Repressed TGF-β signaling through CagA-Smad3 interaction as pathogenic mechanisms of Helicobacter pylori-associated gastritis

Abstract

Helicobacter pylori (H. pylori) infection causes chronic gastric inflammation, peptic ulceration, and gastric carcinogenesis, in which H. pylori cytotoxin-associated gene A (CagA) plays major pathogenic action. Since transforming growth factor-β (TGF-β) and its signaling also are principally implicated in either modulating gastric mucosal inflammatory responses or causing carcinogenesis and are attenuated after H. pylori infection, we hypothesized that dysregulated Smad signaling and repressed TGF-β might be core pathogenic mechanism for H. pylori-associated gastritis or carcinogenesis. Until now, no precise underlying mechanism how deranged TGF-β signaling developed after H. pylori infection relevant to various clinical manifestations remains unclear. In this study, we examined the molecular mechanism about the inhibition of TGF-β signaling by H. pylori CagA protein. H. pylori CagA significantly suppressed TGF-β/Smad transcriptional responses through critical inhibition of Smad3, though CagA interacted constitutively with Smad2, Smad3, and Smad4. CagA inhibited TGF-β-induced suppression of proinflammatory chemokines, such as IL-8, CXCL1 and CXCL3, as well as TGF-β-induced transcription of target genes. In conclusion, repressed TGF-β signaling associated with CagA-positive H. pylori infection could be an important determinant for the outcome of H. pylori infection. Therefore, TGF-β signaling is one of the important determinants to avoid from H. pylori CagA pathogenicity.

Keywords: Cytotoxin-associated gene A; Helicobacter pylori; Smad; TGF-β; inflammation.

Fig. 1

Infection of H. pylori inhibits TGF-β-induced transcriptional activation. AGS cells transfected with SBE4-Luc (A) or 3TP-Lux (B) were infected with H. pylori [ATCC 43504, CagA (+) strain] for 4 h at different MOIs. Luciferase activity was measured after 16 h of TGF-β1 stimulation. CagA was co-transfected into AGS cells with ARE-Luc together with FAST-1 (C) or BRE-Luc (D). Luciferase activity was measured after 16 h of TGF-β1 or BMP-2 stimulation. Data shown are the mean ± SD of three separate experiments. *p<0.05, **p<0.01.

Fig. 2

CagA inhibits TGF-β-induced transcriptional activation. CagA was co-transfected into AGS cells with SBE4-Luc (A), 3TP-Lux (B). Mutant CagA (ABccc) was co-transfected into AGS cells with SBE4-Luc (C), 3TP-Lux (D). Luciferase activity was measured after 16 h of TGF-β1 stimulation. Data shown are the mean ± SD of three separate experiments. *p<0.05, **p<0.01.

Fig. 3

CagA inhibits Smad transcriptional activity and interacts with Smad proteins. (A) AGS cells were cotransfected with GAL4 fusion constructs together with G5E1b-Lux in the presence or absence of CagA. Cells were treated with or without TGF-β1 for 16 h. Cell lysates were analyzed for luciferase activity. Data shown are the mean ± SD of three separate experiments. *p<0.05 compared to Gal4DBD. **p<0.05 compared to Gal4-Smad. (B) HEK293T cells were transfected with HA-tagged CagA and Flag-tagged Smad2, Smad3 or Smad4. Protein lysates were immunoprecipitated with anti-HA and immunoblotted with anti-Flag antibody. (C) HEK293T cells were transfected with Flag-tagged Smad3 and HA-tagged CagA. CagA was detected in Smad3 immunoprecipitates and vice versa.

Fig. 4

CagA interacts with MH2 domain of Smad3. (A) Schematic drawings of Smad3 truncation mutants. (B) HEK293T cells were transfected with Flag-Smad3 deletion mutants and with HA-CagA. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Flag antibody.

Fig. 5

CagA inhibits TGF-β-induced nuclear translocation of Smad3 and formation of Smad3-Smad4 complexes. (A) AGS cells transfected with HA-tagged CagA were treated with TGF-β1 for 1 h. Cell lysates were analyzed by immunoblotting. (B) AGS cells were transfected with Flag-Smad3 and HA-CagA, and then treated TGF-β1 for 1 h. Cells were fixed in 4% PFA, and then stained with anti-Flag or anti-HA antibody followed by Alexa Fluor goat anti-mouse or anti-rabbit IgG. (C) AGS cells were transfected with HA-CagA, and treated TGF-β1 for 1 h. Cell lysates were subjected to immunoprecipitation using anti-Smad3 antibody, followed by immunoblotting with anti-Smad4 antibody.

Fig. 6

CagA antagonizes TGF-β-induced expression of target genes. AGS cells were transfected with HA-CagA, and then treated TGF-β1 for 8 h. Total RNA was isolated and the mRNA expression was analyzed by RT-PCR. (A) CagA represses TGF-β-induced expression of PAI-1, fibronectin, Id1 and Smad7. (B) CagA abrogates TGF-β-induced suppression of IL-8, and chemokines CXCL1, CXCL2 and CXCL3. (C) Schematic summary to explain repressed TGF-β, which is pivotal cancer-suppressive cytokine, through Smads interaction with H. pylori CagA, can be responsible for H. pylori-associated gastritis as well as gastric cancer.