The impact of the rs8005161 polymorphism on G protein-coupled receptor GPR65 (TDAG8) pH-associated activation in intestinal inflammation

Abstract

Background: Tissue inflammation in inflammatory bowel diseases (IBD) is associated with a decrease in local pH. The gene encoding G-protein-coupled receptor 65 (GPR65) has recently been reported to be a genetic risk factor for IBD. In response to extracellular acidification, proton activation of GPR65 stimulates cAMP and Rho signalling pathways. We aimed to analyse the clinical and functional relevance of the GPR65 associated single nucleotide polymorphism (SNP) rs8005161.

Methods: 1138 individuals from a mixed cohort of IBD patients and healthy volunteers were genotyped for SNPs associated with GPR65 (rs8005161, rs3742704) and galactosylceramidase (rs1805078) by Taqman SNP assays. 2300 patients from the Swiss IBD Cohort Study (SIBDC) were genotyped for rs8005161 by mass spectrometry based SNP genotyping. IBD patients from the SIBDC carrying rs8005161 TT, CT, CC and non-IBD controls (CC) were recruited for functional studies. Human CD14+ cells were isolated from blood samples and subjected to an extracellular acidic pH shift, cAMP accumulation and RhoA activation were measured.

Results: In our mixed cohort, but not in SIBDC patients, the minor variant rs8005161 was significantly associated with UC. In SIBDC patients, we observed a consistent trend in increased disease severity in patients carrying the rs8005161-TT and rs8005161-CT alleles. No significant differences were observed in the pH associated activation of cAMP production between IBD (TT, CT, WT/CC) and non-IBD (WT/CC) genotype carriers upon an acidic extracellular pH shift. However, we observed significantly impaired RhoA activation after an extracellular acidic pH shift in IBD patients, irrespective of the rs8005161 allele.

Conclusions: The T allele of rs8005161 might confer a more severe disease course in IBD patients. Human monocytes from IBD patients showed impaired pH associated RhoA activation upon an acidic pH shift.

Keywords: Acidic pH; CD; IBD; Inflammatory bowel diseases; RhoA; UC; cAMP; pH-sensing.

Fig. 1

Formation of cAMP in human CD14+ monocytes upon pH shift from pH 7.6 to pH 6.5. 10 μM of G protein-coupled receptor 65 (GPR65) antagonist was used when indicated (+). Human CD14+ cells were obtained from IBD patients carrying either rs8005161 TT, CT or WT/CC genotype, and non-IBD and WT/CC genotype. Produced cAMP was calculated as a ratio of the respective condition relative to pH 7.6 plus inhibitor. No significant differences between the genotypes were identified. cAMP: cyclic adenosine monophosphate, IBD: inflammatory bowel disease, WT: Wild type

Fig. 2

Formation of RhoA in human CD14+ monocytes upon pH shift from pH 7.6 to pH 6.6. a Activated GTPase RhoA in human CD14+ monocytes upon pH shift from pH 7.6 to pH 6.6 (n = 6). Carriers of rare TT genotype showed the lowest level of RhoA activation compared to heterozygous CT, WT/CC or healthy WT/CC subjects. b Significantly decreased activation of GTPase RhoA in CD14+ monocytes of IBD (WT/CC, CT, TT) patients compared to non-IBD (WT/CC) genotype carriers upon pH shift 7.6 to 6.6. Data points are normalized to pH 7.6 condition (n = 6 non-IBD, n = 18 IBD). Each dot represents a single patient, one-way ANOVA, t-test, * p < 0.05. No significant differences between carriers of different alleles were identified. IBD: inflammatory bowel disease, WT: Wild type