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.

Keywords: Helicobacter pylori; TRPV4; gastric epithelium; methylation silencing; transient receptor potential vanilloid 4.

Figure 1

TRPV4 mRNA and protein expression in human gastric tissues and AGS cells. A, TRPV4 and GAPDH mRNA were examined with (+) and without (‐) RT reaction. The expected sizes of the amplified fragments for human TRPV4 and GAPDH were 543 and 983 bp, respectively. TRPV4 mRNA was detected in human gastric tissues but not in AGS cells. B, TRPV4‐ir was observed in human gastric epithelium. The reaction was diminished without anti‐TRPV4 antibody (Ab) or an antigenic peptide (Ag) (absorption experiment). Bars indicate 50 μm

Figure 2

Demethylation assay. A, TRPV4 mRNA was detected in 5Aza‐treated AGS cells. B, Quantitative RT‐PCR (qRT‐PCR) indicated significantly higher TRPV4 mRNA expression in AGS cells treated with another demethylating agent, 5‐aza‐2′‐deoxycytidine (5‐AzaDC, 1 μM), for 1 wk compared to untreated AGS cells (# P<.01 vs AGS). C, Western blotting (WB) analyses revealed a band at approximately 120 kDa that is representative of the expected size for TRPV4 in AGS cells treated with 5Aza but not in untreated AGS cells. (D) Ca²⁺ imaging traces showed no [Ca²⁺]_i responses to the specific TRPV4 agonist GSK (100 nM) in untreated AGS cells and various [Ca²⁺]_i responses to the same stimuli in 5Aza‐treated AGS cells, with significantly larger responses in 5Aza‐treated AGS cells compared to untreated cells (*P<.05 vs AGS). (E) A luciferin–luciferase assay to evaluate ATP release showed that GSK1016790A (GSK, 100 nM) or 5,6‐EET (500 nM) did not enhance ATP release in AGS cells that had not been treated with 5AzaDC. AGS cells responded to a hypotonic solution as a positive control. GSK induced significantly larger ATP release from 5AzaDC‐treated AGS cells than control cells (*P<.05 vs 5AzaDC+ GSK). Pretreatment with the TRPV4 antagonist RN1734 (100 μΜ) inhibited the response (without statistical significance). 5,6‐EET also induced significantly larger ATP release from 5AzaDC‐treated AGS cells than control cells (# P<.01 vs 5AzaDC+ 5,6‐EET), while RN1734 pretreatment significantly inhibited this response. Values were mean + SEM

Figure 3

Methylation‐specific PCR (MSP). A, Black arrows, aqua boxes, and red lines indicate transcriptional orientations, CpG islands, and TRPV4 exons, respectively. CpG islands are present in the human TRPV4 gene, including the promoter region. B, Methylation‐specific PCR analysis of TRPV4 gene methylation in fully unmethylated genes, GenomiPhi (geno) and AGS cells; M = methylated PCR products, U = unmethylated PCR products. TRPV4 methylation was noted in AGS cells but not in geno. C, Incubation with the demethylating agent 5‐azacytidine (5‐Aza, 10 μM) for 1 wk partially demethylated the TRPV4 gene

Figure 4

TRPV4 DNA methylation silencing in the human normal gastric epithelial cell line GES‐1 with Helicobacter pylori coculture. A, TRPV4 mRNA expression levels in GES‐1 with coculture of two different types of H. pylori (NCTC and 193C). Relative expression levels significantly decreased in a time‐dependent manner with coculture of both H. pylori types (*P<.01 vs control). B, Western blotting (WB) indicated that TRPV4 protein expression also decreased with coculture of H. pylori in a time‐dependent manner. C, MSP indicated that methylated PCR products (M) were observed in coculture GES‐1 samples, but not in the control

Figure 5

TRPV4 DNA methylation silencing in Helicobacter pylori‐infected human gastric stomach tissue. A, MSP for the TRPV4 gene in H. pylori‐negative (HP−), H. pylori‐positive (HP+), or H. pylori‐eradicated (erad) gastric epithelium. Methylated PCR products (M) were observed in some HP+ or HP erad samples, but never in HP− samples. B, Methylation rates (%) were significantly higher in samples from HP+ healthy or HP erad individuals compared to HP− subjects (#P<.05 vs healthy [HP−]). C, TRPV4 mRNA expression levels in human gastric biopsy samples. Significantly higher TRPV4 expression was detected in samples from healthy (HP−) and HP erad individuals than in healthy (HP+) subjects (*P<.05 vs HP+). D, TRPV4 expression levels plotted relative to the duration after HP eradication (y) indicated a weak correlation between the time after HP eradication and TRPV4 expression levels (r=.56, P<.05, n=14)