title: Helicobacter pylori-induced H,K-ATPase alpha-subunit gene repression is mediated by NF-kappaB p50 homodimer promoter binding.
abstract: Infection of human gastric body mucosa by the gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and a transitory hypochlorhydria that progresses in approximately 2% of patients to atrophic gastritis, dysplasia, and gastric adenocarcinoma. We have previously shown that H. pylori infection of cultured gastric epithelial cells (AGS) represses the activity of the transfected alpha-subunit (HKalpha) promoter of H,K-ATPase, the parietal cell enzyme mediating acid secretion. However, the mechanistic details of H. pylori-mediated repression of HKalpha and ensuing hypochlorhydria are unknown. H. pylori is known to upregulate the transcription factor NF-kappaB through the ERK1/2 MAPK pathway. We identified NF-kappaB-binding regions in the HKalpha promoter and found that H. pylori inoculation of AGS cells increased NF-kappaB p50 binding to the transfected HKalpha promoter and repressed its transcriptional activity. Immunoblot and DNA-protein interaction studies showed that although active phosphorylated NF-kappaB p65 is present in H. pylori-infected AGS cells, an NF-kappaB p50/p65 heterodimeric complex fails to bind to the HKalpha promoter. Point mutations at -159 and -161 bp in the HKalpha promoter NF-kappaB binding sequence prevented binding of NF-kappaB p50 and prevented H. pylori repression of point-mutated HKalpha promoter activity in transfected AGS cells. Small interfering RNA-mediated knockdown of NF-kappaB p50 in H. pylori-infected AGS cells also abrogated H. pylori-induced HKalpha repression, whereas NF-kappaB p65 knockdown did not. We conclude that H. pylori inhibits HKalpha gene expression by ERK1/2-mediated NF-kappaB p50 homodimer binding to the HKalpha promoter. This study identifies a novel pathogen-dependent mechanism of H,K-ATPase inhibition and contributes to understanding of H. pylori pathophysiology.
authors.affiliation: Department of Medicine, Medical University of South Carolina, Charleston, SC 29425, USA.
authors.forename: Arindam
authors.initials: A
authors.lastname: Saha
authors.forename: Charles E
authors.initials: CE
authors.lastname: Hammond
authors.forename: Maria
authors.initials: M
authors.lastname: Trojanowska
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Smolka
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D016481:Helicobacter Infections / Q000201:enzymology* / Q000235:genetics*; D016480:Helicobacter pylori; D006801:Humans; D007202:Indicators and Reagents; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D008969:Molecular Sequence Data; D052002:NF-kappa B p50 Subunit / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D013274:Stomach Neoplasms / Q000473:pathology; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D007202:Indicators and Reagents; D052002:NF-kappa B p50 Subunit; D034741:RNA, Small Interfering; D020928:Mitogen-Activated Protein Kinases; D003850:Deoxyribonuclease I; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase