Cdx2 Expression and Intestinal Metaplasia Induced by H. pylori Infection of Gastric Cells Is Regulated by NOD1-Mediated Innate Immune Responses

Abstract

Chronic infection with the bacterial Helicobacter pylori is a major cause of gastric and duodenal ulcer disease, gastric mucosal atrophy, and cancer. H. pylori-induced expression of the intestinal epithelial-specific transcription factor caudal-related homeobox 2 (Cdx2) contributes to intestinal metaplasia, a precursor event to gastric cancer. Given a role for the bacterial pattern recognition molecule nucleotide-binding oligomerization domain 1 (NOD1) in the innate immune response to bacterial infection, we investigated mechanisms used by NOD1 to regulate H. pylori infection and its propensity towards the development of intestinal metaplasia. We found that Cdx2 was induced by H. pylori infection in both normal and neoplastic gastric epithelial cells in a manner that was inversely related to NOD1 signaling. Mechanistic investigations revealed that Cdx2 induction relied upon activation of NF-κB but was suppressed by NOD1-mediated activation of TRAF3, a negative regulator of NF-κB. In vivo, prolonged infection of NOD1-deficient mice with H. pylori led to increased Cdx2 expression and intestinal metaplasia. Furthermore, gastric epithelial cells from these mice exhibited increased nuclear expression of the NF-κB p65 subunit and decreased expression of TRAF3. Overall, our findings illuminated a role for NOD1 signaling in attenuating H. pylori-induced Cdx2 expression in gastric epithelial cells, suggesting a rationale to augment NOD1 signaling in H. pylori-infected patients to limit their risks of accumulating precancerous gastric lesions.

Figure 1

cagPAI⁺ H. pylori induces Cdx2 in human gastric epithelial cells. A, total RNA extracted from GCIY cells infected with 5 × 10⁷ CFU/mL cagPAI⁺ H. pylori (Hp) were subjected to RT-PCR. B and C, cell lysates were extracted from H. pylori (Hp)-infected GCIY cells (B) and GCIY cells stimulated with the indicated H. pylori-related products (C), and were subjected to Western blotting.

Figure 2

H. pylori (Hp) induction of Cdx2 is dependent on NF-κB. A and B, GCIY cells transfected with cdx2-luc or cdx2-N0-luc plasmids with control pRL-TK plasmid were infected with the indicated amount (or 5 ×10⁷ CFU/mL in B) of H. pylori; cell lysates were assessed for luciferase activity. C, nuclear extracts of H. pylori–infected GCIY cells were subjected to EMSA. D, GCIY cells were infected with 5 × 10⁷ CFU/mL H. pylori in the absence or presence of BAY11-7082; cell lysates were subjected to Western blotting. Results shown in A and B indicate means ± SD. *, P < 0.05 as compared with uninfected cells.

Figure 3

NOD1 suppresses Cdx2 expression induced by H. pylori (Hp) infection. A, total RNA extracted from AGS cells transfected with the indicated siRNA and infected with H. pylori was subjected to RT-PCR. B, AGS cells were cotransfected with the indicated siRNA, cdx2-luc reporter vector, and pRL-TK plasmid; cell lysates were subjected to luciferase assay. C, total RNA extracted from GSM06 cell stably expressing either control shRNA or NOD1-shRNA was subjected to qPCR. D, GSM06 cells stably expressing NOD1-shRNA were infected with 5 × 10⁷ CFU/mL H. pylori. Total RNA was extracted and subjected to qPCR. E, GCIY cells were infected with H. pylori with or without preincubation with iE-DAP. Total RNA was extracted and subjected to qPCR. Results shown in B–E indicated as means ± SD. *, P < 0.05 as compared with H. pylori-infected cells transfected with control siRNA (B), control shRNA transfected cells (C and D), and non-pretreated cells (E). AU, arbitrary units.

Figure 4

TRAF3 suppresses NF-κB activation and Cdx2 expression. A, AGS cells transfected with an NF-κB reporter plasmid together with pRL-TK plasmid and cotransfected with either a TRAF3 expression plasmid or TRAF3-siRNA were infected with H. pylori (Hp), and relative luciferase activity was measured. Results, means ± SD. *, P < 0.05 as compared with control plasmid or control siRNA–transfected cells. B, AGS cells transfected with either a control plasmid or a TRAF3 expression plasmid were cultured in either H. pylori–infected or uninfected media. After 24 hours, cell lysates were obtained and subjected to Western blotting.

Figure 5

NOD1 deficiency enhances the formation of gastric intestinal metaplasia during in vivo H. pylori (Hp) infection. A, H&E staining, Alcian blue staining, and immunohistochemistry for Cdx2 of gastric mucosa removed from H. pylori–infected NOD1-deficient and NOD1-intact mice. B, percentage of glands exhibiting intestinal metaplasia (IM) in the stomachs of NOD1-intact and NOD1-deficient mice. C, total RNA extracted from the stomachs of these mice was subjected to qPCR. D, loads of H. pylori in the stomachs of NOD1-intact and NOD1-deficient mice 12 months after initiation of H. pylori infection. E, gastric expression of NF-κB p65 in the stomach of NOD1-intact and NOD1-deficient mice. F, total RNA extracted from the stomachs of chronically infected mice was subjected to qPCR. Results shown in B, C, D, and F indicate means ± SD.*, P < 0.05 as compared with H. pylori–infected NOD1-intact mice. AU, arbitrary units.

Figure 6

Schematic view of the suppressive role of NOD1 on H. pylori infection–induced Cdx2 induction.