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Role of Nicotinamide Adenine Dinucleotide
Phosphate Oxidase 1 in Oxidative Burst
Response to Toll-Like Receptor 5 Signaling in
Large Intestinal Epithelial Cells
Tsukasa Kawahara, Yuki Kuwano, Shigetada
Teshima-Kondo, Ryu Takeya, Hideki Sumimoto, Kyoichi
Kishi, Shohko Tsunawaki, Toshiya Hirayama and Kazuhito
Rokutan
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 172:3051-3058; ;
doi: 10.4049/jimmunol.172.5.3051
http://www.jimmunol.org/content/172/5/3051
The Journal of Immunology
Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase
1 in Oxidative Burst Response to Toll-Like Receptor 5
Signaling in Large Intestinal Epithelial Cells1
Tsukasa Kawahara,* Yuki Kuwano,* Shigetada Teshima-Kondo,* Ryu Takeya,†
Hideki Sumimoto,† Kyoichi Kishi,* Shohko Tsunawaki,‡ Toshiya Hirayama,§ and
Kazuhito Rokutan2*
R
eactive oxygen species (ROS),3 notably superoxide anion
(O2⫺) and hydrogen peroxide, operate on a variety of
physiological processes, including host defense, gene expression, oxygen sensing, regulation of vascular tone, bone resorption, apoptosis, cell growth, and transformation (for reviews, see
Refs. 1–3). The best-known O2⫺-producing enzyme is the phagocyte respiratory burst oxidase that plays a crucial role in a process
of killing microorganisms. The catalytic core of this oxidase is the
membrane-integrated flavocytochrome b558 composed of p22phox
and gp91phox subunits, the latter having binding sites for heme,
flavin adenine dinucleotide (FAD), and NADPH, and transfers an
electron from NADPH to molecular oxygen to generate O2⫺ (4).
*Department of Nutrition, School of Medicine, University of Tokushima, Tokushima,
Japan; †Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; ‡Department of Infectious Diseases, National Research Institute for Child Health and
Development, Tokyo, Japan; and §Department of Bacteriology, Institute of Tropical
Medicine, Nagasaki University, Nagasaki, Japan
Received for publication June 12, 2003. Accepted for publication December 22, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by a grant-in-aid for scientific research from Japan Society
for the Promotion of Science (14370184), and Japan Society for the Promotion of
Science Research Fellowships for Young Scientists (04127).
2
Address correspondence and reprint requests to Dr. Kazuhito Rokutan, Department
of Nutrition, School of Medicine, University of Tokushima, 3-18-15 Kuramoto-cho,
Tokushima 770-8503, Japan. E-mail address: [email protected]
3
Abbreviations used in this paper: ROS, reactive oxygen species; DF, DMEM-Ham’s
F-12; Duox, dual oxidase; FAD, flavin adenine dinucleotide; LIEC, large intestinal
epithelial cell; NBT, nitroblue tetrazolium; Nox, NADPH oxidase; O2⫺, superoxide
anion; PAS, periodic acid-Schiff; PDTC, pyrrolidine dithiocarbamate; PVDF, polyvinylidene difluoride filter; rFliC, recombinant structural protein of flagella filament;
SOD, superoxide dismutase; TAB1, TAK1-binding protein 1; TAK1, TGF-␤-activated kinase 1; TLR, Toll-like receptor.
Copyright © 2004 by The American Association of Immunologists, Inc.
Recently, two families of gp91phox homologues have been identified: NADPH oxidase (Nox) and dual oxidase (Duox) families (5).
The Nox family comprises Nox1 (initially termed Mox1 or NOH1), Nox2 (renamed gp91phox), Nox3, Nox4 (Renox), and Nox5
(6 –10). These homologues conserve binding sites for heme, FAD,
and NADPH of Nox2 (5), and are preferentially expressed in
nonphagocytic cells. The Duox family members are Duox1 and
Duox2 (initially termed ThOX1 and ThOX2, respectively), which
have a peroxidase homology domain plus two EF-hand motifs, as
well as binding sites for heme, FAD, and NADPH (5). Of these
family members, the Nox1 mRNA is predominantly expressed in
human colon tissue and a carcinoma cell line, Caco2 cells (6, 7).
However, physiological roles of Nox1 in large intestinal epithelial
cells (LIEC) are not fully understood.
We previously reported that primary cultures of guinea pig gastric pit cells expressed Nox1 and spontaneously secreted O2⫺ (11,
12). ROS derived from Nox1 in the pit cells were essential for their
growth at least in vitro (12). Furthermore, Helicobacter pylori LPS
stimulated the Toll-like receptor (TLR) 4 signaling and activated
Nox1 (13, 14). The increased O2⫺ production and enhanced activation of NF-␬B resulted in the induction of the TNF-␣ and cyclooxygenase II mRNAs in pit cells themselves (12), suggesting a
potential role of Nox1 in inflammatory and immune responses
against H. pylori.
The flavocytochrome b558 requires cytosolic proteins, p67phox,
p47phox, and a small GTPase Rac for electron-transfer reactions to
form O2⫺ (4). P40phox associates with p67phox and enhances membrane translocation of p67phox and p47phox in stimulated phagocytes (15). However, it remains to be elucidated whether these
cytosolic factors are necessary for activation of the other Nox and
Duox family members.
0022-1767/04/$02.00
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The NADPH oxidase 1 (Nox1) is a gp91phox homologue preferentially expressed in the colon. We have established primary cultures
of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O2ⴚ) of 160 nmol/mg protein/h and expressed the Nox1, p22phox, p67phox, and Rac1 mRNAs, but not the gp91phox,
Nox4, p47phox, p40phox, and Rac2 mRNAs. They also expressed novel homologues of p47phox and p67phox (p41nox and p51nox,
respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22phox, p51nox, and Rac1 mRNAs, but not
the other NADPH component mRNAs, and secreted only small amounts of O2ⴚ (<2 nmol/mg protein/h). Cotransfection of p41nox
and p51nox cDNAs in T84 cells enhanced PMA-stimulated O2ⴚ release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O2ⴚ release in association with the induction of Nox1
protein. The enhanced O2ⴚ production by cotransfection of p41nox and p51nox vectors further augmented the rFliC-stimulated
IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-␤-activated kinase
1 and TGF-␤-activated kinase 1-binding protein 1. A potent inhibitor for NF-␬B (pyrrolidine dithiocarbamate) significantly
blocked the rFliC-primed increase in O2ⴚ production and induction of Nox1 protein. These results suggest that p41nox and p51nox
are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities
among bacteria to appropriately operate Nox1 for the host defense. The Journal of Immunology, 2004, 172: 3051–3058.
3052
In this study, we have established primary cultures of guinea pig
LIEC with 90% purity of surface mucous cells and have found that
these cells also produce O2⫺ even at a higher rate than that of
gastric pit cells. Guinea pig LIEC expressed novel genes encoding
p41nox (a p47phox homologue) and p51nox (a p67phox homologue),
which have recently cloned in mouse and human, being named as
the NOX organizer 1 and NOX activator 1 (16 –18), respectively.
Cotransfection of these two homologues was shown to up-regulate
O2⫺-producing capability of Nox1, and in situ hybridization demonstrated that the p41nox and p51nox transcripts were expressed in
epithelial cells of mouse colonic mucosa (16 –18), suggesting their
crucial roles for Nox1 activity. Using guinea pig LIEC and human
colon cancer cell lines, we molecularly and functionally characterized Nox1 expressed in LIEC.
Materials and Methods
Reagents
Preparation and culture of cells
The present study was approved by the Animal Care Committee of University of Tokushima. Male guinea pigs weighing ⬃250 g were purchased
from Shizuoka Laboratory Animal Center (Shizuoka, Japan). Under general anesthesia with pentobarbital, ascending to sigmoid portions of the
guinea pig colon were resected and extensively washed with PBS. Colonic
mucosa was scraped with a sterile glass slide and finely minced with sterile
surgical blades. The minced pieces were then incubated in DMEM-Ham’s
F-12 (1:1) (DF) medium (Life Technologies, Grand Island, NY) containing
0.03% collagenase S-1 (Nitta Gelatin, Osaka, Japan) and 0.2% BSA for 30
min at 37°C. The digested tissues were next washed three times with DF
medium by centrifugation. The resulting pellets were resuspended in DF
medium containing 10% FBS (PAA Lab., Linz, Austria) and incubated at
37oC for 2 h under 5% CO2. Attached cells were positive for a specific
mAb against macrophages (HAM56 clone; Enzo Diagnostic, Farmingdale,
NY) and used as a macrophage population derived from colonic mucosa.
Collected nonadherent cells were washed with DF medium and then cultured for 24 h in 35-mm-diameter culture dishes that had been coated with
type IV collagen (Nitta Gelatin). The growing cells were used as primarily
cultured guinea pig LIEC. These cells were completely replaced by vimentin-positive fibroblasts after a 2-wk cultivation. Guinea pig and human PBL
were prepared, as previously described (14). T84 and Caco2 cells were
maintained in DMEM medium supplemented with 10% FBS, 100 U/ml
penicillin, and 100 ␮g/ml streptomycin.
The amount of O2⫺ release was measured by the superoxide dismutase
(SOD)-inhibitable reduction of cytochrome c, and O2⫺-producing cells
were cytochemically visualized by detecting blue formazan precipitates of
nitroblue tetrazolium (NBT), as previously described (11).
Immunoblotting
Anti-Nox1 Abs were made by immunizing rabbits with synthetic peptides
corresponding to the 480 – 493 (Nox1-C1) and 544 –556 (Nox1-C2) aa residues of human Nox1 (GenBank accession AF166327). The epitopes for
the two anti-Nox1 Abs were designed not to overlap the amino acid sequences of the other Nox homologues (GenBank accession NM000397,
AF190122, NM016931, and NM024505). Nox1-C1- or Nox1-C2-immunized serum was further purified by affinity chromatography using the Ag
peptide-conjugated agarose (Amersham Pharmacia Biotech, Piscataway,
NJ). The amino acid sequence of Nox1-C1 has 93% homology between
human and guinea pig (GenBank, AB099629), and anti-Nox1-C1 Ab recognized both human and guinea pig Nox1 proteins. In contrast, antiNox1-C2 Ab recognized only human Nox1, because guinea pig Nox1 does
not share this motif. Anti-TLR5 Ab was made by immunizing rabbits with
synthetic peptides corresponding to the 836 – 849 aa residues of human
TLR5. Polyclonal Abs against synthetic peptide of human p22phox (residues 177–195), human p47phox (residues 376 –390), human p40phox (resi-
dues 1–15), and recombinant human p67phox were provided, as previously
described (13). Membrane, cytosolic, and whole cell fractions were prepared from cultured cells, as previously described (11). Each sample of 20
␮g protein per lane was separated by SDS-PAGE using an 8% polyacrylamide gel and transferred to a polyvinylidene difluoride filter (PVDF).
After blocking nonspecific binding sites with 4% purified milk casein, the
PVDF was incubated for 1 h at room temperature with one of the above
primary Abs at a 1/1000 dilution. After being washed with PBS containing
0.05% Tween 20, bound Abs were detected by an ECL Western blotting
detection system (Amersham Pharmacia Biotech). Bound Abs were then
removed, and the PVDF was reblotted with a mAb against ␤-actin (Oncogene Research Products, Cambridge, MA). Phosphorylation of TGF-␤activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) was assayed, as previously described (14).
Cytochemical and immunohistochemical stainings
Mucous granule-containing cells were visualized by the periodic acidSchiff (PAS) reaction. For immunohistochemical analysis, growing LIEC
on the dishes were fixed with 4% paraformaldehyde in PBS for 20 min.
They were then incubated in PBS containing 0.03% Triton X-100 for 2 min
on ice and blocked with 4% purified milk casein. These cells were incubated with a 1/500 dilution of anti-Nox1-C1 Ab for 1 h. After washing,
they were treated with a 1/500 dilution of biotin-linked goat Ab against
rabbit IgG (Amersham Pharmacia Biotech) for 1 h, and were then incubated with a 1/500 dilution of streptavidin-conjugated FITC probe for 30
min at room temperature. The cells were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Fluorescence was
viewed using a confocal laser-scanning microscopy (model Axiovert
25CFL; Leica, Heiderberg, Germany). Fibroblasts and macrophages were
immunocytochemically identified with mAbs against vimentin (Santa Cruz
Biotechnology, Santa Cruz, CA) and a macrophage Ag (HAM56 clone);
vimentin-positive fibroblasts and HAM56-positive macrophages were visualized by diaminobenzidine streptavidin-biotin HRP and Vector red alkaline phosphatase methods (Vector Laboratories), respectively.
Paraffin-embedded guinea pig colon tissues were cut into 3-␮m-thickness sections and deparaffinized. After blocking nonspecific binding sites
with 4% purified milk casein, they were incubated with a 1/1000 dilution
of anti-Nox1-C1 Ab in TBS containing 0.1% Tween 20 and 2% BSA
overnight at 4°C. After washing, bound Abs were visualized using Vector
red alkaline phosphatase kit (Vector Laboratories). Finally, specimens
were counterstained with hematoxylin and mounted with Aquatex (Merck,
Darmstadt, Germany).
RT-PCR
Total RNA was prepared from the indicated cells or tissues with an acid
guanidium-thiocynate-phenol chloroform mixture (14). RT-PCR was performed using the following specific PCR primer sets: Nox1-A, 5⬘-ATGG
GAAACTGGGTGGTTA-3⬘ and 5⬘-TAGCTGAAGTTACCATGAGAA3⬘; Nox1-B, 5⬘-TTCTTGGCTAAATCCCATCCA-3⬘ and 5⬘-TTTCTG
TCCAGTCCCCTGCT-3⬘; Nox2, 5⬘-CATCATCTCTTTGTGATCTTCT-3⬘
and 5⬘-CTTAGGTAGTTTCCACGCATC-3⬘; Nox4, 5⬘-GGTCCTTTTGG
AAGTCCATTTGAGG-3⬘ and 5⬘-CACAGCTGATTGATTCCGCTGAG-3⬘;
p22phox, 5⬘-ATGGGGCAGATCGAGTGGGCCATGT-3⬘ and 5⬘-GTAGATG
CCGCTCGCAATGGCCAG-3⬘; p67phox, 5⬘-TCCCGGATTTGCTTCAAC
ATT-3⬘ and 5⬘-TTGGCCAGCTGAGCCACTT-3⬘; p47phox-A, 5⬘-ATCCG
TCACATCGCCCTGCT-3⬘ and 5⬘-CCAACCGCTCTCGCTCTTCT-3⬘;
p47phox-B, 5⬘-AACAGGATCATCCCCCACCT-3⬘ and 5⬘-CAGGTACAT
GGACGGAAAGT-3⬘; p40phox, 5⬘-TGACATCGAGGAGAGAGGCT-3⬘
and 5⬘-GGAAGATCACATCTCCAGCTTTGA-3⬘; p41nox, 5⬘-TTTGCCT
TCTCTGTGCGCTGG-3⬘ and 5⬘-TCTGGGGTGGGCAGGATCACC-3⬘;
p51nox, 5⬘-CAAGCAGTGACTAAGGACACCTG ⫺3⬘ and 5⬘-CACAC
AGGACATCCACCGTGTC-3⬘; Rac1, 5⬘-TGCAGGCCATCAAGTGT
GTGGT-3⬘ and 5⬘-GCTGAGACATTTACAACAGCAGGCAT-3⬘; Rac2,
5⬘-TGCAGGCCATCAAGTGTGTGGT ⫺3⬘ and 5⬘-TAGAGGAGGCTG
CAGGCGCGCTT-3⬘; and GAPDH, 5⬘-TCATGACCACAGTCCATGC
CATCACT-3⬘ and 5⬘-GCCTGCTTCACCACCTTCTTGATGT-3⬘. PCR
products were sequenced with a DNA sequencer and confirmed to be the
corresponding cDNA fragments.
Expression vectors and cDNA constructs
The cDNA encoding human p51nox was provided, as previously described
(18), and the cDNA encoding p67phox was a gift from H. Nunoi (Miyazaki
Medical School, Miyazaki, Japan). The p41nox cDNA (GenBank,
AF539796) was amplified by PCR using human digestive system multiple
tissue cDNA (Clontech Laboratories, Palo Alto, CA). Full-length p67phox
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A recombinant structural protein of flagella filament (rFliC) of Salmonella
enteritidis was prepared, as previously described (19). Staphylococcus aureus peptidoglycan and LPS from Escherichia coli K-235 were purchased
from Fluka Chemie AG (Buchs, Switzerland) and Sigma-Aldrich (St.
Louis, MO), respectively. Pyrrolidine dithiocarbamate (PDTC) was obtained from Calbiochem (San Diego, CA). Phosphorothioate-stabilized
CpG oligodeoxynucleotide (CpG DNA) (TCCATGACGTTCCTGAT
GCT) (20) was purchased from Hokkaido System Science (Sapporo,
Japan).
Nox1 IN LIEC
The Journal of Immunology
3053
ture dishes at 70 –90% confluence were incubated in 1 ml of serum-free DF
medium for 2 h and then treated with rFliC (5 ␮g/ml) in the absence or
presence of 200 U/ml SOD plus 700 U/ml catalase. After treatment for
24 h, the medium was collected and centrifuged at 1000 ⫻ g for 10 min.
The concentrations of IL-8 in the supernatants were measured using a
human IL-8 ELISA kit (R&D Systems, Abington, U.K.), according to the
manufacturer’s protocol. The amount of IL-8 was expressed as pg/ml per
mg cell protein.
Results
Nox1-expressing and O2⫺-producing cells in guinea pig LIEC
cDNA and hemagglutinin-tagged p41nox and p51nox cDNAs were recombined into pAdTrack-CMV vector (21). T84 cells were plated at a concentration of 5 ⫻ 105 cells/well in 24-well plates and transfected with the
vectors using the FuGENE transfection reagent (Roche Biomedical Laboratories, Burlington, NC).
Measurement of IL-8
T84 cells were transfected with mock (pAdTrack-CMV vector) or p51nox
plus p41nox vectors for 48 h. These cells growing in 35-mm-diameter cul-
FIGURE 2. Expression of Nox1 in
guinea pig LIEC. Primary cultures of guinea
pig LIEC were subjected to the PAS staining
(A), immunocytochemistry with an antiNox1-C1 Ab (B and C), and NBT assay (D),
as described in Materials and Methods. Cellular distribution of Nox1 protein in guinea
pig colonic mucosa is shown in E and F. The
specificity of anti-Nox1-C1 Ab was verified
by a preabsorption test with a 50-fold molar
excess of Ag peptide used for the Ab preparation (G). Scale bars indicate 100 ␮m (A and
B), 10 ␮m (C and D), 200 ␮m (E and G), and
20 ␮m (F), respectively. Total RNA was isolated from guinea pig LIEC, Caco2 cells, human and guinea pig PBL, or guinea pig kidney, and subjected to RT-PCR, as described
in Materials and Methods. Mixture with (⫹)
or without (⫺) reverse-transcriptase reaction
was amplified using the specific primer sets
for the detection of Nox1 (H), Nox2 (I), or
Nox4 (J) mRNA. Data are representative of
four independent experiments.
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FIGURE 1. Identification of O2⫺-generating cells. The majority of
growing guinea pig LIEC at 24 h shows the PAS reaction-positive granules
(A and B). Vimentin-positive fibroblasts growing at 24 h and after 2 wk are
shown in C and D, respectively. HAM56-positive macrophages do not
contaminate the final culture at 24 h (E), confirming the successful
removal of adherent cells in the initial 2-h cultivation (F). Cultured
LIEC were counterstained with propidium iodide to visualize cell nuclei
(right panels in B, C, and E). Scale bars indicate 100 ␮m (A) and 20 ␮m
(B–F), respectively.
Contaminated macrophages were removed as attaching cells during an initial 2-h cultivation of isolated guinea pig colonic mucosal
cells. Nonadherent cells were collected and cultured in DF medium
supplemented with 10% FBS. These cells began to adhere to collagen-coated plates within 6 h and became confluent at ⬃24 h.
After a 48-h culture, they started to undergo spontaneous apoptosis, mirroring their rapid turnover in vivo. At the 24-h cultivation,
90 ⫾ 3% (mean ⫾ SD, n ⫽ 8) of cultured cells possessed PAS
reaction-positive granules characteristic of surface mucous cells
(Fig. 1, A and B). Although vimentin-positive fibroblasts were less
than 5% at 24 h (Fig. 1C), they had grown to be an exclusive
population in 2 wk (Fig. 1D). Macrophages were not detected in
the 24-h LIEC culture (Fig. 1E) after their removal by the adherent
method (Fig. 1F), and the growing LIEC were used in the following experiments.
The cultured guinea pig LIEC spontaneously secreted O2⫺ at
156 ⫾ 9 nmol/mg protein/h (mean ⫾ SD, n ⫽ 12). This rate was
higher than that of cultured gastric pit cells primed with H. pylori
LPS (112 ⫾ 5 nmol O2⫺/mg protein/h; mean ⫾ SD, n ⫽ 12).
O2⫺-producing cells contained granules positive for the PAS reaction (Fig. 2A) and expressed Nox1 protein (Fig. 2B). These
Nox1-expressing cells (Fig. 2C) were identical with the cells covered with precipitates of blue formazan (Fig. 2D). In the guinea pig
colon, surface mucous cells possessed immunoreactive materials
to anti-Nox1-C1 Ab (Fig. 2, E and F), and the synthetic Ag
3054
Nox1 IN LIEC
Roles of p41nox and p51nox in Nox1 activity
Effects of bacterial components on Nox1 activity
FIGURE 3. Expressions of NADPH oxidase components in guinea pig
LIEC. Total RNA was isolated from human PBL or cultured guinea pig
LIEC, and the mRNAs of p22phox, p67phox, p47phox, p40phox, Rac1/2, and
GAPDH were amplified by RT-PCR, as described in Materials and Methods (A). Membranes and cytosol were fractionated from guinea pig PBL
and guinea pig LIEC. The amount of p22phox in membrane fraction and of
p67phox, p47phox, p40phox, and ␤-actin in cytosol were estimated by immunoblot analysis using the corresponding Abs (B). The p51nox and p41nox
mRNAs were amplified by specific primer sets using mixtures with (⫹) or
without (⫺) reverse-transcriptase reaction (C). Similar results were obtained in three separate experiments.
polypeptide completely abolished this immunoreactivity (Fig. 2G).
Thus, surface mucous cells of the guinea pig colon constitutively
expressed Nox1 protein both in vitro and in vivo. We also confirmed that the prepared fibroblasts released O2⫺ at ⬍1 nmol/mg
protein/h, but these amounts were not enough to form visible blue
formazan precipitates even after incubation with 0.1 mM of NBT
for 2 h (data not shown).
RT-PCR with two different primer sets amplified the Nox1
mRNA fragments in guinea pig LIEC and Caco2 (Fig. 2H), and
their nucleotide sequences were identical with the guinea pig and
human Nox1 mRNAs, respectively (data not shown). We also confirmed that guinea pig LIEC did not express the Nox2 nor Nox4
mRNA (Fig. 2, I and J).
Expression of NADPH oxidase components in guinea pig LIEC
We next screened the expression of Nox components. As shown in
Fig. 3A, guinea pig LIEC expressed the p22phox, p67phox, and Rac1
transcripts, while the p47phox, p40phox, and Rac2 mRNAs were not
detected. Immunoblot analysis showed that they had p67phox and
p22phox proteins, but not p47phox and p40phox in line with the RTPCR results (Fig. 3B). Moreover, we have cloned the p41nox (GenBank, AB105906) and p51nox (GenBank, AB105907) mRNAs expressed in guinea pig LIEC (Fig. 3C). Guinea pig PBL also
expressed a small amount of p51nox mRNA, but not p41nox.
To elucidate physiological functions of Nox1 in LIEC, we explored possible up-regulator(s) of the oxidase. Mirroring the rapid
turnover of surface mucous cells in vivo, guinea pig LIEC in primary culture appeared to be already in a fully matured and activated status; therefore, we investigated whether T84 cells overexpressing p41nox and p51nox could be primed with bacterial
components for O2⫺ generation. LPS from H. pylori or E. coli was
demonstrated to act as a potent stimulator of Nox1 in primary
cultures of guinea pig gastric mucosal cells (14). Although T84
cells express the TLR4 mRNA (data not shown), they were insensitive to LPS priming: E. coli LPS up to 20 ␮g/ml did not change
the basal O2⫺ generation (Fig. 5A). Neither peptidoglycan from S.
aureus nor CpG DNA increased the O2⫺ production (Fig. 5A). In
contrast, rFliC from S. enteritidis at 2 ␮g/ml or higher concentrations significantly enhanced O2⫺ generation within 12 h (Fig. 5, B
and C). Boiling did not affect the priming action of rFliC, but
treatment with trypsin completely abolished it (Fig. 5A), which are
well-known characteristics of FliC (19). T84 cells expressed the
TLR5 mRNA (Fig. 5D), and TLR5 protein was mainly present in
the membrane fraction (Fig. 5E). TAK1 is a member of mitogenactivated protein kinase kinase kinase. TAB1 is a specific activator
for TAK1. Phosphorylation of TAK1 and TAB1 is a crucial event
for NF-␬B activation through the TLR and IL-1R signaling pathways (23, 24). We confirmed that the treatment of T84 cells with
rFliC promptly phosphorylated TAK1 and TAB1 within 10 min
(Fig. 5F).
ROS-dependent IL-8 release from T84 cells
To address physiological roles of Nox1-derived ROS, we tested
whether O2⫺ production up-regulated IL-8 secretion from T84
cells. Treatment of T84 cells with rFliC for 24 h increased IL-8
release (Fig. 5G). Overproduction of p41nox and p51nox in T84
cells enhanced O2⫺ generation (Fig. 5, A–C), and consequently
augmented the rFliC-stimulated IL-8 production, which was significantly cancelled by inclusion of SOD plus catalase (Fig. 5G).
Induction of Nox1 protein with rFliC
Finally, we studied the mechanisms by which rFliC increased
Nox1 activity in T84 cells. rFliC did not change expression of the
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T84 and Caco2 cells generated only small amounts of O2⫺ (⬍2
nmol/mg protein/h). The low output of O2⫺ in these cells was
mainly due to lower levels of Nox1 expression, compared with that
in guinea pig LIEC or guinea pig gastric pit cells. It may be also
due to the absence or insufficiency of distinct component(s) supportive for the Nox1 activity. The p67phox and p41nox mRNAs
were absent in T84 and Caco2 cells, and the p51nox mRNA level
was much lower than that in guinea pig LIEC (Fig. 4A). As shown
in Fig. 4B, transduction of p67phox or overproduction of p51nox did
not change the spontaneous or PMA-stimulated release of O2⫺
from T84 cells, but p41nox-overexpressing cells significantly increased O2⫺ generation when stimulated by PMA (Fig. 4B). Although transfection of the p41nox-overexpressing cells with the
p67phox vector failed to increase both spontaneous and PMA-stimulated O2⫺ productions, overproduction of p51nox in the p41noxtransfected cells further increased the PMA-responsive O2⫺ generation (Fig. 4B). P67phox is an essential activator for Nox2, and
p51nox has conserved domains that possibly interact with Nox1 in
a similar way as p67phox does with cytochrome b558 in phagocytes
(16 –18, 22). However, our results suggest that p51nox is most
likely a better partner of Nox1 for achieving a high output of O2⫺
production.
The Journal of Immunology
3055
p67phox, p41nox, and p51nox mRNAs (data not shown), but
RT-PCR suggested that treatment with rFliC was likely to stimulate the Nox1 mRNA expression (Fig. 6A). The immunoblotting
clearly demonstrated the rFliC-primed induction of Nox1 protein
(Fig. 6B). The induction of Nox1 was associated with the increase
in O2⫺ generation when primed with rFliC in the presence or absence of PMA (Fig. 6C). Recent reports have shown that TLR5expressing cells including T84 cells activate NF-␬B in response to
bacterial flagellin (25, 26). As shown in Fig. 6, a potent inhibitor
for NF-␬B (PDTC) significantly blocked the rFliC-induced increase in O2⫺ production at concentrations over 1 ␮M (Fig. 6D),
and the induction of Nox1 protein was also concomitantly hampered by PDTC in a dose-dependent manner (Fig. 6E).
Discussion
The Nox1 mRNA is dominantly expressed in the colon (6). Intracellular regions of the Nox family are composed of highly conserved domains; therefore, specific Ab for Nox1 had not been
available. We have developed polyclonal Abs against human Nox1
useful for immunoblot and immunohistochemical analyses, and
found that Nox1 protein was constitutively expressed in surface
mucous cells of the guinea pig colon, supporting an in situ hybridization study showing that the Nox1 transcript was expressed in
epithelial cells of human colonic mucosa (27). A small amount of
Nox1 mRNA is detectable in primary cultures of guinea pig gastric
mucosal cells even in LPS-free conditions (12, 14), while Nox1
protein was absent in normal gastric and small intestinal mucosal
tissues of both guinea pigs and humans (data not shown). The
expression of Nox1 in primary cultures of guinea pig gastric mucosal cells is probably due to oxidative stress during the preparation and cultivation, because the induction of Nox1 is associated
with activation of a redox-sensitive transcription factor NF-␬B
(our unpublished observation). Quiescent guinea pig gastric pit
cells (surface mucous cells) in primary culture produced small
amounts of O2⫺ (⬍10 nmol/mg protein/h), but once primed with
H. pylori LPS, they increased O2⫺ generation 10-fold in association with the induction of Nox1 (13). In contrast, abundant Nox1
protein was constitutively expressed in surface mucous cells of the
guinea pig colon, and these cells in primary culture spontaneously
secreted O2⫺ at a higher rate (⬃160 nmol/mg protein/h), suggesting that Nox1 in the guinea pig colon is constitutively active. In
fact, possible activators, such as IL-1␤, TNF-␣, epidermal growth
factor, TGF-␤, IFN-␥, or bacterial components, did not further
up-regulate the O2⫺ generation (data not shown).
In contrast to primary cultures of guinea pig LIEC, human colon
cancer cell lines (T84 and Caco2 cells) produced only small
amounts of O2⫺ (⬍2 nmol/mg protein/h). RT-PCR analysis
roughly estimated that primarily cultured LIEC appeared to express larger amounts of the Nox1 mRNA than the cell lines. Moreover, freshly isolated and cultured guinea pig LIEC constitutively
expressed p67phox, its homologue p51nox, and a p47phox homologue p41nox, but they were absent or poorly expressed in T84 and
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FIGURE 4. Construction of Nox1 activity in T84 cells. The levels of Nox1, p22phox, p67phox, p41nox, p51nox, Rac1, and GAPDH mRNAs in guinea pig
LIEC, T84 cells, or Caco2 cells were estimated by RT-PCR analysis, as described in Materials and Methods (A). T84 cells were then transfected for 48 h
with the indicated cDNA-containing vectors, and O2⫺ release from the treated cells was determined in the presence or absence of 250 ng/ml PMA (B). The
levels of expressed proteins were assessed by immunoblot analysis with Ab against hemagglutinin or p67phox, and results are shown in the lower panels.
Similar results were obtained in three separate experiments. Values are means ⫾ SD (n ⫽ 8). ⴱ, p ⬍ 0.01, ANOVA and Scheffé’s test.
3056
Nox1 IN LIEC
Caco2 cells. Mouse and human p41nox lack the regulatory domain
corresponding to the aa 286 –340 of human p47phox (16 –18),
which may explain why Nox1 of guinea pig LIEC was in a selfactivated status to generate O2⫺ without any stimulants. When the
p41nox was transfected, T84 cells augmented O2⫺ production in
response to PMA. T84 cells expressed a low level of the p51nox
mRNA, and overproduction of p51nox further increased O2⫺-generating capability, but transfection of the p67phox was not effective
(Fig. 4B). P51nox lacks putative domains to interact with p40phox
(15–18), and colonic epithelial cells did not have p40phox. Based
on these findings, p51nox, rather than p67phox, may be a physiological partner with Nox1 and p41nox in catalyzing an electron
transfer from NADPH to molecular oxygen.
At present, physiological roles of colonic Nox1 are not fully
understood. The finding that transfected Nox1 confers mitogenic
properties on NIH 3T3 cells bore the scenario that Nox1 may be
involved in the process of cell transformation (6). We demonstrated that the terminally differentiated surface mucous cells in the
colon constitutively expressed Nox1 protein, suggesting that Nox1
may have other roles besides mitogenic properties. We examined
whether Nox1-derived ROS exhibited bactericidal activities. For
this purpose, 2 ⫻ 107 or 2 ⫻ 108 CFU/ml of S. enteritidis was cocultured with guinea pig LIEC for different incubation times (up to 8 h),
and the bacterial growth was estimated by colony counts grown on
tryptic soy agar for12 h. Guinea pig LIEC did not affect the growth
rate of bacteria, and inclusion of SOD and catalase in the culture
medium also did not change the growth (data not shown).
Surface mucous cells serve a primary protective role against
irritants by providing mucous coat. Recently, these cells have been
shown to play an important role in host defense as well, producing
proinflammatory mediators after the interaction with pathogenic
microbes (28). In fact, stimulation of TLR4 in guinea pig gastric
mucosal cells by H. pylori LPS activated NF-␬B within 30 min,
followed by up-regulation of Nox1 activity within 8 h (11, 14).
Enhanced production of ROS further augmented NF-␬B activation, leading to prolonged expression of the TNF-␣ and cyclooxygenase II mRNAs (11, 12). T84 cells specifically responded to
rFliC and induced Nox1, although they were insensitive to LPS,
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FIGURE 5. Up-regulation of O2⫺ production from T84 cells by rFliC. T84 cells cotransfected with p51nox and p41nox vectors were treated with native
rFliC (5 ␮g/ml), boiled rFliC (5 ␮g/ml), trypsin-digested rFliC (5 ␮g/ml), LPS (5 ␮g/ml), peptidoglycan (10 ␮g/ml), or CpG DNA (50 ␮g/ml) (A). The
T84 cells were incubated with different concentrations of rFliC for 24 h (B) or treated with 5 ␮g/ml rFliC for the indicated times (C) to measure spontaneous
O2⫺ release. Values are means ⫾ SD (n ⫽ 6). ⴱ, p ⬍ 0.01, as compared with untreated cells (ANOVA and Scheffé’s test). The TLR5 mRNA levels in T84
cells as well as human PBL were measured by RT-PCR analysis (D). Membrane (m.), cytosolic (c.), or whole cell (w.) fractions were prepared from T84
cells and subjected to immunoblot analysis with an anti-TLR5 Ab using human PBL as a control (E). After treatment of T84 cells with rFliC (5 ␮g/ml)
for the indicated times, whole cell proteins were prepared and subjected to immunoblot analysis using anti-TAK1 Ab or anti-TAB1 Ab. p-TAK1,
phosphorylated TAK1; p-TAB1, phosphorylated TAB1 (F). Similar results were obtained in three separate experiments. T84 cells were transfected with
mock or p51nox plus p41nox vectors were primed with rFliC (5 ␮g/ml) for 24 h in the absence or presence of 200 U/ml SOD plus 700 U/ml catalase. Amounts
of IL-8 production from these cells were determined in three separate experiments (G), as described in Materials and Methods. The values are expressed
as pmol/ml/mg protein (means ⫾ SD, n ⫽ 9). #, p ⬍ 0.01, ANOVA and Scheffé’s test.
The Journal of Immunology
3057
CpG DNA, and peptidoglycan. Abreu et al. (29, 30) have also
demonstrated that T84 and Caco2 cells are broadly unresponsive to
TLR2 and TLR4 signalings. When T84 cells transfected with both
p41nox and p51nox vectors were primed with rFliC, they increased
O2⫺-producing ability to higher than 5-fold of the vector alone
control (Fig. 6C). This enhanced O2⫺ production by transfection
of T84 cells with p41nox and p51nox cDNAs significantly enhanced
the rFliC-stimulated IL-8 release from the cells (Fig. 5G). These
results suggest that the TLR5-mediated up-regulation of Nox1 activity may contribute innate immune response by enhancing inflammatory responses of LIEC, rather than by directly killing
pathogenic bacteria.
It has been shown that intestinal epithelial cells, including T84
and Caco2 cells, express TLR5, and bacterial flagellin stimulates
the TLR5 signaling, leading to the activation of proinflammatory
signals, particularly NF-␬B pathway (25, 26). T84 cells are known
to show highly polarized expression of TLR5 on the basolateral
surface (26, 31). Certain flagellated bacteria are capable of translocating flagellin and stimulating TLR5 (31). Gastric pit cells were
sensitive to LPS (11, 13, 14), while T84 cells were not. This difference in the sensitivity may reflect physiological environments:
LIEC are always exposed to Gram-negative bacteria. Thus, surface
mucous cells of the stomach and colon may use different TLR
members to recognize respective pathogenic microbes, activate
Nox1, and finally produce defensive mediators. Rapid phosphorylation of TAK1 and TAB1 with rFliC confirmed that it actually
stimulated TLR5 signaling. Stimulation of TLR5 is suggested to
activate multiple signaling pathways (25, 26, 32). Transduction of
dominant-negative TAK1-adenovirus vector failed to inhibit
rFliC-primed Nox1 induction and O2⫺ increase (data not shown).
PDTC, however, significantly blocked both responses, which suggest an important role of a putative NF-␬B binding site at ⫺253 bp
in the human NOX1 5⬘-flank (GenBank, NT010552). Further studies are necessary to settle this issue.
The present study clearly demonstrated that p41nox and p51nox,
novel p47phox and p67phox homologues, respectively, are essential
components for Nox1 in achieving a potent oxidase activity. Additionally, the Nox1 may constitute early responses in epithelial
cells against pathogens for the host defense.
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