Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells

Tsukasa Kawahara,1 Motoyuki Kohjima,2 Yuki Kuwano,1 Hisano Mino,1 Shigetada Teshima-Kondo,3 Ryu Takeya,2 Shohko Tsunawaki,4 Akihiro Wada,5 Hideki Sumimoto,2 and Kazuhito Rokutan3

Departments of 1Nutritional Physiology and 3Stress Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima; 2Medical Institute of Bioregulation, Kyushu University, Fukuoka; 4Department of Infectious Diseases, National Research Institute for Child Health and Development, Tokyo; and 5Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan

Submitted 6 July 2004 ; accepted in final form 30 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primary cultures of guinea pig gastric mucosal cells express NADPH oxidase 1 (Nox1), a homolog of gp91phox, and produce superoxide anion (O2) at a rate of ~100 nmol·mg protein–1·h–1 in response to Helicobacter pylori (H. pylori) lipopolysaccharide (LPS) from virulent type I strains. The upregulated O2 production also enhances H. pylori LPS-stimulated tumor necrosis factor-{alpha} or cyclooxygenase-2 mRNA expression, which suggests a potential role for Nox1 in the pathogenesis of H. pylori-associated diseases. The H. pylori LPS-stimulated O2 production in cultured gastric mucosal cells was inhibited by actinomycin D as well as cycloheximide, suggesting that the induction is regulated at the transcriptional level. The LPS treatment not only increased the Nox1 mRNA to a greater extent but also induced expression of the message-encoding, Nox-organizing protein 1 (NOXO1), a novel p47phox homolog required for Nox1 activity. In addition, H. pylori LPS activated Rac1; i.e., it converted Rac1 to the GTP-bound state. A phosphoinositide 3-kinase inhibitor, LY-294002, blocked H. pylori LPS-induced Rac1 activation and O2 generation without interfering with the expression of Nox1 and NOXO1 mRNA. O2 production inhibited by LY-294002 was completely restored by transfection of an adenoviral vector encoding a constitutively active Rac1 but not an inactive Rac1 or a constitutively active Cdc42. These findings indicate that Rac1 plays a crucial role in Nox1 activation. Thus the H. pylori LPS-stimulated O2 production in gastric mucosal cells appears to require two distinct events: 1) transcriptional upregulation of Nox1 and NOXO1 and 2) activation of Rac1.

superoxide anion; phosphoinositide 3-kinase; Toll-like receptor 4; inflammation


SUPEROXIDE ANION (O2) PRODUCED by professional phagocytes plays an essential role in the elimination of invading microorganisms. Activation of the responsible enzyme, phagocyte NADPH oxidase, requires the assembly of a membrane-integrated cytochrome b558 (a heterodimer formed by gp91phox and p22phox) with cytosolic components p47phox, p67phox, and the small GTPase Rac (for review, see Ref. 3). Recently, six homologs of gp91phox have been identified and named systematically the NADPH oxidase (Nox)/dual oxidase (Duox) family (for review, see Refs. 26, 27). These novel enzymes are proposed to serve a variety of functions, including regulation of cell growth (36, 38), atherosclerosis (16, 28, 35), thyroid hormone synthesis (7), and host defense (15, 19). Among these homologs, Nox1 is dominantly expressed in the colon among human tissues and in colon adenocarcinoma cell lines (5, 19, 36). Guinea pig gastric mucosal cells in primary culture express Nox1 and spontaneously produce O2 (38, 39). These cells are able to release O2 at much higher rates when exposed to Helicobacter pylori (H. pylori) lipopolysaccharide (LPS) from type I but not from less virulent type II strains, suggesting that Nox1 may be involved in the pathogenesis of H. pylori-associated diseases (21). The molecular mechanism underlying the induction of O2 production by LPS, however, remains to be elucidated.

Recent identification of novel homologs of p47phox and p67phox, designated Nox organizer 1 (NOXO1; also known as p41nox) and Nox activator 1 (NOXA1; also known as p51nox), respectively, has improved the understanding of the molecular mechanism underlying regulation of Nox1 activity (4, 6, 14, 37). Although ectopic expression of Nox1 alone in cultured cells does not result in O2 production, cotransfection with NOXO1 and NOXA1 cDNA leads to generation of a large amount of O2 (4, 6, 14, 37). In addition, O2 production by Nox1 is significantly enhanced by p22phox (37). Thus Nox1 is likely complexed with p22phox and activated by the Nox organizers and activators, as is gp91phox (Nox2). During the activation of the phagocyte oxidase, Rac translocates to the membrane independently of the other cytosolic factors. At the membrane, GTP-bound Rac directly interacts with p67phox and probably also with cytochrome b558 (9, 11, 24), thereby participating in electron transfer from NADPH to molecular O2 (1, 8, 23, 29). On the other hand, it remains unclear whether the other Nox/Duox family members require Rac for their oxidase activities.

Guinea pig gastric mucosal cells constitutively express Nox1, p22phox, p67phox, NOXA1, and Rac1. In this study, we have demonstrated that H. pylori LPS effectively stimulates expression of Nox1 and NOXO1 mRNA and activates Rac1; i.e., it converts Rac to the GTP-bound form. Using a phosphoinositide 3-kinase (PI3K) inhibitor, LY-294002, and an adenoviral vector encoding constitutively active Rac1, we suggest that H. pylori LPS-stimulated O2 production in gastric mucosal cells is controlled by two distinct mechanisms: 1) transcriptional upregulation of Nox1 and NOXO1 and 2) activation of Rac1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of primary cultures of gastric mucosal cells under LPS-free conditions. Specific pathogen-free male Hartley guinea pigs were purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan) and treated in accordance with the guidelines of the National Institutes of Health. The present study also was approved by the Animal Care Committee of the University of Tokushima. Gastric mucosal cells were isolated from fundic glands and prepared under LPS-free conditions (21). Cells were cultured for 2 days in RPMI 1640 medium supplemented with 2 mM glutamine, 10% (vol/vol) fetal bovine serum (FBS), 0.1 mg/ml streptomycin, and 100 U/ml penicillin in 5% CO2-95% air at 37°C. Growing cells were composed of pit cells (>90%), granule-free pit cell precursors (5%), parietal cells (5%), and fibroblasts (<1%) (38, 39), and pit cells were confirmed to be responsible for O2 generation (39). After being washed with RPMI 1640 medium, the cells maintained in the medium supplemented with 10% FBS were used for experiments. Viability of the cells treated with various compounds or transfected with adenoviral vectors was maintained throughout the experiments, which were based on lack of lactase dehydrogenase release into reaction medium, continued Trypan blue exclusion, and adherence to the culture plates. For measurement of O2 production after washing with HBSS, these cells were incubated in HBSS containing 80 µM cytochrome c. The rate of O2 release was determined by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c and expressed in nanomoles per milligram of protein per hour (39).

Purification of H. pylori LPS and lipid A. A clinical strain of type I H. pylori was used in this study (indicated as H. pylori 1 in Ref. 21). H. pylori LPS and lipid A were purified from this strain, and the endotoxin activities of LPS and lipid A were determined to be 109 and 22.1 endotoxin units (EU)/µg, respectively, using the Limulus amoebocyte lysate assay (21).

Reverse transcriptase (RT)-PCR. Total RNA was isolated from the indicated cells with an acid guanidium-thiocyanate-phenol chloroform mixture (38), and cDNA was synthesized with MuLV reverse transcriptase (Applied Biosystems, Foster City, CA). The following primer sets were used to amplify the respective guinea pig cDNA products with GeneAmp PCR system (Applied Biosystems): tumor necrosis factor-{alpha} (TNF-{alpha}), 5'-AAAGTAGACCTGCCCGGACT-3' and 5'-GTACCTCATCTACTCCCAGG-3'; and cyclooxygenase-2 (COX-2), 5'-CCAGTTTGTTGAATCATTCACC-3' and 5'-AAAGTACTCGGCTTCCAGTAG-3'. The primer sets for Nox1, gp91phox, Nox4, NOXO1, p47phox, p67phox, NOXA1, p22phox, Rac1, Rac2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were described previously (19). Amplified PCR products were ligated into a pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA) and sequenced. Each product was confirmed to be the corresponding cDNA fragment.

Northern blot analysis. Total RNA (25 µg/lane) isolated from guinea pig gastric mucosal cells or peripheral blood leukocytes was electrophoretically separated on a 1% agarose formaldehyde gel and transferred to a Hybond N-plus nylon filter (Amersham Pharmacia, Piscataway, NJ). Hybridization reaction was performed with radiolabeled cDNA fragments in ExpressHyb solution (Clontech Laboratories, Palo Alto, CA) according to the manufacturer's protocols. The cDNA probes for guinea pig Nox1, NOXO1, p47phox, NOXA1, and p67phox mRNA were prepared by performing RT-PCR as described above. These mRNA levels were measured by Northern hybridization and standardized by rehybridization with the cDNA probe for guinea pig GAPDH.

Assay for Rac1 activation. A cDNA fragment encoding the Rac-binding domain (RBD; amino acids 66–147) of human p21-activated protein kinase 2 (PAK2; GenBank accession no. U24153) was isolated from the human placenta cDNA from Multiple Tissue cDNA Panel I (Clontech Laboratories) by performing PCR using the following primer set: 5'-AAAAGGATCCAAGAAAGAAAAGGAACGGCCAG-3' and 5'-AAAACTCGAGTTTCTCAGGAGGAGTAAAGCTCAG-3'. The BamHI and XhoI sites, respectively, are underlined. The PCR product was confirmed to be human PAK2 and subcloned into pGEX-4T-1 (Amersham Pharmacia). The glutathione S-transferase (GST)-fused protein was expressed in Escherichia coli JM109 cells and purified by glutathione-Sepharose-4B (Amersham Pharmacia) (2).

After cells growing on 60-mm-diameter culture dishes were exposed to 180 ng/ml (equivalent to 21 EU/ml) of H. pylori LPS for the indicated times, the reaction was terminated by washing with PBS and adding lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 4 mM EGTA, 4 mM EDTA, 100 µM PMSF, and 100 µM leupeptin). After the cell lysate (5 x 106 cells) was centrifuged for 20 s at 12,000 g, the resulting supernatant was mixed with GST- or GST-PAK2-RBD-conjugated glutathione-Sepharose 4B beads and incubated for 5 min on ice. After undergoing three washes with cold PBS, the beads were resuspended in the Laemmli sample buffer. The bound proteins were separated in 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). After being blocked, the membrane was probed with an anti-Rac1 monoclonal antibody (clone 23A8; Upstate Biotechnology, Lake Placid, NY) that does not cross-react with other members of the Rho family (22, 34). The blot was developed using an enhanced chemiluminescence Western blot detection kit (Amersham Pharmacia) to visualize the antibody, and then the membrane was stained with Coomassie brilliant blue to estimate the amount of precipitated GST or GST-PAK2-RBD protein. The amount of total Rac1 in each cell lysate was standardized by immunoblotting with the anti-Rac1 antibody before incubation of the lysate with the GST-fused proteins.

Phosphorylation of Akt. Antibodies against Akt and a phosphorylated form of Akt (Ser473) were purchased from Cell Signaling Technology (Beverly, MA). The amount of total or phosphorylated Akt was measured by immunoblotting as described previously (20).

Adenoviruses. The Adeno-X expression system (Clontech Laboratories) was used to generate an adenovirus encoding Flag-tagged human Rac1, substituting Gly12 with Val (G12V) or Thr17 with Asn (T17N), or encoding Flag-tagged human Cdc42, substituting Gly12 with Val (G12V) (31), according to the manufacturer's protocol. The multiplicity of infection of these viruses was determined by counting surviving human embryonic kidney (HEK)-293 cells after transfection for 12 days. Expression of the transfected proteins was assessed by immunoblotting using an anti-Flag antibody (Sigma-Aldrich, St. Louis, MO).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
H. pylori LPS-induced O2 production and Nox1 mRNA expression in gastric mucosal cells. Guinea pig gastric mucosal cells cultured under LPS-free conditions spontaneously produced O2 at a rate of ~10 nmol·mg protein–1·h–1, while they secreted 10 times greater amounts of O2 when treated with H. pylori LPS at 180 ng/ml or higher (21). As shown in Fig. 1A, after exposure to 180 ng/ml of H. pylori LPS, the rate of O2 production began to increase within 4 h and reached a maximum at 16 h. In response to H. pylori LPS, TNF-{alpha} and Cox-2 mRNA were expressed within 2 h, and these expressions were observed continuously up to 24 h (Fig. 1B). The presence of a flavoprotein inhibitor, diphenylene iodonium (DPI), blocked the LPS-triggered increase in O2 production (Fig. 1A). At the same time, DPI inhibited the LPS-induced expression of TNF-{alpha} and Cox-2 mRNA at 8 h or later (Fig. 1B). At these time points, gastric mucosal cell oxidase was fully activated (Fig. 1A). Inclusion of SOD and catalase did not affect the LPS-stimulated expression of the mRNA within 4 h, while it blocked this expression in the late phase similarly to the way DPI did (Fig. 1B). These results suggest that reactive oxygen species (ROS) produced by the H. pylori LPS-treated cells also may enhance the inflammatory responses of gastric mucosal cells to H. pylori infection.



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Fig. 1. Effects of Helicobacter pylori (H. pylori) lipopolysaccharide (LPS) on O2 production and NADPH oxidase 1 (Nox1) mRNA expression. A: after guinea pig gastric mucosal cells cultured under LPS-free conditions were left untreated or were treated with 5 µM diphenylene iodonium (DPI) for 30 min, they were then exposed to 180 ng/ml [21 endotoxin units (EU)/ml] of H. pylori LPS in RPMI 1640 medium containing 10% fetal bovine serum (FBS) for the indicated times. O2 generation by these cells was assayed by the cytochrome c method. B: after cells were untreated or treated with 180 ng/ml H. pylori LPS for the indicated times in the absence or presence of either 5 µM DPI or 200 U/ml superoxide dismutase (SOD) plus 700 U/ml catalase in the culture medium, levels of tumor necrosis factor-{alpha} (TNF-{alpha}), cyclooxygenase (COX)-2, and GAPDH mRNA in these cells were assessed using reverse transcriptase (RT)-PCR. The number of PCR cycles is 25. C: after pretreatment with 75 µM actinomycin D or 5 µg/ml cycloheximide for 30 min, these cells were exposed to 180 ng/ml of H. pylori LPS for 16 h. Actinomycin D or cycloheximide was present during the 16-h treatment. D: after gastric mucosal cells were left untreated or were treated with 180 ng/ml H. pylori LPS for 4 h, expression of the Nox1 mRNA in the cells was measured using RT-PCR using Caco-2 cells as a positive control. E: expression of gp91phox and Nox4 mRNA in gastric mucosal cells exposed to 180 ng/ml H. pylori LPS for 4 h was assayed using RT-PCR. Human and guinea pig peripheral blood leukocytes (PBL) and guinea pig kidney were used as the corresponding positive controls. The number of cycles is indicated in D and E. Values are means ± SD; n = 8. *P < 0.001 compared with untreated and H. pylori LPS-treated cells (ANOVA and Scheffé's test) (A and C, respectively). These experiments were repeated 3 times with similar results. Hp, H. pylori; GMCs, gastric mucosal cells.

 
H. pylori LPS-triggered O2 production was completely canceled in the presence of actinomycin D as well as cycloheximide (Fig. 1C), suggesting a regulation at the transcriptional level. To test this possibility, we first examined whether H. pylori LPS stimulated expression of mRNA for Nox1 or other O2-producing enzymes. Using the sequence of the guinea pig Nox1 cDNA that we had obtained (GenBank accession no. AB099629), we performed RT-PCR and found that quiescent cells maintained under LPS-free conditions expressed a small amount of the Nox1 mRNA, which was amplified by PCR at 30 cycles but not at 20 cycles (Fig. 1D). Treatment with H. pylori LPS for 4 h increased the Nox1 mRNA level (Fig. 1D). On the other hand, the gp91phox and Nox4 mRNA were not detected in guinea pig gastric mucosal cells even after treatment with H. pylori LPS (Fig. 1E), as well as in the quiescent cells (data not shown). Thus the O2 production induced by LPS appears to involve the enhanced expression of Nox1.

Induction of NOXO1 mRNA expression by H. pylori LPS. We next investigated the expression of mRNA for Nox-activating proteins such as NOXO1 and NOXA1 in gastric mucosal cells untreated or treated with H. pylori LPS. For this purpose, we had cloned the guinea pig NOXO1 cDNA (GenBank accession no. AB105906) (19), the deduced amino acid sequence of which shows 72% identity with that of human NOXO1 (GenBank accession no. AF539796). Northern blot analysis performed with a probe derived from the cDNA revealed that H. pylori LPS stimulated the expression of NOXO1 mRNA, with a peak at 4 h (Fig. 2A), and a similar time course was observed in the upregulation of Nox1 mRNA (Fig. 2A). Lipid A of H. pylori, which is also capable of enhancing O2 generation by gastric mucosal cells (21), stimulated the expression of NOXO1 and Nox1 mRNA (Fig. 2B). The expression of these two mRNA was blocked by polymyxin B (Fig. 2B), an agent that interacts with the lipid A moiety of LPS and inhibits the H. pylori LPS- or lipid A-triggered elevation of O2 generation (21).



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Fig. 2. Expression of mRNA for Nox1 and Nox components. A: gastric mucosal cells were treated with 180 ng/ml H. pylori LPS for the indicated times. The levels of Nox1, NOXO1, p47phox, NOXA1, p67phox, and GAPDH mRNA in these cells as well as guinea pig PBL were measured using Northern blot analysis. B: after 4-h treatment with H. pylori LPS (180 ng/ml), its lipid A (950 ng/ml, equivalent to 21 EU/ml), or the same amount of H. pylori LPS that was preincubated with polymyxin B (100 µg/ml) for 1 h at 37°C, expression of Nox1, NOXO1, p47phox, p67phox, NOXA1, p22phox, and GAPDH mRNA was measured using RT-PCR. The number of cycles is shown in parentheses in B. Similar results were obtained in 3 separate experiments. poly B, polymyxin B.

 
In contrast to the NOXO1 mRNA, the p47phox mRNA does not seem to exist in quiescent guinea pig gastric mucosal cells or in cells stimulated with H. pylori LPS or its lipid A. Although we had previously obtained a very weak signal of a 47-kDa protein immunoreactive to an antibody against human p47phox in guinea pig gastric pit cells (39), the carefully performed Northern blot analysis (Fig. 2A) and RT-PCR (Fig. 2B) in the present study revealed that neither quiescent nor stimulated cells express p47phox mRNA.

We also estimated the level of the mRNA for Nox activators, i.e., NOXA1 and p67phox, in gastric mucosal cells. To do this, we had cloned the cDNA for guinea pig NOXA1 (GenBank accession no. AB105907) and p67phox (GenBank accession no. AB105909) (19) and performed RT-PCR and Northern blot analysis on the basis of the cDNA sequences. As shown in Fig. 2, A and B, the NOXA1 and p67phox mRNA were expressed in quiescent cells, whereas neither H. pylori LPS nor lipid A increased these mRNA levels. This may be inconsistent with our previous finding that in guinea pig gastric mucosal cells, the amount of a 67-kDa protein that cross-reacted with an antibody against human p67phox increased in parallel with elevation of O2 generation after treatment with H. pylori LPS (21). To explore this inconsistency, we developed a novel polyclonal antibody against human recombinant p67phox that recognized the guinea pig p67phox with a molecular mass of 63 kDa, and the amount was not affected by H. pylori LPS (data not shown). On the basis of these findings, we concluded that H. pylori LPS did not stimulate the induction of p67phox. In addition, the mRNA for p22phox, a possible partner of Nox1 (37), was expressed in quiescent gastric mucosal cells, and the amount of the mRNA did not change even after the addition of H. pylori LPS (Fig. 2B). Taken together, gastric mucosal cells constitutively express the mRNA for NOXA1, p67phox, and p22phox, and the treatment with H. pylori LPS specifically induces the transcription of the Nox1 and NOXO1 genes, which is likely involved in the induction of O2 generation.

Effects of LY-294002 on H. pylori LPS-triggered O2 generation. Activation of PI3K plays a crucial role in the activation of the phagocyte oxidase in response to a chemoattractant, N-formylmethionyl-leucyl-phenylalanine (fMLP) (10, 18, 32). In this study, we tested whether a PI3K inhibitor, LY-294002, suppressed H. pylori LPS-triggered elevation of O2 secretion from gastric mucosal cells. Pretreatment with LY-294002 for 30 min dose dependently blocked the LPS-stimulated upregulation of O2 generation, with an IC50 of 7 µM (Fig. 3A). It should be noted that at 0.5 to 50 µM concentrations, LY-294002 did not affect the LPS-stimulated expression of Nox1 and NOXO1 mRNA (Fig. 3B). Phosphorylated lipid products generated by PI3K act as second messengers to activate protein kinases, including Akt (18). We also confirmed that H. pylori LPS phosphorylated Akt at Ser473 and that pretreatment with LY-294002 completely blocked the phosphorylation in H. pylori LPS-treated cells (Fig. 3C). These results suggest that in addition to the expression of Nox1 and NOXO1, a LY-294002-sensitive event appears to be involved in the H. pylori LPS-enhanced O2 generation.



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Fig. 3. Effects of LY-294002 on O2 production triggered by LPS. A: gastric mucosal cells were incubated with 180 ng/ml H. pylori LPS in the presence or absence of the different concentrations of LY-294002 as well as vehicle (DMSO) for 16 h, and the rate of O2 production was measured. Values are means ± SD; n = 8. *P < 0.01, compared with H. pylori LPS-treated cells (ANOVA and Scheffé's test). B: expression of Nox1, NOXO1, and GAPDH mRNA were measured using RT-PCR after 4-h treatment with 180 ng/ml H. pylori LPS in the presence of the indicated concentrations of LY-294002 or vehicle. The number of cycles is 20. C: after cells were incubated with 180 ng/ml H. pylori LPS in the presence or absence of 50 µM LY-294002 for the indicated times, the levels of phosphorylated Akt (p-Akt) and total Akt were measured using immunoblot analysis. Similar results were obtained in 3 separate experiments.

 
Activation of Rac1 by stimulation with H. pylori LPS. It is known that PI3K inhibitors suppress activation of Rac in neutrophils (2), which is likely involved in the blockade of activation of the phagocyte NADPH oxidase. On the other hand, it remains unclear whether Rac plays a role in the activation of the other NADPH oxidases, including Nox1. To elucidate the role of Rac in the H. pylori LPS-induced generation of O2, we first examined the expression of Rac isoforms in guinea pig gastric mucosal cells. As shown in Fig. 4A, gastric mucosal cells expressed only Rac1 mRNA between the two Rac isoforms thus far identified in guinea pigs (25), in contrast to the predominant expression of Rac2 in human neutrophils (17).



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Fig. 4. Effects of H. pylori LPS on Rac1 activation. A: expression of Rac1 and Rac2 mRNA in guinea pig gastric mucosal cells before or after treatment with H. pylori LPS was determined by performing RT-PCR using guinea pig PBL as a control. B: gastric mucosal cells were incubated for 10 min with 180 ng/ml of H. pylori LPS. C: cells were incubated for the indicated times with 180 ng/ml of H. pylori LPS. D: cells were treated with H. pylori LPS at the indicated concentrations. E: after pretreatment with LY-294002 at the indicated concentrations or vehicle (DMSO) for 30 min, cells were incubated with 180 ng/ml of H. pylori LPS for 1 h. The cells were lysed, and GTP-bound Rac1 was precipitated with glutathione S-transferase (GST) (B) or GST- human p21-activated protein kinase 2 (PAK2)-Rac-binding domain (RBD) (BE) bound to glutathione-Sepharose-4B beads. The amounts of Rac1 in the precipitates and total Rac1 in cell lysates were assessed using immunoblotting with the anti-Rac1 antibody. The amount of GST or GST-PAK2-RBD protein in the precipitates was detected by staining with Coomassie brilliant blue (B). Similar results were obtained in 3 separate experiments.

 
We next tested whether H. pylori LPS activated endogenous Rac in gastric mucosal cells by a performing pull-down assay using the Rac-binding domain of the protein kinase PAK2 (PAK2-RBD). PAK2 binds to GTP-bound Rac with high affinity, whereas the affinity for the GDP-bound form is undetectably low (2, 41). As shown in Fig. 4B, Rac1 in guinea pig gastric mucosal cells stimulated by H. pylori LPS bound to GST-fused PAK2-RBD but not to GST alone. This activation occurred within 5 min and continued for up to 16 h (Fig. 4C). H. pylori LPS dose dependently activated Rac1 (Fig. 4D), and its minimum effective concentration of 180 ng/ml was similar to that required for the upregulation of O2 production (21). Furthermore, LY-294002 blocked the LPS-stimulated activation of Rac1 (Fig. 4E) in association with the inhibition of O2 generation (Fig. 3A), suggesting the involvement of Rac1 activation, an event sensitive to LY-294002, in the LPS-induced O2 generation by Nox1.

Restoration of LY-294002-inhibited O2 generation by expression of a constitutively active Rac1. To confirm the role of Rac in H. pylori LPS-stimulated O2 generation, we transduced an adenoviral vector encoding a constitutively active form of Rac1 (G12V) to gastric mucosal cells and tested whether it affected the inhibition of O2 production using LY-294002. As shown in Fig. 5, the active Rac1 (G12V) dose dependently restored O2 production even in the presence of LY-294002. On the other hand, an inactive form of Rac1 (T17N) failed to do so. The findings indicate that activation of Rac1 plays an essential role in LPS-stimulated O2 generation. The role appears to be specific for Rac because a constitutively active form of Cdc42 (G12V), another Rho family small GTPase, had no effect (Fig. 5). Thus expression of the active Rac was required for the induction of O2 generation when LPS-stimulated cells were treated with LY-294002, an agent that did not affect the expression of Nox1 and NOXO1 (Fig. 3B). On the other hand, the constitutively active Rac1 failed to stimulate O2 generation by the quiescent cells, alternatively supporting the importance of the expression of Nox1 and NOXO1 (Fig. 5).



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Fig. 5. Restoration of LY-294002-inhibited O2 production by expression of an active Rac1. Guinea pig gastric mucosal cells were transfected with adenoviral vector encoding constitutively active Rac1, Cdc42 (G12V), or the inactive form of Rac1 (T17N) and then incubated for 24 h. These cells infected with the indicated multiplicity of infection were exposed to 180 ng/ml of H. pylori LPS for 16 h in the presence or absence of 50 µM LY-294002. The rates of O2 production were measured. Values are means ± SD; n = 8. *P < 0.01 compared with LY-294002-pretreated, H. pylori LPS-exposed cells (ANOVA and Scheffé's test). The levels of expressed proteins were assessed using immunoblot analysis with antibody against Flag or {beta}-actin, as shown at bottom. Similar results were obtained in 3 separate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we have shown that H. pylori LPS induced the transcription of the Nox1 and NOXO1 genes and activated the GTPase Rac1 in gastric mucosal cells, and also that the two events played crucial roles in O2 production induced by the bacterial component. The property of the Nox1 gene as an inducible one also has been demonstrated in other types of cells. Interferon-{gamma}, 1{alpha},25-dihydroxyvitamin D3, or Salmonella enteritidis flagellin stimulates the Nox1 transcript expression in colon cancer cell lines (13, 19), and platelet-derived growth factor or angiotensin II also upregulates the expression level of Nox1 mRNA in rat vascular smooth muscle cells (28, 35).

Activation of Nox1 as well as of gp91phox requires both a Nox organizer and a Nox activator; the organizers include p47phox and NOXO1, and the activators include p67phox and NOXA1 (4, 6, 14, 37). Gastric mucosal cells constitutively express the two Nox activators (Fig. 2). Hence, expression of a Nox organizer is required for the oxidase activation in these cells. Indeed, H. pylori LPS induces the transcription of the NOXO1 gene (Fig. 2). To the best of our knowledge, the present study is the first to show that NOXO1 is regulated at the transcriptional level. At present, no specific antibodies for guinea pig Nox1 and NOXO1 are available for immunoblotting; therefore, the levels of these two proteins and the kinetics of their synthesis were not determined. However, the inhibition of the LPS-stimulated O2 production with cycloheximide indirectly supports that synthesis of Nox1 and NOXO1 proteins accompany the expression of their mRNA.

For activation of the phagocyte oxidase containing gp91phox, two switches are required to be turned on at the same time: conformational change of p47phox and activation of Rac. The conformational change of p47phox allows its SH3 domains to bind to p22phox for the oxidase activation; the SH3 domains are normally masked via intramolecular interaction with the autoinhibitory region. Because this region is absent in NOXO1 (4, 6, 14, 37), NOXO1 may exist in a constitutively active form. Indeed, NOXO1 is capable of binding via its SH3 domains to p22phox without any conformational changes (37). p22phox is constitutively expressed in gastric mucosal cells. Therefore, once NOXO1 is synthesized with Nox1 in the LPS-treated gastric mucosal cells, Nox1 assembled with p22phox is expected to constitutively interact with NOXO1, thereby entering into a quasi-activated state. Cheng and Lambeth (6) also have shown the colocalization of NOXO1 and Nox1 in HEK-293 cells overproducing these proteins.

Several lines of evidence demonstrated in the present study suggest that activation of Rac serves as a switch for the activation of Nox1 similarly to that for gp91phox. First, Rac1 activation by H. pylori LPS occurred much earlier than the increase in O2 production; however, Rac1 was still in an active state (Fig. 4B) when gastric mucosal cells increased O2 production (Fig. 1A). Second, LY-294002 inhibited the LPS-induced activation of Rac1 in a similar dose-dependent manner because it blocked O2 generation (Figs. 3 and 4). Finally, expression of a constitutively active Rac1 completely restored O2 production in the presence of LY-294002 (Fig. 5). For the restoration, Cdc42 failed to replace Rac (Fig. 5), although Cdc42 is highly similar to Rac in its amino acid sequence. In agreement with this finding, the Nox activators p67phox (24) and NOXA1 (37) are capable of binding to GTP-bound Rac but not to GTP-bound Cdc42.

The kinetics of activation of Rac2 in fMLP-stimulated neutrophils coincides with rapid and transient generation of O2 in these cells (2). The activity of the small GTPase is regulated by guanine-nucleotide exchange factor (GEF) (40) and GTPase-activating protein (GAP) (30). A recent study (12) suggested that the neutrophil NADPH oxidase might be regulated by GAP, including Breakpoint cluster region protein, p50RhoGAP, and p190RhoGAP. Recently, it was shown that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, a Rac-GEF ({beta}Pix), and Rac1 (33). In gastric mucosal cells, H. pylori LPS activated Rac1 within 5 min, while rapid activation was not linked to enhanced O2 production. The continuous activation of Rac1 and the slower induction of Nox1 and NOXO1 may result in the full activation of the oxidase. The Rac1 activation in the intervening period may exhibit distinct functions besides activation of Nox1. In fact, overproduction of the active Rac1 failed to further increase O2 production in the LPS-untreated cells (Fig. 5). Once guinea pig gastric mucosal cells are primed with H. pylori LPS, they continuously and spontaneously produce O2 for more than 24 h. To understand this unique feature of Nox1, studies to identify GEF and GAP as proteins that are involved in Nox1 activation in guinea pig gastric mucosal cells are underway.

Gastric mucosal cells maintained under LPS-free conditions spontaneously produced ~10 nmol O2/mg of protein/h. DPI and/or LY-294002 partially reduced the production by 40–50%, indicating that the primary cultures may contain a small number of activated cells. However, these cells still have Nox- and Rac1-independent O2-producing capability. At present, the source of the activity is unknown. However, basal O2 generation plays a crucial role in the stimulation of cell growth and the suppression of spontaneous apoptosis after the maturation of pit cells (38). With regard to activation of Nox1, colonic epithelial cells (T84 cells) preferentially use the Toll-like receptor (TLR)5, rather than TLR4, against S. enteritidis infection in vitro (19), whereas TLR4 and its adaptor protein (MD-2) are crucial against H. pylori infection in gastric epithelial cells (21). Thus gastric and colonic epithelial cells may use different TLR members to discern pathogenicities among bacteria, depending on their environment and to activate Nox1 appropriately for host defense. Although the amounts of O2 produced by the gastric and colonic epithelial cells are not enough to directly kill H. pylori and S. enteriditis, respectively, at least in vitro (19, 39), Nox1 may be one of the key molecules representing the initial trigger for host innate and inflammatory responses against microbial pathogens.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This study was supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research 14370184 (to K. Rokutan) and by a Grant-in-Aid for Scientific Research from the 21st Century COE Program, Human Nutritional Science on Stress Control, Tokushima, Japan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. Rokutan, Dept. of Stress Science, Institute of Health Biosciences, Univ. of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan (E-mail: rokutan{at}basic.med.tokushima-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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