Sweat Protects against Contact Hypersensitivity: Transient Sweat Suppression Compromises Skin Barrier Function in Mice.

Although subtle barrier defects may facilitate allergen penetration, thereby enabling allergic sensitization, the relationship between sweating disturbance and skin barrier function is unknown. However, many studies on contact hypersensitivity in mice examined ear skin, which does not sweat, instead of the footpad, where sweating is uniquely present. In this study, we assessed whether sweat suppression in the footpad before hapten application provoked a skin barrier abnormality and reduced inflammatory thresholds to topical haptens. Mice without any genetic skin barrier dysfunction displayed markedly reduced inflammatory thresholds to haptens under transient sweat suppression before hapten application. Epicutaneously applied haptens penetrated the skin more robustly in the presence of sweat suppression compared with that in its absence, although this increase was abolished by exposure to high-humidity conditions. These mice displayed a subtle atopic dermatitis-like inflammation mediated by type 2 response-dominant inflammation and increased IgE responses, mimicking some events occurring in nonlesional atopic dermatitis skin in humans and in murine models. These lesions were dramatically attenuated by exposure to high-humidity conditions. In our model, hapten sensitization does not require mechanical injury, explaining why sensitization occurs through nonlesional atopic dermatitis skin. Awareness of the importance of preserving sweating responses is essential to prevent occupational contact dermatitis and atopic dermatitis.

A fluorogenic substrate for the detection of lipid amidases in intact cells.

Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 µM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 µM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 µM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.

Genetic ablation of Saposin-D in Krabbe disease eliminates psychosine accumulation but does not significantly improve demyelination.

Krabbe disease is an inherited demyelinating disease caused by a genetic deficiency of the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase (GALC). The Twitcher (Twi) mouse is a naturally occurring, genetically and enzymatically authentic mouse model that mimics infantile-onset Krabbe disease. The major substrate for GALC is the myelin lipid GalCer. However, the pathogenesis of Krabbe disease has long been explained by the accumulation of psychosine, a lyso-derivative of GalCer. Two metabolic pathways have been proposed for the accumulation of psychosine: a synthetic pathway in which galactose is transferred to sphingosine and a degradation pathway in which GalCer is deacylated by acid ceramidase (ACDase). Saposin-D (Sap-D) is essential for the degradation of ceramide by ACDase in lysosome. In this study, we generated Twi mice with a Sap-D deficiency (Twi/Sap-D KO), which are genetically deficient in both GALC and Sap-D and found that very little psychosine accumulated in the CNS or PNS of the mouse. As expected, demyelination with the infiltration of multinucleated macrophages (globoid cells) characteristic of Krabbe disease was milder in Twi/Sap-D KO mice than in Twi mice both in the CNS and PNS during the early disease stage. However, at the later disease stage, qualitatively and quantitatively comparable demyelination occurred in Twi/Sap-D KO mice, particularly in the PNS, and the lifespans of Twi/Sap-D KO mice were even shorter than that of Twi mice. Bone marrow-derived macrophages from both Twi and Twi/Sap-D KO mice produced significant amounts of TNF-α upon exposure to GalCer and were transformed into globoid cells. These results indicate that psychosine in Krabbe disease is mainly produced via the deacylation of GalCer by ACDase. The demyelination observed in Twi/Sap-D KO mice may be mediated by a psychosine-independent, Sap-D-dependent mechanism. GalCer-induced activation of Sap-D-deficient macrophages/microglia may play an important role in the neuroinflammation and demyelination in Twi/Sap-D KO mice.

GDE7 produces cyclic phosphatidic acid in the ER lumen functioning as a lysophospholipid mediator.

Cyclic phosphatidic acid (cPA) is a lipid mediator, which regulates adipogenic differentiation and glucose homeostasis by suppressing nuclear peroxisome proliferator-activated receptor γ (PPARγ). Glycerophosphodiesterase 7 (GDE7) is a Ca2+-dependent lysophospholipase D that localizes in the endoplasmic reticulum. Although mouse GDE7 catalyzes cPA production in a cell-free system, it is unknown whether GDE7 generates cPA in living cells. Here, we demonstrate that human GDE7 possesses cPA-producing activity in living cells as well as in a cell-free system. Furthermore, the active site of human GDE7 is directed towards the luminal side of the endoplasmic reticulum. Mutagenesis revealed that amino acid residues F227 and Y238 are important for catalytic activity. GDE7 suppresses the PPARγ pathway in human mammary MCF-7 and mouse preadipocyte 3T3-L1 cells, suggesting that cPA functions as an intracellular lipid mediator. These findings lead to a better understanding of the biological role of GDE7 and its product, cPA.

Assay of NAAA Activity.

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal hydrolase degrading various N-acylethanolamines at acidic pH. NAAA prefers anti-inflammatory and analgesic palmitoylethanolamide to other N-acylethanolamines as a substrate, and its specific inhibitors are shown to exert anti-inflammatory and analgesic actions in animal models. Therefore, these inhibitors are expected as a new class of therapeutic agents. Here, we introduce an NAAA assay system, using [14C]palmitoylethanolamide and thin-layer chromatography. The preparation of NAAA enzyme from native and recombinant sources as well as the chemical synthesis of N-[1'-14C]palmitoyl-ethanolamine is also described.

Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7.

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.

Sphingosine 1-phosphate receptor type 2 positively regulates interleukin (IL)-4/IL-13-induced STAT6 phosphorylation.

Previous reports have demonstrated that sphingosine 1-phosphate receptor type 2 (S1P2) is involved in the activation of signal transducer and activator of transcription (STAT) 6. Additionally, the major signaling pathway of S1P2 is the Rho-Rho kinase pathway. In this study, we examined the role of S1P2 in STAT6 activation in a macrophage (Mφ) model using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA). We established S1P2knockout THP-1 cells using the CRISPR-Cas9 gene editing system. The PMA-treated S1P2knockout THP-1 Mφs showed decreases in IL-4/IL-13-induced phosphorylation of Janus-activated kinase (JAK) 1, JAK2, and STAT6 as well as mRNA expression of the M2 marker ARG1 compared with wild-type THP-1 Mφs. Pretreatment of PMA-treated THP-1 Mφs with the S1P2 antagonist JTE-013, the Rho inhibitor Rhosin or the Rho kinase inhibitor Y27632 inhibited the IL-4/IL-13-induced increase in STAT6 phosphorylation. The expressions of suppressor of cytokine signaling 3 in the S1P2knockout THP-1 Mφs were higher than those in wild-type THP-1 Mφs. In addition, the protein tyrosine phosphatase inhibitor vanadate enhanced IL-4-induced STAT6 phosphorylation in the S1P2knockout THP-1 Mφs, suggesting that S1P2-Rho-Rho kinase inhibited the negative regulation of STAT6. These results suggest that the S1P2-Rho-Rho kinase pathway is necessary for full activation of STAT6 by IL-4/IL-13 in Mφs.

ATF4-mediated transcriptional regulation protects against ß-cell loss during endoplasmic reticulum stress in a mouse model.

OBJECTIVE: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic ß-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient ß-cells to clarify the role of ATF4 in ß-cells under ER stress conditions. METHODS: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established ß-cell-specific Atf4 knockout (ßAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, ß-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in ß-cells, the islets of ßAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 ß-cells. RESULTS: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/ßAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/ßAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in ß-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/ßAtf4-KO mice. CONCLUSIONS: We conclude that transcriptional regulation by ATF4 maintains ß-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.

Involvement of the γ Isoform of cPLA2 in the Biosynthesis of Bioactive N-Acylethanolamines.

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A2 (cPLA2) functions as an N-acyltransferase to form NAPE. Since the cPLA2 family consists of six isoforms (α, ß, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA2 isoforms were labeled with [14C]ethanolamine, the increase in the production of [14C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [14C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [14C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA2γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA2γ is involved in the biosynthesis of NAE by its phospholipase A1/A2 and lysophospholipase activities.

Inhibition of acid ceramidase elicits mitochondrial dysfunction and oxidative stress in pancreatic cancer cells.

Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno-associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy.

Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.

Growth differentiation factor 15 facilitates lung fibrosis by activating macrophages and fibroblasts.

Lung fibrosis is a devastating disease characterized by fibroblast accumulation and extracellular matrix deposition in lungs. However, its molecular and cellular pathogenesis is not fully understood and the current therapeutic strategies are ineffective. Bleomycin-induced lung fibrosis is the most widely used experimental model for research aimed at in-depth analysis of lung fibrosis mechanisms. The present study aimed to analyse the effects of growth differentiation factor 15 (GDF15), which is associated with many diseases, in lung fibrosis. GDF15 mRNA expression was elevated in the lungs of bleomycin-treated mice, revealed by comprehensive gene analysis. Its protein levels were also increased in the lungs, bronchoalveolar lavage fluid, and plasma obtained from bleomycin-treated mice as compared to those in saline-treated mice. Bleomycin administration in mice resulted in a marked increase in senescence-associated ß-galactosidase-positive and p16INK4a-positive lung structural cells including alveolar epithelial cells and macrophages. Immunohistochemical staining using anti-GDF15 antibody and increased mRNA expression of GDF15 in bleomycin-induced senescent A549 cells indicated that GDF15 is produced from alveolar epithelial cells undergoing bleomycin-induced cellular senescence. GDF15 was also implicated in the augmentation of interleukin-4/interleukin-13-induced mRNA expression of M2 markers including arginase 1 and chitinase-3-like protein and was also responsible for increased α-smooth muscle actin expression through the ALK5-Smad2/3 pathway in WI-38 lung fibroblasts. Therefore, GDF15 secreted from senescent alveolar epithelial cells might act as a profibrotic factor through activation of M2 macrophages and fibroblasts. This implies that GDF15 could be a potential therapeutic target and a predictor of lung fibrosis progression.

Intracellular Ca2+-dependent formation of N-acyl-phosphatidylethanolamines by human cytosolic phospholipase A2ε.

N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ) was found to be Ca2+-dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA2ɛ, and used these cells to analyze endogenous substrates and products of cPLA2ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca2+ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA2ɛ Ca2+-dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors.

Chronic Treatment with α-Lipoic Acid Improves Endothelium-Dependent Vasorelaxation of Aortas in High-Fat Diet-Fed Mice.

α-Lipoic acid (ALA) is used as a dietary supplement and known as an anti-oxidant. The present study aimed to examine whether ALA improves endothelial dysfunction in high-fat diet-fed obese mice. After feeding a high-fat diet to Institute of Cancer Research (ICR) mice for 4 weeks, the mice were maintained with a high-fat diet (group HF) or a high-fat diet containing ALA (25 mg/d, group HF + ALA) for an additional 20 weeks. Age-matched normal diet-fed mice were also used (group Normal). Chronic oral treatment with ALA did not affect various plasma parameters or body weights. As compared with the aortas of Normal mice, those from HF mice showed impaired endothelium-dependent relaxation in response to clonidine. However, such an impairment was not observed in the aortas from HF + ALA mice. The plasma levels of thiobarbituric acid reactive substances, an indicator of oxidative stress, were significantly decreased in HF + ALA mice compared with HF mice, confirming the anti-oxidative effects of ALA. In addition, when the impaired clonidine-induced vasorelaxation of aortas from normal mice under high glucose conditions was used as a model of acute oxidative stress, the vasorelaxation responses were improved in the presence of ALA at 100 µM. Our results suggested that the chronic oral administration of ALA improves endothelial dysfunction in high-fat diet-fed obese mice possibly through the reduction in oxidative stress in vivo.

The role of intracellular anionic phospholipids in the production of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ɛ.

N-Acyl-phosphatidylethanolamines (NAPEs) represent a class of glycerophospholipids and serve as the precursors of bioactive N-acylethanolamines, including arachidonoylethanolamide (anandamide), palmitoylethanolamide and oleoylethanolamide. NAPEs are produced in mammals by N-acyltransferases, the enzymes which transfer an acyl chain of glycerophospholipids to the amino group of phosphatidylethanolamine. Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ, also called PLA2G4E) was identified as Ca2+-dependent N-acyltransferase. We showed that the activity is remarkably stimulated by phosphatidylserine (PS) in vitro. In the present study, we investigated whether or not endogenous PS regulates the function of cPLA2ɛ in living cells. When PS synthesis was suppressed by the knockdown of PS synthases in cPLA2ɛ-expressing cells, the cPLA2ɛ level and its N-acyltransferase activity were significantly reduced. Mutagenesis studies revealed that all of C2, lipase and polybasic domains of cPLA2ɛ were required for its proper localization as well as the enzyme activity. Liposome-based assays showed that several anionic glycerophospholipids, including PS, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, enhance the Ca2+-dependent binding of purified cPLA2ɛ to liposome membrane and stimulate its N-acyltransferase activity. Altogether, these results suggested that endogenous PS and other anionic phospholipids affect the localization and enzyme activity of cPLA2ɛ.

Endocannabinoids and related N-acylethanolamines: biological activities and metabolism.

The plant Cannabis sativa contains cannabinoids represented by Δ9-tetrahydrocannabinol, which exert psychoactivity and immunomodulation through cannabinoid CB1 and CB2 receptors, respectively, in animal tissues. Arachidonoylethanolamide (also referred to as anandamide) and 2-arachidonoylglycerol (2-AG) are well known as two major endogenous agonists of these receptors (termed "endocannabinoids") and show various cannabimimetic bioactivities. However, only 2-AG is a full agonist for CB1 and CB2 and mediates retrograde signals at the synapse, strongly suggesting that 2-AG is physiologically more important than anandamide. The metabolic pathways of these two endocannabinoids are completely different. 2-AG is mostly produced from inositol phospholipids via diacylglycerol by phospholipase C and diacylglycerol lipase and then degraded by monoacylglycerol lipase. On the other hand, anandamide is concomitantly produced with larger amounts of other N-acylethanolamines via N-acyl-phosphatidylethanolamines (NAPEs). Although this pathway consists of calcium-dependent N-acyltransferase and NAPE-hydrolyzing phospholipase D, recent studies revealed the involvement of several new enzymes. Quantitatively major N-acylethanolamines include palmitoylethanolamide and oleoylethanolamide, which do not bind to cannabinoid receptors but exert anti-inflammatory, analgesic, and anorexic effects through receptors such as peroxisome proliferator-activated receptor α. The biosynthesis of these non-endocannabinoid N-acylethanolamines rather than anandamide may be the primary significance of this pathway. Here, we provide an overview of the biological activities and metabolisms of endocannabinoids (2-AG and anandamide) and non-endocannabinoid N-acylethanolamines.

Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase.

N-acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N-acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca2+-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A2 (cPLA2ε) was recently identified as a Ca2+-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA2ε function as Ca-NAT. We next purified both mouse recombinant cPLA2ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca2+ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1 mM CaCl2 and lowered the EC50 value of Ca2+ >8-fold. Using a PS probe, we showed that cPLA2ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA2ε with PS in living cells. Finally, we found that the Ca2+-ionophore ionomycin increased [14C]NAPE levels >10-fold in [14C]ethanolamine-labeled cPLA2ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca2+-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca2+-dependent activity and human cPLA2ε isoforms also functioned as Ca-NAT.

Peripheral tissue levels and molecular species compositions of N-acyl-phosphatidylethanolamine and its metabolites in mice lacking N-acyl-phosphatidylethanolamine-specific phospholipase D.

N-acylethanolamines (NAEs), a class of lipid mediators, are produced from N-acyl-phosphatidylethanolamine (NAPE) by several pathways, including the direct release by NAPE-specific phospholipase D (NAPE-PLD) or the multistep pathway via sn-glycero-3-phospho-N-acylethanolamine (Gp-NAE). Using liquid chromatography-tandem mass spectrometry, we compared peripheral tissue levels of NAPE, Gp-NAE and NAE in NAPE-PLD-deficient (NAPE-PLD-/-) and wild type (WT) mice. NAPE-PLD was suggested to play a major role in the NAPE degradation in heart, kidney, and liver, but not in jejunum, because the NAPE levels except jejunum were significantly higher in NAPE-PLD-/- mice than in WT mice. The deletion of NAPE-PLD failed to alter the NAE levels of these tissues, suggesting its limited role in the NAE production. The enzyme assays with tissue homogenates confirmed the presence of NAPE-PLD-independent pathways in these peripheral tissues. Gp-NAE species having an acyl moiety with 22 carbons and 6 double bonds was enriched in these peripheral tissues. As for sn-2 acyl species of NAPE, 18:2-acyl-containing NAPE species were predominant over 18:1-containing species in heart, liver, and jejunum. Our results show that both molecular species composition of NAPE, NAE and Gp-NAE and their dependencies on Napepld are different among the peripheral tissues, suggesting that each tissue has distinct metabolic pathways and these NAE-containing lipids play tissue-specific roles.

An involvement of phospholipase A/acyltransferase family proteins in peroxisome regulation and plasmalogen metabolism.

The H-Ras-like suppressor (HRASLS) is a protein family consisting of five members in humans. Despite their discovery as tumor suppressors, we demonstrated that all these proteins are phospholipid-metabolizing enzymes, such as phospholipase (PL) A1 /A2 and acyltransferase. We thus proposed to rename HRASLS1-5 as PLA/acyltransferase (PLAAT)-1-5. Notably, PLAATs exhibit N-acyltransferase activity to biosynthesize N-acylated ethanolamine phospholipids, including N-acyl-plasmalogen, which serve as precursors of bioactive N-acylethanolamines. Furthermore, the overexpression of PLAAT-3 in animal cells causes disappearance of peroxisomes and a remarkable reduction in plasmalogen levels. This finding might be related to the inhibitory effect of PLAAT-3 on the chaperone activity of the peroxin PEX19. In this article, we will review our recent findings about PLAAT proteins, with special reference to their roles in peroxisome biogenesis and plasmalogen metabolism.