Transient receptor potential vanilloid 4 (TRPV4) silencing in Helicobacter pylori‐infected human gastric epithelium

Abstract Background Helicobacter pylori (HP) infection induces methylation silencing of specific genes in gastric epithelium. Various stimuli activate the nonselective cation channel TRPV4, which is expressed in gastric epithelium where it detects mechanical stimuli and promotes ATP release. As CpG islands in TRPV4 are methylated in HP‐infected gastric epithelium, we evaluated HP infection‐dependent changes in TRPV4 expression in gastric epithelium. Materials and Methods Human gastric biopsy samples, a human gastric cancer cell line (AGS), and a normal gastric epithelial cell line (GES‐1) were used to detect TRPV4 mRNA and protein expression by RT‐PCR and Western blotting, respectively. Ca2+ imaging was used to evaluate TRPV4 ion channel activity. TRPV4 methylation status was assessed by methylation‐specific PCR (MSP). ATP release was measured by a luciferin‐luciferase assay. Results TRPV4 mRNA and protein were detected in human gastric biopsy samples and in GES‐1 cells. MSP and demethylation assays showed TRPV4 methylation silencing in AGS cells. HP coculture directly induced methylation silencing of TRPV4 in GES‐1 cells. In human samples, HP infection was associated with TRPV4 methylation silencing that recovered after HP eradication in a time‐dependent manner. Conclusion HP infection‐dependent DNA methylation suppressed TRPV4 expression in human gastric epithelia, suggesting that TRPV4 methylation may be involved in HP‐associated dyspepsia.

promoters can lead to their silencing. In contrast, CpG hypomethylation has been associated with gene overexpression. Recent studies suggested that H. pylori (HP) infection induces methylation silencing 4,5 and that HP eradication would decrease DNA methylation in a gene-specific manner. 6 The transient receptor potential vanilloid 4 channel (TRPV4) is a nonselective cation channel that is involved in various cellular functions 7 and is activated by several physical stimuli, such as heat and mechanical, as well as endogenous or exogenous stimuli (eg, endogenous arachidonic acid metabolites, 5,6-EET) 8 and the specific agonist GSK1016790A. 9 TRPV4 is widely expressed throughout the gastrointestinal epithelium, including the esophagus, intestine, and gastric epithelium where it contributes to adenosine triphosphate (ATP) release via exocytosis. 10, 11 We recently reported that TRPV4 is expressed in mouse and rat gastric epithelium and contributes to ATP release and gastric emptying. 12 However, to the best of our knowledge, to date, there are no reports on TRPV4 expression analysis in human gastric epithelium.
Recent studies revealed that TRPV4 function is affected by gain-or loss-of-function mutations, membrane trafficking, or gain of channel function itself. 7,[13][14][15][16] In addition, TRPV4 expression is downregulated in some cancers by an unknown mechanism. 17,18 However, epigenetic modulations of TRPV4 expression have not been reported. We thus hypothesized that gastric TRPV4 expression is suppressed by DNA methylation associated with HP infection.

| Cell lines
The AGS cancer cell line (CCL-248; American Type Culture Collection, Manassas, VA, USA) was cultured in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, and 100 U/mL penicillin. AGS cells were maintained in a humidified incubator at 37°C. The GES-1 gastric epithelial cell line was obtained from The University of Texas at Austin. GES-1 cells are derived from a human nontumorigenic gastric mucosa epithelium and immortalized via SV40. 19 GES-1 cells were maintained in RPMI supplemented with 10% fetal bovine serum, 1% glutamate, and 1% penicillin-streptomycin.

| Bacterial strains and culture, coculture conditions
The HP strain 193C originating from a patient with gastric cancer 20 and the HP strain NCTC developed from an ATCC strain at Yamaguchi University Hospital, Japan, 21 were used in this study. HP were cultured in Brucella broth medium (BB) supplemented with 10% fetal bovine serum (FBS) under microaerophilic conditions (5% O 2 , 10% CO 2 , and 85% N 2 at 37°C; Sanyo-Multigas Incubator; SANYO Electric Co., Ltd. Tokyo, Japan) with 100% humidity on a gyratory shaker (Thermo-shaker; Thermonics, Tokyo, Japan) at 160 reciprocations per min. The formula wherein absorbance of 0.1=10 8 bacteria/mL was used to estimate the concentration of bacteria in each culture.
GES-1 and HP coculture was carried out as described previously. 22 Briefly, GES-1 cells were seeded onto 10-cm culture dishes and grown for 24 hours; these cultures were then washed with phosphatebuffered saline (PBS) three times before coculture. Fresh RPMI 1640 medium without antibiotics or FBS was added 1 hour before addition of HP. HP was cultured overnight in BB-FBS 10% under the conditions described above and then washed twice with PBS. Bacteria were then directly added to the gastric cells at a bacterium/cell ratio of 50:1 for the indicated times.

| Human gastric biopsy samples
We obtained gastric biopsy samples from healthy individuals with/ without H. pylori infection or patients in whom HP was successfully eradicated through esophagogastroduodenoscopy at Toyama University Hospital in Japan. HP infection was defined as more than one positive result for antisera, urease assay, or microscopic evaluation, and no infection was defined as at least two negative results and no obvious atrophic gastritis, which was judged by two endoscopists certified by the Japanese Gastroenterological Endoscopy Society.
Tissue sample analysis procedures were approved by the University of Toyama human subjects committee, and written informed consent was obtained from all individuals. We enrolled individuals between ages 37 and 77 to minimize background factors among the three groups (HPnegative, HP-positive, and HP-eradicated groups) ( Table 1).

| Reverse transcription PCR analysis
RT-PCR was performed as previously described. 10

| Immunochemistry and Western blotting
Immunochemistry and Western blotting were performed as previously described 10 using the antibodies summarized in Table S2.
Human gastric (corpus) biopsies were fixed at 4°C for 6 hours. Tissues  (Table S2). Immunopositive bands were visualized with the ECL system (Thermo Fisher Scientific, MA, USA). were calculated by subtracting the mean basal values from peak values.

| TRPV4 expression in normal human gastric epithelium and silencing in gastric cancer cells
Given that TRPV4 was shown to be expressed in the esophagus and colon epithelium, and that we recently showed that TRPV4 is expressed in mouse and rat gastric epithelia 12 and is suppressed in several cancers, we examined TRPV4 mRNA expression in human gastric biopsy samples as well as AGS cells. 10 jsp)562bp. We next examined TRPV4 protein expression in human gastric epithelium using immunohistochemistry and an absorption experiment. Our results confirmed that TRPV4 protein was indeed expressed in human gastric epithelium ( Figure 1B).

| Demethylation assay
As recent studies suggested that H. pylori infection induced methylation silencing in gastric epithelium and gastric cancer cells, 4,5 we examined the effect of the demethylating agent 5-azacytidine (5-Aza, 10 μM) or 5-AzaDC (1 μM) on TRPV4 mRNA expression in AGS cells and found that TRPV4 expression could be recovered with both demethylating agents (Figure 2A,B). TRPV4 protein expression was also recovered with 5Aza ( Figure 2C). These results suggested that TRPV4 expression might be suppressed by a DNA methylationdependent mechanism. We further examined whether the demethylating agents could functionally recover Ca 2+ influx and ATP release in the presence of the specific TRPV4 agonist GSK1016790A (GSK) 9 using a fluorescent Ca 2+ imaging system and luciferin-luciferase assay, respectively. Ca 2+ imaging showed significantly larger AGS cell responses to GSK (100 nM) following 5Aza treatment ( Figure 2D).
AGS cells showed no ATP release in response to GSK or 5,6-EET, but had a normal response to the hypotonic solution that served as a positive control. Meanwhile, ATP release was significantly enhanced by prior 5Aza treatment, with rates that were similar to the response seen with hypotonic solution. The endogenous TRPV4 activator 5,6-EET induced ATP release that could be inhibited by pretreatment with the TRPV4 antagonist RN1734 (10 μM) ( Figure 2E), suggesting that 5,6-EET-induced ATP release was mediated via TRPV4. These data strongly indicated that functional TRPV4 expression could be recovered in AGS cells presumably via a demethylation-dependent mechanism.

| Methylation-specific PCR in AGS cells
Based on recent studies suggesting that H. pylori infection induces methylation silencing in gastric epithelium and gastric cancer cells, 4,5 together with the presence of CpG islands in human TRPV4 ( Figure 3A) and our result showing that demethylating agents could recover functional TRPV4 expression in AGS cells (Figures 1 and 2), we hypothesized that TRPV4 expression in human gastric epithelial cells can be  (Table S2). MSP data clearly showed that TRPV4 is methylated in AGS cells, but not in the fully unmethylated gene, GenomiPhi (negative control) ( Figure 3B). We next evaluated the demethylating effect of 5Aza (10 μM) on gene methylation status and found that TRPV4 was partially demethylated in 5Aza-treated AGS cells (Fig. 3C). These results showed that TRPV4 suppression could be mediated by DNA methylation in gastric epithelial cells.

| HP-induced direct methylation silencing of TRPV4 channel activity in normal human gastric epithelial cells
Next, we evaluated the direct influence of HP coculture on TRPV4 methylation silencing in the noncancerous human gastric epithelial cell line GES-1. Coculture of GES-1 cells with HP (NCTC or 193C) gradually decreased TRPV4 mRNA expression levels ( Figure 4A).
TRPV4 protein levels in GES-1 cells also decreased after 96 hours of HP coculture ( Figure 4B). MSP indicated that TRPV4 in GES-1 cells was originally fully unmethylated, but after 96 hours of HP coculture, TRPV4 methylation was induced ( Figure 4C). These data clearly showed that HP had direct consequences on TRPV4 methylation silencing in human gastric epithelium.

| TRPV4 methylation silencing in human biopsy samples with H. pylori infection
To examine the association between HP infection and TRPV4 methylation silencing in human stomach tissue, we compared TRPV4 methylation status and mRNA expression levels in human biopsy samples obtained from HP-negative (−) and HP-positive (+) healthy individuals, as well as in tissues from patients where HP infection was successfully eradicated (HP erad; patient characteristics are summarized in Table 1). Methylation-specific PCR products (M) were observed in HP+ or HP erad samples, but never in HP-samples ( Figure 5A). Similarly, the methylation rate (%) was significantly higher in samples obtained from HP+ individuals or HP erad patients than HP-individuals ( Figure 5B, P<.05). Meanwhile, significantly lower TRPV4 mRNA expression was noted in samples obtained from HP+ compared to HP-or HP erad patients ( Figure 5C). Plotting TRPV4 expression levels relative to the duration after HP eradication showed a weak proportional relationship ( Figure 5D, n=15, r=.56, P<.05).

| DISCUSSION
We showed TRPV4 expression in human gastric epithelium and the noncancerous gastric epithelial cell line, GES-1, as well as methylationdependent gene silencing in the human gastric cancer cell line, AGS ( Figure 1-3). We also showed that TRPV4 in GES-1 could be directly silenced with HP coculture (Figure 4). Furthermore, TRPV4 suppression in HP-infected individuals might be recovered by HP eradication presumably in a time-dependent manner ( Figure 5).
While there is a growing amount of evidence concerning TRPV4 channelopathies (a heterogeneous group of disorders resulting from ion channel dysfunction), 16 and particularly for hereditary mutations in channel genes that alter channel function, to our knowledge, this is the first report that shows ion channel gene suppression via a HPinduced DNA methylation silencing mechanism.
We previously reported that TRPV4 is expressed in mouse and rat gastric epithelium where it contributes to ATP release and gastric emptying. 12 Current data suggested that HP-infected individuals ex- with a need-to-treat (NNT) number of 15 25 and recent higher resonance rates (NNT: 6) reported in Asian countries. 26,27 More recently, HP-positive dyspepsia patients are diagnosed as having HP-associated dyspepsia (HpD) if successful eradication is followed by long-term sustained remission (6 months or longer). 3,28,29 Although HP infection is not directly related to gastric emptying in human studies, [30][31][32][33][34] several reports demonstrated that symptoms were resolved after HP eradication with normalization of gastric emptying 35,36 .This apparent contradiction could be explained with the thinking that gradual motor dysfunction can be compensated but rapid recovery is more easily detectable. We evaluated gastric biopsy samples from asymptomatic individuals, and our data limit the comparison of TRPV4 expression levels and DNA methylation rates in different individuals. In future studies, HpD patients should be targeted for prospective evaluation, especially in Asian countries. 37 Our results suggest that TRPV4 methylation silencing could be induced not only in other inflammatory conditions in the stomach but also in other cell types. For instance, in the bladder, skin, and muscle, as well as tissues affected by skeletal and neuronal disorders, acquired TRPV4 DNA methylation silencing might be a diagnostic and therapeutic target to treat these disorders. [16][17][18] In conclusion, we showed that TRPV4 is functionally expressed in human gastric epithelium and contributes to ATP release. TRPV4