiation of anterior foregut endoderm.
Development 134: 2521–2531, 2007.
39. Rex M, Orme A, Uwanogho D, Tointon K, Wigmore PM, Sharpe PT,
Scotting PJ. Dynamic expression of chicken Sox2 and Sox3 genes in
ectoderm induced to form neural tissue. Dev Dyn 209: 323–332, 1997.
40. Silberg DG, Sullivan J, Kang E, Swain GP, Moffett J, Sund NJ,
Sackett SD, Kaestner KH. CDX2 ectopic expression induces gastric
intestinal metaplasia in transgenic mice. Gastroenterology 122: 689–696,
41. So EY, Park HH, Lee CE. IFN-gamma and IFN-alpha posttranscription-
ally down-regulate the IL-4-induced IL-4 receptor gene expression. J Im-
munol 165: 5472–5479, 2000.
42. Smythies LE, Waites KB, Lindsey JR, Harris PR, Ghiara P, Smith
PD. Helicobacter pylori-induced mucosal inflammation is Th1 mediated
and exacerbated in IL-4, but not IFN-?, gene-deficient mice. J Immunol
165: 1022–1029, 2000.
43. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell
126: 1–14, 2006.
44. Takeda K, Tanaka T, Shi W, Matsumoto M, Minami M, Kashi-
wamura S, Nakanishi K, Yoshida N, Kishimoto T, Akira S. Essential
role of Stat6 in IL-4 signaling. Nature 380: 627–6230, 1996.
45. Tani Y, Akiyama Y, Fukamachi H, Yanagihara K, Yuasa Y. Tran-
scription factor SOX2 up-regulates stomach-specific pepsinogen A gene
expression. J Cancer Res Clin Oncol 133: 263–269, 2007.
46. Taranova OV, Magness ST, Fagan BM, Wu Y, Surzenko N, Hutton
SR, Pevny LH. SOX2 is a dose-dependent regulator of retinal neural
progenitor competence. Genes Dev 20: 1187–1202, 2006.
47. Temann UA, Prasad B, Gallup MW, Basbaum C, Ho SB, Flavell RA,
Rankin JA. A novel role for murine IL-4 in vivo: induction of MUC5AC
gene expression and mucin hypersecretion. Am J Respir Cell Mol Biol 16:
48. Tsukamoto T, Inada K, Tanaka H, Mizoshita T, Mihara M, Ushijima
T, Yamamura Y, Nakamura S, Tatematsu M. Down regulation of a
gastric transcription factor, Sox2, and ectopic expression of intestinal
homeobox genes, Cdx1 and Cdx2: inverse correlation during progression
from gastric/intestinal-mixed to complete intestinal metaplasia. J Cancer
Res Clin Oncol 130: 135–145, 2004.
49. Wan H, Kaestner KH, Ang SL, Ikegami M, Finkelman FD, Stahlman
MT, Fulkerson PC, Rothenberg ME, Whitsett JA. Foxa2 regulates
alveolarization and goblet cell hyperplasia. Development 131: 953–964,
50. Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin
SH. A protein interaction network for pluripotency of embryonic stem
cells. Nature 444: 364–368, 2006.
51. Zavros Y, Rathinavelu S, Kao JY, Todisco A, Del Valle J, Weinstock
JV, Low MJ, Merchant JL. Treatment of Helicobacter gastritis with IL-4
requires somatostatin. Proc Natl Acad Sci USA 100: 12944–12949, 2003.
DOWNREGULATION OF SOX2 EXPRESSION IN H. PYLORI INFECTION
AJP-Gastrointest Liver Physiol • VOL 297 • AUGUST 2009 • www.ajpgi.org