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Am J Physiol Gastrointest Liver Physiol 292: G1055-G1061, 2007. First published January 4, 2007; doi:10.1152/ajpgi.00338.2006
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MUCOSAL BIOLOGY

IL-1beta modulation of H,K-ATPase {alpha}-subunit gene transcription in Helicobacter pylori infection

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

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

Submitted 26 July 2006 ; accepted in final form 1 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Helicobacter pylori infection of the human gastric body induces hypochlorhydria by perturbing acid secretion. H. pylori inhibits parietal cell H,K-ATPase {alpha}-subunit (HK{alpha}) gene and protein expression, providing a mechanistic basis for clinical hypochlorhydria. Given that H. pylori infection increases gastric mucosal IL-1beta, an acid secretory inhibitor, we investigated the role of IL-1beta in H. pylori-mediated inhibition of HK{alpha} transcription. Human gastric adenocarcinoma (AGS) cells were transfected with promoter-reporter constructs containing human HK{alpha} 5'-flanking sequence deletions. IL-1beta (10 ng/ml) had no effect on the transcriptional activity of six progressively shorter deletion constructs of the HK{alpha} promoter (HK{alpha}2179–HK{alpha}340) and significantly stimulated the activity of HK{alpha}206, HK{alpha}177, HK{alpha}165, and HK{alpha}102 deletion constructs (80%, 100%, 46%, and 35%, respectively). H. pylori inhibited the transcriptional activity of HK{alpha}2179, HK{alpha}206, HK{alpha}177, and HK{alpha}165; IL-1beta relieved the H. pylori inhibition of HK{alpha}2179 and HK{alpha}206 activity but not HK{alpha}177 and HK{alpha}165 activity. AGS cell pretreatment with a MEK1/2 inhibitor prevented the IL-1beta-mediated stimulation, but p38 and JNK pathway inhibitors did not. IL-1beta mRNA levels in AGS cells were low and unaffected by H. pylori, and ELISAs of H. pylori-conditioned AGS culture media showed no measurable IL-1beta secretion. These data indicate that an IL-1beta-dependent cis-response element lies downstream of –206 nt in the HK{alpha} promoter and that IL-1beta-mediated upregulation of HK{alpha} transcription is affected by an ERK1/2 kinase signal pathway. We conclude that an IL-1beta-responsive HK{alpha} cis element positively regulates HK{alpha} gene transcription in shortened deletion constructs and that H. pylori-induced inhibition of HK{alpha} transcription is not mediated by IL-1beta.

interleukin-1beta; gastric acid secretion

THE GRAM-NEGATIVE BACTERIUM Helicobacter pylori is a microaerophilic acid-resistant neutralophile with spiral morphology that thrives in the mucus layer and mucosal epithelial cells of the human stomach. In developing countries, the prevalence of adult H. pylori infection can be over 80%, compared with 20–50% in developed countries, resulting in ~2 billion H. pylori infections globally (36). Although most infected individuals are asymptomatic, ~20% develop H. pylori-related peptic ulcer disease, primary gastritis, gastric mucosa-associated lymphoid tissue lymphoma, or gastric adenocarcinoma (3). Both in vivo and in vitro experimental data have indicated that H. pylori strains possessing a 40-kb cag pathogenicity island promote host epithelial cell secretion of the chemotactic proinflammatory cytokine IL-8 (11, 29, 34, 37, 42). The ensuing recruitment of neutrophils and monocytes secreting the cytokines IL-1beta and TNF-{alpha} amplifies the inflammatory response, provoking a superficial gastritis that, in a significant subset of patients (~2%), may progress to atrophic gastritis and then to intestinal metaplasia, dysplasia, and, eventually, gastric adenocarcinoma (28).

Gastric mucosal infection by H. pylori is known to perturb normal acid secretory mechanisms. Voluntary human self-ingestion reports and studies of acute gastric mucosal lesions attributed to postendoscopic cross-infection have indicated that acute H. pylori infection is associated with hypochlorhydria (13, 19, 21, 22, 30). Clinical studies have indicated that chronic H. pylori infection may either impair acid secretion, increasing the risk of gastric cancer (6, 16, 30), or stimulate acid secretion, decreasing gastric pH and predisposing individuals to duodenal ulcer (7). These contrasting secretory responses may reflect differing anatomic sites of infection. Thus, H. pylori antral gastritis may be associated with hypergastrinemia, hyperacidity, and duodenal ulcers, whereas H. pylori body gastritis may promote hypochlorhydria leading to cancer (20). In addition to its proinflammarory properties, IL-1beta is a powerful inhibitor of gastric acid secretion (2, 38, 40) and may contribute to gastric hypochlorhydria. In rats, the potent H,K-ATPase antisecretory activity of IL-1beta has been shown to be mediated by parietal cell IL-1 receptors interacting with intracellular Ca2+/phospholipid-dependent signaling pathways (32). IL-1beta also mediates activation of the Fos-Jun pathway and upstream p38 MAPK pathways (10, 17, 25, 44). Given that IL-1beta inhibits gastric acid secretion, elevated mucosal IL-1beta would have antisecretory effects, potentially accounting for H. pylori-mediated hypochlorhydria.

Gastric acid secretion is mediated by a Mg2+-dependent, K+-stimulated, H3O+-transporting, P-type ATPase (H,K-ATPase; EC 3.6.1.36 [EC] ) (9, 31). The H,K-ATPase {alpha}-subunit (HK{alpha}; Mr ~94,000, GenBank Accession No. JO5451) is a polytopic integral membrane protein of tubulovesicular and secretory canalicular membranes in acid-secreting gastric epithelial parietal cells. A close interaction of HK{alpha} with the monotopic membrane-integrated glycosylated beta-subunit (HKbeta, Mr ~60,000–80,000, GenBank Accession No. BX537316) is required for functional electroneutral exchange of lumenal K+ for cytoplasmic protons (5). HK{alpha} gene transcription is positively regulated by the gastric secretagogues histamine, gastrin, and acetyl choline (4). HK{alpha} promoter cis-response elements associated with the induction of HK{alpha} transcription bind parietal cell-specific GATA transcription factors (26, 35). Binding of Sp1 a few bases upstream of the TATA box has been shown to activate constitutive HK{alpha} transcription in canine parietal cells (23), and protein binding to a site 100 bp further upstream (homologous to the c-fos serum response element 3' half-site) has been shown to be associated with EGF-induced transcriptional activation of the HK{alpha} gene (14). Significantly, in vitro H. pylori treatment of human gastric epithelial cells transfected with a plasmid containing a 2.2-kb human HK{alpha} 5'-flanking sequence coupled to a luciferase reporter gene downregulated HK{alpha} transcriptional activity by 60% (12).

The host acid secretory status (16) and impact of H. pylori infection on acid secretion (20) are increasingly recognized as critical elements in defining the clinical outcome. The effects of IL-1beta on HK{alpha} transcriptional activity in the presence and absence of H. pylori have not been described.The present study sought to determine whether H. pylori-dependent downregulation of HK{alpha} gene transcription in gastric epithelial cells is mediated by IL-1beta.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells, media, and reagents. Human gastric adenocarcinoma (AGS) cells (CRL1739) and H. pylori (strain ATCC 49503) were purchased from the American Type Culture Collection (Manassas, VA). Ham's F-12 containing L-glutamine was obtained from Mediatech (Herndon, VA), and Opti-MEM was purchased from Invitrogen (Carlsbad, CA). Brucella broth and microaerophilic gas packs were from BD Biosciences (Sparks, MD). FBS was acquired from Atlanta Biological (Norcross, GA). Phorbol 12-myristate 13-acetate (PMA) and JNK inhibitor II were from Calbiochem-Novabiochem (La Jolla, CA), and IL-1beta was purchased from Sigma-Aldrich (St. Louis, MO). Restriction enzymes, MAPK inhibitors PD-98059 and SB-203580, pGL2-Basic Vector transfection plasmid, and luciferase assay substrate were obtained from Promega (Madison, WI). The transfection plasmid pMaxGFP was purchased from Amaxa (Gaithersburg, MD), and Fugene-6 transfection reagent was from Roche Diagnostics (Indianapolis, IN). The iScript cDNA synthesis kit was from Bio-Rad Laboratories (Hercules, CA). All other reagents were of molecular biology grade with maximum possible purity.

Cell and bacterial cultures. AGS cells were grown in Ham's F-12 containing L-glutamine supplemented with 10% FBS at 37°C in a humified incubator with 5% CO2-95% air. AGS cells were used between passages 40 and 50. H. pylori cultures were grown on Brucella broth agar plates containing 10% FBS and 2.4% agar at 37°C using a microaerophilic gas pack system. Cultures were screened by a urease test regularly during subculturing. Only cultures giving a positive urease test were used for cell infection. For AGS cell infection, H. pylori bacteria were harvested after 36 h of culture in Brucella broth agar plates. Bacteria were resuspended in Ham's F-12 containing FBS and enumerated by measuring absorbance at 600 nm (1 optical density unit at 600 nm = 2.4 x 108 bacteria/ml). Multiplicities of infection (MOIs) were calculated based on AGS and bacterial cell counts.

HK{alpha} promoter-reporter plasmid constructs. Genomic DNA representing a portion of the human gastric HK{alpha} 5'-flanking region was a kind gift from 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 (12). Progressively truncated deletion constructs of HK{alpha} were generated either by restriction endonuclease digestion or by PCR amplification using the HK{alpha} 2179 promoter-reporter plasmid as a template (Table 1). Restriction fragments were resolved on 2% agarose gels, excised under long wave-length UV illumination, gel purified using a QIAquick Gel extraction kit from Qiagen (Valencia, CA), and treated with mung bean nuclease to remove 5' overhangs. Blunt-ended linear DNAs were then ligated to appropriately linearized pGL2-Basic Vector plasmid using T4 DNA ligase.


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  		Table 1. HK{alpha} 5'-flanking sequence deletion constructs transiently transfected into gastric adencarcinoma cells in the form of pGL2-Basic luciferase plasmids

 
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 containing 0.2 µg DNA and Fugene-6 transfection reagent 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 pGL2Basic plasmids containing neither the promoter nor enhancer served as negative controls. The ratio of promoter-reporter plasmids to normalization control plasmids was 1:1. After 24 h of transfection, cells were treated with 100 nM PMA in FBS-supplemented Ham's F-12, H. pylori, and/or IL-1beta for different periods of time. When needed, AGS cells were incubated with inhibitors of intracellular signaling pathways for 90 min before IL-1beta treatment. Cells were lysed with 1x passive lysis buffer, and light emission was measured as relative light units (RLUs) in a Victor 1420 Multi-Label Counter (Perkin-Elmer Bio-Sciences) 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 EM Spectra-Fluorometer (Molecular Devices, Sunnyvale, CA). Data from individual wells were normalized to cotransfected pMAX-GFP fluorescence and corrected by subtracting normalized promoterless pGL2-Basic Vector RLU data. Data points are shown as means ± SD of three independent transfection experiments with each deletion construct.

IL-1beta ELISA. AGS cells were cultured to 75–80% confluency in T-75 flasks and harvested after trypsin-EDTA digestion for 15 min followed by the addition of Ham's F-12 medium containing 10% FBS. Cells were centrifuged at 1,500 rpm for 5 min, resuspended in Ham's F-12 medium with 10% FBS, and counted by hemocytometry, and cell viability was measured by trypan blue exclusion. AGS cells (250,000 cells/well) were plated in six-well cell culture plates and grown for 20 h. Cells were transferred to serum-free medium for 15–20 h and then treated for varying periods of time with 100 nM PMA and/or H. pylori at a MOI of 25. The IL-1beta content of aliquots of cell culture supernatant was measured by ELISA using an IL-1beta Duo Set ELISA Development System (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.

Real-time RT-PCR. AGS cells were grown to 75–80% confluence in 24-well cell culture plates, and culture was continued in the absence of FBS for 15–20 h. H. pylori bacteria were then added to the cells at a MOI of 25 for varying periods of time (0–12 h). AGS cell RNA was isolated using RNA STAT-60 reagent from Tel Test (Friendswood, TX) and reverse transcribed using an iScript cDNA synthesis kit according to the manufacturer's protocol. Measurements of AGS cell IL-1beta mRNA were carried out by real-time RT-PCR using an iCycler iQ with iQ SYBR Green Super mix (Bio-Rad, Hercules, CA) and forward (5'-CACGATGCACCTGTACGATCA-3') and reverse (5'-TGGAGGTGGAGAGCTTTCGTT-3') primers specific for human IL-1beta.

Statistical analysis. Data acquired from experiments with multiple samples subjected to each treatment were analyzed by two-way ANOVA. Levels of significance in multiple pairwise comparisons of treatment and control groups were calculated using Bonferroni post test analysis as implemented in the GraphPad PRISM version 4 statistical software package. Data are expressed as means ± pooled SD, and statistical significance was ascribed to P values of <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Regulation by H. pylori of the transcriptional activity of the HK{alpha} 5'-flanking region transfected into cultured human AGS cells has been reported previously (12), and this model system was used in the present study to investigate the effect of exogenously administered IL-1beta on HK{alpha} promoter activity. To test the hypothesis that HK{alpha} promoter inhibition might be mediated by IL-1beta, AGS cells were transiently transfected with a series of HK{alpha} deletion constructs (Table 1), and maximal transcriptional activity was induced by 100 nM PMA. Cells were then treated with 10 ng/ml IL-1beta, and HK{alpha} promoter activity was measured after 24 h. As shown in Fig. 1, constitutive activities of deletion constructs from HK{alpha}2179 through HK{alpha}340 remained relatively constant (P > 0.05) and were unaffected by IL-1beta (P > 0.05). In the absence of IL-1beta, constitutive activities of deletion constructs HK{alpha}206 through HK{alpha}102 increased up to twofold, whereas those of the shortest constructs (HK{alpha}64 and HK{alpha}58) were significantly attentuated. In the presence of IL-1beta, activites of HK{alpha}206, HK{alpha}177, HK{alpha}165, and HK{alpha}102 deletion constructs were significantly increased compared with untreated controls (P < 0.001). Activities of HK{alpha}206 and HK{alpha}177 constructs were stimulated ~2-fold and those of HK{alpha}165 and HK{alpha}102 constructs were stimulated by 1.4-fold relative to constitutive levels.


Figure 1
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  		Fig. 1. Effect of IL-1beta on the transcriptional activity of H,K-ATPase {alpha}-subunit (HK{alpha}) 5'-flanking sequence deletion constructs. Gastic adenocarcinoma (AGS) cells were transiently transfected with luciferase (Luc) reporter constructs (pGL2-Basic Vector) incorporating HK{alpha} 5'-flanking sequences ranging from –2179 to –58 bp relative to the transcription initiation site and then incubated for 24 h with 100 nM PMA with IL-1beta (10 ng/ml; shaded bars) or without IL-1beta (open bars). In this and Figs. 2–6, HK{alpha} promoter activity was measured as relative light units (RLUs) of luciferase activity in AGS cell lysates and was corrected and normalized as described in MATERIALS AND METHODS. ***P < 0.001.

 
Transcriptional activities of HK{alpha}177 and HK{alpha}165 deletion constructs were regulated by IL-1beta in a dose-dependent manner, as shown in Fig. 2. At all doses of IL-1beta, promoter activities of HK{alpha}177 and HK{alpha}165 were significantly increased compared with untreated construct activities (P < 0.05). HK{alpha}177 promoter activities with 10 and 100 ng/ml IL-1beta did not differ but were significantly increased compared with 1 ng/ml IL-1beta (P < 0.05). HK{alpha}165 promoter activities did not differ significantly from one another at any measured concentration of IL-1beta (P > 0.05). These data indicated that a repressor-binding sequence is located between –340 bp and –206 bp in the HK{alpha} 5'-flanking region and that IL-1beta-responsive cis-activation elements are located between –206 and –64 bp.


Figure 2
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  		Fig. 2. Dose-dependent stimulation of the transcriptional activity of HK{alpha}177 and HK{alpha}165 deletion constructs by IL-1beta. AGS cells were transiently transfected with Luc reporter constructs (pGL2-Basic Vector) incorporating HK{alpha} promoter deletion constructs HK{alpha}177-Luc ({blacksquare} and thick solid line) or HK{alpha}165-Luc ({square} and thin solid line) and then incubated for 24 h with 100 nM PMA with increasing concentrations of IL-1beta (1, 10, and 100 ng/ml).

 
To further characterize the sensitivity of HK{alpha}177 and HK{alpha}165 deletion constructs to IL-1beta, the time course of IL-1beta stimulation of both constructs was examined. As shown in Fig. 3, time-dependent HK{alpha}177 and HK{alpha}165 promoter activities as a function of IL-1beta treatment were not significantly different at 0.5, 1, and 4 h (P > 0.05) but were significantly different at 8, 16, and 24 h (P < 0.001). In the absence of IL-1beta, transcriptional activities of both deletion constructs were maximally stimulated at 16 and 24 h. HK{alpha}177 activity at both time points was increased by approximately twofold after treatment of transfected cells with IL-1beta (10 ng/ml; Fig. 3A). In contrast, HK{alpha}165 activity at the same time points increased only 1.4-fold after treatment of transfected cells with IL-1beta (10 ng/ml; Fig. 3B), indicating a partial loss of the IL-1beta-sensitive cis-responsive binding activity in the HK{alpha} 5'-flanking region between –165 and –177 bp.


Figure 3
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  		Fig. 3. Time course of IL-1beta stimulation of the transcriptional activation of HK{alpha}177 and HK{alpha}165 deletion constructs. AGS cells were transiently transfected with HK{alpha}177-Luc (A) or HK{alpha}165-Luc (B) and then incubated with 100 nM PMA with IL-1beta (10 ng/ml; shaded bars) or without IL-1beta (open bars) for varying periods of time.

 
Because these results clearly argued against an IL-1beta-mediated mechanism of H. pylori inhibition of HK{alpha} gene transcription, we next investigated the effects of H. pylori on both of the HK{alpha} deletion constructs maximally activated by IL-1beta (HK{alpha}177 and HK{alpha}165). AGS cells were transiently transfected with HK{alpha}177 and HK{alpha}165 deletion constructs and then incubated for 24 h with differing concentrations of H. pylori. Transcriptional activities of these two HK{alpha} sequences containing putative IL-1beta cis-response elements were not significantly inhibited by H. pylori at a MOI of 10 (P > 0.05) but were significantly inhibited by H. pylori at MOIs of 25 (P < 0.01) and 50 (P < 0.001) compared with untreated controls. For both constructs, activity differences between H. pylori MOIs of 10 and 25 and between MOIs of 25 and 50 were insignificant (P > 0.05); however, differences between H. pylori MOIs of 10 and 50 were significant (P < 0.001). Although maximal inhibition (63%) of both constructs was induced at a H. pylori MOI of 50 (Fig. 4), at this MOI AGS cells showed significant deterioration of normal morphology, with pronounced vacuolization and blebbing, and so H. pylori MOIs were adjusted to 25 in subsequent experiments.


Figure 4
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  		Fig. 4. Effect of Helicobacter pylori on the transcriptional activity of HK{alpha}177 and HK{alpha}165 deletion constructs. AGS cells were transiently transfected with HK{alpha}177-Luc ({blacksquare} and thick solid line) or HK{alpha}165-Luc ({square} and thin solid line) and treated with increasing multiplicities of infection (MOIs) of H. pylori.

 
Given the observed stimulation of HK{alpha}177 and HK{alpha}165 transcriptional activity by IL-1beta, we next studied the effects of IL-1beta on the transcriptional activity of HK{alpha} 5'-deletion constructs in the setting of H. pylori infection of transfected AGS cells. HK{alpha}2179 (full length), HK{alpha}206, HK{alpha}177, and HK{alpha}165 deletion constructs were transiently transfected into AGS cells and incubated for 24 h with 100 nM PMA and H. pylori at a MOI of 25 with and without 10 ng/ml IL-1beta. Activities of all four deletion constructs were significantly inhibited by H. pylori (Fig. 5). Measurements of normalized luciferase RLUs showed that treatment of transfected cells with H. pylori inhibited the promoter activity of HK{alpha}2179 and HK{alpha}206 by 27% and 67%, respectively, and that this inhibition did not occur in the presence of IL-1beta. In contrast, although H. pylori inhibited HK{alpha}177 and HK{alpha}165 by 37% and 31%, respectively, this inhibition was not significantly relieved by IL-1beta (Fig. 5). These data suggest that activation of an IL-1beta-sensitive cis-response element located between –206 and –177 bp relieves H. pylori-mediated regulatory effects on the HK{alpha} 5'-flanking region.


Figure 5
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  		Fig. 5. Effect of IL-1beta on H. pylori-mediated inhibition of transcriptional activity of HK{alpha}2179, HK{alpha}206, HK{alpha}177, and HK{alpha}165 deletion constructs. AGS cells were transiently transfected with HK{alpha}2179-Luc, HK{alpha}206-Luc, HK{alpha}177-Luc, or HK{alpha}165-Luc constructs and then incubated for 24 h with 100 nM PMA without H. pylori or IL-1beta (open bars), with H. pylori (MOI of 25; light shaded bars), or with H. pylori (MOI of 25) together with IL-1beta (10 ng/ml) (dark shaded bars). *P < 0.05; **P < 0.01; ***P < 0.001.

 
Given the presence of IL-1beta-responsive sequence elements in the HK{alpha} promoter and their modulatory role in H. pylori-mediated HK{alpha} inhibition, we investigated the intracellular signaling pathways through which IL-1beta regulates HK{alpha} gene expression. AGS cells were transiently transfected with HK{alpha}2179, HK{alpha}177, HK{alpha}165, or HK{alpha}102 deletion constructs and then incubated with 10 ng/ml IL-1beta and with one of three inhibitors of specific MAPK signaling pathways: PD-98059, an ERK1/2 pathway inhibitor (50 µM); SB-203580, a p38 pathway inhibitor (10 µM); and JNK inhibitor II, an inhibitor of the JNK pathway (100 nM). As shown in Fig. 6, activities of all four deletion constructs were significantly inhibited in the presence of the ERK1/2 inhibitor (PD-98059) compared with untreated control activities or activities in the presence of IL-1beta (P < 0.001). In the cases of the p38 and JNK pathway inhibitors, there were no significant differences in activities compared with untreated control activities or activities in the presence of IL-1beta. These data indicate that HK{alpha} promoter activity in the targeted deletion constructs is dependent on a functional ERK1/2 signaling pathway and IL-1beta stimulation of HK{alpha} promoter activity is mediated through the same pathway; neither the p38 nor JNK signaling pathways are involved in the regulation of HK{alpha} gene expression.


Figure 6
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  		Fig. 6. Effects of pharmacological inhibition of AGS cell signaling pathways on IL-1beta stimulation of HK{alpha} deletion constructs. AGS cells were transiently transfected with HK{alpha}2179-Luc, HK{alpha}177-Luc, HK{alpha}165-Luc, or HK{alpha}102-Luc constructs and then incubated for 24 h with 100 nM PMA without IL-1beta (open bars), with IL-1beta (10 ng/ml; light shaded bars), with IL-1beta (10 ng/ml) and SB-203580 (p38 pathway inhibitor) (intermediate shaded bars), with IL-1beta (10 ng/ml) and JNK inhibitor II (dark shaded bars), and with IL-1beta (10 ng/ml) and PD-98059 (ERK1/2 pathway inhibitor) (solid bars).

 
At the outset of this study, we had sought to determine whether infection of AGS cells by H. pylori itself induced cellular production and secretion of IL-1beta comparable with the epithelial secretion of IL-8 induced by H. pylori. AGS cells were infected for varying periods of time with H. pylori at a MOI of 25, and the cellular content of IL-1beta mRNA was measured by quantitative RT-PCR and the IL-1beta concentration of cell culture media was measured by ELISA. IL-1beta mRNA levels in control AGS cells were very low and unchanged following H. pylori infection (data not shown). IL-1beta was undetectable by ELISA in the culture media of control or H. pylori-infected cells (data not shown). These results suggest that our observation of IL-1beta effects on HK{alpha} gene expression reflect exogenous, i.e., leukocyte and phagocyte, sources of IL-1beta in the physiological setting rather than local autocrine effects.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We (12) previously reported that in vitro H. pylori infection of cultured gastric epithelial cells transfected with HK{alpha} promoter-reporter constructs resulted in the significant downregulation of HK{alpha} transcriptional activity. In that study, AGS cells were shown to possess functional histamine and EGF receptors whose activation modulated the transcriptional activity of transfected plasmids incorporating a 2.2-kb human HK{alpha} 5'-flanking sequence fused to a luciferase reporter gene. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HK{alpha} promoter activities, and dose dependently inhibited PMA-induced and staurosporine- and calphostin C-sensitive activation of the HK{alpha} promoter. Also, H. pylori dose dependently inhibited EGF activation of HK{alpha} promoter activity. These data suggested that H. pylori infection downregulates HK{alpha} gene expression via intracellular pathways involving protein kinase C and protein tyrosine kinase (12). The possibility that HK{alpha} gene expression was modulated in the transfected AGS cell model by IL-1beta, originating in H. pylori-infected cells and acting in an autocrine capacity, was not addressed in that study.

Mucosal levels of the proinflammatory cytokine IL-1beta are increased following gastric colonization by H. pylori, and IL-1beta is known to be a potent inhibitor of acid secretion. In the present study, we sought to determine whether IL-1beta activates signaling pathways in gastric epithelial cells that ultimately downregulate HK{alpha} transcription. Such a downregulation would lower the expression of functional proton pumps and thereby contribute to gastric hypochlorhydria. Our experiments showed that IL-1beta mRNA levels in AGS cells were low and unresponsive to H. pylori infection and that IL-1beta was undetectable by ELISA in the culture medium of control and infected AGS cells. These findings are inconsistent with a transcriptional level role for IL-1beta in H. pylori-induced HK{alpha} downregulation in the AGS cell model. Thus, in the physiological setting of a H. pylori-infected gastric corpus, any antisecretory effects of IL-1beta originating from exogenous neutrophils and monocytes, and contributing to hypochlorhydria, are unlikely to be mediated by direct perturbation of parietal cell HK{alpha} transcription.

The present study confirms our original observations of H. pylori downregulation of HK{alpha} transcription and extends those findings by investigating the effects of IL-1beta and H. pylori on the transcriptional activity of HK{alpha} 5'-deletion constructs. IL-1beta had no consistent, significant effect on HK{alpha} transcriptional activity until the 5' sequence upstream of –206 bp was discarded, whereupon IL-1beta significantly stimulated activity that was lost only when the 5' sequence upstream of –64 bp was discarded (Fig. 1). Evidently, the human HK{alpha} 5'-flanking region between –206 and –64 bp incorporates cis elements positively responsive to transcription factors mobilized by IL-1beta receptor-coupled signaling pathways (see below). The dose-dependent downregulation by H. pylori of transcriptional activities of the same narrowly circumscribed IL-1beta-sensitive HK{alpha} 5'-flanking region indicates that H. pylori infection disrupts mobilization of these transcription factors. These effects of IL-1beta on HK{alpha} promoter activity in transfected AGS cells are clearly inconsistent with an inhibitory role for IL-1beta in HK gene expression. Thus, although H. pylori effected a 27% inhibition of HK{alpha}2179 transcriptional activity, coincubation of transfected cells with H. pylori and IL-1beta returned HK{alpha} transcription to levels attained in the absence of H. pylori (Fig. 5). This effect of IL-1beta is dependent on the presence of an intact 5'-flanking sequence between –2,179 and –177 bp, as shown in Fig. 5 by the failure of IL-1beta to reverse H. pylori-mediated inhibition of HK{alpha}177 and HK{alpha}165 deletion constructs.

The identity of the IL-1beta-responsive, H. pylori-sensitive transcription factor(s) specifically targeting the HK{alpha} promoter has yet to be defined. Homology domain I of annotated human and rat gastric HK{alpha} 5'-flanking sequences (14, 18, 24, 27) includes the transcription initiation site and ~370 bp of the 5'-flanking sequence. Studies (26, 35) of rat homology domain I have associated the induction of HK{alpha} transcription with binding of GATA-4 and GATA-6 to GATA sequence motifs close to HK{alpha} and HKbeta TATA boxes, with concomitant transcriptional activation of both genes. Deletion analysis of HK{alpha} 5'-flanking sequences transfected into canine parietal cells revealed that Sp1 binding between –54 and –45 bp activates constitutive HK{alpha} transcription (23). Protein binding to a 5'-flanking segment between –162 and –156 bp is associated with EGF-induced transcriptional activation of the HK{alpha} gene (14). Definitive evidence for NF-{kappa}B or cAMP response element-binding protein (CREB) regulation of gastric HK{alpha} gene regulation is lacking, although NF-{kappa}B inhibition (43) and CREB activation (41) of murine inner medullary collecting duct HK{alpha}2 gene regulation have been reported. So far, no empirical evidence links activator protein (AP)-2, AP-4, or STAT binding sites to functional transcription of gastric HK{alpha}.

Our demonstration that an intact functional ERK1/2 signaling pathway is necessary to elicit IL-1beta stimulation of HK{alpha} deletion construct activity (Fig. 6) complements and extends the spectrum of AGS cell signaling pathways induced by IL-1beta receptor activation. Thus, IL-1beta has been shown to dose dependently increase IL-8 secretion by AGS cells, and this effect was significantly reduced by the protein tyrosine kinase inhibitors herbimycin A and genistein; protein kinase C inhibition did not reduce IL-1beta stimulation of IL-8 secretion (3). The transcription factor mobilized by IL-1beta and H. pylori to upregulate IL-8 gene expression is known to be NF-{kappa}B (33). Also, IL-1beta has been shown to dose dependently enhance AGS cell proliferation, and this effect was blocked by an IL-1 receptor antagonist (1). IL-1beta-stimulated proliferation was blocked by genistein and by a specific inhibitor (PD-98059) of the ERK1/2 pathway (1). On the other hand, in the case of IL-1beta dose-dependent stimulation of cyclooxygenase-2 mRNA and protein expression levels and prostaglandin E2 production and secretion in AGS cells, activation of both the ERK1/2 and p38 pathways was shown to be required (8). The involvement of the JNK signaling pathway in IL-1beta-mediated events in AGS cells has not been reported.

The concentration at which IL-1beta exerted regulatory effects on the HK{alpha} promoter in this study is the same (10 ng/ml) as that reported to dose dependently inhibit basal and stimulated acid secretion in isolated rabbit parietal cells (2) and to stimulate gastrin release from rabbit antral G cells (39). Comparable IL-1beta concentrations (25 ng/ml) were reported to stimulate maximal IL-8 release by AGS cells (15). In contrast, in isolated rat parietal cells, acid secretion stimulated by histamine or carbachol was maximally inhibited (by 35% and 67%, respectively) by the preincubation of cells with 2.5 pg/ml IL-1beta (32). These differences in IL-1beta potency with respect to acid secretory regulation may reflect species and/or cell-specific differences in signaling mechanisms regulating acid secretion that are impacted by IL-1beta receptor activation. Thus, in rat parietal cells, IL-1beta treatment has been shown to impair an inositol (1,4,5)-trisphosphate-dependent increase in intracellular Ca2+ concentration and had no significant effect on histamine-stimulated cAMP production (32), whereas, in rabbit parietal cells, IL-1beta inhibited forskolin- but not dibutyryl-cAMP-stimulated acid secretion (2), implicating IL-1beta impairment of cAMP generation but no impairment of protein kinase A activity or protein kinase A-mediated downstream effectors.

Although the mechanism by which IL-1beta inhibits acid secretion at the level of parietal cells remains to be clarified, the present study excludes direct IL-1beta-mediated perturbation of routine transcription factor interactions with the HK{alpha} promoter as a potential mechanism, at least in the AGS cell model investigated here. We can conclude, however, that 1) an IL-1beta-dependent cis-activation sequence is located between –206 and –102 bp in the HK{alpha} promoter and that repressor sequences are located between –206 and –340 bp; 2) IL-1beta modulates H. pylori-mediated HK{alpha} promoter inhibition; and 3) IL-1beta exerts HK{alpha} promoter effects through MEK1/2 MAPK signal transduction pathways.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work 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 for the generous provision of plasmids containing human H,K-ATPase {alpha}-subunit 5'-flanking DNA and Dr. Maria Trojanowska for valuable discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Smolka, Medicine/Gastro CSB 921 E, Medical Univ. of South Carolina, 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
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 REFERENCES
 

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