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Inflammatory bowel disease (IBD) is characterized by chronic, recurring inflammation of the digestive tract. Current therapeutic approaches are limited and include biologics and steroids such as anti-TNFα monoclonal antibodies and corticosteroids, respectively. Significant adverse drug effects can occur for chronic usage and include increased risk of infection in some patients. GPR4, a pH-sensing G protein-coupled receptor, has recently emerged as a potential therapeutic target for intestinal inflammation. We have assessed the effects of a GPR4 antagonist, 2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole (GPR4 antagonist 13, also known as NE 52-QQ57) in the dextran sulfate sodium (DSS)-induced acute colitis mouse model. The GPR4 antagonist 13 inhibited intestinal inflammation. The clinical parameters such as body weight loss and fecal score were reduced in the GPR4 antagonist 13 treatment group compared to vehicle control. Macroscopic disease indicators such as colon shortening, splenic expansion, and mesenteric lymph node enlargement were all reduced in severity in the GPR4 antagonist 13 treated mice. Histopathological features of active colitis were alleviated in GPR4 antagonist 13 treatment groups compared to vehicle control. Finally, inflammatory gene expression in the colon tissues and vascular adhesion molecule expression in the intestinal endothelia were attenuated by GPR4 antagonist 13. Our results indicate that GPR4 antagonist 13 provides a protective effect in the DSS-induced acute colitis mouse model, and inhibition of GPR4 can be explored as a novel anti-inflammatory approach.

The effect of acidosis, a biochemical hallmark of the tumor microenvironment, on cancer progression and metastasis is complex. Both pro- and anti-tumorigenic effects of acidosis have been reported and the acidic microenvironment has been exploited for specific delivery of drugs, imaging agents, and genetic constructs into tumors. In this study we investigate the spreading and focal adhesion of B16F10 melanoma cells that are genetically engineered to overexpress the pH-sensing G protein-coupled receptor GPR4. By using cell attachment assays we found that GPR4 overexpression delayed cell spreading and altered the spatial localization of dynamic focal adhesion complex, such as the localization of phosphorylated focal adhesion kinase (FAK) and paxillin, at acidic pH. The potential G-protein and downstream signaling pathways that are responsible for these effects were also investigated. By using the Rho inhibitor CT04 (C3 transferase), the Rho-associated kinase (ROCK) inhibitors Y27632 and thiazovivin, the myosin light chain kinase (MLCK) inhibitor staurosporine or a G12/13 inhibitory construct, cell spreading was restored whereas the inhibition and activation of the Gq and Gs pathways had little or no effect. Altogether our results indicate that through the G12/13/Rho signaling pathway GPR4 modulates focal adhesion dynamics and reduces cell spreading and membrane ruffling.

Background Extracellular acidosis is a condition found within the tumor microenvironment due to inadequate blood perfusion, hypoxia, and altered tumor cell metabolism. Acidosis has pleiotropic effects on malignant progression; therefore it is essential to understand how acidosis exerts its diverse effects. TDAG8 is a proton-sensing G-protein-coupled receptor that can be activated by extracellular acidosis. Methods TDAG8 gene expression was analyzed by bioinformatic analyses and quantitative RT-PCR in human hematological malignancies. Retroviral transduction was used to restore TDAG8 expression in U937, Ramos and other blood cancer cells. Multiple in vitro and in vivo tumorigenesis and metastasis assays were employed to evaluate the effects of TDAG8 expression on blood cancer progression. Western blotting, immunohistochemistry and biochemical approaches were applied to elucidate the underlying mechanisms associated with the TDAG8 receptor pathway. Results TDAG8 expression is significantly reduced in human blood cancers in comparison to normal blood cells. Severe acidosis, pH 6.4, inhibited U937 cancer cell proliferation while mild acidosis, pH 6.9, stimulated its proliferation. However, restoring TDAG8 gene expression modulated the U937 cell response to mild extracellular acidosis and physiological pH by reducing cell proliferation. Tumor xenograft experiments further revealed that restoring TDAG8 expression in U937 and Ramos cancer cells reduced tumor growth. It was also shown U937 cells with restored TDAG8 expression attached less to Matrigel, migrated slower toward a chemoattractant, and metastasized less in severe combined immunodeficient mice. These effects correlated with a reduction in c-myc oncogene expression. The mechanistic investigation indicated that Gα13/Rho signaling arbitrated the TDAG8-mediated c-myc oncogene repression in response to acidosis. Conclusions This study provides data to support the concept that TDAG8 functions as a contextual tumor suppressor down-regulated in hematological malignancies and potentiation of the TDAG8 receptor pathway may be explored as a potential anti-tumorigenic approach in blood cancers. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1305-6) contains supplementary material, which is available to authorized users.

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the "Warburg effect"), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4- induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis. It has recently been demonstrated that activation of GPR4 by acidosis increases the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and also augments EC-leukocyte adhesion. Inhibition of GPR4 by siRNA or small molecule inhibitors reduces endothelial cell inflammation. As acidotic tissue microenvironments exist in many types of inflammatory disorders, including inflammatory bowel disease (IBD), we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for 7 days to induce acute colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. Collectively, our results suggest GPR4 provides a pro-inflammatory role in the inflamed gut as the absence of GPR4 ameliorates intestinal inflammation in the acute experimental colitis mouse model. Keywords: GPR4; Inflammatory bowel disease (IBD); Acidosis; Endothelial cell; Inflammation

GPR4 is a member of the proton-sensing G protein-coupled receptor family. Within tumor microenvironments, the interstitial acidic pH may activate GPR4 to regulate the behavior of tumor cells. Mouse B16F10 melanoma cells and TRAMP-C1 prostate cancer cells, genetically engineered to overexpress GPR4 or the control vector, were subject to a series of cell migration, invasion and metastasis assays. Upon GPR4 overexpression and activation in an acidic pH, the migration of B16F10 and TRAMP-C1 cells was substantially inhibited in comparison to the vector control. Similar results were observed in the Matrigel invasion and transendothelial invasion assays. At the molecular level, stimulation of GPR4 by acidosis induced the activation of RhoA and the formation of actin stress fibers. In addition, treating B16F10 cells with the known Rho activator CN01 (calpeptin) strongly inhibited cell migration, recapitulating the acidosis/GPR4-induced motility inhibition phenotype. To examine the biological effects in vivo, B16F10 melanoma cells were intravenously injected into syngeneic C57BL/6 mice and pulmonary metastasis was inhibited by approximately 80% in GPR4-overexpressing B16F10 cells in comparison to the vector control. Upon treatment with the Rho activator CN01, the phenotype of the B16F10 vector cells paralleled that of the GPR4-overexpressing cells in cell migration and metastasis assays. These findings suggest that GPR4 activation by an acidic pH inhibits tumor cell migration and invasion, and the Rho GTPase is at least partly responsible for this phenotype.

Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.

Phagocytosis of apoptotic cells is fundamentally important throughout life, because non-cleared cells become secondarily necrotic and release intracellular contents, thus instigating inflammatory and autoimmune responses. Secreted "find-me" and exposed "eat-me" signals displayed by the dying cell in concert with the phagocyte receptors comprise the phagocytic synapse of apoptotic cell clearance. In this scenario, lysophospholipids (lysoPLs) are assumed to act as find-me signals for the attraction of phagocytes. However, both the identity of the lyso-PLs released from apoptotic cells and the nature of the phagocyte receptor are largely unknown. By a detailed analysis of the structural requirements we show here that lysophosphatidylcholine (lysoPC), but none of the lysoPC metabolites or other lysoPLs, represents the essential apoptotic attraction signal able to trigger a phagocyte chemotactic response. Furthermore, using RNA interference and expression studies, we demonstrate that the G-protein-coupled receptor G2A, unlike its relative GPR4, is involved in the chemotaxis of monocytic cells. Thus, our study identifies lysoPC and G2A as the crucial receptor/ligand system for the attraction of phagocytes to apoptotic cells and the prevention of autoimmunity.

Blood vessels in mammalian embryos develop through vasculogenesis and angiogenesis. During the vasculogenesis phase, endothelial progenitor cells cluster to form a vascular plexus, which then undergoes angiogenesis by sprouting and remodeling to form a complex vascular network. Subsequently, recruitment of smooth muscle cells and pericytes to nascent blood vessels is important for the stability and maturation of the vasculature (6). At the molecular level, a myriad of studies demonstrate that cell surface receptors and their ligands, such as vascular endothelial growth factor (VEGF) receptors and VEGF, are critical for vascular formation and function (9). Deficiency in these regulators may lead to various degrees of blood vessel defects. For instance, deletion of VEGF receptors or Tie receptor tyrosine kinases causes abnormal vasculogenesis and angiogenesis at different stages of mouse development (16, 39, 45); lack of platelet-derived growth factor receptor β (PDGFRβ) or PDGF-B results in defective vascular maturation with reduced smooth muscle cell coverage in a subset of blood vessels and deficient mesangial cell recruitment to kidney glomeruli (18, 32, 33, 47). Although protein growth factors and their receptors have been a major focus in angiogenesis studies, many different factors play a positive or negative role in maintaining a delicate balance in vascular formation (7, 38, 52). Tissue pH is one of the factors that can regulate blood vessel formation and function (4, 10). Under various physiological and pathological conditions including exercise, growth of solid tumors, inflammation, ischemia, diabetic ketoacidosis, and renal and respiratory failure, blood vessels form and/or function in a local environment with an acidic pH. Notably, glycolysis and the production of lactic acid by tumor cells in a hypoxic microenvironment lead to a significant reduction of extracellular pH in tumors (17). Studies show that acidic extracellular pH has pleiotropic effects on angiogenesis, including the inhibition of endothelial cell migration, tube formation, and in vitro vascular growth (4, 10). Most previous research has focused on the effects of acidic pH on angiogenic factors. Acidosis has been shown to modulate the expression and activity of both angiogenic and angiostatic factors, which in turn regulate the angiogenesis process (4, 14, 46, 55). Therefore, further understanding of vascular pH responses may help in designing methods to control blood vessel formation and function in various pathologies such as cancer and ischemia. However, little is known about how vascular cells per se directly sense acidic extracellular pH. GPR4 and several related G protein-coupled receptors (GPCRs) have recently been identified as novel proton-sensing receptors (20, 34, 37, 44, 56). Studies have shown that GPR4 is expressed in vascular endothelial and smooth muscle cells, lung, kidney, heart, liver, and other tissues (1, 23, 35, 36, 51). The in vivo function of GPR4 is not well understood, although recent studies indicate that GPR4-related genes in Xenopus laevis are involved in the regulation of embryonic gastrulation (8, 49). Previous in vitro analyses suggest that GPR4 might mediate the sphingosylphosphorylcholine-induced endothelial tube formation and lysophosphatidylcholine-induced impairment of endothelial barrier function (23, 41), but the publication proposing the receptor-ligand relationship between GPR4 and sphingosylphosphorylcholine and lysophosphatidylcholine has been withdrawn (62). The expression of GPR4 in vascular cells and its biochemical function of sensing pH changes have led us to hypothesize that GPR4 is one sensor that blood vessels may use to respond to extracellular acidic pH. Previous cell line overexpression studies demonstrate that acidic pH activates GPR4 to induce the production of cyclic AMP (cAMP) (34, 44). In this respect, cAMP and its downstream kinase protein kinase A have been shown to inhibit angiogenesis (11, 15, 24, 25, 53). The objectives of the present study were twofold: (i) to investigate the in vivo biological role of GPR4 and (ii) to identify whether GPR4 is a functional sensor for blood vessels in response to pH changes. To assess these, we generated GPR4-knockout mice by homologous recombination. Spontaneous hemorrhaging was observed in approximately 17% of GPR4-null embryos and neonates. These mice showed dilated small blood vessels with a decrease of smooth muscle cell coverage and significantly reduced mesangial cells in kidney glomeruli. Direct tissue explants from knockout mice revealed that GPR4 is responsive to acidic extracellular pH to regulate microvessel outgrowth.

Intracellular trafficking and spatial dynamics of membrane receptors critically regulate receptor function. Using microscopic and subcellular fractionation analysis, we studied the localization of the murine G protein-coupled receptor G2A (muG2A). Evaluating green fluorescent protein-tagged, exogenously expressed as well as the endogenous muG2A, we observed that this receptor was spontaneously internalized and accumulated in endosomal compartments, whereas its surface expression was enhanced and stabilized by lysophosphatidylcholine (LPC) treatment. Monensin, a general inhibitor of recycling pathways, blocked LPC-regulated surface localization of muG2A as well as muG2A-dependent extracellular signal-regulated kinase (ERK) activation and cell migration induced by LPC treatment. Mutation of the conserved DRY motif (R→ A) enhanced the surface expression of muG2A, resulting in its resistance to monensin inhibition of ERK activation. Our data suggest that intracellular sequestration and surface expression regulated by LPC, rather than direct agonistic activity control the signaling responses of murine G2A toward LPC.

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value.

Quantification of 3D morphology and measurement of cellular functions were performed on the mouse melanoma cell lines of B16F10 to investigate the intriguing problem of structure–function relations in the genetically engineered cells with GPR4 overexpression. Results of 3D analysis of cells in suspension and phase contrast imaging of adherent cells yield consistent evidence that stimulation of the proton-sensing GPR4 receptor in these cells may modify significantly their morphology with diminishing ability to produce membrane protrusions and to migrate. Examination of the 3D parameters of mitochondria provide further insights on the measured variation of the maximal capacity of oxygen consumption rate among the genetically modified cells, indicating that the proton-sensing receptor may regulate cancer cell metabolism with increased mitochondrial surface area. Our study demonstrates clearly the significant benefits of quantitative 3D morphological study in illuminating cellular functions and development of novel morphology based cell assay methods.

Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2',5'-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a G(i) signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the G(s)/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the G(s)/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells.

T-cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor transcriptionally upregulated by glucocorticoids (GCs) and implicated by overexpression studies in psychosine-mediated inhibition of cytokinesis and in GC-induced apoptosis. To examine the physiological function of TDAG8, we generated knockout (KO) mice by homologous recombination. An enhanced green fluorescent protein reporter was knocked into the disrupted tdag8 locus to allow the analysis of TDAG8 expression in living cells. Interestingly, we found that during thymocyte development, TDAG8 expression resembled the dynamic regulation described for known modulators of GC-induced apoptosis, including Bcl-2, Notch1, and GC receptor. TDAG8 was expressed in double-negative cells, was downregulated at the double-positive transition, and was upregulated in single-positive thymocytes. However, despite this striking expression pattern, maturation and selection of thymocytes, as well as major immune functions, were not affected in TDAG8 KO mice. In contrast to previous overexpression results, TDAG8 was dispensable for psychosine-induced formation of multinucleated cells. Furthermore, TDAG8 KO thymocytes showed normal apoptosis following in vivo and in vitro GC treatment. These results, while establishing a useful reporter strain to study T-lymphocyte maturation, argue against a critical role for TDAG8 in immune development, psychosine-mediated inhibition of cytokinesis, and GC-induced cell death.

G2A is a G-protein–coupled receptor (GPCR) involved in immune regulation. Previous studies have shown that lysophosphatidylcholine (LPC), a bioactive lipid associated with atherosclerosis and autoimmunity, acts through G2A to induce diverse biologic effects. Production of LPC during cell apoptosis serves as a chemotactic signal for macrophage recruitment. Here we demonstrate that macrophage chemotaxis to LPC is dependent on G2A function. Wild-type but not G2A-deficient mouse peritoneal macrophages migrated toward LPC. RNAi-mediated knockdown of G2A in J774A.1 macrophages abolished LPC-induced chemotaxis, whereas overexpression of G2A significantly enhanced this process. Mutation of the conserved DRY motif of G2A resulted in loss of chemotaxis to LPC, suggesting a requirement for G-protein signaling. Unlike most GPCRs, including the chemokine receptors, coupling to Gi is not required for LPC/G2A-mediated chemotaxis, but coupling to Gq/11 and G12/13 is necessary as judged by inhibition with dominant negative forms of these alpha subunits or with regulators of G-protein signaling (RGS) constructs. Collectively, these data establish that pertussis toxin–insensitive G2A signaling regulates macrophage chemotaxis to LPC. Defects in this signaling pathway may be related to the pathogenesis of systemic autoimmune disease.

G2A is an immunoregulatory G protein-coupled receptor predominantly expressed in lymphocytes and macrophages. Ectopic overexpression studies have implicated G2A as a receptor for the bioactive lysophospholipid, lysophosphatidylcholine (LPC). However, the functional consequences of LPC–G2A interaction at physiological levels of receptor expression, and in a cellular context relevant to its immunological role, remain largely unknown. Here, we show impaired chemotaxis to LPC of a T lymphoid cell line in which G2A expression was chronically down-regulated by RNA interference technology. Rescuing this phenotype by reconstitution of the physiological level of receptor expression further supports a functional connection between LPC–G2A interaction and cellular motility. Overexpression of G2A in the T lymphoid cell line significantly enhanced chemotaxis to LPC. It also modified migration toward the LPC-related molecule, lysophosphatidic acid, indicating the possibility of crosstalk between G2A and endogenous lysophosphatidic acid receptors. The role of G2A in LPC-mediated cell migration may be relevant to the autoimmune syndrome associated with genetic inactivation of this G protein-coupled receptor in mice. The experimental system described here can be useful for understanding the structural requirements for LPC recognition by G2A and the signaling pathways regulated by this ligand-receptor pair.