Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393–396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the β-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.
N-Acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide and anandamide, which show anti-inflammatory/analgesic, anorexic and cannabimimetic actions, respectively. These lipid mediators are biosynthesized from N-acyl-phosphatidylethanolamine (NAPE) by NAPE-hydrolyzing phospholipase D (NAPE-PLD) as well as through multi-step pathways via lysoNAPE. We previously showed that genetic deletion of NAPE-PLD markedly increased NAPE levels and significantly decreased NAE levels in brain. Here, we aimed to examine the mechanisms for NAE biosynthesis in peripheral tissues. As compared to wild-type mice, NAPE-PLD–/– mice exhibited increased NAPE levels in heart, kidney and liver, but not in jejunum, suggesting a major role of NAPE-PLD in NAPE degradation in the three tissues. However, the deletion did not affect NAE levels in all the four tissues, showing compensation by other pathway(s). Accordingly, the tissue homogenates from NAPE-PLD–/– mice generated [14C]NAE and [14C]lysoNAPE from [14C]NAPE. These results suggested that the contribution of NAPE-PLD to NAE biosynthesis varies among tissues and might imply the possible presence of the tissue-specific mechanisms.
A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.
Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes.
Lung fibrosis is a devastating disease characterized by fibroblast accumulation and extracellular matrix deposition in lungs, and bleomycin-induced lung fibrosis is the most widely used experimental model. We found that mRNA expression of growth differentiation factor 15 (GDF15) was elevated in the lungs of bleomycin-treated mice by using comprehensive gene analysis. The protein levels of GDF15 also increased in lung tissue, brochoalveolar lavage fluid and plasma acquired from bleomycin-treated mice compared with those from saline-treated mice. Although GDF15 is believed to be associated with stress responses, the role of GDF15 in lung fibrosis is still unknown. Histological analysis with senescence-associated β-galactosidase staining and anti-p16INK4a antibody showed cellular senescence of alveolar epithelial cells and macrophages in bleomycin-induced fibrotic lungs. From immunohistochemical staining using anti-GDF15 antibody and increased mRNA expression of GDF15 in bleomycin-induced senescent A549 cells, GDF15 appears to be produced from alveolar epithelial cells undergoing bleomycin-induced cellular senescence. Macrophages are broadly classified as M1 phenotype with inflammatory effects and M2 phenotype with anti-inflammatory effects. GDF15 augmented interleukin-4/interleukin-13-induced mRNA expression of M2 markers including arginase 1 and chitinase-like 3. On the other hand, it is known that myofibroblasts that produce extracellular matrix in lung fibrosis are increased by activation of fibroblasts. GDF15 also increased in protein expression of α-smooth muscle actin (a myofibroblast marker) through ALK5-Smad2/3 pathway in WI-38 lung fibroblasts. These results suggested that GDF15 secreted from senescent alveolar epithelial cells acts as a profibrotic factor through activations of M2 macrophages and fibroblasts. This provides that GDF15 could be an attractive therapeutic target for treatment and a predictor of progression of lung fibrosis.
Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2α were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.
In the fully customer-oriented design system, design candidates should be evaluated if they reflect appropriately the ambiguous preferences and demands intended by customers and designers or not. The researches in the total human-oriented design system have come up with prototype systems that have functions capable not only of providing an environment for products, but for displaying and evaluating products as well. There are many researches in this field. Here, the technologies to extract the personal room environment models and to control the display system by users' gesture have been introduced.
