Abstract
Helicobacter pylori (H. pylori) infection causes aberrant DNA methylation of various genes in the gastric mucosa. Cyclooxygenases (COX) play a critical role in peptic ulcer development and healing. Human COX-2 has CpG islands (CGIs) in its promoter region, suggesting a possible epigenetic regulation. Here, we evaluated COX-2 promoter methylation in the gastric mucosa of patients with various gastric diseases and found that COX-2 methylation levels in the gastric mucosa were significantly increased in patients with H. pylori infection. We further investigated the roles of COX-2 during the healing of acetic acid-induced gastric ulcers in H. pylori-infected Mongolian gerbils (MGs). While COX-2 mRNA expression levels on the edges of acetic acid-induced gastric ulcers were significantly increased after ulcer induction in MGs in the absence of H. pylori, no such induction was observed in H. pylori-infected gastric mucosa. Cloning of the MG COX-2 gene revealed abundant CGIs in the promoter region. COX-2 mRNA expression in MG-derived gastric carcinoma MGC2 cells was significantly increased by addition of the demethylating agent 5-Aza-dC. Additionally, COX-2 methylation levels were higher in H. pylori-infected MG gastric mucosa than in control mucosa. These results indicated that epigenetic inhibition of COX-2 mRNA induced by H. pylori impairs gastric ulcer healing.
Keywords
- COX-2
- gastric ulcer healing
- Helicobacter pylori
- methylation
- Mongolian gerbils
- MGC2 cells
1. Introduction
2. COX and the gastric mucosal barrier
In 1971, Vane and colleagues first demonstrated that aspirin and other NSAIDs inhibited the synthesis of PGs by blocking COX activity [4]. COX plays pivotal roles in the gastric mucosal barrier [5, 6]. COX catalyzes the conversion of arachidonic acid to the common precursor prostanoids, prostaglandin (PG) H2, and PGG2. The major PGs produced by the human and rodent gastric mucosa are PGE2 and PGI2, with lesser amounts of PGF2 and PGD2. Each of these metabolites binds to a specific G protein-coupled receptor to trigger intracellular responses. PGs have been shown to accelerate ulcer healing in experimental models and humans [7]. COX exists in two isoforms commonly referred to as COX-1 and COX-2. COX-1 is constitutively expressed in various tissues. In the stomach, prostanoids synthesized via the COX-1 pathway are responsible for cytoprotection of the gastric mucosa and the production of thromboxane by platelets. Although COX-2 is generally expressed at very low levels in healthy tissues, including in the stomach, it is expressed at particularly high levels at sites of inflammation.
It was originally thought that only COX-1 was involved in the gastric mucosal defense system; however, several clinical trials have suggested that a COX-2 selective inhibitor produces lesser, but some, gastrointestinal toxicity compared to traditional NSAIDs [8, 9]. In accordance with this finding, animal studies have suggested that both COX-1 and COX-2 are necessary for gastric mucosal healing, and COX-1 inhibition alone, which can be induced pharmacologically by specific inhibitors or genetically by gene targeting [10], does not cause gastric mucosal injury. It has been shown that a combination of selective COX-1 and COX-2 inhibitors is required to cause hemorrhagic erosion of the gastric mucosa, which is comparable to that observed with indomethacin [11]. Prostanoids produced by COX-2, especially PGE2, enhance cell proliferation. The beneficial effects of PGE2 on gastric ulcer healing in rodents appear to be mediated via the EP4 receptor [12]. In addition, COX-2-derived PG stimulates vascular endothelial growth factor (VEGF) release from gastric fibroblasts, which is an important contributor to ulcer healing [13, 14], likely via stimulation of new blood vessel growth. The increase in COX-2 immunoreactivity that is observed in monocytes, macrophages, fibroblasts, and endothelial cells at the ulcer margin is closely correlated, both temporally and spatially, with the increase in cell proliferation [15]. COX-2 appears to represent a second line of defense that is activated during ulcer healing to compensate for the temporary loss of COX-1 in the mucosa adjacent to the ulcer and assists COX-1 in protecting gastric mucosal integrity. The healing-impairment effect of NSAIDs is also observed with selective COX-2 inhibitors [16].
3. H. pylori infection and COX-2 in gastric mucosa
The pathophysiological roles of COX-2 in
4. Acetic acid-induced gastric ulcer healing and COX mRNA levels in Mongolian gerbils with or without H. pylori infection
Gastric ulcers were produced by injecting 25% of acetic acid (0.03 mL) into the submucosal layer of the gastric wall of the antral-oxyntic border in MGs 48 weeks after inoculation with an
5. Effects of COX-2 methylation on its mRNA expression in MGs in vitro
As discussed above,
6. Cloning the MG COX-2 promoter region
As mentioned above, human
7. Role of COX-2 methylation and H. pylori infection in gastric mucosal healing
Nonselective COX inhibitors damage the gastrointestinal mucosa, and gastrointestinal injury represents the most significant side effect of chronic nonsteroidal anti-inflammatory drug (NSAID) use [34]. Thus, selective COX-2 inhibitors have been developed as ideal anti-inflammatory drugs, devoid of GI toxicity, and clinical trials have suggested that selective COX-2 inhibitors produce less gastrointestinal injury than conventional NSAIDs [8, 9]. In support of this, animal studies have also suggested that, in contrast to the initial concept, the importance of COX-2 in the repair of gastric mucosal damage has been recognized [31, 35]. In rat gastric mucosa, markedly elevated levels of
The release of inflammatory cytokines and recruitment of inflammatory cells have been considered the potential factors for delayed ulcer healing in
8. Conclusions
Acknowledgments
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan to HY.
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