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Am J Physiol Gastrointest Liver Physiol 294: G795-G807, 2008. First published January 17, 2008; doi:10.1152/ajpgi.00431.2007
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MUCOSAL BIOLOGY

Helicobacter pylori-induced H,K-ATPase {alpha}-subunit gene repression is mediated by NF-{kappa}B p50 homodimer promoter binding

Arindam Saha, Charles E. Hammond, Maria Trojanowska, and Adam J. Smolka

Department of Medicine, Medical University of South Carolina, Charleston, South Carolina

Submitted 15 August 2007 ; accepted in final form 17 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Infection of human gastric body mucosa by the gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and a transitory hypochlorhydria that progresses in ~2% of patients to atrophic gastritis, dysplasia, and gastric adenocarcinoma. We have previously shown that H. pylori infection of cultured gastric epithelial cells (AGS) represses the activity of the transfected {alpha}-subunit (HK{alpha}) promoter of H,K-ATPase, the parietal cell enzyme mediating acid secretion. However, the mechanistic details of H. pylori-mediated repression of HK{alpha} and ensuing hypochlorhydria are unknown. H. pylori is known to upregulate the transcription factor NF-{kappa}B through the ERK1/2 MAPK pathway. We identified NF-{kappa}B-binding regions in the HK{alpha} promoter and found that H. pylori inoculation of AGS cells increased NF-{kappa}B p50 binding to the transfected HK{alpha} promoter and repressed its transcriptional activity. Immunoblot and DNA-protein interaction studies showed that although active phosphorylated NF-{kappa}B p65 is present in H. pylori-infected AGS cells, an NF-{kappa}B p50/p65 heterodimeric complex fails to bind to the HK{alpha} promoter. Point mutations at –159 and –161 bp in the HK{alpha} promoter NF-{kappa}B binding sequence prevented binding of NF-{kappa}B p50 and prevented H. pylori repression of point-mutated HK{alpha} promoter activity in transfected AGS cells. Small interfering RNA-mediated knockdown of NF-{kappa}B p50 in H. pylori-infected AGS cells also abrogated H. pylori-induced HK{alpha} repression, whereas NF-{kappa}B p65 knockdown did not. We conclude that H. pylori inhibits HK{alpha} gene expression by ERK1/2-mediated NF-{kappa}B p50 homodimer binding to the HK{alpha} promoter. This study identifies a novel pathogen-dependent mechanism of H,K-ATPase inhibition and contributes to understanding of H. pylori pathophysiology.

THE GRAM-NEGATIVE BACTERIUM Helicobacter pylori colonizes the mucus layer and mucosal epithelial cells of the human stomach. In developing countries, the prevalence of adult H. pylori infection can be >80%, compared with 20–50% in developed countries, resulting in ~2 billion H. pylori infections globally (51). Although the majority of infected people are asymptomatic, ~20% develop H. pylori-related primary gastritis and peptic ulcer disease, and a smaller subset (~2%) progress to gastric mucosa-associated lymphoid tissue lymphoma or gastric adenocarcinoma (3). The clinical outcome may reflect differing anatomic sites of infection. Thus H. pylori-mediated antral gastritis is associated with hypergastrinemia, hyperacidity, and duodenal ulcers (12), while H. pylori infection of the gastric body impairs acid secretion (16, 29, 31, 32), and the resulting hypochlorhydria is associated with an increased risk of gastric cancer (11, 26, 30, 39).

Gastric acid secretion is effected by a heterodimeric Mg2+-dependent P-type adenosine triphosphatase (H,K-ATPase, EC 3.6.1.3 [EC] 6; Refs. 13, 44). The polytopic catalytic {alpha}-subunit (HK{alpha}, molecular weight = ~94,000, accession no. J05451) populates tubulovesicular and secretory canalicular membranes in acid-secreting gastric epithelial parietal cells. Close interaction of HK{alpha} with a monotopic glycosylated β-subunit (HKβ; molecular weight = 60,000–80,000, accession no. BX537316) is required for functional electroneutral exchange of lumenal K+ for cytoplasmic H3O+ (9). Acid secretion is positively regulated by the gastric secretagogues histamine, gastrin, and acetyl choline (5). Constitutive HK{alpha} transcription in canine parietal cells is activated by Sp1 binding a few bases upstream of the HK{alpha} TATA box (33), and EGF-induced transcriptional activation of HK{alpha} is associated with protein binding to a site homologous to a c-fos serum response element (22). Parietal cell-specific GATA transcription factors have also been associated with induction of HK{alpha} transcription (36, 50).

The mechanisms underlying acid secretory inhibition after gastric H. pylori infection are unclear. H. pylori induces gastric epithelial cell apoptosis via secreted mediators such as the VacA cytotoxin and lipopolysaccharide (20), damaging epithelial acid-secreting parietal cells (34). H. pylori strains with a 40-kb cag pathogenicity island encoding a type IV secretion system (T4SS) promote host epithelial cell secretion of the chemotactic proinflammatory cytokine IL-8 (14, 40, 47, 52, 56). In response to IL-8, neutrophils and monocytes infiltrate the mucosa and secrete the cytokines IL-1β and TNF-{alpha} amplifying the inflammatory response. In addition to its proinflammatory properties, IL-1β is a powerful inhibitor of gastric acid secretion (2, 54, 55) and may contribute to H. pylori-mediated gastric hypochlorhydria. Sonicates of H. pylori strains were reported to inhibit acid secretion in rabbit isolated gastric epithelial cells, although the inhibitory agent remained poorly characterized (8, 19). Lastly, we (15, 45) previously reported that the transcriptional activity of human HK{alpha} promoter transfected into gastric epithelial cells is inhibited by H. pylori infection (15, 45).

H. pylori infection of gastric epithelial cells is known to activate NF-{kappa}B, a transcription factor that regulates genes involved in innate and adaptive immune response, cell proliferation, adhesion, stress response, inflammation, and apoptosis (41). In unstimulated cells, the five members of the mammalian NF-{kappa}B family, p65 (RelA), RelB, c-Rel, p50/p105 (NF-{kappa}B1), and p52/p100 (NF-{kappa}B2) are inactive homo- or heterodimers bound to members of the I{kappa}B family of proteins through an N-terminally-oriented conserved 300 amino acid Rel homology domain. Cell stimulation, for example by proinflammatory cytokines, activates I{kappa}B kinases (IKK{alpha} and IKKβ), which in turn phosphorylate I{kappa}B N-terminal serines. The ensuing proteasomal degradation of I{kappa}B releases NF-{kappa}B dimers, which are directed to the nucleus by hitherto cryptic nuclear localization signals (17). Binding of NF-{kappa}B dimers to specific sites in the promoter or enhancer regions of target genes is effected by their Rel homology domains. DNA binding of heterodimers incorporating p65, RelB, or c-Rel, each of which contains a C-terminal transactivation domain, may result in increased gene expression, e.g., upregulation of IL-8 (23) and cyclooxygenase-2 (25) in H. pylori-infected gastric epithelial cells, or repression, e.g., downregulation of HK{alpha}2 (colonic/renal H,K-ATPase) in inner medullary collecting duct cells (57). Binding of homodimers (p50/p50 or p52/p52), which lack transactivation domains, may result in decreased gene expression (58); however, p50/p50 homodimer binding to a cis-response element in the IL-10 promoter has recently been reported to activate IL-10 gene expression in macrophages (6).

Given that gastric inflammation and hypochlorhydria are predisposing factors in the progression from gastritis to intestinal metaplasia, dysplasia, and eventually gastric adenocarcinoma (10, 39), we sought to define the molecular mechanism whereby H. pylori mediates inhibition of acid secretion. Here, we tested the hypothesis that H. pylori repression of gastric H,K-ATPase gene expression is effected through NF-{kappa}B binding to HK{alpha} promoter cis-response elements. The results are consistent with H. pylori infection of human gastric adenocarcinoma (AGS) cells mobilizing both p50/p65 heterodimeric and p50/p50 homodimeric NF-{kappa}B to the cell nucleus, where transcriptional activity of HK{alpha} promoter is specifically repressed by binding of homodimeric p50/p50 NF-{kappa}B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell and bacterial cultures. AGS (CRL1739) and H. pylori (strain ATCC 49503) were purchased from the American Type Culture Collection (Manassas, VA). AGS cells were grown for no more than 10 passages in Ham's F-12 containing L-glutamine (Mediatech, Herndon, VA) supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA) at 37°C in a humidified incubator with 5% CO2-95% air. H. pylori cultures were grown on Brucella broth (Difco Laboratories, Detroit, MI) plates containing 10% FBS and 2.4% agar (Brucella-agar plates) at 37°C using a micro-aerophilic gas pack system (BD Biosciences, Sparks, MD). Cultures were screened regularly by urease test during subculturing. Only cultures giving a positive urease test were used for cell infection. For AGS cell infection, H. pylori were harvested after 24 h of culture in Brucella-agar plates. Bacteria were resuspended in Ham's F-12 medium containing FBS and enumerated by measuring absorbance at 600 nm (1 OD600nm= 2.4 x 108 bacteria/ml). Multiplicities of infection (MOI) were calculated based on AGS cell and bacterial cell counts.

Reagents. Restriction enzymes, MAPK inhibitors PD-98059 (V1191) and SB-203580 (V1161), the pGL2-basic vector transfection plasmid, 5x passive lysis buffer, and the luciferase assay substrate were obtained from Promega (Madison, WI). The JNK inhibitor II was purchased from Calbiochem (San Diego, CA). The transfection plasmid pMaxGFP was purchased from Amaxa (Gaithersburg, MD). All other reagents were of molecular biology grade with maximum possible purity.

HK{alpha} promoter-reporter plasmid constructs. Genomic DNA representing a portion of the human gastric H,K-ATPase {alpha}-subunit (HK{alpha}) 5'-flanking region was provided by G. Shull (University of Cincinnati). A 2,179-bp segment of this 5'-flanking region including 20 bp downstream of the transcription initiation site (HK{alpha}2179) was integrated into the luciferase reporter plasmid pGL2-basic vector as described previously (45). Truncated deletion constructs of the 5'-flanking region were generated by PCR amplification using the HK{alpha}2179 promoter-Luc reporter plasmid as a template as described previously (45). Point-mutated HK{alpha}206 promoter-Luc reporter construct (double mutant HK{alpha}206) was generated by replacing a segment of HK{alpha} promoter sequence bounded by PstI and Bsu36I restriction sites with a homologous synthetic oligonucleotide duplex incorporating point mutations at –159 bp (A>C, forward strand; T>G, reverse strand) and at –161 bp (G>A, forward strand; C>T, reverse strand). Sequence fidelity of the point-mutated HK{alpha}206 promoter in pGL2-basic vector was verified by dideoxy sequencing. The inducible cis-reporter plasmid pNF-{kappa}B-Luc and a MEK kinase (MEKK) expression plasmid pFC-MEKK as positive control were purchased from Stratagene (#219077, La Jolla, CA).

Transient transfection. AGS cells (105 cells/well) were cultured overnight in 24-well cell culture plates, washed with PBS, and then treated for 24 h with Opti-MEM (Invitrogen, Carlsbad, CA) containing 0.25 µg DNA and Fugene-6 transfection reagent (Roche Diagnostics, Indianapolis, IN) at a DNA mass-to-Fugene-6 volume ratio of 1:6. AGS cells were cotransfected with pMaxGFP to provide a normalization control and a measure of transfection efficiency, and pGL2-basic vector plasmids devoid of promoter or enhancer served as negative controls. The ratio of promoter-reporter plasmids to normalization control plasmids was 4:1. In experiments using MEKK expression plasmids, pFC-MEKK-to-pNF-kB-Luc-to-pMaxGFP transfection ratios were 2:2:1. After 24 h of transfection, cells were treated with 25 MOI of H. pylori for 6 h. When needed, AGS cells were incubated with inhibitors of intracellular signaling pathways for 90 min before treatment. Cells were lysed with 1x passive lysis buffer, and light emission was measured as relative light units (RLU) in a Victor 1420 multi-label counter (Perkin-Elmer Bio-Sciences, Wellesley, MA) using the luciferase assay substrate according to the manufacturer's protocol. Fluorescence of pMaxGFP reporter plasmids was measured at 485 nm excitation and 538 nm emission in a Spectramax Gemini XS spectrofluorimeter (Molecular Devices, Sunnyvale, CA). Luciferase RLUs were normalized to cotransfected pMaxGFP fluorescence and corrected by subtracting corresponding normalized promoterless pGL2-basic vector RLU data. Data points are shown as means ± SD of three independent transfection experiments with each deletion construct.

Real time RT-PCR. AGS cells were grown to 75–80% confluence in 24-well cell culture plates and were then serum deprived for 15–20 h. Total RNA was isolated using STAT-60 reagent (Tel Test, Friendswood, TX) and reverse-transcribed using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. Measurement of AGS cell NF-{kappa}B p105 (NF-{kappa}B1) and p65 subunit mRNA was carried out by real-time RT-PCR using an iCycler iQ with iQ SYBR Green Super mix (Bio-Rad, Hercules, CA) and NF-{kappa}B1 (PPH01812A) and RelA (PPH00204A) primer mixes from SuperArray Bioscience (Frederick, MD).

Preparation of nuclear extract. AGS cells (75–80% confluent) were serum starved for 15–20 h, treated with H. pylori (25 MOI) for 1 h, washed and harvested in ice-cold PBS, and centrifuged at 520 g for 5 min. The cells were resuspended in ice-cold PBS, centrifuged at 12,000 g for 30 s, and resuspended and incubated in buffer A (10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, and 200 µM PMSF) for 10 min at 4°C. The cells were vortexed for 30 s and centrifuged at 12,000 g for 2 min, and the pellet (nuclear fraction) was resuspended in buffer C (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and 700 µM PMSF) and incubated at 4°C for 20 min. After centrifugation at 12,000 g for 2 min, the nuclear extract supernatant was aliquoted and stored at –80°C until used for EMSA. For immunoblot assays, nuclear fractions were boiled in equal volumes of SDS-PAGE sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 5 mM β-mercaptoethanol, and 10% glycerol). Protein contents of nuclear extracts were measured with Bradford reagent.

EMSA. Synthetic oligonucleotides encoding a consensus NF-{kappa}B-binding oligonucleotide (forward, 5'-CGAAGTTGAGGGGACTTTCCCAGGC-3'; reverse, 5'-CGAGCCTGGGAAAGTCCCCTCAACT-3'), and specific HK{alpha} 5'-flanking sequences (Fig. 1A) were obtained from Sigma Genosys (St. Louis, MO). Single-stranded oligonucleotide pairs were dissolved in 1x TE (10 mM Tris pH 8.0 and 1 mM EDTA) and annealed in 1x TE and 50 mM NaCl by being heating at 95°C for 4 min and then slowly cooled to room temperature. Annealed oligonucleotide probes (1 µg) were desalted and then phosphorylated with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) at 37°C for 30 min with [{gamma}32P]ATP (Perkin Elmer, Wellesley, MA). Radiolabeled probes were isolated on a 2% agarose gel and purified using an Ultrafree-DA DNA purification kit (Millipore, Bedford, MA). Remaining impurities in the probe preparations were removed by buffer exchange using Microcon centrifugal filters (Millipore). Probe specific activities (1–2 x 106 cpm/ng) were calculated from {gamma}32P scintillation counting and Quant-It Picogreen dsDNA quantitation (Invitrogen). Nuclear extracts (8 µg protein) were incubated with {gamma}32P-labeled oligonucleotide probes (15,000 cpm) and poly(dI-dC) (0.3 µg) in binding buffer (10 mM Tris, pH 7.9, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM DTT, and 700 µM PMSF) at 4°C for 45 min. For supershift studies, antibodies (1 µg) were incubated with binding reactions overnight at 4°C before addition of {gamma}32P-labeled probes. Binding reaction volumes were adjusted to 25 µl and resolved on 5% native polyacrylamide gels in 0.5x Tris-Borate-EDTA pH 8.0. Gels were dried and exposed for 24 h on phosphoimager screens.


Figure 1
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  		Fig. 1. Human H, K-ATPase {alpha}-subunit (HK{alpha}) promoter. A: nucleotide sequence of 205 bp of 5'-flanking sequence of human HK{alpha} gene. Greyed-out base pairs represent the 2 NF-{kappa}B binding sites detected in this study by DNase I footprinting. Vertical arrows indicate positions of mutations at –159 and –161 (A->C, G->A in forward strand and T->G, C->T in reverse strand). Horizontal arrow indicates the transcription initiation site. Solid horizontal lines depict the sequences of double-stranded synthetic oligonucleotide probes used in EMSA experiments designed to identify functional HK{alpha} promoter NF-{kappa}B binding sequences. B: schematic depiction of 7 progressively truncated human HK{alpha} 5'-flanking sequence deletion constructs. Numbering represents base pair position with respect to transcription initiation site (TIS) shown as bent arrows. HK{alpha}2179 shows 5' terminus of homology domain I at –322 bp. NF-{kappa}B1 and NF-{kappa}B2 represent the first and second NF-{kappa}B binding regions detected in this study by DNaseI footprinting (Fig. 3). Putative AP-1 and Sp1 binding sites detected by DNase I footprinting (data not shown) are also identified.

 
Immunoblotting analysis. Serum-deprived AGS cells (75–80% confluent) were treated with H. pylori (25 MOI) for 1 h, washed and harvested in ice-cold PBS, and centrifuged at 520 g for 5 min. For analysis of whole cell proteins, the cells were sonicated (20 kHz, 60% power, 6 s) in 50 mM Tris, pH 7.4, 1 mM PMSF, and 5 mM EGTA. For analysis of nuclear fractions, the cells were resuspended and incubated in buffer A for 10 min at 4°C. The cells were vortexed for 30 s and centrifuged at 120 g for 2 min, and the pellet (nuclear fraction) was sonicated as described for whole cells. Protein content of cell and nuclear sonicates was measured with Bradford reagent. Whole cell or nuclear fraction sonicates (10–30 µg protein) were boiled in equal volumes of SDS-PAGE sample buffer, resolved in 4–20% Tris-glycine precast gel, and transferred to polyvinylidene difluoride membrane (Millipore). The replicas were incubated for 1 h in 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween-20 (TTBS), washed three times in TTBS, and incubated overnight at 4°C with antibodies against NF-{kappa}B p50, NF-{kappa}B p65, Ser337-phospho NF-{kappa}B p50, lamin A/C (Santa Cruz Biotechnology, Santa Cruz, CA), Ser536-phospho NF-{kappa}B p65 (Cell Signaling Technology, Danvers, MA), or β-actin (Sigma-Aldrich). The replicas were washed three times in TTBS and incubated at room temperature for 1 h with goat anti-mouse or goat anti-rabbit horseradish peroxidasae-conjugated secondary antibodies, and chemiluminescent signals were detected using ECL substrate (Amersham Biosciences, Pittsburgh, PA). Immunoblot experiments were replicated three times, and signal intensities were quantitated using Image J image processing software (http://rsb.info.nih.gov/ij/). Variance in densitometric measurements in replicate experiments was calculated using Student's t-test, and statistical significance was ascribed to P values <0.05.

Small interfering RNA transfection. Small interfering RNAs (siRNAs) against NF-{kappa}B p105 (J-003520-09), NF-{kappa}B p65 (J-003533–08), and nontargeting siRNA (D-001810–03) were acquired from Dharmacon RNA Technology (Lafayette, CO) and were solubilized according to manufacturer's protocol. AGS cells (105, 75–85% confluent) were incubated with Dharmafect transfection reagent 1 (T-2001–07) and siRNA (100 nM) according to the manufacturer's protocol. NF-{kappa}B p105 and p65 mRNA levels were measured by real time RT-PCR, and the protein levels of expressed transcription factors were estimated by immunoblotting of whole AGS cell extracts. To study the effect of siRNA on HK{alpha} transfection at the 48-h time point, AGS cells were transiently transfected with HK{alpha}206 for 24 h. Transfected cells were then treated with or without H. pylori (25 MOI) for 6 h, and the transcriptional activities of the HK{alpha}206 deletion construct were measured as described earlier.

DNAase I footprint assay. A 297-bp footprinting probe was generated by PCR and {gamma}32P-end-labeled primers using the cloned pGL2-basic vector-HK{alpha} 206 deletion construct as substrate. The PCR product included 48 bp of vector sequence distally and 23 bp proximally. The concentration of the purified probe was measured using Quant-It PicoGreen dsDNA reagent. The {gamma}32P-labeled probe (0.075 fmol) and 0.8 µg recombinant human NF-{kappa}B p50 (Promega) were incubated for 30 min at room temperature in 100 µl of NF-{kappa}B binding buffer (10 mM HEPES pH 7.9, 50 mM KCl, 100 mM EDTA, 25 mM DTT, 10% glycerol, and 0.05% NP-40). Three microliters of NF-{kappa}B binding buffer containing 4 mM MgCl2 and 2 mM CaCl2 were added, and incubation was continued for 1 min at 23°C. RQ1 DNAse (2.7 Units; Promega) was added, and incubation was continued for 1 min at 23°C. The digestion reaction was stopped by adding 180 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, and 0.1 mg/ml yeast tRNA). The digested DNA was extracted with a phenol/chloroform mixture, ethanol-precipitated overnight at –80°C, rinsed with chilled 75% ethanol, dried, and dissolved in sequencing gel loading dye (95% formamide, 10 mM EDTA pH 11, and 0.25 mg/ml bromophenol blue). Sequencing reactions for the footprinting probe were carried out with the SequiTherm Excel II sequencing kit (Epicentre) according to the manufacturer's instructions. DNAse I digests and sequencing reactions were electrophoresed on 8% acrylamide, 7 M urea gels in 1x Tris-Borate-EDTA buffer at 60 W for 2 h. The gels were dried and placed on X-ray film overnight at –80°C.

Statistical analysis. Levels of significance of differences between control and treatment groups were assessed by two-way analysis of variance using the Bonferroni posttest method as implemented in the statistical software package GraphPad PRISM version 4. Statistical significance was ascribed to P values <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
H. pylori-sensitive domains are present in the promoter region of HK{alpha}. Having previously reported that H. pylori infection of AGS cells transfected with a 2,179-bp HK{alpha} promoter-reporter construct represses HK{alpha} promoter activity (15), we sought by means of deletion analysis to further localize the H. pylori-sensitive site(s) in the HK{alpha} 5'-flanking sequence. AGS cells were transfected with one of seven progressively truncated HK{alpha} constructs ranging in size from 2,179 to 37 bp fused to luciferase reporter genes (Fig. 1B). The RLU activity of these HK{alpha}-Luc reporter constructs was measured in control and H. pylori-infected AGS cell lysates. As shown in Fig. 2, H. pylori infection of transfected cells significantly repressed the activity of HK{alpha} deletion constructs longer than 102 bp. Deletion constructs smaller than HK{alpha}102 showed no significant H. pylori repression of HK{alpha} activity, indicating that sites conferring H. pylori sensitivity in the HK{alpha} promoter are located 5' to –102 bp.


Figure 2
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  		Fig. 2. Helicobacter pylori-sensitive domains are present in the promoter region of HK{alpha}. Human gastric adenocarcinoma (AGS) cells were independently transfected with HK{alpha} deletion-Luc constructs and then incubated with H. pylori [multiplicity of infection (MOI) = 25] for 6 h. HK{alpha} promoter activity was expressed as normalized relative light units (RLU) of luciferase activity. Open bars: control AGS cells; filled bars: H. pylori-treated AGS cells. Data represent mean ± SD of 3 independent experiments. For HK{alpha} deletion fragments 2,179, 206, 177, and 165, control and H. pylori treatments differed significantly (***P < 0.001). For HK{alpha} deletion fragments 102, 64, and 37, control and H. pylori treatments were not significantly different.

 
NF-{kappa}B p50 binds to two sites on the HK{alpha} promoter sequence. Given the documented role of NF-{kappa}B in H. pylori-mediated upregulation of proinflammatory IL-8 gene expression in gastric epithelial cells, we hypothesized that NF-{kappa}B also plays a role in regulating HK{alpha} gene expression. In silico analysis of human HK{alpha} 5'-flanking sequence using MatInspector v7.4.2 (www.genomatix.de; Ref. 7) revealed the presence of putative NF-{kappa}B binding motifs within homology domain I of the HK{alpha} promoter. This domain extends from –322 bp to the transcription initiation site (35) and encompasses 5'-flanking sequences sensitive to H. pylori as shown in Fig. 1B. To determine whether NF-{kappa}B actually binds to the HK{alpha} promoter sequence, we carried out DNAse I footprinting assays using a 206-bp region of the HK{alpha} 5'-flanking sequence as substrate. As shown in Fig. 3, inclusion of recombinant human NF-{kappa}B p50 subunit in the binding reactions revealed protected and hypersensitive regions extending from –187 to –205 bp and from –117 to –170 bp (lane 6); no such sites were detected when NF-{kappa}B was omitted from the binding reactions (lane 5). We conclude there are two NF-{kappa}B binding sites within the proximal 206 bp of the HK{alpha} 5'-flanking sequence.


Figure 3
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  		Fig. 3. Two NF-{kappa}B p50 binding sites are located within first 206 bp of HK{alpha} promoter sequence. Representative DNA sequencing gel electrophoretogram showing DNase I footprinting of the proximal 206 bp of HK{alpha} 5'-flanking sequence with recombinant human NF-{kappa}B p50 protein. Protected and hypersensitive regions are identified by base pair numbering with respect to transcription start site. Lanes 1–4 are the sequencing lanes; the products of DNase I digestion of HK{alpha} 5'-flanking sequence are shown in the absence (lane 5) and presence (lane 6) of NF-{kappa}B p50 recombinant protein.

 
ERK1/2 signaling pathway contributes to H. pylori-mediated activation of NF-{kappa}B. Having demonstrated that NF-{kappa}B binding sites are present in the HK{alpha} promoter, we next investigated by means of immunoblot assays the activation of NF-{kappa}B subunits in AGS cells in response to H. pylori infection. Total cellular levels of p50 and p65 were unaffected by H. pylori infection (Fig. 4A, right, top, and middle; β-actin loading controls are shown at right, bottom), indicating some degree of constitutive expression of both NF-{kappa}B subunits in our AGS cell model. However, as shown in Fig. 4A, left, top, nuclear fractions of infected AGS cells showed significantly increased levels of phosphorylated p50 (10.4-fold, SD ± 4.6, P < 0.05) compared with uninfected controls. In contrast, levels of phosphorylated p65 were induced only 2.4-fold (SD ± 1.07, P < 0.05; Fig. 4A, left, middle; lamin A/C loading controls are shown are left, bottom). These data indicate that H. pylori induces in AGS cells an asymmetrical activation of NF-{kappa}B subunits, with significantly more marked activation and nuclear localization of the p50 subunit than of the p65 subunit.


Figure 4
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  		Fig. 4. ERK1/2 signaling pathway contributes to H. pylori-mediated activation of NF-{kappa}B in AGS cells. A: AGS cells were incubated with H. pylori (MOI = 25) for 30 min and cellular and nuclear content of NF-{kappa}B p50 and p65 subunits was assayed by immunoblotting using antibodies recognizing phosphorylated and nonphophosphorylated p50 and p65. Left: nuclear content of phosphorylated p50 and p65 in the presence and absence of H. pylori (with lamin A/C as a loading control). Right: total cellular content of p50 and p65 (with β-actin as a loading control; representative data from 3 independent experiments). B: AGS cells were treated with H. pylori (MOI 25) for 1 h and the nuclear extracts were assayed by EMSA and supershift analysis using a consensus NF-{kappa}B binding probe and NF-{kappa}B p50 and p65 specific antibodies. Lane 1, free probe; lanes 2 and 3, DNA:protein interactions in the absence and presence of H. pylori, respectively; lanes 4 and 5, p50 supershifts; lanes 6 and 7, p65 supershifts; lanes 8 and 9, nonimmune rabbit IgG (representative data from 3 independent experiments). C and D: AGS cells were transfected with cis-reporter plasmid pNF-{kappa}B-Luc and then treated with pyrrolidine dithiocarbamate (PDTC; 10 mM) for 1 h (C) or with PD-98059 (50 µM; D), followed by incubation with H. pylori (MOI = 25) for 6 h. E: AGS cells were cotransfected with cis-reporter plasmid pNF-{kappa}B-Luc and MEK kinase expression plasmid (pFC-MEKK) followed by incubation with H. pylori (MOI = 25) for 6 h. HK{alpha} promoter activity was expressed as normalized RLU of luciferase activity (control is the HK{alpha} promoter activity in the absence of H. pylori and pharmacological inhibitors). Data represent mean ± SD from 3 independent experiments (***P < 0.001).

 
We characterized the dimeric forms of NF-{kappa}B mobilized by H. pylori by means of EMSA. Incubation of a radiolabeled consensus NF-{kappa}B-binding probe with control AGS cell nuclear extracts yielded two relatively low-mobility protein:DNA complexes (Fig. 4B, lane 2, long arrows) and a diffusely migrating, relatively high-mobility complex (lane 2, short arrow). Incubation of the probe with H. pylori-infected AGS cell nuclear extracts substantially increased the intensity of the two low-mobility complexes (lane 3). Addition of p50-specific antibody to the binding reactions supershifted both lower-mobility bands (lanes 4 and 5). A p65 antibody supershifted the slower of the lower-mobility bands (lanes 6 and 7, arrowhead). Neither H. pylori nor NF-{kappa}B antibodies affected the mobility or the intensity of the diffusely-migrating, relatively high-mobility complex (short arrow) The specificity of p50 or p65 interaction with the NF-{kappa}B consensus sequence was confirmed by absence of supershifts when specific antibodies were replaced with nonimmune rabbit IgG (lanes 8 and 9). Addition of 200x molar excess of the unlabeled consensus NF-{kappa}B-binding probe to binding reactions eliminated all mobility-shifted bands (data not shown). These EMSA and supershift data indicated that H. pylori infection of AGS cells promotes activation and interaction of NF-{kappa}B p50/p65 heterodimers with consensus NF-{kappa}B-binding DNA sequences.

To confirm that NF-{kappa}B dimers mobilized in AGS cells in response to H. pylori infection are transcriptionally competent, AGS cells were transiently transfected with the inducible reporter plasmid pNF-{kappa}B-Luc containing a luciferase reporter gene driven by a TATA box promoter element and a fivefold repeat of an NF-{kappa}B enhancer element (TGGGGACTTTCCGC). Treatment of transfected AGS cells with H. pylori (25 MOI) led to a 2.2-fold increase in the transcriptional activity of the NF-{kappa}B cis-reporter plasmid (Fig. 4C), and this activity was abrogated by cotreatment of transfected cells with H. pylori and the NF-{kappa}B activation inhibitor pyrrolidine dithiocarbamate (PDTC; 10 mg/ml). Addition of the inhibitor alone had no effect on the constitutive activity of the NF-{kappa}B cis-reporter plasmid.

The role of the ERK1/2 MAPK pathway in the activation of NF-{kappa}B caused by H. pylori infection was studied in AGS cells transfected with an inducible NF-{kappa}B cis-reporter plasmid. The H. pylori-dependent transcriptional activity of the plasmid was measured in the presence of the ERK1/2 pathway inhibitor PD-98059. As shown in Fig. 4D, treatment of transfected AGS cells with 50 µM PD-98059 abrogated the H. pylori-dependent stimulation of the NF-{kappa}B cis-reporter plasmid; PD-98059 alone had no effect on the baseline activity of the transfected promoter sequence. Finally, evidence implicating MEK1/2 in mediation of H. pylori effects on AGS cell NF-{kappa}B activation was acquired from AGS cells cotransfected with a MEKK expression plasmid and the inducible NF-{kappa}B cis-reporter plasmid. Transcriptional activity of the latter plasmid was measured with and without H. pylori (25 MOI) infection. In uninfected AGS cells, the presence of the MEKK expression plasmid conferred 150-fold higher NF-{kappa}B cis-reporter activity (Fig. 4, D and E, open bars), compared with activity when the MEKK expression plasmid was omitted, reflecting the capacity of overexpressed MEKK to phosphorylate MEK. After H. pylori infection, cotransfected AGS cells showed an approximately twofold upregulation of the NF-{kappa}B cis-reporter plasmid compared with uninfected cells (Fig. 4E), confirming that the upstream mediator of H. pylori activation of NF-{kappa}B in AGS cells is the ERK1/2 signaling pathway, secondary to activation of MEK1/2 activity. Taken together, the immunoblot, EMSA, and transfection data indicate that H. pylori infection of AGS cells leads to activation and nuclear localization of transcriptionally competent NF-{kappa}B through MEKK phosphorylation and activation of the ERK1/2 pathway. The activated NF-{kappa}B takes the form of a p50/p65 heterodimer when interacting with the consensus NF-{kappa}B-binding sequence, and this interaction leads to transcriptional regulation of cognate gene products.

H. pylori enhances binding of NF-{kappa}B p50 to a defined region of the HK{alpha} promoter. To establish more precisely the limits of the HK{alpha} promoter NF-{kappa}B binding sites revealed by DNase I footprinting assay (Fig. 3) and to determine whether H. pylori-activated NF-{kappa}B subunits in AGS cells show increased binding to HK{alpha} promoter sequence, we prepared 20 synthetic ~20-bp oligonucleotide duplexes (T1-T20) representing successive 10-bp overlapping domains of the HK{alpha} 5'-flanking sequence from –1 bp to –205 bp (Fig. 1A). Nuclear extracts of H. pylori-infected and uninfected AGS cells were incubated with {gamma}32P-labeled consensus NF-{kappa}B-binding probe together with 200x molar excess of five-member pools of the unlabeled T1-T20 oligonucleotide duplexes, and the binding reactions were analyzed by EMSA. Only one pool of oligonucleotides (T16-T20) significantly attenuated formation of NF-{kappa}B p50 bands (data not shown). {gamma}32P-labeled oligos T16 through T20 were separately incubated with AGS cell nuclear extracts, and subsequent EMSAs showed that only oligonucleotide T18 formed NF-{kappa}B-specific complexes (Fig. 5A, arrows). H. pylori-infected AGS cell nuclear extracts showed significant augmentation of two low-mobility bands compared with uninfected AGS cell nuclear extracts (Fig. 5A, lanes 2 and 3, arrows). Attempts to identify the mobility-shifting protein by incubating the binding reactions with NF-{kappa}B p50-specific antibody yielded suggestive data in the form of a single supershifted band (Fig. 5A, lanes 4 and 5, arrowhead), with concomitant intensity decreases in the two low-mobility bands (Fig. 5A, lanes 4 and 5, arrows). Incubation of binding reactions with nonimmune rabbit IgG did not elicit the suggestive supershifted band (Fig. 5A, lanes 6 and 7). To address the possibility that NF-{kappa}B p50 levels in H. pylori-treated AGS cell nuclear extracts were close to the detection limit of EMSAs, recombinant human (rh) NF-{kappa}B p50 subunit (rh p50) was incubated with radiolabeled T18 or a point-mutated T18 oligonucleotide probe [forward, 5'-GACATGGGGA(–161)GC(–159)TCTGGGCATCTGGGCA-3'; reverse, 5'-TGCCCAGAGCTCCCCATGTC-3'; cf. Fig. 1A], and the resulting DNA:protein interactions were analyzed by EMSA. As shown in Fig. 5B, the T18 probe formed a single band complex on interacting with rh p50 (lane 2, arrow), which was supershifted in its entirety with antibody against p50 (lane 3, arrowhead). Nonimmune rabbit IgG failed to supershift the T18:rh p50 complex (lane 4). In contrast, the point-mutated T18 probe did not form a complex with rh p50 (Fig. 5B, lane 6), indicating that nucleotide residues encompassing –161 and –159 bp of HK{alpha} promoter constitute a core binding site for NF-{kappa}B p50 interaction with HK{alpha}. The NF-{kappa}B2 footprint region was also independently used as a {gamma}32P-labeled synthetic oligonucleotide probe in EMSA supershifts using the same p50-specific antibody (Fig. 5C). No supershifts were observed. Taken together, these data indicate that NF-{kappa}B p50 mobilized by H. pylori infection of AGS cells binds to a 20-bp domain (–171 to –150 bp) of HK{alpha} 5'-flanking sequence (cf. Fig. 1A, oligonucleotide T18).


Figure 5
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  		Fig. 5. H. pylori enhances NF-{kappa}B p50 binding to HK{alpha} promoter. AGS cells were incubated with H. pylori (MOI = 25) for 1 h. Interactions of {gamma}32P-labeled HK{alpha}-specific oligonucleotide probes with AGS cell nuclear extract proteins or with recombinant human NF-{kappa}B p50 subunit were assayed by EMSA and supershift analysis with antibody against NF-{kappa}B p50 subunit. A: nuclear extracts incubated with T18 (–151 to –170 bp) probe; B: recombinant human NF-{kappa}B p50 subunit incubated with T18 probe (lanes 1–4) or double-mutated T18 probe (lanes 5–8); C: nuclear extracts incubated with a probe encompassing the second footprint region (–187 to –205 bp). EMSA data in A and B represent 3 independent experiments; data in C represent 2 independent experiments.

 
HK{alpha} promoter repression by H. pylori is abolished by mutation of the NF-{kappa}B-binding site. To test the hypothesis that H. pylori-induced NF-{kappa}B p50 binding to the HK{alpha} promoter is functionally responsible for HK{alpha} repression, AGS cells were transfected with a wild-type HK{alpha}-Luc deletion construct encompassing the proximal 206 bp of the HK{alpha} promoter including both NF-{kappa}B footprint regions (HK{alpha}206) or with an HK{alpha}206-Luc deletion construct containing nucleotide substitutions at –159 and –161 bp. The AGS cells were then inoculated for 6 h with H. pylori (25 MOI). As shown in Fig. 6, H. pylori inoculation of AGS cells expressing the wild-type HK{alpha}206-Luc deletion construct caused 54% downregulation of HK{alpha} promoter activity compared with uninoculated control cells. In contrast, no significant downregulation of HK{alpha} promoter activity was observed in AGS cells transfected with the point-mutated HK{alpha}206-Luc deletion construct. The abrogation of the H. pylori-induced HK{alpha} promoter repression by point mutation of two nucleotides in a proximal HK{alpha} NF-{kappa}B binding site indicates that binding of NF-{kappa}B p50 to the HK{alpha} promoter at a binding site encompassing nucleotides –161 and –159 bp is a mechanistic necessity for downregulation of HK{alpha} gene expression.


Figure 6
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  		Fig. 6. Mutation of the core NF-{kappa}B binding site of HK{alpha}206 abrogates H. pylori-induced HK{alpha} repression. AGS cells were transfected with an HK{alpha}206-Luc construct or with a double-mutated HK{alpha}206-Luc construct (mutated at –161 and –159 bp) and then incubated with (filled bars) or without (open bars) H. pylori (25 MOI) incubation for 6 h. HK{alpha}206 promoter activities were expressed as normalized RLU of luciferase activity. Data represent means ± SD from 3 independent experiments (***P < 0.001).

 
H. pylori-induced HK{alpha}206 repression is mediated by NF-{kappa}B and is a downstream effect of ERK1/2 signaling pathway activation. If H. pylori inhibits HK{alpha} gene transcription by means of NF-{kappa}B binding to a cognate cis-response element on the HK{alpha} 5'-flanking sequence, then inhibition of NF-{kappa}B activation should abrogate this effect of H. pylori. AGS cells were transiently transfected with wild-type HK{alpha}206-Luc deletion construct, treated with PDTC (10 mg/ml), and inoculated with H. pylori (MOI = 25). As shown in Fig. 7A, the activity of the HK{alpha}206 deletion construct was inhibited 50% by H. pylori, and this inhibition was circumvented by PDTC treatment of the AGS cells before infection. The activity of the construct was unaffected by PDTC alone, indicating that NF-{kappa}B plays no role in the constitutive transcriptional activity of the proximal HK{alpha} promoter. The signaling pathway of H. pylori-induced NF-{kappa}B-mediated HK{alpha} repression was identified by transfecting AGS cells with the same HK{alpha}206-Luc deletion construct, pretreating the cells with specific signaling pathway inhibitors, and measuring HK{alpha}206 activity after H. pylori infection. NF-{kappa}B-mediated inhibition of HK{alpha}206 activity was not circumvented by treatment of the cells with 10 µM SB-203580, a specific inhibitor of the p38 pathway, or with 100 nM JNK inhibitor II, an inhibitor of the JNK pathway. However, treatment of the cells with 50 µM PD-98059, an inhibitor of the ERK1/2 pathway, almost completely prevented inhibition of HK{alpha}206 activity by H. pylori (Fig. 7B). These data indicate that NF-{kappa}B-mediated transcriptional regulation of HK{alpha} promoter activity is dependent on a functional ERK1/2 signaling pathway; neither the p38 nor the JNK signaling pathways are involved in NF-{kappa}B-mediated inhibition of HK{alpha} activity.


Figure 7
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  		Fig. 7. H. pylori-induced HK{alpha}206 repression is mediated by NF-{kappa}B activated through the ERK1/2 signaling pathway. AGS cells were transfected with HK{alpha}206 and incubated with H. pylori (MOI 25) for 6 h after pretreatment with PDTC (10 mg/ml; A) or MAPK inhibitors PD-98059 (50 µM; B), SB-203580 (10 µM), or JNK inhibitor II (100 nM). HK{alpha}206 promoter activity was expressed as normalized RLU of luciferase activity. Data represent means ± SD from 3 independent experiments (**P < 0.01, ***P < 0.001).

 
NF-{kappa}B p50 siRNA derepresses H. pylori-mediated inhibition of HK{alpha}206 transcriptional activation. To gain additional insights into NF-{kappa}B regulation of HK{alpha}, knockdowns of either NF-{kappa}B p50 or p65 subunits were performed using specific siRNAs. AGS cells were transfected with p105 NF-{kappa}B siRNA, and the subsequent levels of cellular p105 mRNA levels were measured by real-time RT-PCR. Figure 8A shows that after 48 h of siRNA transfection, levels of p105 mRNA had fallen by threefold compared with levels in mock-transfected cells or cells transfected with nontargeting siRNA. Concomitant assessment of NF-{kappa}B p50 expression in transfected cells by immunoblotting of whole AGS cell lysates showed virtual elimination of the NF-{kappa}B p50 signal, confirming p105 NF-{kappa}B siRNA attenuation of NF-{kappa}B p50 expression in AGS cells (Fig. 8B). Finally, the siRNA-transfected cells were transfected for 24 h with the HK{alpha}206 deletion construct followed by H. pylori infection. As shown in Fig. 8C, control AGS cells (mock transfected with siRNA, followed by HK{alpha} transfection) showed the usual ~50% inhibition by H. pylori; in contrast, HK{alpha}206 activity in siRNA-transfected cells infected with H. pylori was no different from activity in control cells. Taken together, these data further establish the essential participation of NF-{kappa}B p50 subunit in the mechanism of HK{alpha}206 inhibition secondary to cell infection by H. pylori.


Figure 8
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  		Fig. 8. NF-{kappa}B p50 siRNA de-represses H. pylori-mediated inhibition of HK{alpha}206 activity. A: AGS cells were transfected with NF-{kappa}B p105-specific siRNA for 48 h and the cellular content of p105 mRNA was measured by real-time RT-PCR using β-microglobulin as the normalizing control. Control represents mock-transfected AGS cells, siRNA represents siRNA transfected AGS cells, and scrambled siRNA represents nontargetting siRNA used as siRNA negative control. Control and scrambled siRNA-treated cells were harvested at the same time point as specific siRNA treatment. Data represent means ± SD of 3 independent experiments. B: AGS cells were transfected with NF-{kappa}B p105-specific siRNA and the cellular content of NF-{kappa}B p50 subunit after 72 h was assayed by immunoblotting. β-actin was used as internal control. Control represents the cellular content of NF-{kappa}B p50 (or β-actin) 72 h after mock transfection of AGS cells. siRNA represents cellular content of NF-{kappa}B p50 (or β-actin) 72 h after siRNA transfection of AGS cells. C: AGS cells were transfected with NF-{kappa}B p105-specific siRNA for 48 h, then transfected with HK{alpha}206 for 24 h, and then incubated with H. pylori (MOI 25) for 6 h. HK{alpha} promoter activity was expressed as normalized RLU of luciferase activity. Data represent means ± SD of 3 independent experiments (*P < 0.05, ***P < 0.001).

 
NF-{kappa}B p65 is not involved in H. pylori-induced HK{alpha} promoter repression. Having identified the role of NF-{kappa}B p50 in H. pylori-induced HK{alpha} promoter repression, we examined the role of NF-{kappa}B p65 subunit in HK{alpha} repression. EMSA and supershift analysis with HK{alpha}T18 promoter region and NF-{kappa}B p65-specific antibodies showed no NF-{kappa}B p65 binding to the HK{alpha} promoter in the presence or absence of H. pylori (Fig. 9A, lane 2 vs. 4, and lane 3 vs. 5). To further investigate the role of NF-{kappa}B p65 subunit in HK{alpha} expression, AGS cells were first transfected with NF-{kappa}B p65-specific siRNA and then transfected with HK{alpha}206 followed by H. pylori infection. As shown in Fig. 9B, siRNA-mediated downregulation of NF-{kappa}B p65 mRNA in AGS cells was confirmed by qRT-PCR; transfection of the cells with nontargeting siRNA showed no repression of p65 mRNA. NF-{kappa}B p65 protein levels were also repressed by AGS cell transfection with p65 siRNA (Fig. 9C). In contrast to results obtained with NF-{kappa}B p50 siRNA, NF-{kappa}B p65 siRNA-transfected cells infected with H. pylori did not relieve H. pylori- induced HK{alpha} repression (Fig. 9D). siRNA pretreatment of AGS cells not infected by H. pylori had no significant effect on HK{alpha} promoter activity. Taken together, these results clearly indicate that NF-{kappa}B p65 is not involved in H. pylori-induced HK{alpha} repression. The participation of NF-{kappa}B p50 subunit and the exclusion of NF-{kappa}B p65 subunit lead us to conclude that NF-{kappa}B p50 homodimer binding to HK{alpha} promoter is the mechanistic basis for H. pylori-induced HK{alpha} repression in gastric epithelial cells.


Figure 9
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  		Fig. 9. NF-{kappa}B p65 plays no role in H. pylori-mediated HK{alpha} inhibition. AGS cells were incubated with H. pylori (MOI 25) for 1 h and nuclear extracts were assayed by EMSA and NF-{kappa}B p65 supershift analysis with HK{alpha}-specific oligonucleotide probe T18 (A; –151 to –170 bp). Lane 1, free probe; lanes 2 and 3, DNA:protein interactions in the absence and presence of H. pylori, respectively; lanes 4 and 5, binding reaction supplemented with NF-{kappa}B p65-specific antibody. B: AGS cells were transfected with NF-{kappa}B Rel A (p65)-specific siRNA for 48 h, and the cellular content of p65 mRNA was measured by real-time RT-PCR using β-microglobulin as the normalizing control. Control represents mock-transfected AGS cells, siRNA represents siRNA transfected AGS cells and scrambled siRNA represents nontargetting siRNA used as siRNA negative control. Control and scrambled siRNA-treated cells were harvested at the same time-point as specific siRNA treatment. Data represent mean ± SD of three independent experiments. C: AGS cells were transfected with NF-{kappa}B Rel A (p65)-specific siRNA and the cellular content of NF-{kappa}B p65 subunit after 72 h was assayed by immunoblotting using β-actin as the internal control. Control represents the cellular content of NF-{kappa}B p65 (or β-actin) 72 h after mock transfection of AGS cells. siRNA represents cellular content of NF-{kappa}B p50 (or β-actin) 72 h after siRNA transfection of AGS cells. D: AGS cells were transfected with NF-{kappa}B Rel A (p65)-specific siRNA for 48 h, then transfected with HK{alpha}206 for 24 h, and then incubated with H. pylori (MOI = 25) for 6 h. HK{alpha} promoter activity was expressed as normalized RLU of luciferase activity. Data represent means ± SD of 3 independent experiments (***P < 0.001).

 

   DISCUSSION
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Hypochlorhydria and gastritis are consistent features of acute H. pylori infection of human gastric mucosa. Given that hyposecretion and gastritis initiate a pathological progression that may culminate in gastric adenocarcinoma, understanding their mechanistic basis clarifies disease etiology and illustrates a novel bacterial adaptation to a hostile ecological niche. Self-administration experiments and reports of putative and confirmed H. pylori-induced hypochlorhydric gastritis (28) show that gastric juice pH begins to increase 3 days after infection, becoming neutral ~13 days later. Antral and corpus mucosal inflammation follows the same time course. Subsequently, both gastric pH and corpus inflammation decrease, with pH returning to <2.0 by day 128; antral inflammation persists throughout infection. Transient H. pylori-mediated hypochlorhydria is not caused by parietal cell ablation, because histologically normal parietal cells are abundant in gastric biopsies of hypochlorhydric epidemic gastritis patients, and gastric permeability is normal (43). Also, acute H. pylori infection in Mongolian gerbils, which mimics the pathophysiological course of human H. pylori infection, causes concurrent hypochlorhydria and gastritis with no glandular atrophy (49). Hypochlorhydria has been ascribed to IL-1β, a potent inhibitor of acid secretion (2); gastric mucosal levels of IL-1β are increased in H. pylori-induced gastritis (37), and IL-1β polymorphisms are associated with increased risk of H. pylori-induced hypochlorhydria (10). However, H. pylori suppresses acid secretion in isolated human oxyntic glands (21), and we (45) previously reported that IL-1β does not repress transcription of HK{alpha} promoter constructs transfected into gastric epithelial cells. Thus IL-1β exerts its antisecretory effects at a posttranslational level, most probably through inositol (1,4,5)-triphosphate-dependent increase in intracellular Ca2+ concentration (46) and an alternative mechanism must underlie H. pylori-induced repression of HK{alpha} transcription.

The mechanistic basis for H. pylori modulation of HK{alpha} gene expression at the level of transcription factor interactions with the HK{alpha} promoter has not been previously addressed. We showed earlier that H. pylori inoculation of AGS cells transiently transfected with HK{alpha} promoter-reporter constructs significantly downregulated HK{alpha} transcriptional activity via intracellular pathways involving PKC and protein tyrosine kinase (15). In the present study, we explored the role of NF-{kappa}B in transcriptional regulation of HK{alpha} gene expression in response to H. pylori infection. Our data show that H. pylori-induced, ERK1/2-mediated NF-{kappa}B activation in AGS cells leads to interaction of NF-{kappa}B with at least one of two cis-response elements in the HK{alpha} promoter. Furthermore, the data show that the NF-{kappa}B p50 subunit homodimer is the functionally active form of NF-{kappa}B in this context and that p50 homodimer is necessary and sufficient to repress HK{alpha} gene expression in HK{alpha} deletion-Luc contructs containing NF-{kappa}B binding sites.

These findings are based on several lines of experimental evidence. First, deletion analysis of a 2,179-bp human HK{alpha} 5'-flanking sequence showed that H. pylori infection of transfected AGS cells significantly repressed activity of deletion constructs 165 bp and longer; further deletions of HK{alpha} promoter abrogated H. pylori repression. Second, DNase I footprints of recombinant NF-{kappa}B p50 subunit on a 206-bp length of HK{alpha} promoter showed the presence of two p50 binding sites. Third, H. pylori infection of AGS cells increased nuclear levels of phosphorylated p50 and p65 subunits, both of which formed complexes with a consensus NF-{kappa}B-binding sequence, and the transcriptional competence of which was shown by PDTC-sensitive upregulation of an NF-{kappa}B cis-reporter plasmid. Fourth, PD-98059-mediated abrogation of H. pylori-induced NF-{kappa}B upregulation and repression of HK{alpha} promoter activity confirmed the functional engagement of the ERK1/2 signaling pathway; p38 and JNK pathway inhibitors were without effect. Fifth, a synthetic 20-mer oligonucleotide based on the putative proximal HK{alpha} NF-{kappa}B cis-response sequence bound recombinant human NF-{kappa}B p50 and NF-{kappa}B p50 in nuclear extracts of H. pylori-infected AGS cells; p65 complexes were not detected, and mutation of two nucleotides in the putative core region of this NF-{kappa}B-binding sequence abolished the binding of NF-{kappa}B p50. Sixth, H. pylori failed to repress the transcriptional activity of the HK{alpha}206 promoter point mutated at the same two nucleotides. Finally, H. pylori-mediated inhibition of HK{alpha} activity was prevented by siRNA knockdown of NF-kB p50 but was unaffected by siRNA knockdown of NF-{kappa}B p65.

The differing degrees of H. pylori-mediated repression of our HK{alpha} deletion constructs were consistent with a functional role for NF-{kappa}B in regulation of the HK{alpha} promoter. Both NF-{kappa}B footprint regions (NF-{kappa}B1 and NF-{kappa}B2) were present in HK{alpha}2179 and HK{alpha}206, only one (NF-{kappa}B1) was present in HK{alpha}177, a partially-deleted NF-{kappa}B1 was present in HK{alpha}165, and neither was present in HK{alpha}102 and shorter constructs (Fig. 1B). Deletion constructs with partially or completely deleted NF-{kappa}B1 showed either minimal (HK{alpha}165) H. pylori repression or none at all (HK{alpha}102, HK{alpha}64, and HK{alpha}37). The comparable, maximal H. pylori repression of HK{alpha}206 and HK{alpha}177, both containing NF-{kappa}B1, implicates the NF-{kappa}B1 binding site as a primary regulator of HK{alpha} gene repression, rather than NF-{kappa}B2. The less-than-maximal H. pylori repression of the full-length promoter (HK{alpha}2179) indicates that other regulatory elements may have roles in H. pylori-mediated HK{alpha} gene repression.

Our immunoblot and EMSA data showed that NF-{kappa}B p50 and p65 subunits were differentially activated by H. pylori infection of AGS cells, both were translocated to the nucleus, and both cooperated in binding to a consensus NF-{kappa}B-binding probe. However, when this consensus binding sequence was replaced with a putative HK{alpha}-specific NF-{kappa}B-binding sequence identified by footprint analysis, only NF-{kappa}B p50 subunits and not p65 subunits were found to bind. This result, and the complementary siRNA data confirming that this binding causes inhibition of HK{alpha} activity, contrasts with reported involvement of NF-{kappa}B in repression of the closely related HK{alpha}2 gene found in colonic and renal epithelia (57). In that study, supershift analyses of NF-{kappa}B interactions with HK{alpha}2 promoter-specific putative NF-{kappa}B-binding sequence were interpreted in terms of p50/p65 heterodimer, not p50/p50 homodimer, interaction with the sequence causing inhibition of gene expression. Clearly the specific promoter context plays an important role in defining relative affinities of NF-{kappa}B heterodimers and homodimers for cis-response elements. Also, the endogenous expression of HK{alpha}2 in immortalized inner medullary collecting duct cells involves the participation in cis-regulatory events of a spectrum of factors the number and composition of which must differ significantly in the transiently transfected AGS cells used in this study.

As noted above, we have previously shown that H. pylori-induced inhibition of HK{alpha} transcription in AGS cells is not mediated by IL-1β. (45). Specifically, we reported that IL-1β upregulates the activity of HK{alpha} deletion constructs incorporating sequence that we identify in the present study as an NF-{kappa}B cis-response element. The deletion constructs in question also incorporate putative binding site(s) for Sp1 transcription factor. We propose that IL-1β-mediated HK{alpha} transcriptional activation is effected at least in part by Sp1 binding to cognate response element(s) in these deletion constructs, whereas H. pylori-mediated HK{alpha} transcriptional repression is effected by p50/p50 NF-{kappa}B homodimers, as we report in this study. This reconciliation of diametrically opposed outcomes of transcription factor binding to the same HK{alpha} deletion constructs is supported by recent data, based on AGS cell nuclear extract EMSA supershift experiments, that IL-1β treatment of AGS cells significantly increases Sp1 binding to a proximal HK{alpha} promoter probe but H. pylori inoculation does not (unpublished observations).

The repressor activity of NF-{kappa}B p50 homodimers described in this study, attributable to the absence of strong transactivation domains which are found in p65, RelB, and c-Rel, has been noted previously in other contexts. Thus macrophages secrete an autocrine factor which decreases TNF{alpha} expression by inducing p50 binding to the TNF{alpha} promoter (1), and in metastatic tumor cells, NF-{kappa}B p50 homodimers repress H-2Kb gene expression (42). The mechanism by which p50 homodimers repress gene expression may require p50 interaction with histone deacetylase-1 (HDAC-1) and subsequent binding of the complex to DNA resulting in gene inactivation (58). In this paradigm, the repressive complex may be displaced from DNA by p50/p65 heterodimers containing phosphorylated NF-{kappa}B p65, with consequent gene activation. In the present study, nonphosphorylated and phosphorylated p65 and p50 subunits in AGS cells, the levels of which were modulated by H. pylori, interacted as p50/p65 heterodimers with consensus NF-{kappa}B DNA-binding sequence or as p50/p50 homodimers with HK{alpha}-specific sequence. Clearly, the latter sequence displays a higher affinity for the p50 homodimer than the p50/p65 heterodimer, resulting in HK{alpha} repression. The probability that AGS cell NF-{kappa}B complexes are associated with corepressor HDACs may introduce a further level of transcriptional control, depending on stimulus-specific association of the complexes with different HDACs, and the relative concentrations of the resulting NF-{kappa}B complexes.

H. pylori mobilizes NF-{kappa}B by at least three distinct mechanisms: T4SS-mediated transfer of CagA into host epithelial cells leading to NF-kB activation via ERK1/2 dependent protein kinase pathways (4); T4SS-mediated delivery of peptidoglycan into host cells, stimulation of the intracellular receptor Nod1, and direct activation of NF-{kappa}B (53); and stimulation of host cell Toll-like receptors (TLR2 and TLR5) by H. pylori lipopolysaccharide and/or flagellins (48), leading to activation of NF-{kappa}B-inducing kinase, which interacts with TRAF2 and TRAF6 to activate IKK{alpha} and IKKβ (27). MAP kinases have been implicated in H. pylori-mediated, NF-{kappa}B-upregulated IL-8 production (24, 38), and nonphosphorylated CagA is known to activate the Ras ERK pathway (18). Our finding that the ERK1/2 signaling pathway participates in H. pylori-mediated HK{alpha} gene repression is consistent with CagA-dependent activation of NF-{kappa}B. We have found that certain H. pylori isogenic mutants deficient in genes encoding T4SS structural elements fail to repress HK{alpha} gene expression, whereas TLR2/TLR5-related and peptidoglycan-related isogenic mutants do not (unpublished observations), suggesting that CagA-mediated activation of NF-{kappa}B underlies HK{alpha} repression by H. pylori.

Based as they are on acute inoculation of gastric epithelial cells with H. pylori, our findings in this study relate to the transient hypochlorhydria that accompanies human gastric infection by H. pylori, and it is only in the context of facilitating H. pylori colonization of the gastric corpus that this transient hypochlorhydria predisposes the mucosa to later development of atrophy and possible eventual progression to neoplastic lesions. The transitory hypochlorhydria induced by H. pylori may represent an important adaptive response facilitating colonization of the otherwise hostile gastric mucosal environment. From a pharmacological perspective, the acid inhibitory mechanism utilized by the bacterium is also of interest, focusing not on classical posttranslational pathways (as exemplified by H2-receptor antagonists or omeprazole proton pump inhibition) but on targeted HK{alpha} transcriptional repression.

In summary, this study has demonstrated that gastric epithelial cell H. pylori-mediated repression of human gastric HK{alpha} gene expression is effected by ERK1/2-mediated NF-{kappa}B activation and nuclear localization, where homodimeric NF-{kappa}B p50 binding to a defined cis-response element between –150 and –170 bp in the HK{alpha} promoter is a necessary and sufficient condition for downregulation of promoter activity.


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 ABSTRACT
 MATERIALS AND METHODS
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 GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-064371 (to A. J. Smolka).


   ACKNOWLEDGMENTS
 
We thank Y. Hannun, L. Luttrell (Medical University of South Carolina), and A. Chaudhuri (Genentech, South San Francisco, CA) for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Smolka, Medical Univ. of South Carolina, CSB921E, 96 Jonathan Lucas St., Charleston, SC 29425 (e-mail: smolkaaj{at}musc.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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A. Saha, C. E. Hammond, M. Gooz, and A. J. Smolka
The role of Sp1 in IL-1{beta} and H. pylori-mediated regulation of H,K-ATPase gene transcription
Am J Physiol Gastrointest Liver Physiol, November 1, 2008; 295(5): G977 - G986.
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