Mechanisms of Helicobacter pylori-induced inhibition of gastric H,K-ATPase expression

Helicobacter pylori infection of the gastric mucosa leads to transient hypochlorhydria that may facilitate development of gastric adenocarcinoma. The H. pylori cytotoxin-associated gene (cag) pathogenicity island (PAI), a genetic locus encoding virulence factors, is known to cause development of pre-cancer phenotype. However, the relationship between the cag PAI and hypochlorhydria is unknown. This dissertation addresses mechanistic aspects of H. pylori-induced H,K-ATPase alpha subunit (HKalpha) repression, testing the general hypothesis that Helicobacter pylori represses gastric acid secretion through cag PAI-mediated NF-kappaB activation leading to repression of HKalpha gene transcription. This study is organized into three Specific Aims. (1) To define specific transcription factors responsible for H. pylori-induced HKalpha repression. Hypothesis. H. pylori -induced NF-kappaB p50 homodimer binding represses HKalpha promoter activity. Gastric adenocarcinoma cells were transiently transfected with HKalpha promoter deletion constructs to locate the HKalpha promoter regions sensitive to H. pylori infection. Using DNase I footprint assay, EMSA and supershift analysis, NF-kappaB p50 but not p65 binding to HKalpha promoter was identified. siRNA knock-down of p50 and p65 followed by transient transfection experiments with HKalpha promoter deletion constructs indicated that p50 homodimer binding causes HKalpha repression. (2) To investigate the role of IL-1beta in H. pylori-induced HKalpha repression. Hypothesis. H. pylori-induced HKalpha transcriptional repression is mediated through IL-1beta secretion. Transient transfection, DNase I footprints, EMSA, supershift experiments and immunoblot experiments showed that IL-1beta activates HKalpha promoter activity through ERK-1/2 mediated Sp1 activation. In addition Sp1 was shown to interact with a Sp1 binding site and with a NF-kappaB binding site of HKalpha promoter. (3) To identify specific H. pylori genes responsible for HKalpha repression. Hypothesis. A functional H. pylori cag PAI is necessary for H. pylori-mediated HKalpha repression. Transiently transfected AGS cells and human gastric biopsies were infected with wild type H. pylori or their corresponding isogenic mutant strains, and HKalpha promoter activity, mRNA level, protein level and acid secretion were measured. The results indicate that the cag PAI specific genes cagA, cagE, cagM, and cagL are necessary for HKalpha repression. The study establishes a mechanistic link between the H. pylori cag PAI and hypochlorhydria, both of which underlie the emergence of gastric adenocarcinoma.