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Am J Physiol Gastrointest Liver Physiol 295: G977-G986, 2008. First published September 4, 2008; doi:10.1152/ajpgi.90338.2008
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MUCOSAL BIOLOGY

The role of Sp1 in IL-1β and H. pylori-mediated regulation of H,K-ATPase gene transcription

Arindam Saha, Charles E. Hammond, Monika Gooz, and Adam J. Smolka

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

Submitted 15 May 2008 ; accepted in final form 30 August 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Helicobacter pylori infection of the gastric body induces transient hypochlorhydria and contributes to mucosal progression toward gastric carcinoma. Acid secretion is mediated by parietal cell H,K-ATPase, in which the catalytic {alpha}-subunit (HK{alpha}) promoter activity in transfected gastric epithelial [gastric adenocarcinoma (AGS)] cells is repressed by H. pylori through NF-{kappa}B p50 homodimer binding to the promoter. IL-1β, an acid secretory inhibitor whose mucosal level is increased by H. pylori, upregulates HK{alpha} promoter activity in AGS cells. Because IL-1β also activates NF-{kappa}B signaling, we investigated disparate HK{alpha} regulation by H. pylori and IL-1β, testing the hypothesis that IL-1β-induced HK{alpha} promoter activation is mediated by the transcription factor Sp1. DNase I footprinting revealed Sp1 binding to the HK{alpha} promoter at –56 to –39 bp. IL-1β stimulated the activity of three HK{alpha} promoter constructs containing NF-{kappa}B and Sp1 sites transfected into AGS cells and also stimulated a construct containing only an Sp1 site. This stimulation was abrogated by mutating the HK{alpha} promoter Sp1 binding site. Gelshift assays showed that IL-1β increased Sp1 but not p50 binding to cognate HK{alpha} probes and that Sp1 also interacts with an HK{alpha} NF-{kappa}B site when bound to its cognate HK{alpha} cis-response element. H. pylori did not augment Sp1 binding to an HK{alpha} Sp1 probe, and small interfering RNA-mediated knockdown of Sp1 expression abrogated IL-1β-induced HK{alpha} promoter stimulation. We conclude that IL-1β upregulates HK{alpha} gene transcription by inducing Sp1 binding to HK{alpha} Sp1 and NF-{kappa}B sites and that the H. pylori perturbation of HK{alpha} gene expression is independent of Sp1-mediated basal HK{alpha} transcription.

adenosine 5'-triphosphatase; Helicobacter pylori; interleukin-1β

THE GRAM-NEGATIVE MICROAEROPHILIC bacterium Helicobacter pylori is a class I carcinogen that colonizes the human gastric epithelium, causing gastritis and perturbing acid secretion and potentially triggering epithelial progression to carcinoma. In particular, H. pylori gastritis of the body of the stomach is strongly correlated with reduced acid secretion (10, 12). Both this hypochlorhydria and gastritis are associated with increased gastric epithelial cell proliferation, which in a significant subset (~2%) of infected individuals may progress to distal gastric adenocarcinoma. The importance of hypochlorhydria in establishing gastric mucosal susceptibility to carcinoma was emphasized in a recent study of gastrin-deficient mice (38). In the absence of the potent acid secretagogue gastrin, G–/– mice were rendered hypochlorhydric, and their stomachs became chronically inflamed, progressing to atrophy of the fundic mucosa, intestinal metaplasia, dysplasia, and eventually antral carcinoma (38). This recapitulation of the phenotypic progression to gastric cancer (7), triggered not by H. pylori but by perturbation of gastric pH, provides the rationale for investigating the mechanisms whereby H. pylori inhibits acid secretion, thereby setting the stage for the pathological sequelae of infection.

Gastric acid secretion is mediated by K+-stimulated, proton-translocating H,K-ATPase, an integral heterodimeric protein of the apical membrane of gastric parietal cells. The enzyme consists of catalytic {alpha}-subunits (HK{alpha}; Mr, 94,000) and regulatory β-subunits (HKβ; Mr, 60,000–80,000), which acquire H+/K+ counter-transport activity when transferred from parietal cell tubulovesicular membranes to secretory canalicular membranes upon stimulation of acid secretion. HK{alpha} transcription is positively regulated by the gastric acid secretagogues histamine, gastrin, and acetyl choline (5). A number of transcription factors associated with HK{alpha} promoter activity have been identified. In rat gastric parietal cells, the zinc finger proteins GATA-GT1 and GATA-GT2 colocalize with HK{alpha} mRNA, suggesting a role for these transcription factors in the regulation of proton pump gene expression (21). In canine parietal cells, constitutive HK{alpha} transcriptional activity is regulated by Sp1 binding to the HK{alpha} promoter –54 to –45 bp upstream of the transcription initiation site (20), whereas an EGF-responsive factor binds to the promoter between –156 and –162 bp (16). More recently, our laboratory has shown that binding of an NF-{kappa}B p50 subunit homodimer to the –150 to –170 bp region of the human HK{alpha} promoter inhibits HK{alpha} transcriptional activity (25).

Studies of the H. pylori perturbation of host cell signaling mechanisms have revealed several pathways by which NF-{kappa}B may be activated. H. pylori strains with a cag pathogenicity island encoding a type IV secretion system (T4SS) inject cytotoxin-associated gene (CagA) oncoprotein into host cells, activating Ras-mediated mitogen-activated protein kinase (MAPK) signaling and the activation of the transcription factor NF-{kappa}B. Consequent upregulation of the interleukin (IL)-8 gene and increased epithelial cell secretion of chemotactic IL-8 recruits activated neutrophils and monocytes into the lamina propria where they secrete the proinflammatory cytokines IL-1β and TNF-{alpha}. In addition, T4SS-mediated delivery of peptidoglycan into host gastric adenocarcinoma (AGS) cells stimulates the intracellular receptor Nod1, leading to the direct activation of NF-{kappa}B (33). H. pylori lipopolysaccharide interaction with gastric epithelial cell Toll-like receptors 2 and 5 also culminates in NF-{kappa}B activation (29). In AGS cells, H. pylori-induced ERK1/2 signaling cascade activates NF-{kappa}B and also activates Sp1, leading to the activation of angiogenic growth factor VEGF-A (30).

IL-1β is an important factor in H. pylori-induced mucosal inflammation and perturbation of acid secretion. H. pylori-positive peptic ulcer disease patients showed a marked increase in IL-1β mRNA in fundic mucosa and increased gastric pH, which reverted to normal levels after H. pylori eradication (22, 35, 36). CagA-positive H. pylori strains are associated with higher mucosal IL-1β levels than CagA-negative strains (2). Both IL-1β and TNF-{alpha} activate NF-{kappa}B, leading to the upregulation of IL-8 and cyclooxygenase-2, and in the presence of H. pylori, certain IL-1β gene polymorphisms are associated with increased risk of gastric adenocarcinoma (11). IL-1β is also a potent inhibitor of gastric acid secretion (34) and thus may contribute to the transient hypochlorhydria associated with acute H. pylori infection. In some cell culture models, IL-1β inhibits activation of Sp1, leading to repression of promoter activities (4, 23), but in AGS cells IL-1β activates Sp1 phosphorylation through ERK1/2 pathways (9). We recently reported that in AGS cells IL-1β activates, rather than represses, phorbol ester-induced transcriptional activity of several human HK{alpha} promoter-reporter constructs through ERK1/2 signaling (24).

In the present study, we sought to clarify the mechanistic details of IL-1β-induced effects on HK{alpha} gene transcription. Although H. pylori and IL-1β are both inflammatory mediators and known activators of the NF-{kappa}B signaling cascade, H. pylori inoculation of AGS cells represses transfected HK{alpha} promoter activity (13, 25), whereas IL-1β activates HK{alpha} transcription (24). We resolved this apparent anomaly by focusing our investigation on Sp1 regulation of the transcriptional activity of the HK{alpha} promoter. Our data indicate that the IL-1β enhancement of HK{alpha} transcription is mediated by Sp1 transactivation of the HK{alpha} Sp1 cis-response element and that a proximal NF-{kappa}B site in the HK{alpha} promoter interacts with Sp1 and other nuclear factors, leading to enhanced HK{alpha} transcription.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells, bacteria, and reagents. Human AGS (CRL1739) and wild-type H. pylori (strain ATCC 49503) were from American Type Culture Collection (Manassas, VA). AGS cells were grown for ≤10 passages in Ham's F-12 containing L-glutamine (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (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, Detroit, MI) plates containing 10% FBS and 2.4% agar (Brucella-agar plates) at 37°C using a microaerophilic gas pack system (BD Biosciences, Sparks, MD). Cultures were routinely urease tested during subculturing, and only positive cultures were used for cell infection. For AGS cell infection, H. pylori were harvested after 24 h of culture. Bacteria were resuspended in Ham's F-12 medium containing FBS and enumerated by absorbance at 600 nm (1 optical density at 600 nm = 2.4 x 108 bacteria/ml). Multiplicities of infection (MOI) were calculated based on AGS cell and bacterial cell counts. IL-1β was purchased from Chemicon (Billerica, MA). Restriction enzymes, the pGL2-basic vector transfection plasmid, 5x passive lysis buffer, and the luciferase assay substrate were obtained from Promega (Madison, WI). 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 HK{alpha} 5'-flanking region was provided by Dr. Gary 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 previously described (13). 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 previously described (24). An HK{alpha}206 construct containing a two-nucleotide mutation in the NF-{kappa}B site (HK{alpha}206{Delta}NF-{kappa}B1) was generated as described (25). An HK{alpha}206 construct containing a six-nucleotide mutation in the Sp1 site (HK{alpha}206{Delta}Sp1) was generated using QuikChange Lightning Site-Directed Mutagenesis kit (Stratagene), following the manufacturer's protocol.

Transient transfection. AGS cells (1.25 x 105 cells/well) were cultured overnight in 24-well cell culture plates, washed with phosphate-buffered saline (PBS), and then transfected with HK{alpha} promoter-Luc reporter constructs as described previously (24). After 24 h, the cells were incubated for a further 24 h with IL-1β (10 ng/ml) or H. pylori (24 h culture; MOI = 25) or F-12 medium alone (vehicle control) and then lysed for measurement of reporter activities. Luciferase relative light units were normalized to cotransfected pMaxGFP fluorescence and corrected by subtracting corresponding normalized promoterless pGL2-basic vector relative light units data. Data points are shown as means ± SD of three independent transfection experiments with each deletion construct.

Immunoblotting analysis. Serum-deprived AGS cells (75–80% confluent) were preincubated as required with the ERK1/2 inhibitor PD-98059 (1 h) and then treated with IL-1β (10 ng/ml) for 4 h. The cells were washed and harvested in ice-cold PBS and centrifuged at 520 g for 5 min. The PBS was discarded, and the cells were dissolved in 1x Triton lysis buffer (Boston Bioproducts, Worcester, MA) containing 1x protease inhibitor cocktail (P8340; Sigma, St. Louis, MO) and 1x Halt phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL). The cells were lysed in a rotating shaker at 4°C for 15 min. Cell lysates (30–40 µg protein as measured with Bradford reagent) 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, Bedford, MA). The replicas were incubated for 1 h in 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween 20 (TTBS), washed 3x in TTBS, and incubated overnight at 4°C with antibodies against Sp1, NF-{kappa}B p50, NF-{kappa}B p65, Ser337-phospho NF-{kappa}B p50 (Santa Cruz Biotechnology, Santa Cruz, CA), p44/42 MAP kinase, Thr202, and Tyr204-phospho p44/42-MAP kinase, Ser536-phospho NF-{kappa}B p65 (Cell Signaling Technology, Danvers, MA), Thr453-phospho Sp1 (Abcam, Cambridge, MA), or β-actin (A5441; Sigma). The replicas were washed 3x in TTBS and incubated at room temperature for 1 h with goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies, and chemiluminescent signals were detected using enhanced chemiluminescence substrate (Amersham Biosciences, Pittsburgh, PA).

DNAase I footprint assay. A 297-bp footprint probe was generated by PCR using 32P-end-labeled primers and 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 32P-labeled probe (2.8 fmol in 13 µl H2O) was combined with 25 µl binding buffer, containing (in mM) 50 Tris-HCl (pH 8.0), 100 KCl, 12.5 MgCl2, 1 EDTA, and 1 DTT and 20% glycerol, and with 12 µl Sp1 dilution buffer, containing 12 mM HEPES (pH 7.5), 50 mM KCl, 6 mM MgCl2, 5 µM ZnSO4, 2 mM DTT, 0.1% Nonidet P-40, and 50% glycerol, containing 1.92 µg recombinant human Sp1 (Promega). After 10 min incubation on ice, 50 µl Ca2+/Mg2+ solution containing (in mM) 5 CaCl2 and 10 MgCl2 was added, and incubation continued at 23°C for 1 min. RQ1 DNase (0.29 units; Promega) diluted in 3 µl Sp1 dilution buffer was added, and incubation was continued at 23°C for 1 min. The reaction was stopped by the addition of 90 µl stop solution containing 200 mM NaCl, 20 mM EDTA, 1% SDS, and 100 µg/ml yeast tRNA. Reactions were extracted with 200 µl phenol-chloroform-isoamyl alcohol (25:24:1) equilibrated with 10 mM Tris (pH 8.0), 1 mM EDTA, and 500 mM NaCl. DNA was ethanol precipitated from the aqueous phase, rinsed with 70% ethanol, and dissolved in sequencing gel-loading dye of 95% formamide, 10 mM EDTA (pH 11.0), and 0.25 mg/ml bromophenol blue. Sequencing reactions for the footprint probe were carried out using a SequiTherm Excel II sequencing kit (Epicentre Biotechnologies, Madison, WI) 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 (TBE) buffer at 60 W for 2 h. The gels were dried and placed on X-ray film overnight at –80°C.

Electrophoretic mobility shift assay. AGS cells (75–80% confluent) were serum deprived for 15–20 h, treated with IL-1β for 1–24 h, washed, and harvested in ice-cold PBS, and nuclear extract was prepared as described previously (25). Synthetic oligonucleotides (Table 1) were obtained from Sigma Genosys (St. Louis, MO). Single-stranded oligonucleotide pairs were dissolved in 1x Tris-EDTA buffer containing (in mM) 10 Tris (pH 8.0) and 1 EDTA and annealed in 1x Tris-EDTA buffer, 50 mM NaCl, by 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 45 min with {gamma}-32PATP (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 counts per minute (cpm)/ng] were calculated from 32P scintillation counting and Quant-It Picogreen dsDNA quantitation (Invitrogen). Nuclear extracts (8 µg protein) were incubated with 32P-labeled oligonucleotide probes (15,000 cpm) and poly(dI-dC) (0.3 µg) in binding buffer containing 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 for 45 min before the addition of radioactive probes. Binding reaction volumes were adjusted to 25 µl, resolved on 5% native polyacrylamide gels in 0.5x TBE (pH 8.0). Gels were dried and exposed for 24 h on phosphoimager screens.


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  		Table 1. Synthetic oligonucleotide sequences of electrophoretic mobility shift assay probes

 
Small interfering RNA transfection. Four different small interfering RNAs (siRNAs) against human Sp1 (J-026959-05-08), and nontargeting siRNA (D-001810-03) were acquired from Dharmacon RNA Technology (Lafayette, CO). AGS cells (105; 75–85% confluent) were incubated with Dharmafect transfection reagent 1 (T-2001-07), and either pooled or individual siRNAs (100 nM) were solubilized according to the manufacturer's protocol. Sp1 mRNA content of the cells was measured by real time RT-PCR using β2-microglobin as the internal control, and the protein levels of expressed Sp1 were estimated by the immunoblotting of whole AGS cell extracts using β-actin as the internal control. To study the effect of siRNA on HK{alpha} promoter activity, AGS cells were transiently transfected with HK{alpha}206 for 24 h. Transfected cells were then treated with or without IL-1β for 24 h, and the transcriptional activities of the HK{alpha}206 deletion construct were measured as described earlier.

Real time RT-PCR. AGS cells were grown to 75–80% confluence in 24-well cell culture plates and then serum deprived for 15–20 h. Total RNA was isolated using STAT-60 reagent (Tel Test, Friendswood, TX) and reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. Measurement of AGS cell Sp1 mRNA was carried out by real-time RT-PCR using an iCycler iQ with iQ SYBR Green Supermix (Bio-Rad) and Sp1 primer mix (PPH01482A) from SuperArray Bioscience (Frederick, MD). The primer sequences for β2-microglobin used as internal control were as follows: 5'-AGATGAGTATGCCTGCCGTGTG-3' (forward) and 5'-TCAAACCTCCATGATGCTGCTTAC-3' (reverse).

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


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Basal HK{alpha} promoter activity is increased by IL-1β and repressed by H. pylori. We have previously reported that IL-1β upregulates the phorbol ester-induced activity of HK{alpha} promoter-reporter constructs that are otherwise repressed by H. pylori (24). To determine the effect of IL-1β on constitutive HK{alpha} promoter activity, AGS cells were independently transiently transfected with a panel of seven HK{alpha} promoter-Luc reporter deletion constructs, which also shows the relative positions of Sp1 and NF-{kappa}B binding sites on the HK{alpha} promoter (Fig. 1A). The cells were treated for 24 h with IL-1β (10 ng/ml), H. pylori (MOI = 25), or medium alone (vehicle control), and the resulting IL-1β-dependent, H. pylori-dependent, and basal (no treatment) HK{alpha} activities were measured by luminometry and normalized to the fluorometric expression levels of cotransfected green fluorescent protein reporter plasmids. As shown in Fig. 1B, the transcriptional activity of the longest HK{alpha} promoter-reporter construct (HK{alpha}2179) was increased ~40% by IL-1β and decreased ~60% by H. pylori. IL-1β significantly increased the activity of deletion constructs HK{alpha}206, HK{alpha}177, HK{alpha}165, and HK{alpha}102 compared with HK{alpha}2179, whereas deletion constructs smaller than HK{alpha}102 showed no significant activation by IL-1β. In contrast, H. pylori significantly repressed the transcriptional activity of all HK{alpha} deletion constructs except HK{alpha}102, HK{alpha}58, and HK{alpha}37. The data indicate that cis-response elements in HK{alpha} 5'-flanking regions downstream of –206 bp are responsible for both the IL-1β-induced activation and H. pylori-induced repression of the promoter. Interestingly, deletion of the proximal NF-{kappa}B1 site caused a 36% decrease in IL-1β-stimulated activity and completely abrogated H. pylori repression of HK{alpha} activity without significantly affecting basal HK{alpha} activity (compare HK{alpha}177 and HK{alpha}102 activities in Fig. 1B). These data suggest that the NF-{kappa}B1 and Sp1 binding sites contribute to both basal and IL-1β-stimulated HK{alpha} transcriptional activity. The persistence of IL-1β-stimulated HK{alpha} activity after deletion of the NF-{kappa}B1 binding site (HK{alpha}102) suggests the involvement of an Sp1 binding site; however, deletion of 44 bp upstream of the Sp1 site (HK{alpha}58) abrogated IL-1β stimulation, suggesting that additional sequence-dependent interactions participate in IL-1β stimulation. The significantly increased basal and IL-1β-dependent activities of HK{alpha}206 compared with HK{alpha}2179 are also consistent with the presence of hitherto unidentified repressor sites upstream of –205 bp in the HK{alpha} promoter.


Figure 1
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  		Fig. 1. A: schematic depiction of 7 progressively truncated human catalytic {alpha}-subunit of H,K-ATPase (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, NF-{kappa}B2, and Sp1 represent the 2 NF-{kappa}B and 1 Sp1 binding regions in HK{alpha} promoter. B: HK{alpha} promoter activity is increased by IL-1β and decreased by Helicobacter pylori. Human gastric adenocarcinoma (AGS) cells were independently transfected with HK{alpha} deletion-Luc constructs followed by incubation with H. pylori [25 multiplicity of infection (MOI); 6 h] or IL-1β (10 ng/ml; 24 h). HK{alpha} promoter activity was expressed in terms of relative luciferase activity. White bar, control AGS cells; light gray bar, IL-1β-treated AGS cells; dark gray bar, H. pylori-treated AGS cells. Data are shown as means ± SD of 3 separate experiments. For HK{alpha}206, HK{alpha}177, HK{alpha}165, and HK{alpha}102 deletion constructs, IL-1β-induced promoter activity differed significantly from control (P < 0.001). For shorter HK{alpha}58 and HK{alpha}37 deletion constructs, IL-1β-induced and control promoter activity did not differ significantly. For HK{alpha}2179, HK{alpha}206, HK{alpha}177, and HK{alpha}165 deletion constructs, H. pylori-induced promoter activity differed significantly from control (P < 0.001). For HK{alpha}102, HK{alpha}58, and HK{alpha}37 deletion constructs, IL-1β-induced and basal promoter activity did not differ significantly.

 
IL-1β activates both NF-{kappa}B and Sp1 through ERK1/2 signaling. Our laboratory has previously shown that H. pylori upregulation of NF-{kappa}B is mediated by the MAPK ERK 1/2 (25). To identify signaling pathways and transcription factors putatively involved in the IL-1β upregulation of proximal HK{alpha} promoter deletion constructs, AGS cells were treated with IL-1β (10 ng/ml; 4 h) in the presence or absence of the ERK1/2 inhibitor PD-98059 (50 µM; added 1 h before IL-1β treatment). The cellular content of phosphorylated and total ERK1/2, NF-{kappa}B p50, and Sp1 was determined by immunoblot analysis of the cell lysates. As shown in Fig. 2, IL-1β upregulates the phosphorylation of ERK1/2, Sp1, and NF-{kappa}B p50, while their total amounts remain constant. The preincubation of cells with PD-98059 abrogated IL-1β-mediated increases in ERK1/2, Sp1, and NF-{kappa}B p50 phosphorylation. The data indicated that ERK1/2 signaling to both NF-{kappa}B and Sp1 provides a basis for IL-1β perturbation of basal HK{alpha} promoter activity mechanistically comparable with H. pylori-induced NF-{kappa}B p50 homodimer repression of the HK{alpha} promoter (25). In addition, the data also indicate that IL-1β may induce changes in the transactivating capacity of Sp1 and NF-{kappa}B p50 subunits by regulating their posttranslational phosphorylation status.


Figure 2
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  		Fig. 2. IL-1β activates both Sp1 and NF-{kappa}B in AGS cells. AGS cells were treated with IL-1β (10 ng/ml) for 4 h, and cellular content of ERK1/2, Sp1, and NF-{kappa}B p50 subunits was assayed by immunoblotting using antibodies recognizing phosphorylated (p) and nonphophosphorylated forms of ERK1/2, Sp1, and NF-{kappa}B p50 subunit. Left: content of p-ERK1/2, Sp1, and NF-{kappa}B p50 in untreated, IL-1β-treated, and PD-98059 + IL-1β-treated AGS cells. Right: cellular content of total ERK1/2, Sp1, and NF-{kappa}B p50 subunits, acting as normalization control for left. Representative data are from 3 independent sets of experiments. Con, control.

 
Sp1 binding region is located upstream of TATA box in HK{alpha} promoter. Given that IL-1β activated Sp1 in AGS cells, we sought to identify HK{alpha} promoter nucleotides serving as an Sp1 binding site. In silico analysis (www.genomatrix.de/online_help/help_matinspector/matinspector_help.html) identified several putative Sp1 binding regions within a 200-bp domain 5' of the HK{alpha} transcription initiation site (analysis not shown). As shown in Fig. 3, DNase I footprint analysis of a 218-bp HK{alpha} promoter substrate incubated with recombinant human Sp1 protein revealed single complementary footprints on both the forward and reverse strands of the substrate DNA (compare lanes 1 and 2 for the forward strand footprint and lanes 3 and 4 for the reverse strand footprint). Comparison of these footprints with the sequencing lanes (G, A, T, and C) defined –56 bp to –39 bp on the HK{alpha} promoter as the Sp1 binding site.


Figure 3
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  		Fig. 3. Sp1 binding region is located upstream of TATA box region in HK{alpha} promoter. Representative DNA sequencing gel electropherogram showing DNase I footprinting of the HK{alpha} 5'-flanking sequence with recombinant human Sp1 protein is shown. Protected and hypersensitive regions are identified by base pair numbering with respect to transcription initiation site. DNase I digestion products in the absence of recombinant Sp1 are shown in lanes 1 and 3, and digestion products in the presence of recombinant Sp1 are shown in lanes 2 and 4. Lanes G, A, T, and C represent the sequencing lanes.

 
Binding of Sp1 to the HK{alpha} promoter is increased by IL-1β and not H. pylori. Although IL-1β clearly increases the level of phosphorylated Sp1 and phosphorylated NF-{kappa}B subunits in AGS cells as shown in Fig. 2, we were particularly interested in the IL-1β modulation of Sp1 and NF-{kappa}B binding to HK{alpha} cis-reponse elements. We have previously reported that NF-{kappa}B p50 homodimer binding to the HK{alpha} promoter downregulates HK{alpha} transcriptional activity (25) and that IL-1β upregulates HK{alpha} activity (24). To delineate more precisely IL-1β-induced effects of NF-{kappa}B and Sp1 binding to HK{alpha} promoter, we turned to EMSA using nuclear extracts of AGS cells treated from 1 to 24 h with or without IL-1β (10 ng/ml) and 32P-labeled HK{alpha} promoter sequences as DNA probes [T18 for NF-{kappa}B1 (25) and the Sp1 footprint region defined in Fig. 3 for Sp1]. When incubated with the Sp1 probe, AGS cell nuclear extracts treated with IL-1β for 4 h showed the enhancement of three DNA-protein complexes (Fig. 4A; compare lanes 2 and 3, long and short arrows). The slowest migrating band (long arrow) was significantly supershifted by the addition of Sp1 antibody but not by the addition of nonimmune rabbit IgG (Fig. 4A, arrowhead; compare lanes 4 and 5 with lanes 6 and 7). Nuclear extracts of AGS cells treated with IL-1β for 12 h also showed a significant enhancement of the Sp1-specific DNA; protein complex but 1, 2, or 24 h IL-1β treatments did not (data not shown). The insignificant supershifting of the two faster migrating bands (short arrows) by Sp1 antibody suggests that other AGS cell nuclear factors in addition to Sp1 interact with the HK{alpha} Sp1 binding sequence.


Figure 4
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  		Fig. 4. IL-1β but not H. pylori increases binding of Sp1 to the HK{alpha} promoter. AGS cells were treated with IL-1β (10 ng/ml; A–C) or with H. pylori (25 MOI) for 4 h (D). Interactions of AGS cell nuclear extract proteins with 32P-labeled HK{alpha}-specific Sp1 oligonucleotide probes were assessed by EMSA and supershift analysis with Sp1 antibody (Ab) or nonimmune rabbit IgG. A: EMSA and supershift analysis with 32P-labeled HK{alpha} Sp1 probe or HK{alpha}{Delta}Sp1 probe (6 nucleotide mutation in the core Sp1 binding region), IL-1β-treated AGS cells, and Sp1-specific antibody or nonimmune rabbit IgG. B: densitometric quantitation of DNA-protein complexes from lanes 1, 2, 7, and 8 in A. Data are shown as means ± SD of 3 independent experiments. C: EMSA with 32P-labeled HK{alpha} Sp1probe (lanes 1 and 2) and with 200x molar excess of cold (nonradioactive) HK{alpha} Sp1 probe (lanes 3 and 4). D: EMSA and supershift analysis with HK{alpha} Sp1 probe and H. pylori-treated AGS cells and Sp1-specific antibody. Gels are representative of 3 replicates for A and 2 replicates for experiments shown in C and D. Long and short arrows indicate DNA-protein complexes; arrowheads indicate supershifted complexes.

 
We sought to identify the site of Sp1 binding to the HK{alpha} promoter by EMSA supershift assays using an HK{alpha} DNA probe ({Delta}Sp1) mutated at six nucleotides corresponding to an Sp1 binding motif encompassed by the Sp1 footprint shown in Fig. 3 (–43 to –38 bp). As shown in Fig. 4A, nuclear extracts of untreated and treated (IL-1β; 10 ng/ml; 4 h) AGS cells yielded mobility-shifted bands of comparable signal intensity (compare lanes 7 and 8, long arrow). Sp1 antibody supershifted these {Delta}Sp1 DNA-protein complexes (lanes 9 and 10, arrowhead), but nonimmune rabbit IgG did not (lanes 11 and 12). These data contrast with the IL-1β-induced enhancement of Sp1 binding to a wild-type HK{alpha} Sp1 probe (Fig. 4A, lane 2), indicating that HK{alpha} promoter sequence –43 bp to –38 bp forms the Sp1 core binding region and is essential for IL-1β-induced enhanced binding of Sp1 to the HK{alpha} promoter. Densitometric quantitation of IL-1β-mediated changes in band intensity of DNA-Sp1 complexes is shown in Fig. 4B, and the specificity of the HK{alpha} Sp1 probe was demonstrated by cold competition (Fig. 4C). The fact that DNA-Sp1 complexes persist despite the mutation of six nucleotides suggests that residues flanking the mutated domain may show Sp1 binding affinity but cannot accommodate a functionally competent Sp1 complex formed by IL-1β-mediated signaling.

Given the IL-1β activation of Sp1 and ensuing upregulation of HK{alpha} promoter activity, we sought to establish whether H. pylori also modulated Sp1 activity while exerting its NF-{kappa}B-mediated repression of HK{alpha} promoter activity. Using EMSA and supershift analysis, we assessed the effect of H. pylori on Sp1 binding to the HK{alpha} Sp1 footprint region. In the absence of H. pylori, significant binding of Sp1 to HK{alpha} promoter, confirmed by Sp1 antibody supershift, was observed (Fig. 4D, compare lanes 1 and 3). No significant change in Sp1 binding to the HK{alpha} Sp1 probe was observed in the presence of H. pylori (25 MOI; 4 h; compare lanes 1 and 2, long arrow), and the Sp1 supershifts were comparable with or without H. pylori (compare lanes 3 and 4, arrowhead). The significant attenuation of the slowest migrating band (long arrow) by Sp1 antibody identifies this band as the DNA-Sp1 complex; the two faster migrating bands are complexes of two other nuclear factors with the Sp1 DNA probe. These data confirm our previous result that Sp1 binds to HK{alpha} promoter in absence of any external stimulus and also show that H. pylori infection does not modulate Sp1 binding to the HK{alpha} promoter region.

Sp1 interacts with NF-{kappa}B binding site of HK{alpha} promoter. Because our HK{alpha} promoter deletion analysis (Fig. 1) had indicated that the NF-{kappa}B1 binding site contributes to both basal and IL-1β-stimulated HK{alpha} activity, we sought to assess the effects of IL-1β on transcription factor interactions with the HK{alpha} NF-{kappa}B1 binding site, hypothesizing that Sp1 may exhibit promiscuous binding to an NF-{kappa}B site. Nuclear extracts of AGS cells were assayed by EMSA using the T18 HK{alpha} promoter DNA probe containing the NF-{kappa}B1 binding site (25). As shown in Fig. 5A, IL-1β treatment of AGS cells caused no change in DNA-protein complex band intensities (compare lanes 1 and 2), and neither NF-{kappa}B p50 subunit antibody (lanes 3 and 4) nor Sp1 antibody (lanes 5 and 6) produced a supershift of any of these complexes. The data indicate that IL-1β does not modulate nuclear factor interactions with the HK{alpha} NF-{kappa}B1 binding site and that Sp1 is not a constituent of the DNA-protein complexes formed by the T18 probe of the HK{alpha} NF-{kappa}B1 binding site. Reasoning that Sp1 may interact with an NF-{kappa}B site if bound simultaneously to the Sp1 binding site, we incubated nuclear extracts of AGS cells with a radioactive NF-{kappa}B1 T18 probe and a cold (nonradioactive) HK{alpha} Sp1 probe. As shown in Fig. 5B, the resulting EMSA banding distributions differed from those acquired with the T18 probe alone (long and short arrows; lanes 1 and 2). Significantly, Sp1 antibody now supershifted the slowest migrating (long arrow) DNA-protein complex, but the supershifted bands (arrowhead; lanes 3 and 4) were unaffected by IL-1β treatment. Coincubation of the T18 probe and cold HK{alpha} Sp1 probe with recombinant human Sp1, rather than with AGS cell nuclear extracts, yielded no mobility-shifted bands and no Sp1 supershift (lanes 5 and 6, respectively). Taken together, these data indicate that, in AGS cells, basal and IL-1β-mediated HK{alpha} activation involves the interaction of Sp1 and other essential AGS nuclear protein(s) with both the HK{alpha} Sp1 and HK{alpha} NF-{kappa}B1 binding sites.


Figure 5
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  		Fig. 5. IL-1β modulates Sp1 binding to NF-{kappa}B binding region in HK{alpha} promoter. A: interactions of AGS cell nuclear extract (NE) with and without IL-1β treatment (10 ng/ml) with 32P-labeled HK{alpha}-specific NF-{kappa}B1 oligonucleotide probe (T18; Ref. 25) by EMSA and supershift analysis with NF-{kappa}B p50 antibody (lanes 3 and 4) or Sp1 antibody (lanes 5 and 6). B: interactions of AGS nuclear extracts (lanes 1-4) or recombinant (r) human Sp1 protein (lanes 5 and 6) with an equimolar mixture of 32P-labeled NF-{kappa}B1 T18 probe and cold (nonradioactive) HK{alpha} Sp1 probe. Long and short arrows indicate DNA-protein complexes; arrowheads indicate supershifted complexes. Note the supershifts induced by addition of Sp1 antibody in lanes 3 and 4 (change in long arrow band to arrowheads). The gel is a representative EMSA gel from 2 independent experiments.

 
IL-1β-induced increase in HK{alpha} promoter activity is primarily regulated by Sp1. We next investigated the functional consequences of the IL-1β-mediated increased Sp1 binding to a cognate HK{alpha} promoter cis-response element. AGS cells were transfected with wild-type HK{alpha}206 deletion construct, {Delta}NF-{kappa}B HK{alpha}206 with two mutated nucleotides in the NF-{kappa}B1 binding region as described previously (25) or {Delta}Sp1 HK{alpha}206 bearing a six-nucleotide mutation in the Sp1 cis-response element. Cells were treated with IL-1β (10 ng/ml; 24 h), and the activity of the transfected promoter-reporter constructs was measured by luminometry. When compared with wild-type HK{alpha}206, the activity of the {Delta}Sp1 HK{alpha}206 construct was reduced by 81% and that of the {Delta}NF-{kappa}B1 HK{alpha}206 construct was reduced by 40% (Fig. 6). These data show that both Sp1 and NF-{kappa}B1 cis-response elements confer HK{alpha} transcriptional activity. An essential requirement for this activity is an intact Sp1 cis-response element, in the absence of which an intact NF-{kappa}B1 cis-response element confers neither basal nor IL-1β-stimulated HK{alpha} transcriptional activity. IL-1β caused a twofold increase in wild-type HK{alpha}206 promoter activity, had no effect on the significantly reduced activity of the {Delta}Sp1 HK{alpha}206 construct, and caused a twofold increase in activity of the {Delta}NF-{kappa}B1 HK{alpha}206 construct (Fig. 6). These data confirm the functional participation of Sp1 binding in IL-1β-induced HK{alpha} promoter upregulation and additionally emphasize a requirement for a functional NF-{kappa}B1 binding site on the HK{alpha} promoter for maximal IL-1β stimulation of HK{alpha} promoter activity.


Figure 6
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  		Fig. 6. IL-1β-induced increase in HK{alpha} promoter activity is regulated by Sp1 and not NF-{kappa}B. AGS cells were transiently transfected with either wild-type HK{alpha}206 or double-mutated HK{alpha}206 (HK{alpha} NF-{kappa}B1) or Sp1 core-mutated HK{alpha}206 (HK{alpha}{Delta}Sp1) and incubated with (gray bars) or without (white bars) IL-1β (10 ng/ml) for 24 h. HK{alpha}206 promoter activity was measured as normalized relative light units. Data are shown as means ± SD of 3 independent experiments. ***P < 0.001.

 
Sp1 downregulation abrogates IL-1β-induced increase in HK{alpha} promoter activity. Further confirmation that Sp1 mediates IL-1β-induced HK{alpha} promoter activation was obtained by transfecting AGS cells with Sp1-specific siRNA and subsequently measuring transfected HK{alpha} promoter activity in response to IL-1β treatment. Sp1 mRNA levels in AGS cells 48 h after transfection with a pool of four different Sp1 siRNAs, or with one of three individual siRNA members of the pool, were <35% of the levels measured in AGS cells transfected with either nontargeting siRNA (nt siRNA) or transfection reagent alone (control), as measured by RT-PCR (Fig. 7A). Of the three individual siRNAs, siRNA 3 showed maximum knockdown of Sp1 mRNA (>90%). Sp1 protein levels 72 h after transfection with Sp1 siRNA 3 were similarly downregulated as measured by Sp1-specific antibody immunoblotting of AGS cell lysates (Fig. 7B). AGS cell transfections with the other two Sp1-specific siRNAs caused comparable downregulation of Sp1 protein expression (data not shown). Finally, AGS cells were transfected with Sp1 siRNA 3 for 48 h and then transfected with the HK{alpha}206 deletion construct for 24 h. The cells were then incubated with IL-1β (10 ng/ml; 24 h). As shown in Fig. 7C, IL-1β treatment failed to elicit the activation of the HK{alpha} construct. Interestingly, the basal activity of HK{alpha}206 deletion construct was reduced by 22% in Sp1-specific siRNA-transfected AGS cells compared with non-siRNA-transfected AGS cells, a finding consistent with significantly reduced levels of cellular Sp1 being sufficient to promote HK{alpha} transcriptional activity and with the possibility that cis-activation sites other than Sp1 sites may contribute to HK{alpha} basal transcription.


Figure 7
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  		Fig. 7. Sp1 downregulation abrogates IL-1β-induced increase in HK{alpha} promoter activity. A: AGS cells were transfected with Sp1-specific small interfering RNA (siRNA) for 48 h, and the cellular content of Sp1 in presence and absence of siRNA was measured by real-time PCR using β2-microglobin as the internal control. Control, mock-transfected AGS cells; siRNA pool, AGS cells transfected with a mixture of 4 different siRNAs; siRNA 1, siRNA 2, and siRNA 3, AGS cells transfected with individual siRNAs; nontargeting (nt) siRNA, AGS cells transfected with nt siRNA as a negative control. Control and nt siRNA-transfected cells were harvested at the same time point as siRNA-treated cells. Data are shown as means ± SD of 3 independent experiments (***P < 0.001 compared with control). B: AGS cells were transfected with Sp1-specific siRNA 3, and the cellular content of Sp1 protein was measured after 72 h by immunoblotting using β-actin as the internal control. Control, mock-transfected AGS cells; siRNA, AGS cells transfected with siRNA 3 for 72 h. C: AGS cells were transfected with Sp1-specific siRNA 3 for 48 h, transfected with HK{alpha}206 for 24 h, and then treated with IL-1β for 24 h. HK{alpha}206 promoter activity was expressed as normalized relative light units (RLU) of luciferase activity. Data are shown as means ± SD of 3 independent experiments. ***P < 0.001.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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The present study explored the mechanistic basis of IL-1β perturbation of HK{alpha} promoter activity at the level of Sp1 and NF-{kappa}B transcription factor interactions with the promoter. Our recent report of IL-1β activation, and not inhibition, of human HK{alpha} promoter-Luc reporter constructs transfected into AGS cells (24) suggests that IL-1β inhibits acid secretion at a posttranslational rather than transcriptional level. As a potent inhibitor of gastric acid secretion, IL-1β probably contributes to H. pylori-induced hypochlorhydria through impairment of inositol (1,4,5)-triphosphate-dependent increase in intracellular Ca2+ concentration (26) and consequent failure of parietal cell tubulovesicular fusion and H,K-ATPase activation. Evidence that H. pylori interferes with the normal regulation of HK{alpha} transcription has accrued from studies examining the effects of H. pylori in the HK{alpha} promoter-transfected AGS cell model (13, 25), and we have recently reported that the mechanistic basis of HK{alpha} downregulation by H. pylori is ERK1/2-mediated NF-{kappa}B p50 homodimer binding to an NF-{kappa}B1 cis-response element at –170 to –150 bp on the HK{alpha} 5'-flanking sequence (25).

We find in the present study that although IL-1β promotes Sp1 binding to a defined HK{alpha} cis-response element, the consequent activation of HK{alpha} promoter activity is mediated by Sp1 interaction with both the Sp1 and 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 IL-1β treatment of transfected AGS cells significantly upregulated the activity of deletion constructs encompassing only NF-{kappa}B and Sp1 binding sites, and this activation was sustained though less pronounced with progressive deletion of the NF-{kappa}B sites; H. pylori treatment of the same constructs inhibited their transcriptional activity. Second, IL-1β increased the expression of phospho-ERK, phospho-Sp1, and phospho-p50 NF-{kappa}B subunits without affecting expression of total ERK1/2, Sp1 and NF-{kappa}B p50. Third, DNase I footprints of recombinant Sp1 subunit on a 206-bp length of HK{alpha} promoter showed the presence of an Sp1 binding site from –56 bp to –39 bp. Fourth, IL-1β increased binding of Sp1 to the proximal HK{alpha} promoter, whereas NF-{kappa}B p50 interaction with NF-{kappa}B1 site was not detected; in contrast, H. pylori had no effect on Sp1 binding to the promoter. Fifth, Sp1 was detected as a constituent of the NF-{kappa}B1 DNA-protein complex by the coincubation of HK{alpha} Sp1 and NF-{kappa}B binding site probes with AGS cell nuclear extracts. Sixth, the core mutation of the HK{alpha} Sp1 binding site abrogated IL-1β-induced activation of the HK{alpha} promoter, whereas mutation of two nucleotides in the HK{alpha} NF-{kappa}B binding site did not. Finally, the IL-1β-induced activation of HK{alpha} promoter was prevented by siRNA knockdown of Sp1.

These results illuminate aspects of the regulation of normal HK{alpha} expression and the mechanisms of H. pylori and IL-1β perturbation of this regulation. In isolated canine parietal cells transfected with HK{alpha} promoter deletion constructs, basal transcriptional activity of H,K-ATPase {alpha}-subunit promoter was shown to be conferred by Sp1 binding to –54 to –45 bp of the promoter (20). The human HK{alpha} Sp1 site we identified is 80% homologous to the canine HK{alpha} Sp1 binding sequence, and its 5' addition to the proximal 37 bp of an HK{alpha} promoter construct confers a 5.5-fold increase in basal HK{alpha} transcriptional activity in transfected AGS cells. Further additions of HK{alpha} sequence confer progressively greater basal HK{alpha} activity, culminating in the 15-fold activation exerted by a 206-bp HK{alpha} promoter construct. Since 5' extension of the HK{alpha} promoter construct from –102 bp to –206 bp adds two NF-{kappa}B1 binding sites, the resulting enhancement of HK{alpha} basal transcriptional activity may be attributed to cooperative binding of nuclear factors to both the Sp1 site and at least one of the NF-{kappa}B sites. This was confirmed by deletion of two nucleotides in the core recognition motif of the proximal NF-{kappa}B1 sequence, which reduced HK{alpha} basal activity by 40% (Fig. 6). We detected no NF-{kappa}B p50 subunit in control AGS cell nuclear extracts, consistent with the insignificant activation of NF-{kappa}B in resting gastric epithelial cells in vivo in the absence of proinflammatory stimuli. We propose therefore that the cooperative participation of the HK{alpha} NF-{kappa}B1 binding site in basal HK{alpha} transcriptional activity reflects the interaction of Sp1 with this site. Functional interaction of Sp1 with NF-{kappa}B sites has been reported in HeLa cells transfected with P-selectin promoter-reporter constructs and treated with TNF-{alpha} (15). Sp1 and NF-{kappa}B sites also cooperate in mediating basal and interferon {gamma}-stimulated CD98 transcription in transiently transfected Caco-BBE epithelial cells (37).

The same nuclear factor binding sites were implicated in IL-1β activation of HK{alpha} promoter constructs. IL-1β caused the 26-fold activation of the 206-bp HK{alpha} construct compared with the 37-bp construct. Although the progressive 5' extension of the HK{alpha} promoter included both a partial (HK{alpha}165) and a complete (HK{alpha}177) NF-{kappa}B1 binding site, NF-{kappa}B subunits themselves play no role in IL-1β-induced HK{alpha} activation as shown by the failure of a mutated NF-{kappa}B1 binding site to abrogate this activation. As appears to be the case in the regulation of basal HK{alpha} transcription, Sp1 binding to both Sp1 and NF-{kappa}B cis-response elements may underlie IL-1β-mediated upregulation of HK{alpha} transcription. Sp1 binding to the HK{alpha} NF-{kappa}B1 site would prevent the interaction of NF-{kappa}B p50 homodimers with that site and preempt consequent repression of HK{alpha} promoter activity (25). With the use of a consensus NF-{kappa}B probe, the significant activation of both NF-{kappa}B p50 and p65 subunits was reported in AGS cells exposed to 25 ng/ml IL-1β for 2 h (17), presumably allowing the formation of p50/p65 heterodimers and p50/p50 homodimers. In the present study, using a HK{alpha}-specific NF-{kappa}B1 probe (T18), we detected no binding of p50 subunits in nuclear extracts of AGS cells exposed to 10 ng/ml IL-1β for 4 h, and so the failure of IL-1β to repress HK{alpha} transcription is consistent with the inability of NF-{kappa}B p50 homodimers to bind to the NF-{kappa}B1 site due to site occupancy by Sp1.

The functional significance in a physiological setting (i.e., in vivo gastric mucosa) of nuclear factor interactions with the HK{alpha} promoter described in this article cannot be readily assessed. AGS cells are useful in studies of H. pylori pathophysiology because acquisition and maintenance of human gastric parietal cells in culture in a functionally competent state for longer than 3 or 4 days is extremely difficult. As transformed dedifferentiated cells originating in a gastric adenocarcinoma (1), AGS cells differ in many respects from terminally differentiated gastric parietal cells, not least in their lack of proton pump expression. Accordingly, H. pylori or IL-1β-driven interactions of AGS cell transcription factors with transfected proton pump promoter constructs may not faithfully recapitulate IL-1 receptor-mediated and H. pylori-mediated perturbations of parietal cell intracellular signaling. The net impact on parietal cell HK{alpha} transcription of the integrated contributions of both perturbations is presumably modulated by numerous other considerations. These include relative concentrations, phosphorylation status, and access of transcription factors and related accessory proteins to promoter DNA, appropriate assembly of transcriptional complexes on the promoter, acetylation status of nucleosomal histones, etc., all conditions that are unique to the parietal cell and are necessarily different in the AGS cell model used in this study. However, reported parallels between AGS cells and parietal cells include the presence of functional histamine H2 receptors (13), EGF receptors (18), components of proinflammatory signaling pathways (19), IL-8 secretory capacity (8, 28), and sensitivity to H. pylori and IL-1β (17). That Sp1 confers basal transcriptional activity on transfected HK{alpha} promoter-reporter constructs both in AGS cells (this article) and in primary canine parietal cells (20) lends further credence to AGS cells as an experimental surrogate for human parietal cells.

Our demonstration that IL-1β enhances Sp1 binding to the HK{alpha} promoter and that the consequent activation of the promoter is further enhanced by the presence of the NF-{kappa}B1 cis-response element, without detectable increased binding of NF-{kappa}B p50 subunit, suggests that IL-1β does not exert an acid inhibitory effect at the level of HK{alpha} transcription. Our previous report that NF-{kappa}B p50 homodimer binding to the HK{alpha} promoter NF-{kappa}B1 site is responsible for H. pylori-mediated HK{alpha} gene repression (25) in AGS cells suggests that the same mechanism plays a role in H. pylori-mediated clinical hypochlorhydria. Interestingly, EGF activation of HK{alpha} gene transcription in canine parietal cells (16) has been attributed to the activation of the Akt signal transduction pathway (32) and binding of cis-regulatory protein to a canine HK{alpha} cis-response element between –162 and –156 bp (5'-GACATGG-3') (16) that exhibits 100% sequence homology with the human HK{alpha} NF-{kappa}B1 site mediating the H. pylori repression of HK{alpha} promoter activity (25). We have also reported the EGF activation of human HK{alpha} full-length promoter-Luc construct in AGS cells, although its mechanistic basis was not explored (13). EGF is known to activate NF-{kappa}B in nonsmall cell lung carcinoma cell lines (27), in breast cancer cells (3), in A-431 carcinoma cells and mouse embryo fibroblasts (31), and in human proximal tubule cells (14). Accordingly, we hypothesize that the EGF activation of NF-{kappa}B in gastric epithelial cells favors the assembly of NF-{kappa}B p50/p65 heterodimers that activate HK{alpha} gene expression, in contrast with the H. pylori induction of NF-{kappa}B p50/p50 homodimers that repress HK{alpha} gene expression. Also of interest is the observation that EGF activates gastrin promoter constructs transfected into AGS cells by inducing the phosphorylation of Sp1 (6), and so cooperative interactions between Sp1 and NF-{kappa}B and their cognate cis-response elements may have significant regulatory consequences in EGF-stimulated signaling in gastric epithelial cells.

In conclusion, we have found that IL-1β upregulates the activity of human H,K-ATPase {alpha}-subunit proximal promoter sequences through Sp1 transactivation of a cis-response element located between –56 bp to –39 bp upstream of the HK{alpha} transcription start site that also confers basal HK{alpha} transcriptional activity. Our data are consistent with the model of Sp1 interaction with the HK{alpha} promoter shown in Fig. 8, whereby Sp1 confers basal and IL-1β-mediated HK{alpha} promoter activity by direct binding to the HK{alpha} Sp1 cis-response element and indirect binding to the HK{alpha} NF-{kappa}B1 binding site via the concurrent recruitment of nuclear cofactor(s). NF-{kappa}B subunits do not participate in this transactivation complex. IL-1β augments Sp1 binding to the HK{alpha} Sp1 cis-response element through the activation of an ERK1/2 signaling pathway, enhancing HK{alpha} promoter activity. Sp1 transactivation of HK{alpha} promoter is not perturbed by H. pylori, which represses HK{alpha} transcription by the mobilization of NF-{kappa}B p50 homodimers. These findings provide a mechanistic understanding of the differential effects of H. pylori and IL-1β on H,K-ATPase {alpha}-subunit gene expression and uncover a novel mechanism of IL-1β-induced HK{alpha} promoter regulation.


Figure 8
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  		Fig. 8. Schematic diagram of proposed interaction of Sp1 transcription factor with Sp1 and NF-{kappa}B binding sites on the proximal HK{alpha} promoter. The proximal 5'-flanking sequence of the HK{alpha} promoter is represented by the black hairpin; gray boxes represent transcription factor binding sites, and white circles represent Sp1 protein and putative cofactor(s) required for Sp1-mediated coupling of the Sp1 and NF-{kappa}B binding sites.

 

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


   ACKNOWLEDGMENTS
 
We thank Dr. Gary Shull (University of Cincinnati) for the generous provision of a plasmid containing 2,199 bp of human gastric H,K-ATPase {alpha}-subunit 5'-flanking DNA and Dr. Richard M. Peek, Jr. (Vanderbilt University School of Medicine), 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
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 DISCUSSION
 GRANTS
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