**3.** *H. pylori* **infection and COX-2 in gastric mucosa**

individuals infected with *H. pylori* remain asymptomatic throughout their life, essentially all infected individuals develop chronic inflammation. Patients with antral-predominant gastritis are predisposed to duodenal ulcers, while patients with corpus-predominant gastritis and multifocal atrophy are more likely to have gastric ulcers. Eradication of *H. pylori* drastically lowers the recurrence of *H. pylori*-associated peptic ulcers. In addition, the observed delayed ulcer healing has been reported to involve *H. pylori*-induced inflammation, increased apoptosis of epithelial cells at the ulcer margin, overexpression of inflammatory cytokines, and reduced gastric microcirculation [2]. Cyclooxygenase (COX) is a membrane-bound glycoprotein that functions as the rate-limiting enzyme in prostaglandin (PG) synthesis. PGs increase the resistance of the gastric mucosa to injury by downregulating inflammatory responses. Two major COX isoforms have been identified, COX-1, which is constitutively expressed and considered a housekeeping enzyme, and COX-2, whose mRNA levels rise rapidly in response to inflammatory and mitogenic stimuli. There is a known synergism between *H. pylori* infection and nonsteroidal anti-inflammatory drug (NSAID) use in the development of peptic ulcers and ulcer bleeding [3]. These findings indicate possible interactions between *H. pylori* infection, COX activity, and ulcerogenesis. Aberrant methylation of 5′-CpG islands (CGIs) has been implicated in the transcriptional silencing of a wide range of genes involved in various diseases, such as cancer. Human *COX-2* has CG-rich CGIs in its promoter region, which suggests epigenetic regulation. Here, we report and discuss our recent results on the epigenetic regulation of *COX-2* activity in *H. pylori*-infected gastric mucosa of humans and Mongolian gerbils (MGs), and the

possible relationship between *COX-2* methylation and delayed gastric ulcer healing.

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 precur-

, and PGG<sup>2</sup>

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

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

and PGI<sup>2</sup>

. The major PGs produced by the human

and PGD<sup>2</sup>

. Each

, with lesser amounts of PGF<sup>2</sup>

**2. COX and the gastric mucosal barrier**

sor prostanoids, prostaglandin (PG) H<sup>2</sup>

and rodent gastric mucosa are PGE<sup>2</sup>

inflammation.

208 Chromatin and Epigenetics

The pathophysiological roles of COX-2 in *H. pylori*-infected gastric mucosa are intriguing. *H. pylori* has been implicated as an inducer of COX-2 in the stomach [17–19]. In addition, COX-2 expression is elevated within *H. pylori*-induced gastritis and malignant lesions [20], and *H. pylori* induced COX-2 expression and enhanced PGE<sup>2</sup> production in a human gastric carcinoma cell line. Both *H. pylori* infection and NSAID use independently and significantly increase the risk of peptic ulcers and ulcer bleeding. While COX-2 is necessary for gastric mucosal healing [21], *H. pylori* infection is the leading cause of gastric ulceration. To understand the bimodal effects of *H. pylori* infection on COX-2 induction during ulcer healing, we explored the effects of epigenetic regulation and *H. pylori* infection on the induction of COX-2 *in vivo* and *in vitro.*

*H. pylori* infection causes aberrant DNA methylation of various genes in the gastric mucosa, including *COX-2* [22–25]. Human *COX-2* has CGIs in its promoter region. Thus, we compared *COX-2* promoter methylation levels in the gastric mucosa in *H. pylori*-positive and *H. pylori*-negative cases [26]. As mentioned above, in qualitative experiments, *COX-2* gene promoter methylation levels were significantly higher in *H. pylori*-positive cases than in *H. pylori*-negative cases (**Figure 1**). *COX-2* promoter methylation levels were significantly lower in patients with *H. pylori* eradication than in those with *H. pylori* infection. We then investigated the effects of *COX-2* promoter methylation on *COX-2* mRNA expression *in vitro* using the human gastric adenocarcinoma cell line Kato III, in which the *COX-2* promoter is densely methylated [22]. *COX-2* mRNA expression was not observed in these cells, despite the addition of the protein kinase C stimulator α-phorbol 12,13-dibutyrate (PDBu). However, *COX-2* expression was observed after the addition of the demethylating agent 5-Aza-dC, and expression was enhanced by adding PDBu (**Figure 2**) [26]. These results indicate that *H. pylori* infection causes reversible *COX-2* promoter methylation in the gastric mucosa, and that *COX-2* mRNA expression is regulated through an epigenetic mechanism.

**4. Acetic acid-induced gastric ulcer healing and** *COX* **mRNA levels** 

*H. pylori* infection in humans is best modeled in Mongolian gerbils (MGs), and chronic infection with *H. pylori* induces inflammatory cell infiltration in the gastric mucosa in MGs (**Figure 3A**). It was previously shown that *H. pylori* infection significantly delayed acetic acid-induced ulcer healing in mice and MGs [27–29]. *H. pylori* infection induces aberrant DNA methylation of several CGIs in MGs [30]. As described above, COX-2-derived PGs are important for gastric ulcer healing, and the *COX-2* promoter is densely methylated in the human gastric mucosa in the presence of *H. pylori* infection. To investigate the roles of *COX-2*

Role of *COX-2* Promoter Methylation and *Helicobacter pylori* Infection in Impaired Gastric Ulcer…

http://dx.doi.org/10.5772/intechopen.79973

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**Figure 3.** A. Microscopic features of *H. pylori*-infected gastric mucosa of Mongolian gerbils (MGs). Sections were stained with hematoxylin and eosin. B. Gross appearance 10 days after gastric ulcer induction of *H. pylori*-infected MGs. C. Microphotograph 10 days after gastric ulcer induction in *H. pylori*-infected MGs. D. Serial changes in acetic acidinduced gastric ulcer areas in MGs with or without *H. pylori* infection. *H. pylori* (ATCC43504; American Type Culture Collection, Rockville, MD) was grown in Brucella broth (Becton Dickinson, Cockeysville, MD) containing 10% v/v horse

(MGs/Sea) were purchased from Kyudo (Saga, Japan). At 11 weeks of age, *H. pylori* (0.8 mL samples of Brucella broth

Gastric ulcers were induced experimentally in MGs according to the method described by Wang et al. [40]. Briefly, after anesthetization with ketalar, the abdomen was opened through a midline incision, and 50 μL of 25% acetic acid was injected in the subserosa of the anterior wall of the stomach. The MGs were killed at5, 10, and 20 days after ulcer induction, and the stomach was dissected and removed. The maximum and minimum diameters of the ulcers were measured, and the ulcer area, which was approximately elliptical, was calculated and was compared between MGs with and without *H. pylori* infection. Values are the mean ± SE. The animal care committee of St. Marianna University approved the experimental design, and the animals were cared for in accordance with institutional guidelines.

colony-forming units) was delivered intragastrically using an oral catheter after fasting for 24 h.

) and high humidity with shaking (150 rpm). Male MGs

serum for 40 h at 37°C under microaerobic conditions (15% CO2

containing 1.0 × 109

**in Mongolian gerbils with or without** *H. pylori* **infection**

**Figure 1.** *COX-2* DNA methylation levels in patients with or without *H. pylori* infection, and patients previously with successfully eradicated *H. pylori* infection. The figure is modified from Ref. [26].

**Figure 2.** Effects of a PKC stimulator (α-phorbol 12,13-dibutyrate; PDBu) on COX-2 mRNA expression with or without 5-aza-dC in the human gastric adenocarcinoma cell line KATO-III. KATO-III cells were treated with vehicle (1 μmol/L) with or without 5-Aza-dC for 5 days. The figure is modified from Ref. [26].
