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

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.

The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment.

The pH-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1; GPR68) is expressed in the gut. Inflammatory bowel disease is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated, pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration, and proliferation were measured using electric cell-substrate impedance-sensing technology. Localization of the tight junction proteins zonula occludens protein 1 and occludin and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability and protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant upregulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. The deconstruction of OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms.

Lack of the pH-sensing Receptor TDAG8 [GPR65] in Macrophages Plays a Detrimental Role in Murine Models of Inflammatory Bowel Disease.

BACKGROUND: Tissue inflammation in inflammatory bowel diseases [IBD] is associated with local acidification. Genetic variants in the pH-sensing G protein-coupled receptor 65, also known as T cell death-associated gene 8 [TDAG8], have been implicated in IBD and other autoimmune diseases. Since the role of TDAG8 in intestinal inflammation remains unclear, we investigated the function of TDAG8 using murine colitis models. METHODS: The effects of TDAG8 deficiency were assessed in dextran sodium sulphate [DSS], IL-10-/-, and T cell transfer colitis murine models. RNA sequencing of acidosis-activated TDAG8-/- and wild-type [WT] peritoneal macrophages [MΦs] was performed. RESULTS: mRNA expression of IFN-γ, TNF, IL-6, and iNOS in TDAG8-/- mice increased significantly in colonic lymphoid patches and in colonic tissue in acute and chronic DSS colitis, respectively. In transfer colitis, there was a trend towards increased IFN-γ, iNOS, and IL-6 expression in mice receiving TDAG8-/- T cells. However, absence of TDAG8 did not lead to changes in clinical scores in the models tested. Increased numbers of infiltrating MΦs and neutrophils, but not CD3+ T cells, were observed in DSS-treated TDAG8-/- mice. No differences in infiltrating CD3+ T cells were observed between mice receiving TDAG8-/- or WT naïve T cells in transfer colitis. RNA sequencing showed that acidosis activation of TDAG8 in MΦs modulated the expression of immune response genes. CONCLUSIONS: TDAG8 deficiency triggers colonic MΦ and neutrophil infiltration, and expression of pro-inflammatory mediators in DSS colitis models. In transfer colitis, mice receiving TDAG8-/- T cells presented a significantly higher spleen weight and a tendency towards increased expression of pro-inflammatory markers of monocyte/MΦ activity.

The ESRP1-GPR137 axis contributes to intestinal pathogenesis.

Aberrant alternative pre-mRNA splicing (AS) events have been associated with several disorders. However, it is unclear whether deregulated AS directly contributes to disease. Here, we reveal a critical role of the AS regulator epithelial splicing regulator protein 1 (ESRP1) for intestinal homeostasis and pathogenesis. In mice, reduced ESRP1 function leads to impaired intestinal barrier integrity, increased susceptibility to colitis and altered colorectal cancer (CRC) development. Mechanistically, these defects are produced in part by modified expression of ESRP1-specific Gpr137 isoforms differently activating the Wnt pathway. In humans, ESRP1 is downregulated in inflamed biopsies from inflammatory bowel disease patients. ESRP1 loss is an adverse prognostic factor in CRC. Furthermore, generation of ESRP1-dependent GPR137 isoforms is altered in CRC and expression of a specific GPR137 isoform predicts CRC patient survival. These findings indicate a central role of ESRP1-regulated AS for intestinal barrier integrity. Alterations in ESRP1 function or expression contribute to intestinal pathology.

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein-Coupled Receptor OGR1 (GPR68).

BACKGROUND & AIMS: A novel family of proton-sensing G-protein-coupled receptors, including ovarian cancer G-protein-coupled receptor 1 (OGR1) (GPR68) has been identified to play a role in pH homeostasis. Hypoxia is known to change tissue pH as a result of anaerobic glucose metabolism through the stabilization of hypoxia-inducible factor-1α. We investigated how hypoxia regulates the expression of OGR1 in the intestinal mucosa and associated cells. METHODS: OGR1 expression in murine tumors, human colonic tissue, and myeloid cells was determined by quantitative reverse-transcription polymerase chain reaction. The influence of hypoxia on OGR1 expression was studied in monocytes/macrophages and intestinal mucosa of inflammatory bowel disease (IBD) patients. Changes in OGR1 expression in MonoMac6 (MM6) cells under hypoxia were determined upon stimulation with tumor necrosis factor (TNF), in the presence or absence of nuclear factor-κB (NF-κB) inhibitors. To study the molecular mechanisms involved, chromatin immunoprecipitation analysis of the OGR1 promoter was performed. RESULTS: OGR1 expression was significantly higher in tumor tissue compared with normal murine colon tissue. Hypoxia positively regulated the expression of OGR1 in MM6 cells, mouse peritoneal macrophages, primary human intestinal macrophages, and colonic tissue from IBD patients. In MM6 cells, hypoxia-enhanced TNF-induced OGR1 expression was reversed by inhibition of NF-κB. In addition to the effect of TNF and hypoxia, OGR1 expression was increased further at low pH. Chromatin immunoprecipitation analysis showed that HIF-1α, but not NF-κB, binds to the promoter of OGR1 under hypoxia. CONCLUSIONS: The enhancement of TNF- and hypoxia-induced OGR1 expression under low pH points to a positive feed-forward regulation of OGR1 activity in acidic conditions, and supports a role for OGR1 in the pathogenesis of IBD.

G Protein-coupled pH-sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation.

BACKGROUND: A novel family of proton-sensing G protein-coupled receptors, including OGR1, GPR4, and TDAG8, was identified to be important for physiological pH homeostasis and inflammation. Thus, we determined the function of proton-sensing OGR1 in the intestinal mucosa. MTEHODS: OGR1 expression in colonic tissues was investigated in controls and patients with IBD. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied for more than 200 days. Microarray profiling was performed using Ogr1 and Ogr1 (WT) residential peritoneal macrophages. RESULTS: Patients with IBD expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant upregulation of OGR1 expression, which could be reversed by the presence of NF-κB inhibitors. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1/Il-10 mice. These mice displayed significantly less rectal prolapses. Upregulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell-adhesion gene pathways. CONCLUSIONS: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF-κB inhibition reverses the induction of OGR1 expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 knockout model. Our data indicate a pathophysiological role for pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation.