Abstract
Both the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, and infection with Helicobacter pylori are major causes of gastric ulcers. Although some clinical studies suggest that infection with H. pylori increases the risk of developing NSAID-induced gastric lesions, the molecular mechanism governing this effect is unknown. We recently found that in cultured gastric cells, expression of endoplasmic reticulum (ER) chaperones (such as 150-kDa oxygen-regulated protein (ORP150) and glucose-regulated protein 78 (GRP78)) is induced by NSAIDs and confers protection against NSAID-induced apoptosis, which is important in the development of NSAID-induced gastric lesions. In this study we have found that co-culture of gastric cells with H. pylori suppresses the expression of ER chaperones. This suppression was regulated at the level of transcription and accompanied by a reduction in the level of activating transcription factor 6 (ATF6), one of the transcription factors for ER chaperone genes. In vivo, inoculation of mice with H. pylori suppressed the expression of ER chaperones at gastric mucosa both with and without administration of indomethacin. Inoculation with H. pylori also stimulated formation of indomethacin-induced gastric lesions and mucosal cell death. In addition, we found that heterozygous ORP150-deficient mice are sensitive to the development of indomethacin-induced gastric lesions and mucosal cell death. The results of this study suggest that H. pylori exacerbates NSAID-induced gastric lesions through suppression of expression of ER chaperones, which stimulates NSAID-induced mucosal cell death.
Highlights
An inhibitory effect of non-steroidal anti-inflammatory drugs (NSAIDs) on cyclooxygenase (COX) activity and the resulting decrease in the gastric level of prostaglandins (PGs), especially PGE2, was believed to be the only explanation for the gastric side effects of NSAIDs because PGE2 is a strong protective factor for gastric mucosa [10]
We have recently demonstrated that NSAIDs induce apoptosis in cultured gastric mucosal cells and at gastric mucosa in a manner independent of COX inhibition [12,13,14,15,16] and have suggested that both COX inhibition and gastric mucosal apoptosis are required for the formation of NSAID-induced gastric lesions (16 –18)
We show that inoculation of mice with H. pylori suppresses the expression of endoplasmic reticulum (ER) chaperones at gastric mucosa and exacerbates NSAID-induced gastric lesions and mucosal cell death
Summary
Chemicals and Animals—RPMI 1640 and Helicobacter selection agar were obtained from Nissui Pharmaceutical Co (Osaka, Japan). The plasmid pCMVshort-EGFP-ATF6␣ (a gift from Dr Mori, Kyoto University) [42] was transfected into AGS cells, according to the manufacturer’s protocols. Cells were fixed in 4% paraformaldehyde for 20 min, blocked with goat serum for 15 min, incubated for 12 h with antibody against GRP94 in the presence of 2.5% bovine serum albumin before being incubated for 2 h with Alexa Fluor 594 goat anti-rat IgG. Total RNA was extracted from gastric tissues or cultured cells using an RNeasy kit according to the manufacturer’s protocol. Sections were blocked with 2.5% goat serum for 10 min, incubated for 12 h with antibody against ORP150 or GRP78 in the presence of 2.5% bovine serum albumin, and incubated for 2 h with Alexa Fluor 488 goat anti-rabbit immunoglobulin G in the presence of DAPI (5 mg/ml). Differences were considered to be significant for values of p Ͻ 0.05
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