Keisuke Yanagida

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.

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 p2y9/GPR23 as a novel fourth LPA receptor, LPA4 (Noguchi, K., Ishii, S., and Shimizu, T. (2003) J. Biol. Chem. 278, 25600-25606). To assess the functions of LPA4 in neuronal cells, we used rat neuroblastoma B103 cells that lack endogenous responses to LPA. In B103 cells stably expressing LPA4, we observed Gq/11-dependent calcium mobilization, but LPA did not affect adenylyl cyclase activity. In LPA4 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 LPA4 as well as LPA1 couple to Gq/11 and G12/13, whereas LPA4 differs from LPA1 in that it does not couple to Gi/o. Through neurite retraction and cell aggregation, LPA4 may play a role in neuronal development such as neurogenesis and neuronal migration.

LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis

Lysophosphatidic acid (LPA) is a potent lipid mediator with a wide variety of biological actions mediated through G protein-coupled receptors (LPA(1-6)). LPA(4) has been identified as a G(13) protein-coupled receptor, but its physiological role is unknown. Here we show that a subset of LPA(4)-deficient embryos did not survive gestation and displayed hemorrhages and/or edema in many organs at multiple embryonic stages. The blood vessels of bleeding LPA(4)-deficient embryos were often dilated. The recruitment of mural cells, namely smooth muscle cells and pericytes, was impaired. Consistently, Matrigel plug assays showed decreased mural cell coverage of endothelial cells in the neovessels of LPA(4)-deficient adult mice. In situ hybridization detected Lpa4 mRNA in the endothelium of some vasculatures. Similarly, the lymphatic vessels of edematous embryos were dilated. These results suggest that LPA(4) regulates establishment of the structure and function of blood and lymphatic vessels during mouse embryogenesis. Considering the critical role of autotaxin (an enzyme involved in LPA production) and Gα(13) in vascular development, we suggest that LPA(4) provides a link between these 2 molecules.

Current progress in non-Edg family LPA receptor research.

Lysophosphatidic acid (LPA) is the simplest phospholipid yet possesses myriad biological functions. Until 2003, the functions of LPA were thought to be elicited exclusively by three subtypes of the endothelial differentiation gene (Edg) family of G protein-coupled receptors - LPA(1), LPA(2), and LPA(3). However, several biological functions of LPA could not be assigned to any of these receptors indicating the existence of one or more additional LPA receptor(s). More recently, the discovery of a second cluster of LPA receptors which includes LPA(4), LPA(5), and LPA(6) has paved the way for new avenues of LPA research. Analyses of these non-Edg family LPA receptors have begun to fill in gaps to understand biological functions of LPA such as platelet aggregation and vascular development that could not be ascribed to classical Edg family LPA receptors and are also unveiling new biological functions. Here we review recent progress in the non-Edg family LPA receptor research, with special emphasis on the pharmacology, signaling, and physiological roles of this family of receptors. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

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.

Interplay between CXCR2 and BLT1 Facilitates Neutrophil Infiltration and Resultant Keratinocyte Activation in a Murine Model of Imiquimod-Induced Psoriasis

Psoriasis is an inflammatory skin disease with accelerated epidermal cell turnover. Neutrophil accumulation in the skin is one of the histological characteristics of psoriasis. However, the precise mechanism and role of neutrophil infiltration remain largely unknown. In this article, we show that orchestrated action of CXCR2 and leukotriene B4 receptor BLT1 plays a key role in neutrophil recruitment during the development of imiquimod (IMQ)-induced psoriatic skin lesions in mice. Depletion of neutrophils with anti–Ly-6G Ab ameliorated the disease severity, along with reduced expression of proinflammatory cytokine IL-1β in the skin. Furthermore, CXCR2 and BLT1 coordinately promote neutrophil infiltration into the skin during the early phase of IMQ-induced inflammation. In vitro, CXCR2 ligands augment leukotriene B4 production by murine neutrophils, which, in turn, amplifies chemokine-mediated neutrophil chemotaxis via BLT1 in autocrine and/or paracrine manners. In agreement with the increased IL-19 expression in IMQ-treated mouse skin, IL-1β markedly upregulated expression of acanthosis-inducing cytokine IL-19 in human keratinocytes. We propose that coordination of chemokines, lipids, and cytokines with multiple positive feedback loops might drive the pathogenesis of psoriasis and, possibly, other inflammatory diseases as well. Interference to this positive feedback or its downstream effectors could be targets of novel anti-inflammatory treatment.

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 LPA1 and LPA3, 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. (Cancer Sci 2012; 103: 1099–1104)

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 A2 (PLA2) 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 PLA2s, group V secretory PLA2, 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 PLA2 is likely to be responsible for the increased PLA2 activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA2/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.

Size-selective opening of the blood–brain barrier by targeting endothelial sphingosine 1–phosphate receptor 1

Significance The blood–brain barrier (BBB) poses a major obstacle for drug delivery to the central nervous system (CNS). This study revealed that genetic or pharmacological targeting of sphingosine 1–phosphate receptor-1 (S1P1) facilitates a small-molecule-selective BBB opening, without major signs of CNS inflammation or injury. This size-selective BBB opening could be attributed to changes in the cytoskeletal association of tight junction proteins. Importantly, BBB opening by pharmacological blockage of S1P1 was reversible, suggesting that targeting S1P1 may be a promising strategy for the safe delivery of therapeutic agents into the CNS to treat neurodegenerative and neuroinflammatory diseases and neurological cancers. The vasculature of the central nervous system (CNS) forms a selective barrier termed the blood–brain barrier (BBB). Disruption of the BBB may contribute to various CNS diseases. Conversely, the intact BBB restricts efficient penetration of CNS-targeted drugs. Here, we report the BBB-regulatory role of endothelial sphingosine 1–phosphate (S1P) receptor-1, a G protein-coupled receptor known to promote the barrier function in peripheral vessels. Endothelial-specific S1pr1 knockout mice (S1pr1iECKO) showed BBB breach for small-molecular-mass fluorescence tracers (<3 kDa), but not larger tracers (>10 kDa). Chronic BBB leakiness was associated with cognitive impairment, as assessed by the novel object recognition test, but not signs of brain inflammation. Brain microvessels of S1pr1iECKO mice showed altered subcellular distribution of tight junctional proteins. Pharmacological inhibition of S1P1 function led to transient BBB breach. These data suggest that brain endothelial S1P1 maintain the BBB by regulating the proper localization of tight junction proteins and raise the possibility that endothelial S1P1 inhibition may be a strategy for transient BBB opening and delivery of small molecules into the CNS.

Vascular and Immunobiology of the Circulatory Sphingosine 1-Phosphate Gradient

Vertebrates are endowed with a closed circulatory system, the evolution of which required novel structural and regulatory changes. Furthermore, immune cell trafficking paradigms adapted to the barriers imposed by the closed circulatory system. How did such changes occur mechanistically? We propose that spatial compartmentalization of the lipid mediator sphingosine 1-phosphate (S1P) may be one such mechanism. In vertebrates, S1P is spatially compartmentalized in the blood and lymphatic circulation, thus comprising a sharp S1P gradient across the endothelial barrier. Circulatory S1P has critical roles in maturation and homeostasis of the vascular system as well as in immune cell trafficking. Physiological functions of S1P are tightly linked to shear stress, the key biophysical stimulus from blood flow. Thus, circulatory S1P confinement could be a primordial strategy of vertebrates in the development of a closed circulatory system. This review discusses the cellular and molecular basis of the S1P gradients and aims to interpret its physiological significance as a key feature of the closed circulatory system.