Korean J Gastroenterol.  2016 Oct;68(4):186-194. 10.4166/kjg.2016.68.4.186.

Role of Inhibitory Transforming Growth Factor-β Signal Smad7 in Helicobacter pylori-associated Gastric Damage

• Lee HJ
• Park JM
• Hahm KB

• KMID: 2383514
• DOI: http://doi.org/10.4166/kjg.2016.68.4.186

Abstract

BACKGROUND/AIMS
Transforming growth factor-beta (TGF-β) is a cytokine implicated in the susceptibility, development, and progression of gastrointestinal cancer and certain other neoplasms. In the later stages of cancer, TGF-β not only acts as a bystander of host-immune response, but also contributes to cell growth, invasion, and metastasis. In the current study, we generated gastric mucosal cells that stably express Smad7, and explored the Helicobacter pylori-associated biological changes between mock-transfected and Smad7-transfected RGM1 cells.
METHODS
RGM1 cells stably transfected with Smad7 were infected with H. pylori, and molecular changes in apoptotic markers and inflammatory mediators were examined. Several candidate genes were explored in Smad7-overexpressing cells after H. pylori infection.
RESULTS
Overexpression of Smad7 in RGM1 cells significantly increased the H. pylori-induced cytotoxicity compared to mock-transfected cells. Exaggerated increases in inflammatory mediators, cyclooxygenase 2, inducible NO synthase, and augmented apoptosis were noted in Smad7-overexpressing cells, whereas mitigated heme oxygenase 1 was noted in Smad7- overexpressing cells. These phenomena were reversed in cells transfected with Smad7 siRNA.
CONCLUSIONS
These data suggest that inhibition of Smad7 is a possible target for mitigating H. pylori-associated inflammation.

Keyword

Helicobacter pylori; Transforming growth factor beta; Smad7; Gastritis

MeSH Terms

Apoptosis
Cyclooxygenase 2
Gastritis
Gastrointestinal Neoplasms
Helicobacter pylori
Helicobacter*
Heme Oxygenase-1
Inflammation
Neoplasm Metastasis
Nitric Oxide Synthase
RNA, Small Interfering
Transforming Growth Factor beta
Cyclooxygenase 2
Heme Oxygenase-1
Nitric Oxide Synthase
RNA, Small Interfering
Transforming Growth Factor beta

Figure

• 

  Fig. 1. Cytotoxicity against Helicobacter pylori in Smad7 overexpressed cells. (A) Stable expression of Smad7 in RGM1 cells was detected with Flag-tag (upper) and Smad7 (lower) antibodies. (B) MTT assay in mock- or Smad7-transfected RGM1 cells after treatment with 100 multiplicity of infection (MOI) H. pylori. (C) mock- or Smad7-transfected RGM1 cells were treated with H. pylori (100 MOI) for 24 hours, followed by Annexin V staining. Cells were stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. Bars represent mean±SD (n=3). (D) Western blot analysis of PARP and Bax after treatment with 100 MOI H. pylori in mock-transfected or Smad7-transfected RGM1 cells. (E) Expression of XBP-1, GADD34, CHOP, and Smad7 mRNA after treatment with 100 MOI H. pylori in mock-transfected or Smad7-transfected RGM1 cells.

• 

  Fig. 2. Inflammation mediators against Helicobacter pylori in Smad7 over-expressed cells. (A) Expression of Cox-2, iNOS, TNF-, and Smad7 mRNA after treatment with 100 multiplicity of infection (MOI) H. pylori in mock-transfected or Smad7-transfected RGM1 cells. (B) Western blot analysis of COX-2 and iNOS after treatment with 100 MOI H. pylori in mock-transfected or Smad7-transfected RGM1 cells. (C) Expression of HO-1 and Smad7 mRNA after treatment with 100 MOI H. pylori in mock-transfected or Smad7-transfected RGM1 cells.

• 

  Fig. 3. Inflammation mediators against Helicobacter pylori in Smad7 knock-down cells. (A) Target-specific siRNA for Smad7 (Smad7 siRNA) or a negative control RNA (mock) was transfected into RGM1 cells for 24 hours. Expression of Cox-2 and Smad7 mRNA was checked by RT-PCR. (B) Expression of COX-2 and Smad7 were compared between mock-transfected and Smad7 siRNA-transfected cells. (C) Expression of IL-6, TNF-, and iNOS mRNA were compared between mock-transfected and Smad7 siRNA-transfected cells.

• 

  Fig. 4. Schematic summary. The current study raises hope that inhibiting Smad7 can be a strategy to alleviate Helicobacter pylori-induced gastric damage. ER stress, endoplasmic reticulum stress.

Reference

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