GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4(+) T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

SUMMARY GPR15 is a G protein-coupled receptor proposed to play a role in mucosal immunity that also serves as entry cofactor for HIV and SIV. To discover novel endogenous GPR15 ligands, we screened a hemofiltrate-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of Cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2 and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that Cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this G protein-coupled receptor.

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.

Pulmonary alveolar homeostasis is dependent upon balanced airway and tissue surfactant pools. Quantitative and qualitative alterations in alveolar surfactant pools are associated with inflammation and tissue destruction in severe lung diseases including infant respiratory distress syndrome, acute lung injury and pulmonary alveolar proteinosis. Identification of a physiologically-dominant molecular pathway within alveolar epithelial cells that senses and regulates endogenous alveolar surfactant pools, coupled with the ability to pharmacologically modulate it both positively and negatively, would be a major therapeutic advance for patients with lung diseases associated with pulmonary surfactant disorders. We and others have previously shown that Gpr116 is a critical regulator of surfactant homeostasis in mice. Here we extend this work to show that human and mouse Gpr116 proteins are highly conserved at the amino acid level, are expressed on the plasma membrane of alveolar type II cells and functionally couple to intracellular G proteins when activated. Further, we have identified a synthetic peptide, GAP16, that is capable of activating mouse and human Gpr116 in vitro, resulting in increased Gq/11-dependent inositol phosphate conversion and calcium mobilization, cortical F-actin stabilization, and increased impedance of cell monolayers. Administration of GAP16 suppressed surfactant secretion from primary type II cells in vitro and nebulization of GAP16 to wild type mice was sufficient to suppress surfactant secretion from alveolar type II cells in vivo. These data provide proof-of-concept that Gpr116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools in humans to pulmonary diseases associated with surfactant dysfunction.

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.

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-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-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 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.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

index, or below 150 points in the CDAI index.Results: 40 controls and 41 patients with IBD that initiate treatment with IFX (19 CD, 6 UC) or with ADA (15 CD, 1 UC) were included.70% of patients were in concomitant treatment with corticoids and 77% with thiopurines.In 66.7% of cases there was response to IFX/ADA therapy.At week 14, 72% of patients were in clinical remission.No significant differences of AP serum concentrations between patients with IBD and healthy controls were found.Apart from PlGF (p<0.05),concentrations of VEGF, Ang1, Ang2 and Tie2, were unchanged during treatment with IFX/ ADA.There were no significant differences between CD and UC, IFX and ADA treatment or between responders and non-responders to IFX/ADA treatment.Conclusions: PlGF serum levels and it pro-inflammatory activity seem to be down-regulated by antiTNF therapy.Nevertheless, the effectiveness of IFX and ADA treatment is not be related to modifications in the concentrations of the other angiogenic factors (VEGF, Ang1, Ang2 and Tie2).* Significant differences (p<0.05)respect to pre-treatment values.

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.

Background: A single layer of epithelial cells creates a physical barrier between the external lumen and the internal milieu of the gastrointestinal tract. During inflammation, which is typically associated with a decrease in local pH, the intestinal barrier can be disrupted. Defects in barrier integrity have been implicated in the pathophysiology of inflammatory bowel disease (IBD). However, the molecular mechanisms mediating the responses to changes in pH are not well understood. G-protein-coupled receptor 68 (GPR68), also known as ovarian cancer GPR1 (OGR1), is a proton-sensing receptor, which upon acidification stimulates second messenger signaling, such as intracellular Ca2+ flux, inositol phosphate (IP) formation, and extracellular signal-regulated kinase (ERK) phosphorylation. We have investigated OGR1-mediated signaling pathways in response to lowered pH in intestinal epithelialcells. The colon carcinoma-derived-Caco2 cell monolayers can be used as In Vitro cell culture models of the human intestinal epithelium. Methods/Results: The OGR1 cDNA fragment was generated by PCR, and cloned into the pcDNA3.1 vector carrying a neomycin resistance gene. Clonal selection of stably transfected Caco2s was by limiting dilution or cloning cylinders, using the selection agent G418 (400 mg/ml). OGR1-overexpressing clones were selected by positive mRNA/protein expression and immunofluorescence, and those exhibiting strong IP formation upon acidification were chosen for further studies. The selected Caco2-OGR1 clones showed a typical pH-dependent GPCR response, in both intracellular Ca2+ signaling and label-free assays (EPIC). In addition, they exhibited weak but significant formation of cAMP upon extracellular acidification. When compared to the Caco2 vector control clone, the Caco2-OGR1 clones showed ERK phosphorylation after a pH shift from pH 7.9 to pH 6.8 for 5 minutes, by Western blotting. No pH-dependent phosphorylation remained present after 30 minutes. The Caco2-OGR1 clones exhibited significant serum response factor (SRF) activity at acidic pH but not at alkaline or neutral pH, in dual luciferase assays using SRE-dependent promoter constructs. Consistent with this, mRNA expression of a SRF target gene c-FOS was elevated in parallel. Electric Cell-Substrate Impedance Sensing (ECIS) assays showed tightening of the barrier in the Caco2-OGR1 monolayers at acidic pH, but not at neutral or alkaline pH. Conclusion: We have created Caco2 cell clones stably overexpressing OGR1, and have shown that these serve as valid tools to study OGR1 function and OGR1-mediated signaling. OGR1 overexpression in intestinal epithelial cells enhanced ERK signaling, led to increased tight junction formation, and increased SRF activity. Unravelling OGR1-dependent signaling may aid our understanding of the pathophysiology of IBD.