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Tissue acidification resulting from dysregulated cellular bioenergetics accompanies various inflammatory states. GPR68, along with other members of proton-sensing G protein-coupled receptors, responds to extracellular acidification and has been implicated in chronic inflammation-related diseases such as ischemia, cancer, and colitis. The present study examined the role of extracellular acidification on human pulmonary endothelial cell (EC) permeability and inflammatory status per se and investigated potential synergistic effects of acidosis on endothelial dysfunction caused by bacterial lipopolysaccharide (LPS, Klebsiella pneumoniae). Results showed that medium acidification to pH 6.5 caused a delayed increase in EC permeability illustrated by a decrease in transendothelial electrical resistance and loss of continuous VE-cadherin immunostaining at cell junctions. Likewise, acidic pH induced endothelial inflammation reflected by increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1, upregulated mRNA transcripts of tumor necrosis factor-α, IL-6, IL-8, IL-1β, and CXCL5, and increased secretion of ICAM-1, IL-6, and IL-8 in culture medium monitored by ELISA. Among the GPCRs tested, acidic pH selectively increased mRNA and protein expression of GPR68, and only the GPR68-specific small molecule inhibitor OGM-8345 rescued acidosis-induced endothelial permeability and inflammation. Furthermore, acidic pH exacerbated LPS-induced endothelial permeability and inflammatory response in cultured lung macrovascular as well as microvascular endothelial cells. These effects were suppressed by OGM-8345 in both EC types. Altogether, these results suggest that GPR68 is a critical mediator of acidic pH-induced dysfunction of human pulmonary vascular endothelial cells and mediates the augmenting effect of tissue acidification on LPS-induced endothelial cell injury. [ABSTRACT FROM AUTHOR]

An interoceptive homeostatic reflex monitors levels of CO2/H+ to maintain blood gas homeostasis and rapidly regulate tissue acid–base balance by driving lung ventilation and CO2 excretion—this CO2-evoked increase in respiration is the hypercapnic ventilatory reflex (HCVR). Retrotrapezoid nucleus (RTN) neurons provide crucial excitatory drive to downstream respiratory rhythm/pattern-generating circuits, and their activity is directly modulated by changes in CO2/H+ . RTN neurons express GPR4 and TASK-2, global deletion of which abrogates CO2/H+ activation of RTN neurons and the HCVR. It has not been determined if the intrinsic pH sensitivity of these proton detectors is required for these effects. We used CRISPR/Cas9 genome editing to generate mice with mutations in either of two pH-sensing histidine residues in GPR4 to determine effects on RTN neuronal CO2/H+ sensitivity and the HCVR. In global GPR4(H81F) and GPR4(H167F) mice, CO2-stimulated breathing and CO2-induced RTN neuronal activation were strongly blunted, with no effect on hypoxia-stimulated breathing. In brainstem slices from GPR4(H81F) mice, peak firing of RTN neurons during bath acidification was significantly reduced compared with GPR4 wild-type mice, and a subpopulation of RTN neurons was rendered pH-insensitive, phenocopying previous results from GPR4-deleted mice. These effects were independent of changes in RTN number/distribution, neuronal excitability or transcript levels for GPR4 and TASK-2. CO2-stimulated breathing was reduced to a similar extent in GPR4(H81F) and TASK-2-deleted mice, with combined mutation yielding no additional deficit in the HCVR. Together, these data demonstrate that the intrinsic pH sensitivity of GPR4 is necessary for full elaboration of the HCVR. [ABSTRACT FROM AUTHOR]

The precise regulation of pH homeostasis is crucial for normal physiology. However, in tissue microenvironments, it can be impacted by pathological conditions such as inflammation and cancer. Due to the overproduction and accumulation of acids (protons), the extracellular pH is characteristically more acidic in inflamed tissues and tumors in comparison to normal tissues. A family of proton-sensing G-protein-coupled receptors (GPCRs) has been identified as molecular sensors for cells responding to acidic tissue microenvironments. Herein, we review the current research progress pertaining to these proton-sensing GPCRs, including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), in inflammation and cancer. Growing evidence suggests that GPR4 and GPR68 are mainly pro-inflammatory, whereas GPR65 is primarily anti-inflammatory, in various inflammatory disorders. Both anti- and pro-tumorigenic effects have been reported for this family of receptors. Moreover, antagonists and agonists targeting proton-sensing GPCRs have been developed and evaluated in preclinical models. Further research is warranted to better understand the roles of these proton-sensing GPCRs in pathophysiology and is required in order to exploit them as potential therapeutic targets for disease treatment. [ABSTRACT FROM AUTHOR]

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation‐related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment‐induced chemokines C‐X3‐C motif chemokine ligand 1 (CX3CL1) and C‐X‐C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM‐1) increased in HDLECs after acid treatment in a time‐dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid‐sensing receptors, HDLECs basally expressed G protein‐coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM‐1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti‐VCAM‐1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid‐sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer. [ABSTRACT FROM AUTHOR]

Arteriovenous malformations (AVMs) are fast-flow vascular malformations and refer to important causes of intracerebral haemorrhage in young adults. Getting deep insight into the genetic pathogenesis of AVMs is necessary. Herein, we identified two vital missense variants of G protein-coupled receptor (GPCR) associated sorting protein 1 (GPRASP1) in AVM patients for the first time and congruously determined to be loss-of-function variants in endothelial cells. GPRASP1 loss-of-function caused endothelial dysfunction in vitro and in vivo. Endothelial Gprasp1 knockout mice suffered a high probability of cerebral haemorrhage, AVMs and exhibited vascular anomalies in multiple organs. GPR4 was identified to be an effective GPCR binding with GPRASP1 to develop endothelial disorders. GPRASP1 deletion activated GPR4/cAMP/MAPK signalling to disturb endothelial functions, thus contributing to vascular anomalies. Mechanistically, GPRASP1 promoted GPR4 degradation. GPRASP1 enabled GPR4 K63-linked ubiquitination, enhancing the binding of GPR4 and RABGEF1 to activate RAB5 for conversions from endocytic vesicles to endosomes, and subsequently increasing the interactions of GPR4 and ESCRT members to package GPR4 into multivesicular bodies or late endosomes for lysosome degradation. Notably, the GPR4 antagonist NE 52-QQ57 and JNK inhibitor SP600125 effectively rescued the vascular phenotype caused by endothelial Gprasp1 deletion. Our findings provided novel insights into the roles of GPRASP1 in AVMs and hinted at new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Simple Summary: Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a debilitating condition with chronic inflammation in the digestive tract. Patients with IBD are at higher risk of developing colitis-associated colorectal cancer (CAC) compared with the general population. The etiology of IBD is not well understood, but both genetic and environmental factors have been implicated. In this study, we investigated the role of the pH-sensing GPR4 receptor in colitis and CAC mouse models. GPR4 knockout alleviated intestinal inflammation, reduced tumor angiogenesis, and impeded CAC development. Our data suggest that the inhibition of GPR4 may be explored as a potential therapeutic approach for IBD treatment and CAC prevention. GPR4 is a proton-sensing G protein-coupled receptor highly expressed in vascular endothelial cells and has been shown to potentiate intestinal inflammation in murine colitis models. Herein, we evaluated the proinflammatory role of GPR4 in the development of colitis-associated colorectal cancer (CAC) using the dextran sulfate sodium (DSS) and azoxymethane (AOM) mouse models in wild-type and GPR4 knockout mice. We found that GPR4 contributed to chronic intestinal inflammation and heightened DSS/AOM-induced intestinal tumor burden. Tumor blood vessel density was markedly reduced in mice deficient in GPR4, which correlated with increased tumor necrosis and reduced tumor cell proliferation. These data demonstrate that GPR4 ablation alleviates intestinal inflammation and reduces tumor angiogenesis, development, and progression in the AOM/DSS mouse model. [ABSTRACT FROM AUTHOR]

Previous studies have indicated that the development of preeclampsia (PE) involves the regulation of circular RNA (circRNA). However, the role of hsa_circ_0014736 (circ_0014736) in PE remains unknown. Thus, the study proposes to reveal the function of circ_0014736 in the pathogenesis of PE and the underlying mechanism. The results showed that circ_0014736 and GPR4 expression were significantly upregulated, while miR-942-5p expression was downregulated in PE placenta tissues when compared with normal placenta tissues. circ_0014736 knockdown promoted the proliferation, migration, and invasion of placenta trophoblast cells (HTR-8/SVneo) and inhibited apoptosis; however, circ_0014736 overexpression had the opposite effects. circ_0014736 functioned as a sponge for miR-942-5p and regulated HTR-8/SVneo cell processes by interacting with miR-942-5p. Additionally, GPR4, a target gene of miR-942-5p, was involved in miR-942-5p-mediated actions in HTR-8/SVneo cells. Moreover, circ_0014736 stimulated GPR4 production through miR-942-5p. Collectively, circ_0014736 inhibited HTR-8/SVneo cell proliferation, migration, and invasion and induced cell apoptosis through the miR-942-5p/GPR4 axis, providing a possible target for the treatment of PE.

Due to its high metastatic potential, malignant melanoma is one of the deadliest skin cancers. In melanoma as well as in other cancers, acidification of the tumor microenvironment (=TME, inverse pH‐gradient) is a well‐known driver of tumor progression and metastasis. Membrane‐bound receptors, such as the proton‐sensitive GPCR (pH‐GPCR) GPR4, are considered as potential initiators of the signalling cascades relevant to malignant transformation. In this study, we investigated the pH‐dependent migration of GPR4 wildtype/overexpressing SK‐Mel‐28 cells using an impedance‐based electrical wounding and migration assay and classical Boyden chamber experiments. Migration of GPR4 overexpressing SK‐Mel‐28 cells was enhanced in a range of pH 6.5–7.5 as compared to controls in the impedance‐based electrical wounding and migration assay. In Boyden chamber experiments, GPR4 overexpression only increased migration at pH 7.5 in a Matrigel‐free setup, but not at pH 6.5. Results indicate that GPR4 is involved in the migration of melanoma cells, especially in the tumor periphery, and that this process is affected by pH in the TME. [ABSTRACT FROM AUTHOR]

Previous studies have indicated that the development of preeclampsia (PE) involves the regulation of circular RNA (circRNA). However, the role of hsa_circ_0014736 (circ_0014736) in PE remains unknown. Thus, the study proposes to reveal the function of circ_0014736 in the pathogenesis of PE and the underlying mechanism. The results showed that circ_0014736 and GPR4 expression were significantly upregulated, while miR-942-5p expression was downregulated in PE placenta tissues when compared with normal placenta tissues. circ_0014736 knockdown promoted the proliferation, migration, and invasion of placenta trophoblast cells (HTR-8/ SVneo) and inhibited apoptosis; however, circ_0014736 overexpression had the opposite effects. circ_0014736 functioned as a sponge for miR-942-5p and regulated HTR-8/SVneo cell processes by interacting with miR-942-5p. Additionally, GPR4, a target gene of miR-942-5p, was involved in miR942-5p-mediated actions in HTR-8/SVneo cells. Moreover, circ_0014736 stimulated GPR4 production through miR942-5p. Collectively, circ_0014736 inhibited HTR-8/SVneo cell proliferation, migration, and invasion and induced cell apoptosis through the miR-942-5p/GPR4 axis, providing a possible target for the treatment of PE. [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysm (AAA) is a severe cardiovascular disease (CVD) that is partly attributable to endothelial dysfunction, inflammatory response, and angiogenesis. G protein-coupled receptor 4 (GPR4), a proton-sensitive G protein-coupled receptor that is abundantly expressed in vascular endothelial cells, has been associated with numerous physiological functions. Nevertheless, its potential involvement in the development of AAA remains unexplored. In this study, we examined the impact of GPR4 deletion on the development of AAA in ApoE-deficient mice. The mice were categorized into four distinct groups: the ApoE-/- with saline group, the ApoE-/-GPR4-/- with saline group, the ApoE-/- with Ang II group, and the ApoE-/-GPR4-/- with Ang II group. AAA were induced in the ApoE-/- mice through the perfusion of angiotensin II (Ang II). Notably, GPR4 was substantially elevated in the AAA tissues from both human subjects and experimental mice. The deletion of GPR4 substantially decreased the formation of Ang II-induced AAA, damages to elastin, and the expression of aortic inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), as well as vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 (VEGF-A/VEGF-R2), in ApoE-/- mice. Human aortic endothelial cells (HAECs) were transfected with lenti-viral GPR4 shRNA and subsequently stimulated with Ang II. Our findings indicate that the knockout of GPR4 attenuated Ang II-induced angiogenic tube formation in HAECs by decreasing the expression of VEGF-A and VEGF-R2. Furthermore, GPR4 knockout also hindered the activation of specificity protein-1 (SP-1) by reducing its expression and transcriptional activity. Notably, the overexpression of SP-1 reversed the inhibitory effects of GPR4 knockout on angiogenic tube formation and the expression of VEGF-A/VEGF-R2. This suggests that the protective effects of GPR4 knockout are achieved through the inhibition of SP-1. In summary, the absence of GPR4 impeded AAA formation, indicating that GPR4 could potentially serve as a therapeutic target for AAA., (© 2025 Wiley Periodicals LLC.)

Myocardial ischemia/reperfusion (MI/R) injury is a detrimental disease with high mortality worldwide. We aimed to explore the role of G protein-coupled receptor 4 (GPR4) and lysophosphatidic acid receptor 1 (LPAR1) in MI/R injury in vitro. H9c2 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) conditions to simulate the MI/R injury and GPR4 expression was detected. Then, GPR4 was knocked down and cell viability was examined with a CCK-8 assay. The activities of LDH, CK and CK-MB were detected to evaluate the damage of OGD/R-induced H9c2 cells. ELISA kits and TUNEL staining were used to examine the inflammation and apoptosis of H9c2 cells exposed to OGD/R conditions. Western blot was employed to detect the expression of proteins related to apoptosis and NLRP3 inflammasome signaling. Additionally, Co-IP analyzed the binding between GPR4 and LPAR1. Finally, LPAR1 was overexpressed to conduct the rescue experiments. Results revealed that GPR4 was upregulated in OGD/R-treated H9c2 cells and GPR4 knockdown attenuated the damage of H9c2 cells. OGD/R induced inflammation and apoptosis were markedly inhibited by GPR4 silencing, as evidenced by the decreased TNF-α, IL-6 and IL-8 levels as well as the elevated Bcl-2 expression and reduced Bax and cleaved caspase3 expression. Moreover, GPR4 bound to LPAR1 and upregulated LPAR1 expression. Interference with GPR4 inactivated the NLRP3 inflammasome signaling. Besides, LPAR1 overexpression abrogated the effects of GPR4 silencing on the damage, inflammation and apoptosis of H9c2 cells induced by OGD/R. Particularly, LPAR1 upregulation promoted the activation of NLRP3 inflammasome signaling in GPR4-silenced H9c2 cells induced by OGD/R. To be concluded, GPR4 deficiency inactivates NLRP3 inflammasome signaling by inhibiting LPAR1 expression to ameliorate OGD/R -induced inflammation and apoptosis of cardiomyocytes. • GPR4 was upregulated in OGD/R-treated H9c2 cells and GPR4 knockdown attenuated the damage of H9c2 cells. • GPR4 knockdown alleviated the inflammation and apoptosis of H9c2 cells exposed to OGD/R conditions. • GPR4 knockdown inactivated NLRP3 inflammasome signaling in OGD/R-treated H9c2 cells. • GPR4 bound to LPAR1 and upregulated LPAR1 expression in H9c2 cells exposed to OGD/R conditions. • LPAR1 overexpression restored the activation of NLRP3 inflammasome signaling in GPR4-silenced H9c2 cells induced by OGD/R. [ABSTRACT FROM AUTHOR]

Local acidification is a common feature of many disease processes such as inflammation, infarction, or solid tumor growth. Acidic pH is not merely a sequela of disease but contributes to recruitment and regulation of immune cells, modifies metabolism of parenchymal, immune and tumor cells, modulates fibrosis, vascular permeability, oxygen availability, and consumption, invasiveness of tumor cells, and impacts on cell survival. Thus, multiple pH-sensing mechanisms must exist in cells involved in these processes. These pH sensors play important roles in normal physiology and pathophysiology, and hence might be attractive targets for pharmacological interventions. Among the pH-sensing mechanisms, OGR1 (GPR68), GPR4 (GPR4), and TDAG8 (GPR65) have emerged as important molecules. These G protein-coupled receptors are widely expressed, upregulated in inflammation and tumors, sense changes in extracellular pH in the range between pH 8 and 6, and are involved in modulating key processes in inflammation, tumor biology, and fibrosis. This review discusses key features of these receptors and highlights important disease states and pathways affected by their activity. [ABSTRACT FROM AUTHOR]

Abstract Background Patients with coronary artery disease (CAD) are characterized by a decline in vascular regeneration, which is related to the dysfunction of endothelial progenitor cells (EPCs). G-protein-coupled receptor 4 (GPR4) is a proton-sensing G-protein-coupled receptor (GPCR) that contributes to neovascularization in acidic microenvironments. However, the role of GPR4 in regulating the angiogenic capacity of EPCs from CAD patients in response to acidity generated in ischemic tissue remains completely unclear. Methods The angiogenic capacity of EPCs collected from CAD patients and healthy subjects was evaluated in different pH environments. The GPR4 function of regulating EPC-mediated angiogenesis was analyzed both in vitro and in vivo. The downstream mechanisms were further investigated by genetic overexpression and inhibition. Results Acidic environment prestimulation significantly enhanced the angiogenic capacity of EPCs from the non-CAD group both in vivo and in vitro, while the same treatment yielded the opposite result in the CAD group. Among the four canonical proton-sensing GPCRs, GPR4 displays the highest expression in EPCs. The expression of GRP4 was markedly lower in EPCs from CAD patients than in EPCs from non-CAD individuals independent of acid stimulation. The siRNA-mediated knockdown of GPR4 with subsequent decreased phosphorylation of STAT3 mimicked the impaired function of EPCs from CAD patients at pH 6.4 but not at pH 7.4. Elevating GPR4 expression restored the neovessel formation mediated by EPCs from CAD patients in an acidic environment by activating STAT3/VEGFA signaling. Moreover, the beneficial impact of GPR4 upregulation on EPC-mediated angiogenic capacity was abrogated by blockade of the STAT3/VEGFA signaling pathway. Conclusions Our present study demonstrated for the first time that loss of GPR4 is responsible for the decline in proton sensing and angiogenic capacity of EPCs from CAD patients. Augmentation of GPR4 expression promotes the neovessel formation of EPCs by activating STAT3/VEGF signaling. This finding implicates GPR4 as a potential therapeutic target for CAD characterized by impaired neovascularization in ischemic tissues.

Hormone-secreting pituitary adenomas show unregulated hormonal hypersecretion and cause hyperpituitarism. However, the mechanism of the unregulated hormone production and secretion has not yet been fully elucidated. Solid tumors show reduced extracellular pH, partly due to lactate secretion from anaerobic glycolysis. It is known that extracellular acidification affects hormone secretion. However, whether and how the extracellular acidification influences the unregulated hormone production and secretion remain unknown. In the present study, we found that GPR4, a proton-sensing G protein-coupled receptor, was highly expressed in MtT/S cells, a growth hormone-producing and prolactin-producing pituitary tumor cell line. When we reduced the extracellular pH, growth hormone and prolactin mRNA expressions increased in the cells. Both increased expressions were partially suppressed by a GPR4 antagonist. We also found that extracellular acidification enhanced growth hormone-releasing factor-induced growth hormone secretion from MtT/S cells. These results suggest that GPR4 may play a role in hypersecretion of the hormone from hormone-producing pituitary tumors. A GPR4 antagonist will be a useful tool for preventing the hypersecretion.

Aim: Type A intercalated cells of the renal collecting duct participate in the maintenance of the acid/base balance through their capacity to adapt proton secretion to homeostatic requirements. We previously showed that increased proton secretion stems in part from the enlargement of the population of proton secreting cells in the outer medullary collecting duct through division of fully differentiated cells, and that this response is triggered by growth/differentiation factor 15. This study aimed at deciphering the mechanism of acid load-induced secretion of Gdf15 and its mechanism of action. Methods: We developed an original method to evaluate the proliferation of intercalated cells and applied it to genetically modified or pharmacologically treated mice under basal and acid-loaded conditions. Results: Gdf15 is secreted by principal cells of the collecting duct in response to the stimulation of vasopressin receptors. Vasopressin-induced production of cAMP triggers activation of AMP-stimulated kinases and of Na,K-ATPase, and induction of p53 and Gdf15. Gdf15 action on intercalated cells is mediated by ErbB2 receptors, the activation of which triggers the expression of cyclin d1, of p53 and anti-proliferative genes, and of Egr1. Conclusion: Acidosis-induced proliferation of intercalated cells results from a cross talk with principal cells which secrete Gdf15 in response to their stimulation by vasopressin. Thus, vasopressin is a major determinant of the collecting duct cellular homeostasis as it promotes proliferation of intercalated cells under acidosis conditions and of principal cells under normal acid-base status. [ABSTRACT FROM AUTHOR]

Coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first emerged in late 2019 and has since rapidly become a global pandemic. SARS-CoV-2 infection causes damages to the lung and other organs. The clinical manifestations of COVID-19 range widely from asymptomatic infection, mild respiratory illness to severe pneumonia with respiratory failure and death. Autopsy studies demonstrate that diffuse alveolar damage, inflammatory cell infiltration, edema, proteinaceous exudates, and vascular thromboembolism in the lung as well as extrapulmonary injuries in other organs represent key pathological findings. Herein, we hypothesize that GPR4 plays an integral role in COVID-19 pathophysiology and is a potential therapeutic target for the treatment of COVID-19. GPR4 is a pro-inflammatory G protein-coupled receptor (GPCR) highly expressed in vascular endothelial cells and serves as a “gatekeeper” to regulate endothelium-blood cell interaction and leukocyte infiltration. GPR4 also regulates vascular permeability and tissue edema under inflammatory conditions. Therefore, we hypothesize that GPR4 antagonism can potentially be exploited to mitigate the hyper-inflammatory response, vessel hyper-permeability, pulmonary edema, exudate formation, vascular thromboembolism and tissue injury associated with COVID-19.

Background: Patients with coronary artery disease (CAD) are characterized by a decline in vascular regeneration, which is related to the dysfunction of endothelial progenitor cells (EPCs). G-protein-coupled receptor 4 (GPR4) is a proton-sensing G-protein-coupled receptor (GPCR) that contributes to neovascularization in acidic microenvironments. However, the role of GPR4 in regulating the angiogenic capacity of EPCs from CAD patients in response to acidity generated in ischemic tissue remains completely unclear. Methods: The angiogenic capacity of EPCs collected from CAD patients and healthy subjects was evaluated in different pH environments. The GPR4 function of regulating EPC-mediated angiogenesis was analyzed both in vitro and in vivo. The downstream mechanisms were further investigated by genetic overexpression and inhibition. Results: Acidic environment prestimulation significantly enhanced the angiogenic capacity of EPCs from the non-CAD group both in vivo and in vitro, while the same treatment yielded the opposite result in the CAD group. Among the four canonical proton-sensing GPCRs, GPR4 displays the highest expression in EPCs. The expression of GRP4 was markedly lower in EPCs from CAD patients than in EPCs from non-CAD individuals independent of acid stimulation. The siRNA-mediated knockdown of GPR4 with subsequent decreased phosphorylation of STAT3 mimicked the impaired function of EPCs from CAD patients at pH 6.4 but not at pH 7.4. Elevating GPR4 expression restored the neovessel formation mediated by EPCs from CAD patients in an acidic environment by activating STAT3/VEGFA signaling. Moreover, the beneficial impact of GPR4 upregulation on EPC-mediated angiogenic capacity was abrogated by blockade of the STAT3/VEGFA signaling pathway. Conclusions: Our present study demonstrated for the first time that loss of GPR4 is responsible for the decline in proton sensing and angiogenic capacity of EPCs from CAD patients. Augmentation of GPR4 expression promotes the neovessel formation of EPCs by activating STAT3/VEGF signaling. This finding implicates GPR4 as a potential therapeutic target for CAD characterized by impaired neovascularization in ischemic tissues. [ABSTRACT FROM AUTHOR]

Abstract 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. [ABSTRACT FROM AUTHOR]

Information on the distribution and biology of the G-protein coupled receptor 4 (GPR4) in the brain is limited. It is currently thought that GPR4 couples to G s proteins and may mediate central respiratory sensitivity to CO 2 . Using a knock-in mouse model, abundant GPR4 expression was detected in the cerebrovascular endothelium and neurones of dorsal raphe, retro-trapezoidal nucleus locus coeruleus and lateral septum. A similar distribution was confirmed using RNAscope in situ hybridisation. In HEK293 cells, overexpressing GPR4, it was highly constitutively active at neutral pH with little further increase in cAMP towards acidic pH. The GPR4 antagonist NE 52-QQ57 effectively blocked GPR4-mediated cAMP accumulation (IC 50 26.8 nM in HEK293 cells). In HUVEC which natively express GPR4, physiological acidification (pH 7.4–7.0) resulted in a cAMP increase by ∼55% which was completely prevented by 1 μM NE 52-QQ57. The main extracellular organic acid, l -lactic acid (LL; 1–10 mM), suppressed pH dependent activation of GPR4 in HEK293 and HUVEC cells, suggesting allosteric negative modulation. In unanaesthetised mice and rats, NE 52-QQ57 (20 mg kg −1 ) reduced ventilatory response to 5 and 10% CO 2 . In anaesthetised rats, systemic administration of NE 52-QQ57 (up to 20 mg kg −1 ) had no effect on hemodynamics, cerebral blood flow and blood oxygen level dependent responses. Central administration of NE 52-QQ57 (1 mM) in vagotomised anaesthetised rats did not affect CO 2 -induced respiratory responses. Our results indicate that GPR4 is expressed by multiple neuronal populations and endothelium and that its pH sensitivity is affected by level of expression and LL. NE 52-QQ57 blunts hypercapnic response to CO 2 but this effect is absent under anaesthesia, possibly due to the inhibitory effect of LL on GPR4. [ABSTRACT FROM AUTHOR]

Background and Aims: During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. Methods: The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. Results: Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/- /Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+ /Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. Conclusions: Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD. [ABSTRACT FROM AUTHOR]

The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, noncholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed in situ hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker, Neuromedin B mRNA (Nmb). Most (∼75%) RTN neurons express low-to-moderate levels of Nmb and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, TASK-2 and Gpr4. These Nmb-low RTN neurons also express varying levels of transcripts for Gal, Penk, and Adcyap1, and receptors for substance P, orexin, serotonin, and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of Nmb mRNA. These Nmb-high RTN neurons do not express Fos after hypercapnia and have low-to-undetectable levels of Kcnk5 or Gpr4 transcripts; they also express Adcyap1, but are essentially devoid of Penk and Gal transcripts. In male rats, Nmb is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum, Nmb is a selective marker of the RTN in rodents; Nmb-low neurons, the vast majority, are central respiratory chemoreceptors, whereas Nmb-high neurons likely have other functions. [ABSTRACT FROM AUTHOR]

GPR4, a G-protein coupled receptor, functions as a proton sensor being activated by extracellular acidic pH and has been implicated in playing a key role in acidosis associated with a variety of inflammatory conditions. An orally active GPR4 antagonist 39c was developed, starting from a high throughput screening hit 1 . The compound shows potent cellular activity and is efficacious in animal models of angiogenesis, inflammation and pain. [ABSTRACT FROM AUTHOR]

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1. [ABSTRACT FROM AUTHOR]

Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8) and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4-/- showed altered expression of PPARα, IL-6, IL-10, TNFα, and TGF-1β in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4+/+ and Gpr4-/- mice. In contrast, in aged mice (12 months old), the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues.

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. [ABSTRACT FROM AUTHOR]

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 G 12/13 inhibitory construct, cell spreading was restored whereas the inhibition and activation of the G q and G s pathways had little or no effect. Altogether our results indicate that through the G 12/13 /Rho signaling pathway GPR4 modulates focal adhesion dynamics and reduces cell spreading and membrane ruffling. [ABSTRACT FROM AUTHOR]

Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. [ABSTRACT FROM AUTHOR]

Protons (hydrogen ions) are the simplest form of ions universally produced by cellular metabolism including aerobic respiration and glycolysis. Export of protons out of cells by a number of acid transporters is essential to maintain a stable intracellular pH that is critical for normal cell function. Acid products in the tissue interstitium are removed by blood perfusion and excreted from the body through the respiratory and renal systems. However, the pH homeostasis in tissues is frequently disrupted in many pathophysiologic conditions such as in ischemic tissues and tumors where protons are overproduced and blood perfusion is compromised. Consequently, accumulation of protons causes acidosis in the affected tissue. Although acidosis has profound effects on cell function and disease progression, little is known about the molecular mechanisms by which cells sense and respond to acidotic stress. Recently a family of pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), has been identified and characterized. These GPCRs can be activated by extracellular acidic pH through the protonation of histidine residues of the receptors. Upon activation by acidosis the pH-sensing GPCRs can transduce several downstream G protein pathways such as the Gs, Gq/11, and G12/13 pathways to regulate cell behavior. Studies have revealed the biological roles of the pH-sensing GPCRs in the immune, cardiovascular, respiratory, renal, skeletal, endocrine, and nervous systems, as well as the involvement of these receptors in a variety of pathological conditions such as cancer, inflammation, pain, and cardiovascular disease. As GPCRs are important drug targets, small molecule modulators of the pH-sensing GPCRs are being developed and evaluated for potential therapeutic applications in disease treatment. [ABSTRACT FROM AUTHOR]

The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

Currently, the mechanism(s) responsible for the regulation of urea transporter B (UT-B) expression levels in the epithelium of the rumen remain unclear. We hypothesized that rumen fermentation products affect ruminal UT-B expression. Therefore, the effects of short-chain fatty acids (SCFA), pH, ammonia, and urea on mRNA and protein levels of UT-B were assayed in primary rumen epithelial cell cultures and in rumen epithelium obtained from intact goats. In vitro, SCFA and acidic pH were found to synergetically stimulate both mRNA and protein expression of UT-B, whereas NH4Cl decreased mRNA and protein levels of UT-B at pH 6.8. Treatment with urea increased both levels at pH 7.4. When goats received a diet rich in nitrogen (N) and nonfiber carbohydrates (NFC), their rumen epithelium had higher levels of UT-B, and the rumen contained higher concentrations of SCFA and NH3-N with a lower pH. An increase in plasma urea-N concentration was also observed compared with the plasma of the goats that received a diet low in N and NFC. In a second feeding trial, goats that received a NFC-rich, but isonitrogenous, diet had higher mRNA and protein levels of UT-B, and higher levels of G proteincoupled receptor (GPR) 41 and GPR4, in their rumen epithelium. The ruminal concentrations of SCFA and NH3-N also increased, while a lower pH was detected. In contrast, the serum urea-N concentrations remained unchanged. These data indicate that ruminal SCFA and pH are key factors, via GPR4 and GPR41, in the dietary regulation of UT-B expression, and they have priority over changes in plasma urea. [ABSTRACT FROM AUTHOR]

The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention. [ABSTRACT FROM AUTHOR]

Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8) and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4-/- showed altered expression of PPARα, IL-6, IL-10, TNFα, and TGF-1β in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4+/+ and Gpr4-/- mice. In contrast, in aged mice (12 months old), the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Under ischemic and inflammatory circumstances, such as allergic airway asthma, rheumatoid arthritis, atherosclerosis, and tumors, extracellular acidification occurs due to the stimulation of anaerobic glycolysis. An acidic microenvironment has been shown to modulate pro-inflammatory or anti-inflammatory responses, including cyclooxygenase-2 (COX-2) expression, prostaglandin synthesis, and cytokine expression, in a variety of cell types, and thereby to exacerbate or ameliorate inflammation. However, molecular mechanisms underlying extracellular acidic pH-induced actions have not been fully understood. Recent studies have shown that ovarian cancer G protein-coupled receptor 1 (OGR1)-family G protein-coupled receptors (GPCRs) can sense extracellular pH or protons, which in turn stimulates intracellular signaling pathways and subsequent diverse cellular responses. In the present review, I discuss extracellular acidic pH-induced inflammatory responses and related responses in inflammatory cells, such as macrophages and neutrophils, and non-inflammatory cells, such as smooth muscle cells and endothelial cells, focusing especially on proton-sensing GPCRs. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

The H+-K+-ATPase α-subunit (HKα2) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα2 plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing Gs-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα2 expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO2. PKA activity and HKα2 protein were monitored by immunoblot analysis, and HKα2 mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα2 mRNA; however, PKA activity and HKα2 protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα2 expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα2 protein by increasing PKA activity without altering HKα2 mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα2 and acid-base balance. [ABSTRACT FROM AUTHOR]

The H+-K+-ATPase α-subunit (HKα2) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα2 plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing Gs-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα2 expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO2. PKA activity and HKα2 protein were monitored by immunoblot analysis, and HKα2 mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα2 mRNA; however, PKA activity and HKα2 protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα2 expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα2 protein by increasing PKA activity without altering HKα2 mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα2 and acid-base balance. [ABSTRACT FROM AUTHOR]

Abstract: GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways, including the Gs-protein/cAMP, G12/13-protein/Rho, and Gq-protein/phospholipase C pathways. In the present study, we examined whether extracellularly located histidine residues of GPR4 sense extracellular protons and, if so, whether a certain histidine residue is critical for coupling to the single or multiple signaling pathway(s). We found that the mutation of histidine residue at 79, 165, or 269 from the N-terminal of GPR4 to phenylalanine shifted the half-maximal effective concentration (EC50) of proton-induced signaling activities to the right, including cAMP accumulation, SRE promoter activity reflecting Rho activity, and NFAT promoter activity reflecting phospholipase C signaling activity, without an appreciable change in the maximal activities. These results suggest that the protonation of each one of histidine residues at 79, 165, and 269 in GPR4 may be critical for conformational change of the receptor for coupling to multiple intracellular signaling pathways through G-proteins. [Copyright &y& Elsevier]

Abstract: GPR4 was initially identified as a receptor for sphingosylphosphorylcholine and lysophosphatidylcholine; however, lipid actions have not always been confirmed. Instead, ligand-independent actions have sometimes been observed in GPR4- and other OGR1 family receptor-expressing cells. Here, we examined the possible involvement of extracellular protons, which have recently been proposed as another ligand for GPR4. At pH 7.4, the epidermal growth factor-induced extracellular signal-regulated kinase activity was lower in GPR4-transfected RH7777 cells, in association with increased cAMP accumulation, than in vector-transfected cells. The serum response element (SRE)-driven transcriptional activity was also clearly higher in GPR4-expressing HEK293 cells than in vector-transfected cells at pH 7.4. These apparent ligand-independent actions were very small at alkalinic 7.8. The SRE activity was further increased by extracellular acidification in a manner dependent on the G13 protein/Rho signaling pathway in HEK293 cells expressing GPR4 or other OGR1 receptor family members. GPR4-expressing cells also showed a calcineurin-dependent nuclear factor of activated T cell (NFAT) promoter activation at pH 7.4, and this activity was further increased by pH below 7.2 in association with inositol phosphate production. In contrast to the cAMP and SRE responses, however, alkalinization to pH 7.8 hardly affected the high basal activity. Finally, the expression of GPR4 hardly modulated the sphingosylphosphorylcholine- or lysophosphatidylcholine-induced action. These results suggest that an extracellular proton play a role as a ligand in some of previously postulated ligand-independent actions through GPR4 receptors. Moreover, GPR4 may be a multi-functional receptor coupling to Gs, G13, and Gq/11 proteins in response to extracellular acidification. [Copyright &y& Elsevier]

Abundant evidence documents the highly proinflammatory actions of lysophosphatidylcholine (LPC). Further, LPC, found in high amounts in oxidized low-density lipoprotein (LDL), is implicated as an atherogenic factor. In endothelial cells, LPC impairs endothelial barrier function through GPR4, a novel receptor hypothesized to be sensitive to LPC and protons. The authors investigated the stimulation by LPC or low pH of GPR4 in human brain microvascular endothelial cells (HBMECs) and whether the activated GPR4 regulates in vitro monocyte transmigration. The results indicated that HBMECs stimulated by LPC (5 μM), but not low pH, showed a twofold increase in monocyte transmigration. Using retroviruses containing siRNA to GPR4, a > 60% reduction of GPR4 expression resulted in blockade of the LPC-stimulated transmigration. The inhibited response was restored by co-expression with an small interference RNA (siRNA)-resistant, but functional, GPR4 mutant construct. To investigate potential signaling mechanisms, the siRNA-mediated knockdown of GPR4 also prevented LPC-induced RhoA activation. C3 transferase, a Rho inhibitor, prevented ∼ ∼ 65% of the LPC-stimulated transmigration. LPC also increased MLC phosphorylation by 5 min, which was inhibited by the Rho kinase inhibitor, Y-27632 (10 μM) or ML-7 (myosin light chain kinase (MLCK) inhibitor). The findings indicate that the proinflammatory and atherogenic LPC stimulated endothelial GPR4, which promoted monocyte transmigration through a RhoA-dependent pathway. [ABSTRACT FROM AUTHOR]

Abstract: OGR1, GPR4, G2A, and TDAG8 share 40% to 50% homology with each other and seem to form a family of GPCRs. They have been described as receptors for lipid molecules such as sphingosylphosphorylcholine, lysophosphatidylcholine, and psychosine. Recent studies, however, have revealed that these receptors also sense extracellular protons or pH through histidine residues of receptors and stimulate a variety of intracellular signaling pathways through several species of hetero-trimeric G-proteins, including Gs, Gi, Gq, and G12/13. Thus, this family of GPCR seems to recognize both lipid molecules and protons as ligands. Although our knowledge of proton-sensing and lysolipid-sensitive GPCRs is preliminary, the receptor levels and ligand levels especially protons are both sensitively modulated in response to a variety of microenvironmental changes. These results suggest a multiple role of proton-sensing GPCRs in a variety of physiological and pathophysiological states. [Copyright &y& Elsevier]

The GPR4 subfamily consists of four G protein-coupled receptors that share significant sequence homology. In addition to GPR4, this subfamily includes OGR1, TDAG8 and G2A. G2A has previously been shown to be a potent transforming oncogene for murine 3T3 cells. Here we show that GPR4 also malignantly transforms NIH3T3 cells and that TDAG8 malignantly transforms the normal mammary epithelial cell line NMuMG. Overexpression of GPR4 or TDAG8 in HEK293 cells led to transcriptional activation from SRE- and CRE-driven promoters, independent of exogenously added ligand. TDAG8 and GPR4 are also overexpressed in a range of human cancer tissues. Our results suggest that GPR4 and TDAG8 overexpression in human tumors plays a role in driving or maintaining tumor formation.Oncogene (2004) 23, 6299-6303. doi:10.1038/sj.onc.1207838 Published online 28 June 2004 [ABSTRACT FROM AUTHOR]