Inverse agonism of lysophospholipids with cationic head groups at Gi-coupled receptor GPR82.

GPR82 is an orphan G protein-coupled receptor (GPCR) that has been implicated in lipid storage in mouse adipocytes. However, the intracellular signaling as well as the specific ligands of GPR82 remain unknown. GPR82 is closely related to GPR34, a GPCR for the bioactive lipid molecule lysophosphatidylserine. In this study, we screened a lipid library using GPR82-transfected cells to search for ligands that act on GPR82. By measuring cyclic adenosine monophosphate levels, we found that GPR82 is an apparently constitutively active GPCR that leads to Gi protein activation. In addition, edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine), an artificial lysophospholipid with a cationic head group that exerts antitumor activity, inhibited the Gi protein activation by GPR82. Two endogenous lysophospholipids with cationic head groups, lysophosphatidylcholine (1-oleoyl-sn-glycero-3-phosphocholine) and lysophosphatidylethanolamine (1-oleoyl-sn-glycero-3-phosphoethanolamine), also exhibited GPR82 inhibitory activity, albeit weaker than edelfosine. Förster resonance energy transfer imaging analysis consistently demonstrated that Gi protein-coupled GPR82 has an apparent constitutive activity that is edelfosine-sensitive. Consistent data were obtained from GPR82-mediated binding analysis of guanosine-5'-O-(3-thiotriphosphate) to cell membranes. Furthermore, in GPR82-transfected cells, edelfosine inhibited insulin-induced extracellular signal-regulated kinase activation, like compounds that function as inverse agonists at other GPCRs. Therefore, edelfosine is likely to act as an inverse agonist of GPR82. Finally, GPR82 expression inhibited adipocyte lipolysis, which was abrogated by edelfosine. Our findings suggested that the cationic lysophospholipids edelfosine, lysophosphatidylcholine and lysophosphatidylethanolamine are novel inverse agonists for Gi-coupled GPR82, which is apparently constitutively active, and has the potential to exert lipolytic effects through GPR82.

Quantification of Fatty Acids in Mammalian Tissues by Gas Chromatography-Hydrogen Flame Ionization Detection.

In mammalian organisms, fatty acids (FAs) exist mostly in esterified forms, as building blocks of phospholipids, triglycerides, and cholesteryl esters, while some exist as non-esterified free FAs. The absolute quantification of FA species in total lipids or in a specific lipid class is critical in lipid-metabolism studies. To quantify FAs in biological samples, gas chromatography-hydrogen flame ionization detection (GC-FID)-based methods have been used as highly robust and reliable techniques. Prior to GC-FID analysis, FAs need to be derivatized to volatile FA methyl esters (FAMEs). The derivatization of unsaturated FAs using classical derivatization methods that rely on high reaction temperature requires skill; consequently, the quantification results are often unreliable. The recently available FA-methylation procedure rapidly and reliably derivatizes a variety of FA species, including poly-unsaturated FAs (PUFAs). To analyze FAs in mammalian tissue samples, lipid extraction and fractionation are also critical for robust analysis. In this report, we describe a whole protocol for the GC-FID-based FA quantification of mammalian tissue samples, including lipid extraction, fractionation, derivatization, and quantification. The protocol is useful when various FAs, especially unsaturated FAs, need to be reliably quantified.

Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo.

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors in vitro under near-physiological conditions.

Polyunsaturated fatty acids promote Plasmodium falciparum gametocytogenesis.

The molecular triggers of sexual differentiation into gametocytes by blood stage Plasmodium falciparum, the most malignant human malaria parasites, are subject of much investigation for potential transmission-blocking strategies. The parasites are readily grown in vitro with culture media supplemented by the addition of human serum (10%) or by a commercially available substitute (0.5% AlbuMAX). We found better gametocytemia with serum than AlbuMAX, suggesting suboptimal concentrations of some components in the commercial product; consistent with this hypothesis, substantial concentration differences of multiple fatty acids were detected between serum- and AlbuMAX-supplemented media. Mass spectroscopy analysis distinguished the lipid profiles of gametocyte- and asexual stage-parasite membranes. Delivery of various combinations of unsaturated fatty-acid-containing phospholipids to AlbuMAX-supported gametocyte cultures improved gametocyte production to the levels achieved with human-serum-supplemented media. Maturing gametocytes readily incorporated externally supplied d5-labeled glycerol with fatty acids into unsaturated phospholipids. Phospholipids identified in this work thus may be taken up from extracellular sources or generated internally for important steps of gametocyte development. Further study of polyunsaturated fatty-acid metabolism and phospholipid profiles will improve understanding of gametocyte development and malaria parasite transmission.

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.

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.