Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1.

Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.

Ponesimod inhibits astrocyte-mediated neuroinflammation and protects against cingulum demyelination via S1P1 -selective modulation.

Ponesimod is a sphingosine 1-phosphate (S1P) receptor (S1PR) modulator that was recently approved for treating relapsing forms of multiple sclerosis (MS). Three other FDA-approved S1PR modulators for MS-fingolimod, siponimod, and ozanimod-share peripheral immunological effects via common S1P1 interactions, yet ponesimod may access distinct central nervous system (CNS) mechanisms through its selectivity for the S1P1 receptor. Here, ponesimod was examined for S1PR internalization and binding, human astrocyte signaling and single-cell RNA-seq (scRNA-seq) gene expression, and in vivo using murine cuprizone-mediated demyelination. Studies confirmed ponesimod's selectivity for S1P1 without comparable engagement to the other S1PR subtypes (S1P2,3,4,5 ). Ponesimod showed pharmacological properties of acute agonism followed by chronic functional antagonism of S1P1 . A major locus of S1P1 expression in the CNS is on astrocytes, and scRNA-seq of primary human astrocytes exposed to ponesimod identified a gene ontology relationship of reduced neuroinflammation and reduction in known astrocyte disease-related genes including those of immediate early astrocytes that have been strongly associated with disease progression in MS animal models. Remarkably, ponesimod prevented cuprizone-induced demyelination selectively in the cingulum, but not in the corpus callosum. These data support the CNS activities of ponesimod through S1P1 , including protective, and likely selective, effects against demyelination in a major connection pathway of the brain, the limbic fibers of the cingulum, lesions of which have been associated with several neurologic impairments including MS fatigue.

Fingolimod: Lessons Learned and New Opportunities for Treating Multiple Sclerosis and Other Disorders.

Fingolimod (FTY720, Gilenya) was the first US Food and Drug Administration-approved oral therapy for relapsing forms of multiple sclerosis (MS). Research on modified fungal metabolites converged with basic science studies that had identified lysophospholipid (LP) sphingosine 1-phosphate (S1P) receptors, providing mechanistic insights on fingolimod while validating LP receptors as drug targets. Mechanism of action (MOA) studies identified receptor-mediated processes involving the immune system and the central nervous system (CNS). These dual actions represent a more general theme for S1P and likely other LP receptor modulators. Fingolimod's direct CNS activities likely contribute to its efficacy in MS, with particular relevance to treating progressive disease stages and forms that involve neurodegeneration. The evolving understanding of fingolimod's MOA has provided strategies for developing next-generation compounds with superior attributes, suggesting new ways to target S1P as well as other LP receptor modulators for novel therapeutics in the CNS and other organ systems.

Lysophosphatidic acid (LPA)-antibody (504B3) engagement detected by interferometry identifies off-target binding.

BACKGROUND: Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that acts through its six cognate G protein-coupled receptors. As a family, lysophospholipids have already produced medicines (e.g., sphingosine 1-phosphate) as is being pursued for LPA through the use of specific antibodies that reduce ligand availability. METHODS: The binding properties of a commercially available, reportedly specific, monoclonal LPA antibody named 504B3 that is related to the clinical candidate Lpathomab/LT3015 were reexamined using a free solution assay (FSA) measured in a compensated interferometric reader (CIR). RESULTS: Measurement of 504B3 binding properties with an FSA-CIR approach revealed similar binding affinities for 504B3 against LPA as well as the non-LPA lipids, phosphatidic acid (PA) and lysophosphatidylcholine (LPC). CONCLUSIONS: Antibody binding specificity and sensitivity, particularly involving lipid ligands, can be assessed in solution and without labels using FSA-CIR. These findings could affect interpretations of both current and past basic and clinical studies employing 504B3 and related anti-LPA antibodies.

A Soluble Epoxide Hydrolase Inhibitor, 1-TrifluoromethoxyPhenyl-3-(1-Propionylpiperidin-4-yl) Urea, Ameliorates Experimental Autoimmune Encephalomyelitis.

Polyunsaturated fatty acids (PUFAs) are essential FAs for human health. Cytochrome P450 oxygenates PUFAs to produce anti-inflammatory and pain-resolving epoxy fatty acids (EpFAs) and other oxylipins whose epoxide ring is opened by the soluble epoxide hydrolase (sEH/Ephx2), resulting in the formation of toxic and pro-inflammatory vicinal diols (dihydroxy-FAs). Pharmacological inhibition of sEH is a promising strategy for the treatment of pain, inflammation, cardiovascular diseases, and other conditions. We tested the efficacy of a potent, selective sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Prophylactic TPPU treatment significantly ameliorated EAE without affecting circulating white blood cell counts. TPPU accumulated in the spinal cords (SCs), which was correlated with plasma TPPU concentration. Targeted lipidomics in EAE SCs and plasma identified that TPPU blocked production of dihydroxy-FAs efficiently and increased some EpFA species including 12(13)-epoxy-octadecenoic acid (12(13)-EpOME) and 17(18)-epoxy-eicosatrienoic acid (17(18)-EpETE). TPPU did not alter levels of cyclooxygenase (COX-1/2) metabolites, while it increased 12-hydroxyeicosatetraenoic acid (12-HETE) and other 12/15-lipoxygenase metabolites. These analytical results are consistent with sEH inhibitors that reduce neuroinflammation and accelerate anti-inflammatory responses, providing the possibility that sEH inhibitors could be used as a disease modifying therapy, as well as for MS-associated pain relief.

Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader.

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.

Introduction: Druggable Lipid Signaling Pathways.

Lipids are essential for life. They store energy, constitute cellular membranes, serve as signaling molecules, and modify proteins. In the long history of lipid research, many drugs targeting lipid receptors and enzymes that are responsible for lipid metabolism and function have been developed and applied to a variety of diseases. For example, non-steroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed medications for fever, pain, and inflammation. The NSAIDs block prostaglandin production by inhibiting cyclooxygenases. A recent innovative breakthrough in drug discovery for the lipid biology field was the development of the sphingosine 1-phosphate receptor modulators (fingolimod, siponimod and ozanimod) for the treatment of multiple sclerosis, which were approved by the United States Food and Drug Administration in 2010, 2019 and 2020, respectively. This review series of "Druggable Lipid Signaling Pathways" provides 9 outstanding reviews that summarize the currently available drugs that target lipid signaling pathways and also outlines future directions for drug discovery. The review chapters include lipid signaling pathways (prostanoids, leukotrienes, epoxy fatty acids, sphingolipids, lysophospholipids, endocannabinoids, and phosphoinositides) and lipid signaling proteins (lysophospholipid acyltransferases, phosphoinositide 3-kinase, and G protein-coupled receptors (GPCRs)). Drugs targeting lipid signaling pathways promise to be life changing magic for the future of human health and well-being.

Druggable Lipid GPCRs: Past, Present, and Prospects.

G protein-coupled receptors (GPCRs) have seven transmembrane spanning domains and comprise the largest superfamily with ~800 receptors in humans. GPCRs are attractive targets for drug discovery because they transduce intracellular signaling in response to endogenous ligands via heterotrimeric G proteins or arrestins, resulting in a wide variety of physiological and pathophysiological responses. The endogenous ligands for GPCRs are highly chemically diverse and include ions, biogenic amines, nucleotides, peptides, and lipids. In this review, we follow the KonMari method to better understand druggable lipid GPCRs. First, we have a comprehensive tidying up of lipid GPCRs including receptors for prostanoids, leukotrienes, specialized pro-resolving mediators (SPMs), lysophospholipids, sphingosine 1-phosphate (S1P), cannabinoids, platelet-activating factor (PAF), free fatty acids (FFAs), and sterols. This tidying up consolidates 46 lipid GPCRs and declutters several perplexing lipid GPCRs. Then, we further tidy up the lipid GPCR-directed drugs from the literature and databases, which identified 24 clinical drugs targeting 16 unique lipid GPCRs available in the market and 44 drugs under evaluation in more than 100 clinical trials as of 2019. Finally, we introduce drug designs for GPCRs that spark joy, such as positive or negative allosteric modulators (PAM or NAM), biased agonism, functional antagonism like fingolimod, and monoclonal antibodies (MAbs). These strategic drug designs may increase the efficacy and specificity of drugs and reduce side effects. Technological advances will help to discover more endogenous lipid ligands from the vast number of remaining orphan GPCRs and will also lead to the development novel lipid GPCR drugs to treat various diseases.

Lysophospholipid G protein-coupled receptor binding parameters as determined by backscattering interferometry.

Lysophosphatidic acid (LPA) activates cognate G protein-coupled receptors (GPCRs) to initiate biological signaling cascades. Lysophospholipid (LP) receptor binding properties remain incompletely assessed because of difficulties with ligand lipophilicity and lipid "stickiness." These inherent attributes produce high levels of nonspecific binding within cell-membrane preparations used to assess GPCRs, as has been shown in classical binding assays using radiolabeled ligands, making accurate measurements of lipid binding kinetics difficult to achieve. Backscattering interferometry (BSI) is an optical technology that measures molecular binding interactions by reporting changes in the refractive index of a solution after binding events. Here, we report the use of BSI to assess LPA1 for its ability to bind to naturally occurring lipids and a synthetic LPA1 antagonist (ONO-9780307), under both primary- and competition-binding conditions. Assessment of 12 different lipids demonstrated that the known LP ligand, 1-oleoyl-LPA, as well as an endocannabinoid metabolite, anandamide phosphate, are specific ligands for LPA1, whereas other LPs tested were not. Newly determined dissociation constants (Kd values) for orthosteric lipid ligands approximated 10-9 M, substantially lower (i.e., with higher affinity) than measured Kd values in classical binding or cell-based assays. These results demonstrate that BSI may have particular utility in assessing binding interactions between lipid receptors and their lipid ligands and could provide new screening approaches for lipid receptor identification and drug discovery.

Systematic Understanding of Bioactive Lipids in Neuro-Immune Interactions: Lessons from an Animal Model of Multiple Sclerosis.

Bioactive lipids, or lipid mediators, are utilized for intercellular communications. They are rapidly produced in response to various stimuli and exported to extracellular spaces followed by binding to cell surface G protein-coupled receptors (GPCRs) or nuclear receptors. Many drugs targeting lipid signaling such as non-steroidal anti-inflammatory drugs (NSAIDs), prostaglandins, and antagonists for lipid GPCRs are in use. For example, the sphingolipid analog, fingolimod (also known as FTY720), was the first oral disease-modifying therapy (DMT) for relapsing-remitting multiple sclerosis (MS), whose mechanisms of action (MOA) includes sequestration of pathogenic lymphocytes into secondary lymphoid organs, as well as astrocytic modulation, via down-regulation of the sphingosine 1-phosphate (S1P) receptor, S1P1, by in vivo-phosphorylated fingolimod. Though the cause of MS is still under debate, MS is considered to be an autoimmune demyelinating and neurodegenerative disease. This review summarizes the involvement of bioactive lipids (prostaglandins, leukotrienes, platelet-activating factors, lysophosphatidic acid, and S1P) in MS and the animal model, experimental autoimmune encephalomyelitis (EAE). Genetic ablation, along with pharmacological inhibition, of lipid metabolic enzymes and lipid GPCRs revealed that each bioactive lipid has a unique role in regulating immune and neural functions, including helper T cell (TH1 and TH17) differentiation and proliferation, immune cell migration, astrocyte responses, endothelium function, and microglial phagocytosis. A systematic understanding of bioactive lipids in MS and EAE dredges up information about understudied lipid signaling pathways, which should be clarified in the near future to better understand MS pathology and to develop novel DMTs.

Lysophosphatidic acid receptor, LPA6, regulates endothelial blood-brain barrier function: Implication for hepatic encephalopathy.

Cerebral edema is a life-threatening neurological condition characterized by brain swelling due to the accumulation of excess fluid both intracellularly and extracellularly. Fulminant hepatic failure (FHF) develops cerebral edema by disrupting blood-brain barrier (BBB). However, the mechanisms by which mediator induces brain edema in FHF remain to be elucidated. Here, we assessed a linkage between brain edema and lysophosphatidic acid (LPA) signaling by utilizing an animal model of FHF and in vitro BBB model. Azoxymethane-treated mice developed FHF and hepatic encephalopathy, associated with higher autotaxin (ATX) activities in serum than controls. Using in vitro BBB model, LPA disrupted the structural integrity of tight junction proteins including claudin-5, occludin, and ZO-1. Furthermore, LPA decreased transendothelial electrical resistances in in vitro BBB model, and induced cell contraction in brain endothelial monolayer cultures, both being inhibited by a Rho-associated protein kinase inhibitor, Y-27632. The brain capillary endothelial cells predominantly expressed LPA6 mRNA, whose knockdown blocked the LPA-induced endothelial cell contraction. Taken together, the up-regulation of serum ATX in hepatic encephalopathy may activate the LPA-LPA6-G12/13-Rho pathway in brain capillary endothelial cells, leading to enhancement of BBB permeability and brain edema.

Randomised controlled trial of long-term maintenance corticosteroid therapy in patients with autoimmune pancreatitis.

OBJECTIVE: Corticosteroid has been established as the standard therapy for autoimmune pancreatitis (AIP), but the requirement for maintenance corticosteroid therapy is controversial. We conducted a randomised controlled trial to clarify the efficacy of maintenance corticosteroid therapy in patients with AIP. DESIGN: We conducted a multicentre, tertiary setting, randomised controlled trial. After the induction of remission with the initial oral prednisolone (PSL) treatment, maintenance therapy with PSL at 5-7.5 mg/day was continued for 3 years or withdrawn at 26 weeks. The primary endpoint was relapse-free survival over 3 years and the secondary endpoint was serious corticosteroid-related complications. All analyses were performed on an intention-to-treat basis. RESULTS: Between April 2009 and March 2012, 49 patients with AIP were randomly assigned to the maintenance therapy group (n=30) or the cessation group (n=19). Baseline characteristics were not different between the two groups. Relapses occurred within 3 years in 11 out of 19 (57.9%) patients assigned to the cessation group, and in 7 of 30 (23.3%) patients in the maintenance therapy group. The relapse rate over 3 years was significantly lower in the maintenance therapy group than that in the cessation group (p=0.011). The relapse-free survival was significantly longer in the maintenance therapy group than that in the cessation group (p=0.007). No serious corticosteroid-related complications requiring discontinuation of PSL were observed. CONCLUSIONS: Maintenance corticosteroid therapy for 3 years may decrease relapses in patients with AIP compared with those who discontinued the therapy at 26 weeks. TRIAL REGISTRATION NUMBER: UMIN000001818; Results.

Lysophospholipid receptors in drug discovery.

Lysophospholipids (LPs), including lysophosphatidic acid (LPA), sphingosine 1-phospate (S1P), lysophosphatidylinositol (LPI), and lysophosphatidylserine (LysoPS), are bioactive lipids that transduce signals through their specific cell-surface G protein-coupled receptors, LPA1-6, S1P1-5, LPI1, and LysoPS1-3, respectively. These LPs and their receptors have been implicated in both physiological and pathophysiological processes such as autoimmune diseases, neurodegenerative diseases, fibrosis, pain, cancer, inflammation, metabolic syndrome, bone formation, fertility, organismal development, and other effects on most organ systems. Advances in the LP receptor field have enabled the development of novel small molecules targeting LP receptors for several diseases. Most notably, fingolimod (FTY720, Gilenya, Novartis), an S1P receptor modulator, became the first FDA-approved medicine as an orally bioavailable drug for treating relapsing forms of multiple sclerosis. This success is currently being followed by multiple, mechanistically related compounds targeting S1P receptor subtypes, which are in various stages of clinical development. In addition, an LPA1 antagonist, BMS-986020 (Bristol-Myers Squibb), is in Phase 2 clinical development for treating idiopathic pulmonary fibrosis, as a distinct compound, SAR100842 (Sanofi) for the treatment of systemic sclerosis and related fibrotic diseases. This review summarizes the current state of drug discovery in the LP receptor field.

TDAG8 activation inhibits osteoclastic bone resorption.

Although the roles of acids in bone metabolism are well characterized, the function of proton-sensing receptors in bone metabolism remains to be explored. In this study, we evaluated the role of proton-sensing receptor T-cell death-associated gene 8 (TDAG8) in osteoclastic activity during bone loss after ovariectomy. Through observations of bone mineral content, we found that pathological bone resorption was significantly exacerbated in mice homozygous for a gene trap mutation in the Tdag8 gene. Furthermore, osteoclasts from the homozygous mutant mice resorbed calcium in vitro more than the osteoclasts from the heterozygous mice did. Impaired osteoclast formation under acidic conditions was ameliorated in cultures of bone marrow cells by Tdag8 gene mutation. Extracellular acidification changed the cell morphology of osteoclasts via the TDAG8-Rho signaling pathway. These results suggest that the enhancement of TDAG8 function represents a new strategy for preventing bone resorption diseases, such as osteoporosis.

Modeling of eicosanoid fluxes reveals functional coupling between cyclooxygenases and terminal synthases.

Eicosanoids, including prostaglandins (PG) and leukotrienes, are lipid mediators derived from arachidonic acid. A quantitative and biochemical level understanding of eicosanoid metabolism would aid in understanding the mechanisms that govern inflammatory processes. Here, we present a combined experimental and computational approach to understanding the biochemical basis of eicosanoid metabolism in macrophages. Lipidomic and transcriptomic measurements and analyses reveal temporal and dynamic changes of the eicosanoid metabolic network in mouse bone marrow-derived macrophages (BMDM) upon stimulation of the Toll-like receptor 4 with Kdo2-Lipid A (KLA) and stimulation of the P2X7 purinergic receptor with adenosine 5'-triphosphate. Kinetic models were developed for the cyclooxygenase (COX) and lipoxygenase branches of arachidonic acid metabolism, and then the rate constants were estimated with a data set from ATP-stimulated BMDM, using a two-step matrix-based approach employing a constrained least-squares method followed by nonlinear optimization. The robustness of the model was validated through parametric sensitivity, uncertainty analysis, and predicting an independent dataset from KLA-primed ATP-stimulated BMDM by allowing the parameters to vary within the uncertainty range of the calculated parameters. We analyzed the functional coupling between COX isozymes and terminal enzymes by developing a PGH2-divided model. This provided evidence for the functional coupling between COX-2 and PGE2 synthase, between COX-1/COX-2 and PGD2 synthase, and also between COX-1 and thromboxane A2 synthase. Further, these functional couplings were experimentally validated using COX-1 and COX-2 selective inhibitors. The resulting fluxomics analysis demonstrates that the "multi-omics" systems biology approach can define the complex machinery of eicosanoid networks.

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.

Molecular and neuroimmune pharmacology of S1P receptor modulators and other disease-modifying therapies for multiple sclerosis.

Multiple sclerosis (MS) is a neurological, immune-mediated demyelinating disease that affects people in the prime of life. Environmental, infectious, and genetic factors have been implicated in its etiology, although a definitive cause has yet to be determined. Nevertheless, multiple disease-modifying therapies (DMTs: including interferons, glatiramer acetate, fumarates, cladribine, teriflunomide, fingolimod, siponimod, ozanimod, ponesimod, and monoclonal antibodies targeting ITGA4, CD20, and CD52) have been developed and approved for the treatment of MS. All the DMTs approved to date target immunomodulation as their mechanism of action (MOA); however, the direct effects of some DMTs on the central nervous system (CNS), particularly sphingosine 1-phosphate (S1P) receptor (S1PR) modulators, implicate a parallel MOA that may also reduce neurodegenerative sequelae. This review summarizes the currently approved DMTs for the treatment of MS and provides details and recent advances in the molecular pharmacology, immunopharmacology, and neuropharmacology of S1PR modulators, with a special focus on the CNS-oriented, astrocyte-centric MOA of fingolimod.

FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis.

Vitamin B12 (B12) deficiency causes neurological manifestations resembling multiple sclerosis (MS); however, a molecular explanation for the similarity is unknown. FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analog approved for MS therapy that can functionally antagonize S1P1. Here, we report that FTY720 suppresses neuroinflammation by functionally and physically regulating the B12 pathways. Genetic and pharmacological S1P1 inhibition upregulates a transcobalamin 2 (TCN2)-B12 receptor, CD320, in immediate-early astrocytes (ieAstrocytes; a c-Fos-activated astrocyte subset that tracks with experimental autoimmune encephalomyelitis [EAE] severity). CD320 is also reduced in MS plaques. Deficiency of CD320 or dietary B12 restriction worsens EAE and eliminates FTY720's efficacy while concomitantly downregulating type I interferon signaling. TCN2 functions as a chaperone for FTY720 and sphingosine, whose complex induces astrocytic CD320 internalization, suggesting a delivery mechanism of FTY720/sphingosine via the TCN2-CD320 pathway. Taken together, the B12-TCN2-CD320 pathway is essential for the mechanism of action of FTY720.

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.