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
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ABSTRACT |
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superoxide anion; phosphoinositide 3-kinase; Toll-like receptor 4; inflammation
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.
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MATERIALS AND METHODS |
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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- (TNF-
), 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 66147) 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).
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RESULTS |
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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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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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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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DISCUSSION |
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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 (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 4050%, 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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GRANTS |
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FOOTNOTES |
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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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