Tanida, Hiromi Kataoka, Takeshi Kamiya, JS Alexander and Takashi

Transcription

Takashi Mizushima, Makoto Sasaki, Tomoaki Ando, Tsuneya Wada, Mamoru
Tanaka, Yasuyuki Okamoto, Masahide Ebi, Yosikazu Hirata, Kenji Murakami,
Tsutomu Mizoshita, Takaya Shimura, Eiji Kubota, Naotaka Ogasawara, Satoshi
Tanida, Hiromi Kataoka, Takeshi Kamiya, J. S. Alexander and Takashi Joh
Am J Physiol Gastrointest Liver Physiol 298:255-266, 2010. First published Nov 25, 2009;
doi:10.1152/ajpgi.00264.2009
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Am J Physiol Gastrointest Liver Physiol 298: G255–G266, 2010.
First published November 25, 2009; doi:10.1152/ajpgi.00264.2009.
Blockage of angiotensin II type 1 receptor regulates TNF-␣-induced
MAdCAM-1 expression via inhibition of NF-␬B translocation to the nucleus
and ameliorates colitis
Takashi Mizushima,1 Makoto Sasaki,2 Tomoaki Ando,1 Tsuneya Wada,1 Mamoru Tanaka,1
Yasuyuki Okamoto,1 Masahide Ebi,1 Yosikazu Hirata,1 Kenji Murakami,1 Tsutomu Mizoshita,1
Takaya Shimura,1 Eiji Kubota,1 Naotaka Ogasawara,2 Satoshi Tanida,1 Hiromi Kataoka,1
Takeshi Kamiya,1 J. S. Alexander,3 and Takashi Joh1
1
Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya;
and 2Department of Gastroenterology, Aichi Medical University School of Medicine, Aichi, Japan; and 3Department
of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
Submitted 7 July 2009; accepted in final form 18 November 2009
mucosal addressin cell adhesion molecule-1; inflammatory bowel
disease; nuclear factor-␬B
molecules (ECAMs) play essential
roles in the development of chronic inflammation by recruiting
leukocytes, especially lymphocytes, to tissues and by supporting their adhesive behavior, including rolling, firm adhesion,
ENDOTHELIAL CELL ADHESION
Address for reprint requests and other correspondence: M. Sasaki, Dept. of
Gastroenterology, Aichi Medical Univ. School of Medicine, 21 Karimata,
Yasago, Nagakute, Aichi-gun, Aichi 480-2295, Japan (e-mail: msasaki@aichi-med-u.
ac.jp).
http://www.ajpgi.org
and extravasation (20). Infiltration of tissues by leukocytes is a
common hallmark of many chronic inflammatory states, including inflammatory bowel diseases (IBD), ulcerative colitis
(UC), and Crohn‘s disease (CD), as well as of coronary heart
disease and renal disease. The expression of ECAMs, such as
intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and mucosal addressin cell
adhesion molecule-1 (MAdCAM-1), plays an important role at
the onset of IBD in experimental models of colitis (10, 35, 36, 47).
These adhesion molecules are also upregulated in the inflamed
human colon of CD and UC patients (6). MAdCAM-1 is believed
to be the most important of the ECAMs for the development of
colitis (10, 47). MAdCAM-1 is preferentially expressed on
endothelial cells in intestinal mucosa, submucosa, and Peyer’s
patches and plays a central role in leukocyte homing to the
mucosal immune compartment (6). MAdCAM-1 orchestrates
both rolling and firm adhesion of lymphocytes to gut endothelial cells through binding of L-selectin or ␣4␤7-integrin, expressed on immunocytes (4). MAdCAM-1 expression is dramatically amplified during inflammation (10, 42, 44, 47). So
either immunoneutralization of MAdCAM-1, or its ligand,
␣4␤7-integrin, has been the focus of anti-MAdCAM-based
therapy of IBD (19, 27, 55). Recently, humanized monoclonal
antibodies to ␣4␤7- and ␣4-integrin, vedolizumab and natalizumab, respectively, were approved for the therapy of IBD (18,
40, 54). Other adhesion molecules may also be involved in colitis,
as an antibody against VCAM-1 has been reported to prevent
colitis in animal models (3, 21, 41, 53).
In the last decade, many reports have shown that angiotensin
II (ANG II) has significant proinflammatory actions in the
vascular wall, inducing the production of reactive oxygen
species (ROS), inflammatory cytokines, and adhesion molecules via nuclear factor-␬B (NF-␬B) and janus kinase/signal
transducer and activator of transcription (5, 46). An ANG II
type 1 receptor (AT1R) blocker (ARB) regulates arteriosclerosis, cardiovascular death, stroke, and myocardial infarction
via improvement of blood vessel endothelial cell function,
plaque involution, and control of macrophage permeation via
control of inflammation caused by ANG II (11, 39). Recently,
ANG II receptor blockage has been shown to prevent endothelial
inflammation by modulation of TNF-␣-induced VCAM-1 expression via regulation of NF-␬B activation (8, 9). These reports
suggest pleiotropic effects of ARB, such as antioxidant effects,
although the details of these effects are unclear.
0193-1857/10 $8.00 Copyright © 2010 the American Physiological Society
G255
Mizushima T, Sasaki M, Ando T, Wada T, Tanaka M, Okamoto Y, Ebi M, Hirata Y, Murakami K, Mizoshita T, Shimura T,
Kubota E, Ogasawara N, Tanida S, Kataoka H, Kamiya T,
Alexander JS, Joh T. Blockage of angiotensin II type 1 receptor
regulates TNF-␣-induced MAdCAM-1 expression via inhibition of
NF-␬B translocation to the nucleus and ameliorates colitis. Am J
Physiol Gastrointest Liver Physiol 298: G255–G266, 2010. First
published November 25, 2009; doi:10.1152/ajpgi.00264.2009.— Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is an
important target in the treatment of inflammatory bowel disease (IBD).
Recently, treatment of IBD with an antibody to ␣4␤7-integrin, a ligand
for MAdCAM-1, has been an intense focus of research. Our aim was to
clarify the mechanism by which MAdCAM-1 is regulated via angiotensin
II type 1 receptor (AT1R), and to verify if AT1R might be a novel target
for IBD treatment. The role of AT1R in the expression of MAdCAM-1
in SVEC (a murine high endothelial venule cell) and MJC-1 (a mouse
colonic endothelial cell) was examined following cytokine stimulation.
We further evaluated the effect of AT1R on the pathogenesis of immunemediated colitis using AT1R-deficient (AT1R⫺/⫺) mice and a selective
AT1R blocker. AT1R blocker significantly suppressed MAdCAM-1
expression induced by TNF-␣, but did not inhibit phosphorylation of
p38 MAPK or of I␬B that modulate MAdCAM-1 expression. However, NF-␬B translocation into the nucleus was inhibited by these
treatments. In a murine colitis model induced by dextran sulfate
sodium, the degree of colitis, judged by body weight loss, histological
damage, and the disease activity index, was much milder in
AT1R⫺/⫺ than in wild-type mice. The expression of MAdCAM-1
was also significantly lower in AT1R⫺/⫺ than in wild-type mice.
These results suggest that AT1R regulates the expression of MAdCAM-1 under colonic inflammatory conditions through regulation of
the translocation of NF-␬B into the nucleus. Furthermore, inhibition
of AT1R ameliorates colitis in a mouse colitis model. Therefore,
AT1R might be one of new therapeutic target of IBD via regulation of
MAdCAM-1.
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ROLE OF AT1R IN THE REGULATION OF MAdCAM-1 AND COLITIS
The renin-angiotensin system has become a target of digestive disease research, and it has been shown that the angiotensin-converting enzyme inhibitor attenuates pancreatic inflammation (28). Moreover, it has been reported that ANG II is
increased in colonic mucosa of CD patients (24), and, in animal
experiments of colitis, the degree of colitis is attenuated either
by administration of angiotensin-converting enzyme inhibitors
(7, 50, 57) or by using angiotensin gene-deficient mice (23).
These reports suggest the involvement of the renin-angiotensin
system in colonic inflammation. The importance of AT1R for
colitis was recently demonstrated in a colitis model using
AT1R-knockout mice (26). However, the precise mechanism
by which AT1R influences colitis is unclear.
In this paper, we focused on elucidation of the regulation of
AT1R and MAdCAM-1, which are key molecules in the
pathogeneses of colitis.
tions, with light-dark cycles of 12:12 h. The study protocol was
approved by the Animal Care Committee of Nagoya City University
(approval number: 05106).
Experimental Design
Female mice of 9 wk of age were divided into three groups: an
AT1R knockout (AT1R⫺/⫺) colitis group [AT1R⫺/⫺ dextran sulfate sodium (DSS) group], a wild-type (AT1R⫹/⫹) colitis group
(wild DSS group), and a wild-type (AT1R⫹/⫹) noncolitis group
(control group) (n ⫽ 8 per group). The mice in the colitis group were
treated with 2% DSS (MP Biomedicals, Morgan, CA), dissolved in
their drinking water, which was provided ad libitum, and were killed
at the end of 5 days of DSS treatment. The mice in the noncolitis
group were given plain drinking water, provided ad libitum, for 5
days.
For the ARB treatment study, female mice at 9 wk of age were
randomly designated to receive an intraperitoneal injection of either
placebo (normal saline, 0.5 ml) or the ARB, candesartan (Takeda; 0.4
MATERIALS AND METHODS
Reagents
Antibodies to ICAM-1 and VCAM-1 were purchased from R&D
systems (Minneapolis, MN), an antibody to MAdCAM-1 (clone
MECA 367) was purchased from Pharmingen (San Diego, CA), an
antibody to AT1R was purchased from Santa Cruz Biotechnology
(Delaware, CA), and antibodies to phopsho-p38 mitogen-activated
protein kinase (MAPK) (Thr180/Tyr182), p38 MAPK, NF-␬B p65,
IkB-␣, and phospho-I␬B-␣ (Ser32) were purchased from Cell Signaling (Danvers, MA). Recombinant mouse TNF-␣ was purchased from
Sigma (St. Louis, MO). ANG II was purchased from the Peptide
Institute (Minoh-shi, Osaka, Japan). Candesartan (ARB) was purchased from Takeda (Osaka, Japan).
Cell Culture
The SVEC4-10 cell line is an endothelial cell line derived by SV40
(strain 4A) transformation of murine small vessel endothelial cells
originally isolated from the axillary lymph node vessels of an adult male
C3H/Hej mouse (31). This cell line was maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum
and 1% streptomycin-penicillin and incubated at 37°C in an atmosphere of 95% air and 5% CO2. The MJC-1 cell line is a colon
endothelial cell line derived from the mouse strain H-2Kb-tsA58
(Immortomouse) (2). This cell line was maintained in MEM-D-valine
(Promo Cell, Heidelberg, Germany) supplemented with 10% fetal
bovine serum, 2 mM L-glutamine (Life Technologies, Rockville,
MD), 1% nonessential amino acids (Life Technologies), 1% MEM
vitamins solution (Life Technologies), 1% antibiotics/antimycotics
(Life Technologies), and 10 U/ml of IFN-␥ (Endogen, Woburn, MA).
The cells were plated onto 25-cm2 flasks (precoated with 1% gelatin).
The addition of IFN-␥ was used to activate the myosin heavy chain
H-2Kb class I promoter, which regulates the level of the large T
antigen protein in the Immortomouse-derived cells (25). This cell line
was maintained at the permissive temperature (33°C) in an atmosphere of 95% air and 5% CO2, and the cells were cultured at 37°C for
24 h before all experiments to inactive the SV40 large T antigen.
Mice
Female wild-type C57BL/6J (AT1R ⫹/⫹) mice were purchased
from SLC (Hamamatsu, Japan). AT1R knockout male mice
(Agtr1atm1Unc) were purchased from Jackson Laboratory (Bar Harbor,
ME). After being backcrossed at least seven times to the C57BL/6
strain, we bred AT1R⫹/⫹ (littermate control) and AT1R⫺/⫺ (AT1R
knock out) by crossing AT1R⫹/⫺ and AT1R⫹/⫺ mice. The mice
were bred and maintained in the Animal Care Committee of Nagoya
City University, under temperature- and humidity-controlled condiAJP-Gastrointest Physiol • VOL
Fig. 1. Effect of candesartan [a selective angiotensin II type 1 receptor (AT1R)
blocker] on TNF-␣-induced mucosal addressin cell adhesion molecule-1
(MAdCAM-1) protein expression in SVEC and MJC-1 cells. A: SVEC cells
were pretreated with or without candesartan at a concentration of 0, 10, 30, or
50 ␮M for 1 h and stimulated with or without TNF-␣ (20 ng/ml) for 24 h
(n ⫽ 4), and then MAdCAM-1 protein expression was analyzed by Western
blotting using actin blotting as a loading control. Blots were quantified by
densitometry, and the data are presented as fold of internal control. TNF-␣
dramatically increased MAdCAM-1 protein expression, which was inhibited
by candesartan in a dose-dependent manner. Values are means ⫾ SE. *P ⬍
0.05 compared with TNF-␣. #P ⬍ 0.05 compared with control. B: MJC-1 cells
were pretreated with or without candesartan (50 ␮M) for 1 h and stimulated
with TNF-␣ (20 ng/ml) for 24 h, and then inhibition of MAdCAM-1 expression by candesartan was analyzed by Western blotting.
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mg/kg, injection volume 0.5 ml) once a day (n ⫽ 8 per group). The
treatment (placebo or candesartan) was administered daily for as
many days as the mice received DSS. Mice were given 2% DSS
dissolved in their drinking water, which was provided ad libitum, and
were killed at the end of 5 days of DSS treatment.
Data and Specimen Collection
Body weight, stool blood, and the stool form of each mouse were
recorded daily. The presence of occult blood was measured by using
a guaiac-card test (Shionogi, Osaka, Japan). At the end of 5 days, mice
were euthanized with a high dose of ketamine/xylazine, and laparotomy with total colectomy was immediately performed. The colon was
washed with phosphate-buffered saline (PBS) to remove fecal materials.
Western Blotting
Cells and distal colon tissues of mice were washed with PBS (⫺)
and subsequently dissolved in 1⫻ cell lysis buffer (Cell Signaling
Technology) containing 20 mmol/l Tris · HCl (pH 7.5), 150 mmol/l
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NaCl, 1 mmol/l Na2EDTA, 1 mmol/l EGTA, 1% Triton, 2.5 mmol/l
sodium pyrophosphate, 1 mmol/l ␤-glycerophosphate, 1 mmol/l
Na3VO4, and 1 ␮g/ml leupeptin. After disruption using a Bio-ruptor
sonicator (Cosmo Bio) or a homogenizer for 15 s (cells) or for 30 s
(colon tissues), respectively, lysates were centrifuged at 20,000 g for
10 min (cells) or for 30 min (colon tissues) at 4°C. On the other hand,
nuclear extracts of SVEC cells were prepared by using Thermo
Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents
(Thermo Scientific, Rockford, IL), according to the manufacturer’s
instructions. Each sample was normalized on an equal protein concentration by using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Protein samples from cells (75 ␮g each) or from colon
tissues (60 ␮g each) were separated by 10% SDS-PAGE and then
transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). For all blots, the membrane was blocked
with 5% skimmed milk in PBS (⫺) for 1 h at room temperature. The
membrane was then incubated with the primary antibodies indicated
in the text, either anti-MAdCAM-1, ICAM-1, or VCAM-1 for 2 h at
room temperature, or with AT1R, phospho-p38 MAPK, p38 MAPK,
AJP-Gastrointest Physiol • VOL
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Fig. 2. A: knockdown of the AT1R protein using
short interfering RNA (siRNA). SVEC and MJC-1
cells were transiently transfected with one of two
different siRNAs against AT1R (no. 1 or no. 2), or
with the respective scrambled siRNA. Thirty-six
hours after transfection, AT1R protein expression
was analyzed by Western blotting. Protein expression of AT1R was downregulated by both siRNAs
in both cell lines. B: effect of AT1R-siRNA knockdown on TNF-␣-induced MAdCAM-1 protein expression in SVEC and MJC-1 cells. AT1R knockdown by either of the AT1R siRNAs (no. 1 or no.
2) inhibited MAdCAM-1 protein induction by
TNF-␣ (20 ng/ml, 24 h) in both SVEC and MJC-1
cells, as assessed by Western blotting analysis (n ⫽
4). Blots were quantified by densitometry, and the
data are presented as fold of internal control. Values are means ⫾ SE. *P ⬍ 0.05 compared with
scrambled siRNA, TNF-␣ (⫹). #P ⬍ 0.05 compared with scrambled siRNA, TNF-␣ (⫺).
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phospho-I␬B-␣, I␬B-␣, or NF-␬B p65 overnight at 4°C and then
washed with 0.05% Tween 20 in PBS (⫺) three times at 5-min
intervals. The membrane was incubated with secondary antibody for
1 h at room temperature, followed by three washes with 0.05% Tween
20 in PBS (⫺) three times at 5-min intervals. The membrane was then
treated with enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotechnology) for 1 min at room temperature and
exposed to scientific imaging films (Eastman Kodak), and protein
bands were visualized. Filters were stripped and reprobed with a
monoclonal ␤-actin antibody (Abcam Plc., Cambridge, MA), or an
anti-proliferating cell nuclear antigen antibody (Cell Signaling) as
internal controls. The expression of MAdCAM-1, ICAM-1, VCAM-1,
and ␤-actin was quantified by analyzing the density of the scanned gel
band with imaging analyzer. The densitometric result of MAdCAM-1,
ICAM-1, and VCAM-1 was normalized against that of ␤-actin in each
lane, and the data were presented as fold of control.
Real-time Reverse Transcription Polymerase Chain Reaction
Transfection of Short Interfering RNA Oligonucleotides
Two short interfering RNAs (siRNAs) were used for AT1R knockdown. The first siRNA [AT1R no. 1 (AT1 siRNA sc-29751)] was
purchased from Santa Cruz Biotechnology, and the second siRNA
[AT1R no. 2 (Agtr1a siRNA ID: s62143)] was purchased from
Applied Biosystems. Silencer Negative Control no. 1 (scrambled)
siRNA (Applied Biosystems) was used as control siRNA. The day
before transfection, cells were trypsinized, diluted with fresh medium
without antibiotics, and transferred to 24-well plates. Transient transfection of siRNAs was carried out using Oligofectamine (Invitrogen)
according to the manufacturer’s instructions, resulting in a final
siRNA concentration of 200 nM added to the cells. Cells were usually
assayed 36 –72 h after transfection. Specific silencing was confirmed
by at least three independent experiments.
Immunofluorescence Microscopy
Subconfluent SVEC cells were preincubated with or without candesartan (50 ␮M) for 1 h and then stimulated with TNF-␣ (20 ng/ml)
for 1 h, following which the subcellular localization of NF-␬B p65
was analyzed by immunofluorescence. For this assay, the cells were
fixed with ethanol and acetone. Incubation with primary antibodies
against NF-␬B p65 was generally carried out in PBS containing 0.1%
milk at room temperature. Sections were then incubated with the
appropriate secondary antibody, and all sections were counterstained
with 4=,6-diamidino-2-phenylindole (Kirkegaard and Perry Laborato-
Fig. 3. Lack of effect of candesartan on TNF-␣-induced
phosphorylation of p38 mitogen-activated protein (MAP)
kinase, and on the phosphorylation and degradation of
I␬B, in SVEC (A) and MJC-1 (B) cells. The endothelial
cells were pretreated with or without candesartan (50
␮M) for 1 h and then stimulated with TNF-␣ (20 ng/ml)
for 0, 2, 5, 10, or 30 min. Transient phosphorylation of
p38 MAPK and of IkB, as well as degradation of IkB, in
SVEC (A) and MJC-1 (B) cells were assayed by Western
blot and were unaffected by candesartan treatment.
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MAdCAM-1 mRNA expression in response to TNF-␣ treatment in
SVEC and MJC-1 cells, or mRNA levels of monocyte chemoattractant protein-1 (MCP-1), TNF-␣, and MAdCAM-1 in the mice colon
were measured by real-time reverse-transcription polymerase chain
reaction (real-time RT-PCR). For this assay, SVEC and MJC-1 were
first pretreated for 1 h with or without 50 ␮M candesartan and were
then incubated for 4 h with or without 20 ng/ml TNF-␣. Total RNA
was extracted from SVEC and MJC-1 cells, or from distal colon tissue
of mice by using Trizol reagent (Invitrogen, Carlsbad, CA), and 2 ␮g
RNA were reverse transcribed into complementary DNA (cDNA)
using High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, Tokyo, Japan), according to the manufacturer’s instructions.
Primers for mouse MAdCAM-1, TNF-␣, MCP-1, and control mouse glyceraldehydes-3-phosphate dehydrogenase were obtained from Applied Biosystems (MAdCAM-1, Mm00522088_m1; TNF-␣ Mm00443258_m1;
MCP-1, Mm00441242_m1; glyceraldehydes-3-phosphate dehydrogenase,
4352339E). Real-time RT-PCR was carried out using the ABI 7500 Fast
Real-Time PCR system (Applied Biosystems). Each experiment was
carried out in a 20-␮l reaction volume containing 18 ␮l TaqMan Fast
Universal PCR Master Mix (Applied Biosystems), 1 ␮l cDNA, and 1 ␮l
primers. Uniform amplification of the products was rechecked by analysis of the melting curve of the amplified products. All reactions were
carried out in triplicate to assess reproducibility of the data. The data were
presented as fold of internal control.
ries, Gaithersburg, MD). Images were obtained using an Eclipse 80i
fluorescence microscope (Nikon, Tokyo, Japan).
Histological Scoring
The 5-mm tissue of the distal colon was used for histological
assessment. Hematoxylin-eosin (H&E) staining was performed using
common methods. Inflammation and crypt damage in the H&Estained sections were assessed by using the criteria described by
Dieleman et al. (14). The crypt was scored on a scale of 0 to 4 grade:
grade 0, intact crypt; grade 1, loss of the basal one-third of the crypt;
grade 2, loss of two-thirds of the crypt; grade 3, loss of entire crypt
with surface epithelium remaining intact; grade 4, loss of the entire
crypt and surface epithelium. These changes were quantified as to the
percent involvement by the disease process: grade 1, 1–25%; grade 2,
26 –50%; grade 3, 51–75%; grade 4, 76 –100%. The crypt damage
score was determined as the multiplication of the grade of the crypt
and percent area score (0 –16). Inflammation severity was subjectively
evaluated on a scale of 0 to 3 grade: grade 0, none; grade 1, slight;
grade 2, moderate; grade 3, severe. The extent of disease involvement
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was estimated as follows: grade 0, none; grade 1, mucosa; grade 2,
mucosa and submucosa; grade 3, transmural. The inflammation score
was determined as the multiplication of the grade of the inflammation
severity and inflammation extent (0 –9). Histological scoring was
performed in a blind manner.
Statistical Analysis
Results are expressed as the mean ⫾ SE. Data were analyzed using
Kruskal-Wallis test and Mann-Whitney’s U-test. P values ⬍ 0.05
were considered as statistically significant.
RESULTS
Candesartan Prevented TNF-␣-induced MAdCAM-1
Expression in SVEC and MJC-1 Cells
To evaluate the effect of AT1R on the regulation of MAdCAM-1
expression, we first assayed the effect of the selective inhibitor of
AT1R, candesartan, on TNF-␣-induced MAdCAM-1 protein ex-
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Fig. 4. Effect of candesartan on TNF-␣stimulated translocation of nuclear factor
(NF)-␬B p65 to the nucleus. SVEC cells were
pretreated with or without 50 ␮M candesartan for
1 h and then stimulated with TNF-␣ (20 ng/ml, 1
h). A: candesartan inhibited the TNF-␣-induced
increase in intranuclear NF-␬B, as judged by
Western blot analysis. Proliferating cell nuclear
antigen (PCNA) and actin were assayed as loading controls. B: candesartan completely blocked
the TNF-␣-induced translocation of NF-␬B to the
nucleus when assayed by immunofluorescence.
4=,6-Diamidino-2-phenylindole (DAPI) staining
was used to stain the nuclei.
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with TNF-␣ (20 ng/ml) for 6 h, following which ICAM-1 and
VCAM-1 expression was detected by Western blotting analysis
and quantified by analyzing the density of the scanned ICAM-1
and VCAM-1 gel band with an imaging analyzer (n ⫽ 4).
TNF-␣-induced ICAM-1 and VCAM-1 expression was significantly prevented by AT1R knockdown [ICAM-1: scrambled
siRNA, TNF-␣ (⫹) vs. AT1R siRNA, TNF-␣ (⫹), 0.40 ⫾ 0.04
vs. 0.20 ⫾ 0.03-fold of control, P ⬍ 0.05; VCAM-1: scrambled siRNA, TNF-␣ (⫹) vs. AT1R siRNA, TNF-␣ (⫹): 0.40 ⫾
0.01 vs. 0.30 ⫾ 0.03-fold of control, P ⬍ 0.05].
Candesartan Did Not Inhibit TNF-␣-induced
Phosphorylation of p38 MAPK Nor TNF-␣-induced
Phosphorylation and Degradation of I␬B in SVEC or MJC-1 Cells
To determine the mechanism by which ARB inhibits
MAdCAM-1 expression, we examined the effect of candesartan on TNF-␣-induced phosphorylation of p38 MAPK
and of I␬B, which play key roles in TNF-␣ induction of
MAdCAM-1. SVEC and MJC-1 cells were pretreated with or
without candesartan (50 ␮M) for 1 h and then stimulated with
TNF-␣ (20 ng/ml) for the times indicated in Fig. 3. TNF-␣
induced transient phosphorylation of p38 MAPK and of I␬B,
Candesartan Prevented TNF-␣-induced ICAM-1 and
VCAM-1 Expression in SVEC Cells
We next assayed the effect of candesartan on TNF-␣-induced
ICAM-1 and VCAM-1 protein expression. SVEC cells were
pretreated with or without candesartan (50 ␮M) and then stimulated with TNF-␣ (20 ng/ml) for 6 h (n ⫽ 4). ICAM-1 and
VCAM-1 protein expression was detected by Western blot analysis and quantified by analyzing the density of the scanned
ICAM-1 and VCAM-1 gel band with an imaging analyzer. TNF␣-induced ICAM-1 and VCAM-1 expression was significantly
inhibited by candesartan (ICAM-1: TNF-␣ vs. TNF-␣ ⫹ candesartan, 0.40 ⫾ 0.02 vs. 0.29 ⫾ 0.02-fold of control, P ⬍ 0.05;
VCAM-1: TNF-␣ vs. TNF-␣ ⫹ candesartan, 0.40 ⫾ 0.03 vs. 0.30 ⫾
0.03-fold of control, P ⬍ 0.05).
AT1R Knockdown Inhibited TNF-␣-induced MAdCAM-1
Expression in SVEC and MJC-1 Cells
To clarify the effect of the specific blockage of AT1R on
TNF-␣-induced MAdCAM-1 expression, the AT1R gene was
knocked down using an siRNA technique. Figure 2A show the
knockdown of the AT1R gene by two different AT1R siRNAs
(no. 1 or no. 2) in SVEC and MJC-1 cells, respectively.
Thirty-six hours after siRNA transfection, SVEC and MJC-1
cells were stimulated with TNF-␣ (20 ng/ml) for 24 h; following this, MAdCAM-1 expression was detected by Western
blotting analysis and quantified by analyzing the density of
the scanned MAdCAM-1 gel band with an imaging analyzer
(n ⫽ 4). TNF-␣-induced MAdCAM-1 expression was significantly prevented by AT1R knockdown in both cell types
(Fig. 2B).
AT1R Knockdown Inhibited TNF-␣-induced ICAM-1 and
VCAM-1 Expression in SVEC Cells
To clarify the effect of the specific blockage of AT1R on
TNF-␣-induced ICAM-1 and VCAM-1 expression, the AT1R
gene was knocked down using an siRNA technique. Thirty-six
hours after siRNA transfection, SVEC cells were stimulated
Fig. 5. Effects of AT1R-gene knockout in a mouse dextran sulfate sodium
(DSS)-colitis model. Disease activity index (DAI; A) and body weight loss
(B) were assayed in AT1R-gene knockout (AT1R⫺/⫺) and wild-type mice
following DSS-induced colitis and in control, nontreated mice. DAI is the
average score of body weight loss, stool blood, and stool consistency. The
DAI score, as well as the degree of body weight loss, were significantly
lower in AT1R⫺/⫺ mice than in wild-type mice. The values are means ⫾
SE. **P ⬍ 0.01 compared with wild DSS mice. ##P ⬍ 0.01 compared with
control mice.
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pression. SVEC cells were pretreated with or without candesartan
for 1 h at concentrations varying from 10 to 50 ␮M and were then
stimulated with TNF-␣ (20 ng/ml) for 24 h (n ⫽ 4). MAdCAM-1
protein expression was detected by Western blot analysis and
quantified by analyzing the density of the scanned MAdCAM-1
gel band with an imaging analyzer. TNF-␣-induced MAdCAM-1
expression was significantly inhibited by candesartan in a dosedependent manner (Fig. 1A). To confirm the effect of candesartan
on MAdCAM-1 expression, a similar experiment was carried out
using another endothelial cell line, MJC-1, which was isolated
from mouse colon. Candesartan also inhibited TNF-␣-induced
MAdCAM-1 expression in MJC-1 cells (Fig. 1B). Second, we evaluated the effect of candesartan on TNF-␣-induced MAdCAM-1
mRNA expression in SVEC and MJC-1 cells by real-time
RT-PCR analysis. The endothelial cells were pretreated with or
without 50 ␮M candesartan for 1 h and then stimulated with
TNF-␣ (20 ng/ml) for 4 h. Candesartan significantly inhibited
TNF-␣-induced MAdCAM-1 mRNA expression in both SVEC
(TNF-␣ vs. TNF-␣ ⫹ candesartan: 0.00029 ⫾ 0.00004 vs.
0.00016 ⫾ 0.00003-fold of control, P ⬍ 0.05) and MJC-1 cells
(TNF-␣ vs. TNF-␣ ⫹ candesartan: 0.00012 ⫾ 0.00002 vs.
0.00006 ⫾ 0.00001-fold of control, P ⬍ 0.05).
but phosphorylation of these proteins was not influenced by
candesartan in either SVEC (Fig. 3A) or MJC-1 (Fig. 3B) cells.
TNF-␣-induced I␬B degradation was also unaffected by candesartan treatment (Fig. 3, A and B).
Candesartan Prevented TNF-␣-induced NF-␬B Activation by
Inhibition of NF-␬B Translocation to the Nucleus
with TNF-␣. Candesartan completely blocked this translocation of NF-␬B to the nucleus (Fig. 4B).
DSS-induced Colitis Was Ameliorated in AT1R⫺/⫺ Mice
Body weight loss, disease activity, and colon shortening
were lower in AT1R⫺/⫺ than in wild-type mice. Our previous
in vitro results suggested that AT1R blockage might be a new
therapeutic approach for treatment of IBD. To confirm that this
was the case, we compared DSS-induced colitis in AT1R⫺/⫺
and wild-type mice. Symptoms of acute colitis developed in
the mice following 5 days treatment with 2% DSS, with
diarrhea being observed first, followed by rectal bleeding and
severe weight loss. It is important to note that there was no
difference in the consumption of drinking water between wildtype and AT1R⫺/⫺ mice. The disease activity index, a combinatorial index of body weight loss, stool blood, and stool
consistency (15), was significantly lower in AT1R⫺/⫺ than in
wild-type mice (Fig. 5A). The change in body weight induced
by DSS, which is presented as the percent change from the
baseline value of day 0, was significantly ameliorated in
AT1R⫺/⫺ mice compared with wild-type mice (Fig. 5B).
Concomitant with these changes, DSS treatment induced a
significant decrease in colon length in both wild-type and
AT1R⫺/⫺ mice. Colon shortening was significantly lower in
the AT1R⫺/⫺ mice (81.3 ⫾ 2.6 mm) than in the wild-type
mice (71.9 ⫾ 5.4 mm).
Fig. 6. Effect of AT1R-gene knockout on colon
damage in the mouse DSS-colitis model. A: colon
damage in control (a) and in 5-day DSS-treated
wild-type (b) and AT1R⫺/⫺ (c) mice was assessed histologically. Differences in mucosal
thickness, infiltration of inflammatory cells, and
crypt damage were observed between DSStreated wild-type and control mice. The DSSinduced colonic changes were much less in
AT1R⫺/⫺ than in wild-type mice. In AT1R⫺/⫺
mice, both the inflammation score (B) and the
crypt damage score (C) were significantly lower
than those of wild-type mice. The values are
means ⫾ SE. **P ⬍ 0.01 and *P ⬍ 0.05 compared with wild DSS mice. ##P ⬍ 0.01 compared
with control mice.
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We further analyzed the mechanism by which candesartan
inhibits MAdCAM-1 expression by determination of its
potential effect on the translocation of NF-␬B that is induced by activation of I␬B and that plays an important role
in the regulation of MAdCAM-1 expression. SVEC were
pretreated with or without 50 ␮M candesartan for 1 h and
then stimulated with TNF-␣ (20 ng/ml) for 1 h. NF-␬B
cellular localization was analyzed by immunofluorescence
microscopy and by Western blotting of the whole cell and of
the nuclear compartment. Following TNF-␣ stimulation, the
expression of intranuclear NF-␬B was increased, although
no change in the total cellular level of NF-␬B protein was
detected by Western blotting analysis. These results indicated that TNF-␣ stimulation induced NF-␬B translocation
to the nucleus. Candesartan had significantly blocked the
increase of intranuclear expression of NF-␬B (Fig. 4A). By
immunofluorescence analysis, NF-␬B was observed to localize in the cytoplasm under resting conditions, but to
translocate into the nucleus following cellular activation
G261
G262
AT1R blockage prevents induction of colitis by regulation of
the endothelial expression of MAdCAM-1 via inhibition of
NF-␬B activation.
It has been reported that ANG II activates NF-␬B through
nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-derived ROS and that this activation induces ICAM-1 and
VCAM-1 expression (30, 49). In preliminary experiments,
ANG II induced ICAM-1 and VCAM-1, but not MAdCAM-1
expression (data not shown). This result means that the expression of MAdCAM-1 is regulated by a different pathway than
that by which ICAM-1 and VCAM-1 are regulated. This
possibility is supported by our laboratory’s previous report that
TNF-␣-induced MdCAM-1 expression is controlled by cytochrome P-450-derived ROS, not by NADPH oxidase-derived
ROS (45). We, therefore, focused our study on the relationship
between AT1R and MAdCAM-1 expression, since it was a
potentially novel mechanism for the regulation of MAdCAM-1
expression.
In this experiment, we used two vascular endothelial cell
lines, because MAdCAM-1 is preferentially expressed on only
endothelial cells in intestinal mucosa, submucosa, and Peyer’s
DSS-induced Colitis Was Ameliorated in ARB-treated Mice
Then, we investigated whether the blockage of AT1R by
using ARB might be a new therapeutic approach for treatment
of IBD or not. We further compared DSS-induced colitis in
candesartan-treated mice and nontreated mice. Symptoms of
acute colitis developed in the mice following 5 days treatment
with 2% DSS. Disease activity index was significantly lower in
candesartan-treated mice than in nontreated mice (Fig. 9A).
The change in body weight induced by DSS, which is presented as the percent change from the baseline value of day 0,
was significantly ameliorated in candesartan-treated mice compared with nontreated mice (Fig. 9B).
DISCUSSION
This is the first report concerning the relationship between
AT1R and the expression of MAdCAM-1, one of the most
important pathogenic molecules in IBD. We have shown that
Fig. 7. Effect of AT1R-gene knockout on cytokine and chemokine production
in the mouse DSS-colitis model. The mRNA expression of the Th-1 cytokine,
TNF-␣ (A), and the chemokine, monocyte chemoattractant protein-1 (MCP-1;
B) in the colon of control or DSS-treated wild-type and AT1R⫺/⫺ mice,
compared with that of an internal control GAPDH, was assessed using
real-time RT-PCR. The data are presented as fold of internal control. TNF-␣
and MCP-1 expression were significantly lower in AT1R⫺/⫺ than in DSStreated wild-type mice. Values are means ⫾ SE. **P ⬍ 0.01 and *P ⬍ 0.05
compared with wild DSS mice. ##P ⬍ 0.01 and #P ⬍ 0.05 compared with
control mice.
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Histological damage was milder in AT1R⫺/⫺ than in wildtype DSS-treated mice. The effect of DSS on the colon was
further examined in AT1R⫺/⫺ and wild-type mice by histological assessment of tissue damage. H&E staining of the colon
of wild-type mice at day 5 of DSS treatment showed thicker
mucosa, greater infiltration of inflammatory cells, and a higher
level of crypt damage compared with non-DSS-treated mice
(control). In contrast, the colon of AT1R⫺/⫺ mice appeared
less damaged and inflamed, showing only mild evidence of
crypt damage and inflammatory cell infiltration (Fig. 6A). In
AT1R⫺/⫺ mice, both the inflammation and the crypt damage
scores were significantly lower than those of wild-type mice
(Fig. 6, B and C).
The levels of TNF-␣ and MCP-1 mRNA in the colon were
lower in AT1R⫺/⫺ than in wild-type DSS-treated mice. To
investigate the role of AT1R in mucosal immunity, TNF-␣ and
MCP-1 mRNA levels in the colon were quantified by real-time
RT-PCR. The mRNA levels of the proinflammatory Th1 cytokine TNF-␣ and the chemokine MCP-1 were significantly
elevated in DSS-treated mice compared with control mice (Fig. 7,
A and B, respectively). However, the levels of TNF-␣ and
MCP-1 mRNA were significantly lower in AT1R⫺/⫺ than in
wild-type mice (Fig. 7, A and B, respectively). These results
indicate that AT1R is involved in upregulation of a proinflammatory cytokine and a chemokine in the inflamed colon and
that AT1R⫺/⫺ mice are resistant to DSS-induced colonic
damage.
The expression of MAdCAM-1 in the colon is attenuated in
AT1R⫺/⫺ DSS-treated mice. We next determined whether the
in vitro inhibition of MAdCAM-1 expression that we observed
following AT1R blockage could also be observed in vivo. We,
therefore, evaluated the expression of MAdCAM-1 in DSSinduced inflammatory colon by Western blotting analysis and
real-time RT-PCR. MAdCAM-1 protein expression was detected
by Western blot analysis and quantified by analyzing the density
of the scanned MAdCAM-1 gel band with an imaging analyzer.
In AT1R⫺/⫺ mice, the colonic expression of MAdCAM-1 at
both the protein and the mRNA level was significantly lower
than that of wild-type mice (Fig. 8). Therefore, the combined in vitro and in vivo results indicate that AT1R
blockage induces resistance to DSS induction of colonic
inflammation by inhibition of MAdCAM-1 expression.
G263
Fig. 8. Effect of AT1R-gene knockout on colonic expression of MAdCAM-1
in the mouse DSS-colitis model. A: colonic protein expression of MAdCAM-1
was assayed by Western blotting. Blots were quantified by densitometry, and
the data are presented as fold of internal control. MAdCAM-1 protein expression was significantly attenuated in AT1R⫺/⫺ mice compared with wild-type
mice. B: colonic MAdCAM-1 mRNA expression, compared with that of the
internal control GAPDH, was assayed by real-time RT-PCR. The data are
presented as fold of internal control. MAdCAM-1 mRNA expression was
significantly lower in AT1R⫺/⫺ than in wild-type mice. Values are means ⫾
SE. *P ⬍ 0.05 compared with wild DSS mice. ##P ⬍ 0.01 compared with
control mice.
patches (6). Most experimental studies on MAdCAM-1 have
been reported using a brain-derived endothelioma cell line
(bEnd.3) and SVEC, because MAdCAM-1 is inducible in these
cell lines. We used SVEC (35, 43, 45) in our experiment
concerning MAdCAM-1. However, we thought that SVEC was
not sufficient to study the colitis, because it is the cell line
derived from axillary lymph node. Therefore, we have established a colon endothelial cell line, MJC-1 (2), in which
MAdCAM-1 is also inducible.
Interestingly, we showed in this study that blockage of AT1R
by using the ARB, candesartan, or by AT1R gene knockdown
controlled not only TNF-␣-induced-ICAM-1 and VCAM-1 expression, but also the expression of MAdCAM-1 by SVEC and
MJC-1 cells. Moreover, the degree of inhibition was more dramatic for MAdCAM-1 than for ICAM-1 and VCAM-1. MAdCAM-1,
ICAM-1, and VCAM-1 expression is induced by proinflammatory cytokines, such as TNF-␣ and IL-1␤ (2, 32, 38). It is
believed that TNF-␣ induces MAdCAM-1, ICAM-1, and
VCAM-1 expression via activation of p38 MAPK and NF-␬B
Fig. 9. Effects of AT1R blocker treatment in a mouse DSS-colitis model. DAI
(A) and percent body weight change (B) were assayed in candesartan (a
selective AT1R blocker) treated and nontreated mice following DSS-induced
colitis and in control, non-DSS-induced mice. The DAI score, as well as the
degree of body weight loss, were significantly lower in candesartan-treated
mice than in nontreated mice. The values are means ⫾ SE. **P ⬍ 0.01 and
*P ⬍ 0.05 compared with candesartan nontreated mice. ##P ⬍ 0.01 compared
with control mice.
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(1, 37, 52, 58). It has also been reported that the p38 MAPK/
cAMP response element binding protein pathway plays a
critical role in TNF-␣-induced-VCAM-1 expression (34). In
this study, we showed that candesartan did not prevent TNF␣-induced phosphorylation of p38 MAPK, but strongly prevented NF-␬B activation. This result is consistent with our
laboratory’s previous report that TNF-␣-regulated expression
of MAdCAM-1 involves two separate pathways: one involving
p38 MAPK, and the second involving NF-␬B (37). Moreover,
our findings suggest that the NF-␬B pathway is more critical
for MAdCAM-1 expression than the p38 MAPK pathway.
We also investigated the potential mechanism by which
candesartan inhibits NF-␬B and found that candesartan did not
inhibit TNF-␣ stimulation of I␬B phosphorylation, but did
inhibit translocation of NF-␬B to the nucleus from the cytoplasm. This effect of candesartan was confirmed by both
Western blotting analysis of p65 in nuclear fractions and by an
immunofluorescence microscopic analysis of p65 cellular localization. This result is consistent with our laboratory’s previous data that the effect of candesartan on MAdCAM-1
expression is achieved through modulation of the NF-␬B
pathway and not through the p38 MAPK pathway. NF-␬B
exists as either a heterodimer or a homodimer, among which
G264
thermore, administration of ARB to mice conferred resistance
to DSS-induced colitis. These findings suggest that the AT1R
is involved in one of the pathogenesis of DSS-induced colitis
via the regulation of MAdCAM-1. However, from our additional study to confirm the in vitro result that AT1R blockage
inhibits nuclear translocation of NF-␬B, we could not detect
clear in situ difference of NF-␬B to nuclear translocation
between DSS-treated wild mice and AT1R⫺/⫺ mice. To
clarify the mechanism, further experiments are necessary.
In summary, we have demonstrated a novel mechanism by
which AT1R regulates the expression of MAdCAM-1 by
modulation of the nuclear translocation of NF-␬B. Moreover,
AT1R regulation of MAdCAM-1 expression ameliorates colitis in a mouse colitis model. We conclude that AT1R-regulation of MAdCAM-1 expression potentially provides a promising novel target for the treatment of IBD.
ACKNOWLEDGMENTS
We thank Dr. Takashi Okamoto for helpful suggestions and Yukimi Ito for
expert technical assistance.
DISCLOSURES
There is no financial support to be disclosed.
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the p65/p50 is the most ubiquitous heterodimer. In resting cell,
NF-␬B dimers are sequestered in the cytoplasm through association with inhibitory proteins I␬B (51). Upon treatment with
NF-␬B inducers, such as proinflammatory cytokines, I␬B is
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Tanida, Hiromi Kataoka, Takeshi Kamiya, JS Alexander and Takashi

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