Solange Vidal

The Proton-activated G Protein Coupled Receptor OGR1 Acutely Regulates the Activity of Epithelial Proton Transport Proteins

The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H+-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl2 and CuCl2 that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.

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-&kgr;B 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-&kgr;B 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.

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.

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia

The identification of a natural ligand of the orphan chemoattractant receptor GPR15 provides mechanistic insight into the migration of lymphocytes in the skin. Deorphanizing a chemoattractant receptor The orphan G protein–coupled receptor GPR15 mediates the trafficking of lymphocytes to the colon and the skin and the recruitment of effector T cells to inflamed intestinal tissue. Suply et al. purified a natural ligand of GPR15 (GPR15L) from porcine colonic extracts. In vitro assays showed that GPR15L specifically activated GPR15, but not other chemoattractant receptors. Although migration assays suggested that GPR15L inhibited chemokine-induced T cell migration, mouse skin allotransplantations showed that GPR15L recruited CD8+ T cells to the graft and that loss of the ligand was associated with increased graft protection. Given that GPR15L mRNA is abundant in psoriatic lesions, these data suggest that targeting the GPR15-GPR15L axis may help in the treatment of inflammatory skin conditions. GPR15 is an orphan G protein–coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.

Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling

Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide-binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell-specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116-/- mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.

Tu1351 Functional Consequences of G Protein-Coupled Receptor 68 (GPR68/OGR1) Overexpression in Intestinal Epithelial Cells

effects including improved barrier function. Aims: To evaluate the protective effects of SCFAs on ethanol-induced intestinal barrier dysfunction and to elucidate possible mechanisms. Methods: Caco-2 monolayers were preincubated apically with either medium only or butyrate (2, 10 or 20 mM), propionate (4, 20 or 40 mM) or acetate (8, 40 or 80 mM) for 1 h, followed by ethanol (40 mM) for 3 h. Barrier function was analyzed by the trans-epithelial resistance (TER) and FITC-D4 clearance. Localization of ZO-1, occludin and F-actin was investigated by immunofluorescence. ROS generation, mitochondrial function and ATP levels were determined by dichlorofluorescein diacetate, MTT and bioluminescence assay, respectively. AMP-activated protein kinase activity (AMPK) was assessed by cell based ELISA. Results: Exposure to 40 mM ethanol decreased TER (to 74.1±2.1 %) and increased FITC-D4 clearance (0.097±0.001%) compared to untreated monolayers (105.4±1.4 and 0.017±0.001, resp.; P<0.0001). Pre-incubation with 2, 10 and 20 mM butyrate increased TER (88.8±2.0, 87.3±2.9 and 95.6±2.8%, resp) and decreased FITC-D4 clearance (0.033±0.001, 0.034±0.003 and 0.036±0.001%, resp) compared to ethanol exposed monolayers (74.1±2.1; and 0.097±0.001%, resp.; P<0.001). Similar results were obtained with propionate and 8 and 40 but not 80 mM acetate. SCFAs attenuated ethanol-induced abnormal localization of ZO-1 and occludin and distortion of F-actin. Furthermore, 20 mM butyrate prevented ethanol-induced ROS generation (100.8±4.2 vs. 154.4±4.3%; P<0.0001), improved mitochondrial function (85.7±10.3 vs. 47.2±4.2%; P<0.0001) and increased cellular ATP levels (104.0±1.8 vs. 53.2±1.0%; P<0.0001) compared to ethanol exposed monolayers. Similar data were obtained with 40 mM propionate and 8 mM acetate. SCFAs alone and in the presence of ethanol induced AMPK phosphorylation. Inhibition of AMPK by compound C, reduced the SCFAs-induced improvement of barrier dysfunction. Conclusions: Physiological concentrations of SCFAs attenuate ethanol-induced oxidative and metabolic stress in intestinal cells and thereby improve barrier dysfunction. These effects appear to be partly regulated through AMPK signaling pathway. The data suggest potential of SCFAs as prophylactic and/ or therapeutic agents against ethanol-induced gut leakiness.