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Helicobacter pylori ‐induced inhibition of H,K‐ATPase α subunit gene expression is mediated by NF‐κB
Author(s) -
Saha Arindam,
Hammond Charles E,
Smolka Adam J
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1317-a
Subject(s) - microbiology and biotechnology , transfection , reporter gene , nf κb , biology , protein subunit , gene expression , gene , chemistry , signal transduction , biochemistry
H. pylori inhibits H,K‐ATPase α subunit (HKα) gene expression in gastric epithelial cells, and is also known to activate NF‐κB which up‐regulates IL‐8 gene expression. We tested the hypothesis that H. pylori ‐induced transcriptional inhibition of HKα is mediated by NF‐κB. Human gastric adenocarcinoma (AGS) cell nuclear extracts showed an H. pylori ‐dependent (25 MOI) consensus NF‐κB‐binding DNA sequence:protein complex by EMSA. NF‐κB p50 antibody supershifts confirmed the protein to be NF‐κB. H. pylori infection of AGS cells transfected with an NF‐κB‐responsive promoter‐Luc reporter construct caused a 48% increase in promoter activity, indicating increased NF‐κB functional activation in response to H. pylori . DNase‐I footprinting identified two NF‐κB binding sites (‐205 to ‐187 nt and −170 to −117 nt) on the HKα 5′‐flanking sequence. AGS cell transfection of a 226 bp HKα promoter‐Luc reporter construct encompassing the NF‐κB footprint region caused an H. pylori ‐dependent 54% inhibition of HKα promoter activity, which was blocked by 10 mM PDTC or 50 μM PD‐98059 but not with 10 μM SB‐203580 or 100 nM JNK Inhibitor II. We conclude that H. pylori augments NF‐κB functional activation in gastric epithelial cells; that NF‐κB binds to two sites on HKα 5′‐flanking sequence; and that H. pylori ‐mediated transcriptional down‐regulation of HKα occurs through ERK‐1/2‐dependent NF‐κB activation. (Supported by NIH DK64371 to AJS)