Constitutively active GPR43 is crucial for proper leukocyte differentiation.

The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.

Bitter taste receptor T2R38 is expressed on skin-infiltrating lymphocytes and regulates lymphocyte migration.

Bitter taste receptors (T2Rs) are G protein-coupled receptors involved in the perception of bitter taste on the tongue. In humans, T2Rs have been found in several sites outside the oral cavity. Although T2R38 has been reported to be expressed on peripheral lymphocytes, it is poorly understood whether T2R38 plays immunological roles in inflammatory skin diseases such as atopic dermatitis (AD). Then, we first confirmed that T2R38 gene expression was higher in lesional skin of AD subjects than healthy controls. Furthermore, skin T2R38 expression levels were correlated with serum thymus and activation-regulated chemokine and IgE levels in AD patients. In lesional skin of AD, section staining revealed that CD3+ T cells in the dermis were T2R38 positive. In addition, flow cytometry analysis showed T2R38 expression in skin T cells. Migration assays using T2R38-transduced Jurkat T cell leukemia cells revealed that T2R38 agonists exerted a dose-dependent migration inhibitory effect. Moreover, skin tissue extracts, as well as supernatants of cultured HaCaT keratinocytes, caused T2R38-dependent migration inhibition, indicating that there should be an endogenous ligand for T2R38 in the skin epidermis. These findings implicate T2R38 as a migratory inhibitory receptor on the skin-infiltrating lymphocytes and as a therapeutic target for allergic/inflammatory skin diseases.

Sphingosine-1-phosphate promotes tumor development and liver fibrosis in mouse model of congestive hepatopathy.

BACKGROUND AND AIMS: Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and HCC. However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood. APPROACH AND RESULTS: Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce LSECs capillarization in mice and in vitro. LSEC capillarization was also confirmed in patients with FALD. Mitogenic factor, sphingosine-1-phosphate (S1P), was increased in congestive liver and expression of sphingosine kinase 1, a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR) 1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobial treatment lowered portal blood LPS concentration, LSEC capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver. CONCLUSIONS: In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for patients with RHF with primary or metastatic liver cancer.

Druggable Lysophospholipid Signaling Pathways.

Lysophosphatidic acid (LPA) has major roles as a bioactive signaling molecule, with multiple physiological and pathological roles being described in almost every major organ system. In this review we discuss LPA signaling pathways as emerging drug targets for multiple conditions relevant to human health and disease. LPA signals through the six G protein-coupled receptors LPA1-6, and several of these receptors along with the LPA-producing enzyme including autotaxin (ATX) are now established as therapeutic targets with potential to treat various human diseases as exemplified by several LPA signaling targeting compounds now in clinical trials for idiopathic pulmonary fibrosis and systemic sclerosis. Several crystal structures of LPA receptors and ATX have been solved, which will accelerate development of highly selective and effective LPA signaling targeting compounds. We also review additional bioactive lysophospholipid (LPL) signaling molecules including lysophosphatidylserine and lysophosphatidylinositol, which represent the next wave of LPL druggable targets. An emerging theme in bioactive LPL signaling is that where the ligand is produced and how it is delivered to the cognate receptor are critical determinants of the biological responses. We will also discuss how connecting the production and function of bioactive LPLs will identify new therapeutic strategies to effectively target LPL signaling pathways.

Sphingosine 1-Phosphate Receptor Signaling Establishes AP-1 Gradients to Allow for Retinal Endothelial Cell Specialization.

Transcriptional mechanisms that drive angiogenesis and organotypic vascular endothelial cell specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs). Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent vascular endothelial (VE)-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this TF. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of ß-catenin-dependent genes such as TCF/LEF1 and ZIC3 TFs, transporters, and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 gradient and enabling organotypic endothelial cell specialization of the vascular network.

Stepwise phosphorylation of leukotriene B4 receptor 1 defines cellular responses to leukotriene B4.

Leukotriene B4 (LTB4) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB4 Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB4, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1. Different concentrations of LTB4 induced distinct phosphorylation events, and these ligand-induced modifications facilitated additional phosphorylation events at the basal phosphorylation sites. Because neutrophils migrate toward inflammatory sites along a gradient of LTB4, the degree of BLT1 phosphorylation likely increases in parallel with the increase in LTB4 concentration as the cells migrate. At high concentrations of LTB4, deficiencies in these two types of phosphorylation events impaired chemotaxis and ß-hexosaminidase release, a proxy for degranulation, in Chinese hamster ovary (CHO-K1) and rat basophilic leukemia (RBL-2H3) cells, respectively. These results suggest that an LTB4 gradient around inflammatory sites enhances BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of phagocytic and immune cells and the initiation of local responses, including degranulation.

Orally active lysophosphatidic acid receptor antagonist attenuates pancreatic cancer invasion and metastasis in vivo.

Pancreatic cancer is highly metastatic and has a poor prognosis. However, there is no established treatment for pancreatic cancer. Lysophosphatidic acid (LPA) has been shown to be present in effluents of cancers and involved in migration and proliferation in a variety of cancer cells, including pancreatic cancer cells, in vitro. In the current study, we examined whether an orally active LPA antagonist is effective for pancreatic cancer tumorigenesis and metastasis in vivo. Oral administration of Ki16198, which is effective for LPA(1) and LPA(3), into YAPC-PD pancreatic cancer cell-inoculated nude mice significantly inhibited tumor weight and remarkably attenuated invasion and metastasis to lung, liver, and brain, in association with inhibition of matrix metalloproteinase (MMP) accumulation in ascites in vivo. Ki16198 inhibited LPA-induced migration and invasion in several pancreatic cancer cells in vitro, which was associated with the inhibition of LPA-induced MMP production. In conclusion, Ki16198 is a promising orally active LPA antagonist for inhibiting the invasion and metastasis of pancreatic cancer cells. The inhibitory effects of the antagonist on invasion and metastasis in vivo may be partially explained by the inhibition of motility activity and MMP production in cancer cells.

The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor.

Tumors often are associated with a low extracellular pH, which induces a variety of cellular events. However, the mechanisms by which tumor cells recognize and react to the acidic environment have not been fully elucidated. T-cell death-associated gene 8 (TDAG8) is an extracellular pH-sensing G protein-coupled receptor that is overexpressed in various tumors and tumor cell lines. In this report, we show that TDAG8 on the surface of tumor cells facilitates tumor development by sensing the acidic environment. Overexpression of TDAG8 in mouse Lewis lung carcinoma (LLC) cells enhanced tumor development in animal models and rendered LLC cells resistant to acidic culture conditions by increasing activation of protein kinase A and extracellular signal-regulated kinase in vitro. Moreover, shRNA-mediated knockdown of endogenous TDAG8 in NCI-H460 human non-small cell lung cancer cells reduced cell survival in an acidic environment in vitro as well as tumor development in vivo. Microarray analyses of tumor-containing lung tissues of mice injected with TDAG8-expressing LLC cells revealed up-regulation of genes related to cell growth and glycolysis. These results support the hypothesis that TDAG8 enhances tumor development by promoting adaptation to the acidic environment to enhance cell survival/proliferation. TDAG8 may represent a therapeutic target for arresting tumor growth.

Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA4. Here we report that p2y5 is a novel LPA receptor coupling to the G13-Rho signaling pathway. "LPA receptor-null" RH7777 and B103 cells exogenously expressing p2y5 showed [3H]LPA binding, LPA-induced [35S]guanosine 5'-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and Gs/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA6.

Platelet-activating factor production in the spinal cord of experimental allergic encephalomyelitis mice via the group IVA cytosolic phospholipase A2-lyso-PAFAT axis.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a critical role in inflammatory disorders including experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Although PAF accumulation in the spinal cord (SC) of EAE mice and cerebrospinal fluid of MS patients has been reported, little is known about the metabolic processing of PAF in these diseases. In this study, we demonstrate that the activities of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase (LysoPAFAT) are elevated in the SC of EAE mice on a C57BL/6 genetic background compared with those of naive mice and correlate with disease severity. Correspondingly, levels of groups IVA, IVB, and IVF cytosolic PLA(2)s, group V secretory PLA(2), and LysoPAFAT transcripts are up-regulated in the SC of EAE mice. PAF acetylhydrolase activity is unchanged during the disease course. In addition, we show that LysoPAFAT mRNA and protein are predominantly expressed in microglia. Considering the substrate specificity and involvement of PAF production, group IVA cytosolic PLA(2) is likely to be responsible for the increased PLA(2) activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA(2)/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.

LPA4/p2y9/GPR23 mediates rho-dependent morphological changes in a rat neuronal cell line.

Lysophosphatidic acid (LPA) is a potent lipid mediator that evokes a variety of biological responses in many cell types via its specific G protein-coupled receptors. In particular, LPA affects cell morphology, cell survival, and cell cycle progression in neuronal cells. Recently, we identified p2y(9)/GPR23 as a novel fourth LPA receptor, LPA(4) (Noguchi, K., Ishii, S., and Shimizu, T. (2003) J. Biol. Chem. 278, 25600-25606). To assess the functions of LPA(4) in neuronal cells, we used rat neuroblastoma B103 cells that lack endogenous responses to LPA. In B103 cells stably expressing LPA(4), we observed G(q/11)-dependent calcium mobilization, but LPA did not affect adenylyl cyclase activity. In LPA(4) transfectants, LPA induced dramatic morphological changes, i.e. neurite retraction, cell aggregation, and cadherin-dependent cell adhesion, which involved Rho-mediated signaling pathways. Thus, our results demonstrated that LPA(4) as well as LPA(1) couple to G(q/11) and G(12/13), whereas LPA(4) differs from LPA(1) in that it does not couple to G(i/o). Through neurite retraction and cell aggregation, LPA(4) may play a role in neuronal development such as neurogenesis and neuronal migration.