Phospholipase D2 regulates endothelial permeability through cytoskeleton reorganization and occludin downregulation.

Endothelial permeability is controlled by adhesive strengths which connect cells to each other through interendothelial junctions and by contractile forces associated with cytoskeleton reorganization. Phospholipase D (PLD) activation resulting in the generation of phosphatidic acid (PA) is increasingly recognized as a key event in the initiation of various cell responses. In human umbilical vein endothelial cells (HUV-EC), enhancement of intracellular PA by a variety of approaches increased the permeability of endothelial cell monolayers and induced stress fibre formation. Using adenovirus-mediated overexpression and siRNA silencing, we showed that PLD2 but not PLD1 was involved in the enhancement of basal permeability through cytoskeleton reorganization. Furthermore, PLD2 overexpression induced ERK1/2 activation and downregulated the expression of occludin, a major component of tight junctions. A substantial part of PLD2 protein was associated with the low-density caveolin-rich fractions isolated on sucrose gradients. The Raf-1 specific inhibitor GW-5074 drastically reduced hyperpermeability induced by PLD2 overexpression, and inhibited PA-mediated increase of endothelial permeability and ERK1/2 activation. On the whole, the present results demonstrate the selective role of PLD2 isoform in the control of endothelial permeability through a mechanism involving both stress fibre formation and contraction, and occludin downregulation, possibly resulting from PA-mediated activation of Raf-1.

Effects of increasing docosahexaenoic acid intake in human healthy volunteers on lymphocyte activation and monocyte apoptosis.

Dietary intake of long-chain n-3 PUFA has been reported to decrease several markers of lymphocyte activation and modulate monocyte susceptibility to apoptosis. However, most human studies examined the combined effect of DHA and EPA using relatively high daily amounts of n-3 PUFA. The present study investigated the effects of increasing doses of DHA added to the regular diet of human healthy volunteers on lymphocyte response to tetradecanoylphorbol acetate plus ionomycin activation, and on monocyte apoptosis induced by oxidized LDL. Eight subjects were supplemented with increasing daily doses of DHA (200, 400, 800, 1600 mg) in a TAG form containing DHA as the only PUFA, for 2 weeks each dose. DHA intake dose-dependently increased the proportion of DHA in mononuclear cell phospholipids, the augmentation being significant after 400 mg DHA/d. The tetradecanoylphorbol acetate plus ionomycin-stimulated IL-2 mRNA level started to increase after ingestion of 400 mg DHA/d, with a maximum after 800 mg intake, and was positively correlated (P < 0.003) with DHA enrichment in cell phospholipids. The treatment of monocytes by oxidized LDL before DHA supplementation drastically reduced mitochondrial membrane potential as compared with native LDL treatment. Oxidized LDL apoptotic effect was significantly attenuated after 400 mg DHA/d and the protective effect was maintained throughout the experiment, although to a lesser extent at higher doses. The present results show that supplementation of the human diet with low DHA dosages improves lymphocyte activability. It also increases monocyte resistance to oxidized LDL-induced apoptosis, which may be beneficial in the prevention of atherosclerosis.

Inhibition of de novo ceramide synthesis upregulates phospholipase D and enhances myogenic differentiation.

In L6 skeletal myoblasts induced to differentiate by Arg8-vasopressin treatment, a short-lived lowering of ceramide levels was observed, followed by a long-lasting elevation that was prevented by inhibitors of the de novo synthesis pathway, fumonisin B1 and myriocin. Both inhibitors increased the expression of myogenic differentiation markers and cell fusion rate, whereas short-chain ceramides inhibited these responses. Similar drug effects were observed on primary mouse satellite cell differentiation. Furthermore, bacterial sphingomyelinase overexpression suppressed myogenin nuclear accumulation in L6 cells. These data suggested that endogenous ceramide mediates a negative feedback mechanism limiting myogenic differentiation, and that inhibitors of ceramide synthesis promoted myogenesis by removing this control. Phospholipase D (PLD), a recognized target of ceramide, is required for myogenesis, as shown by the negative effects of PLD1 isoform depletion obtained by siRNA treatment. Fumonisin induced an increase in PLD activity of L6 cells, whereas C6-ceramide decreased it. The expression of PLD1 mRNA transcripts was selectively decreased by C6-ceramide, and increased by ceramide synthesis inhibitors. An early step of myogenic response is the PLD1-dependent formation of actin stress fiber-like structures. C6-ceramide addition or overexpression of sphingomyelinase impaired actin fiber formation. Ceramide might thus regulate myogenesis through downregulation of PLD1 expression and activity.

Electrical probing of endothelial cell behaviour on a fibronectin/polystyrene/thiol/gold electrode by Faradaic electrochemical impedance spectroscopy (EIS).

The electrochemical impedance spectroscopy (EIS) technique has been shown to be an effective tool for monitoring endothelial cell behaviour on a multilayer functionalised gold electrode. Polystyrene, a reproducible model substrate, is deposited as a thin layer on a thiol functionalised gold electrode. Fibronectin, a protein promoting endothelial cell adhesion, is then adsorbed on the polystyrene surface. The different steps of this multilayer assembly are characterized by Faradaic impedance. The charge transfer resistance and the capacitance for the total layer are modified at each step according to the electrical properties of each layer. This gives the endothelial cells' electrical state in terms of its resistive and capacitive properties. In this study, the endothelial cell layer presents a specific charge transfer resistance equal to 1.55 kOmega cm(2) with no large defects in the cell layer, and a specific capacitance equal to few microF cm(-2) explained by the existence of pseudopods. These electrical properties are correlated to the endothelial cell viability, adhesion and cytoskeleton organization.

Phospholipase D as a potential target for the antiproliferative effects of polyunsaturated fatty acids in rat thymocytes.

The effects of various saturated and unsaturated fatty acids (FAs) on the proliferative response and phospholipase D (PLD) activity of rat thymocytes were investigated. When added to culture medium as complexes with albumin, all the FAs tested, except stearic acid, inhibited the ConA-induced thymocyte proliferation, eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids being the most inhibitory. Apart from 22:6n-3 which slightly increased the percentage of late apoptotic and necrotic thymocytes in the presence of mitogen, none of the FAs induced significant apoptosis or necrosis. A short 2-h preincubation of rat thymocytes in the presence of FA-albumin complexes was sufficient to induce a significant enrichment of cell phospholipids with each FA and to stimulate thymocyte PLD activity. However, 20:5n-3 was inactive despite a large enrichment in phospholipids. Furthermore, the PLD activity of activated thymocytes was negatively correlated to the proliferative response, with the exception of 20:5n-3-supplemented cells. These results support further our current hypothesis that PLD activity conveys antiproliferative signals in lymphoid cells, and suggest that 20:5n-3 inhibits thymocyte proliferation by a particular mechanism unrelated to that of the other FAs.

Phospholipase D protects ECV304 cells against TNFalpha-induced apoptosis.

Tumor necrosis factor alpha (TNFalpha), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFalpha-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFalpha-induced apoptosis. TNFalpha/CHX induced a robust increase in ceramide levels after 16 h of treatment when cell death was maximal. PLD activity was increased at early time point (1h) whereas both PLD activity and PLD1 protein were strongly decreased after 24h. TNFalpha/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFalpha-induced cell death.

Effect of dietary argan oil on fatty acid composition, proliferation, and phospholipase D activity of rat thymocytes.

OBJECTIVE: Argan oil is receiving increasing attention due to its potential health benefits in the prevention of cardiovascular risk, but no information to date is available about its possible effect on immune cells and functions. METHODS: To address this issue male rats were fed one of five diets that contained fish oil, argan oil, olive oil, coconut oil, or sunflower oil for 4 wk. The fatty acid composition of plasma and thymocyte lipids was then analyzed in relation to the mitogen-induced proliferation and phospholipase D (PLD) activity of thymocytes. RESULTS: The 18:2omega-6 proportion in thymocyte phospholipids from rats fed argan oil was significantly lower than that observed in phospholipids from rats fed sunflower oil and fish oil but higher than that found in the olive oil and coconut oil groups. Further, a significant positive linear relation was found between thymocyte proliferation and the 18:2omega-6 proportion in thymocyte phospholipids, whatever the diet. The proliferation response of thymocytes to mitogenic activation was also inversely correlated to PLD activity measured in intact thymocytes. Subsequent western blotting experiments indicated that the diet-induced variations in PLD activity mainly reflected variations in the expression of PLD2 protein. CONCLUSIONS: On the whole, the present study shows that the effects of argan oil on immune cells are very similar to those of olive oil, and that, as a consequence, argan oil can be used as a balanced dietary supply without marked adverse effects on immune cell function.

Disruption of lipid rafts stimulates phospholipase d activity in human lymphocytes: implication in the regulation of immune function.

Recent evidence suggests that phospholipase D (PLD) can be regulated through its association/dissociation to lipid rafts. We show here that modifying lipid rafts either by cholesterol depletion using methyl-beta-cyclodextrin and filipin or by conversion of sphingomyelin to ceramide with exogenous bacterial sphingomyelinase (bSMase) markedly activated the PLD of human PBMC. bSMase was the most potent PLD activator, giving maximal 6- to 7-fold increase in PLD activity. Triton X-100-treated lysates prepared from control PBMC and from bSMase-treated cells were fractionated by centrifugation on sucrose density gradient. We observed that bSMase treatment of the cells induced a larger ceramide increase in raft than in nonraft membranes and displaced both the Src kinase Lck and PLD1 out of the raft fractions. In addition, the three raft-modifying agents markedly inhibited the lymphoproliferative response to mitogenic lectin. To examine further the potential role of PLD activation in the control of lymphocyte responses, we transiently overexpressed either of the PLD1 and PLD2 isoforms in Jurkat cells and analyzed the phorbol ester plus ionomycin-induced expression of IL-2 mRNA, which is one of the early responses of lymphocyte to activation. We observed a 43% decrease of IL-2 mRNA level in Jurkat cells overexpressing PLD1 as compared with mock- or PLD2-transfected cells, which indicates that elevated PLD1, but not PLD2, activity impairs lymphocyte activation. Altogether, the present results support the hypothesis that PLD1 is activated by exclusion from lipid rafts and that this activation conveys antiproliferative signals in lymphoid cells.

Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton.

We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.

Phorbol ester-induced differentiation of L6 myogenic cells involves phospholipase D activation.

TPA, a potent PKC activator, inhibits myogenic differentiation and activates phospholipase D (PLD). We evaluated the involvement of PLD in the TPA effects on L6 myoblasts differentiation. TPA, at concentrations inhibiting differentiation of L6 cells, induced a strong, though transient, PLD activation. Surprisingly, at nanomolar concentration, TPA induced both myogenic differentiation and sustained activation of PLD. Differential effect of TPA can be ascribed to PKC downregulation induced by highest TPA concentrations. TPA-induced differentiation was inhibited by 1-butanol, confirming the involvement of PLD in this effect. These data suggest that prolonged elevation of PLD activity is required for myogenic differentiation.

Formation of palmitic acid/Ca2+ complexes in the mitochondrial membrane: a possible role in the cyclosporin-insensitive permeability transition.

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.

Ceramide in the antiapoptotic effect of ischemic preconditioning.

Although the mechanism by which ischemic preconditioning (PC) inhibits myocardial apoptosis during ischemia-reperfusion is unclear, evidence indicates a role for the secondary messenger ceramide. We investigated in vivo whether PC may affect ceramide and sn-1,2-diacylglycerol (DAG) production, and attenuate apoptosis during ischemia. Rabbits underwent 30 min of ischemia, followed by 4 h of reperfusion. Before this, they received either no intervention (control group) or one episode of 5 min of ischemia, followed by 5 min of reperfusion (PC group), or an intravenous administration of the sphingomyelinase inhibitor D609. Myocardial content of ceramide and DAG was measured using the DAG kinase assay at different time points of the experiment. Apoptosis was detected and quantified by a sandwich enzyme immunoassay. Both AR and infarct size were measured using blue dye injection and triphenyltetrazolium chloride staining. Control hearts exhibited a peak of ceramide production at 5 min of the prolonged ischemia, with a mean value averaging 64 +/- 5 ng/mg tissue (P < 0.05 vs. 48 +/- 4 ng/mg at baseline). In contrast, ischemic PC and D609 prevented ceramide increase during the prolonged ischemia. Myocardial DAG content was increased only in PC hearts at 30 min of ischemia. Preconditioned and D609 groups developed less apoptosis, as well as a limited infarct size, compared with the control group. These results suggest that the antiapoptotic effect of PC may be due to a reduced ceramide production during sustained ischemia in the rabbit heart.

Transphosphatidylation activity of Streptomyces chromofuscus phospholipase D in biomimetic membranes.

The phospholipase D (PLD) from Streptomyces chromofuscus belongs to the superfamily of PLDs. All the enzymes included in this superfamily are able to catalyze both hydrolysis and transphosphatidylation activities. However, S. chromofuscus PLD is calcium dependent and is often described as an enzyme with weak transphosphatidylation activity. S. chromofuscus PLD-catalyzed hydrolysis of phospholipids in aqueous medium leads to the formation of phosphatidic acid. Previous studies have shown that phosphatidic acid-calcium complexes are activators for the hydrolysis activity of this bacterial PLD. In this work, we investigated the influence of diacylglycerols (naturally occurring alcohols) as candidates for the transphosphatidylation reaction. Our results indicate that the transphosphatidylation reaction may occur using diacylglycerols as a substrate and that the phosphatidylalcohol produced can be directly hydrolyzed by PLD. We also focused on the surface pressure dependency of PLD-catalyzed hydrolysis of phospholipids. These experiments provided new information about PLD activity at a water-lipid interface. Our findings showed that classical phospholipid hydrolysis is influenced by surface pressure. In contrast, phosphatidylalcohol hydrolysis was found to be independent of surface pressure. This latter result was thought to be related to headgroup hydrophobicity. This work also highlights the physiological significance of phosphatidylalcohol production for bacterial infection of eukaryotic cells.

Mechanisms involved in the stimulation of prostacyclin synthesis by human lymphocytes in human umbilical vein endothelial cells.

1 Endothelial cells play an important role in the modulation of vascular tone because of their ability to produce vasoactive substances such as prostacyclin (PGI(2)). Cell-cell contact between human umbilical vein endothelial cells (HUVEC) and peripheral blood lymphocytes has been shown to stimulate endothelial PGI(2) synthesis by increasing free arachidonic acid availability through endothelial cytosolic phospholipase A2 (cPLA(2)) activation. In this study, we sought to determine whether phospholipase C (PLC) and D (PLD) activation also contributes, besides cPLA(2), to the lymphocyte-induced PGI(2) synthesis in HUVEC, and to delineate further the potential mechanisms of cPLA(2) activation triggered by the interaction of HUVEC with lymphocytes. 2 Pretreatment of endothelial cells with the PI-PLC inhibitor U-73122 before the coincubation with lymphocytes markedly inhibited the PGI(2) output whereas the diacylglycerol (DAG) lipase inhibitor RHC 80267 and ethanol had no effect. These results suggest that PLC may be involved through inositol trisphosphate generation and calcium mobilization, and that neither DAG nor phosphatidic acid (PtdOH) was used as sources of arachidonic acid. 3 The stimulated PGI(2) synthesis was protein kinase C (PKC)-independent but strongly inhibited by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U-0126 and by the Src kinase inhibitor PP1. 4 Immunoblot experiments showed an increased phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) upon lymphocyte addition till 4 h coincubation. Phosphorylation was markedly inhibited by U-0126 and PP1 addition. 5 Collectively, these results suggest that the signaling cascade triggered by lymphocytes in endothelial cells involves an Src kinase/ERK1/2 pathway leading to endothelial cPLA(2) activation.

Influence of very low dietary intake of marine oil on some functional aspects of immune cells in healthy elderly people.

Ageing is a multifactorial process involving decreased antioxidant defences and immune functions. n-3 Polyunsaturated fatty acids have been associated with human health benefits, especially against inflammatory and autoimmune diseases. However, their immunomodulatory effects were usually observed with high dosages (>2 g/d) known to increase lipid peroxidation. In contrast, very low doses, that may prevent lipid peroxidation, might affect the immune system differently. To study the latter hypothesis further, we investigated whether the supplementation of healthy elderly people with very low doses of marine oil (MO), a docosahexaenoate (DHA)- and eicosapentaenoate (EPA)-rich triacylglycerol, was able to affect lymphocyte proliferation and biochemical markers known to be altered with age. In a randomized, double-blind design, twenty healthy elderly subjects were assigned to a placebo group (600 mg sunflower oil/d) or to a group consuming 600 mg MO/d providing 150 mg DHA + 30 mg (EPA) for 6 weeks. At day 42, the proliferative responses of lymphocytes to several mitogens were significantly (P<0.01) decreased in the MO group compared with control values. This was accompanied by a slight lowering of their cytosolic cyclic nucleotide phosphodiesterase (PDE) activity, a marked and significant (P<0.05) increase of their particulate PDE activity (+56-57 %) and a slight but significant (P<0.05) increase in cyclic nucleotide intracellular levels. At the same time, the glutathione peroxidase activity was markedly and significantly (P<0.01) depressed in the MO group. None of these modifications could be seen in the placebo group. Collectively, these results demonstrate that even very low doses of n-3 fatty acids are sufficient to affect the immune responses of elderly subjects.

Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney.

Detergent-resistant membranes (DRM) were prepared from bovine kidney cortex. The criterion used to test their purification was the increase in the activity of a GPI membrane-anchored protein, the alkaline phosphatase. Its association with specific proteins and lipids was tested. Two successive Triton X-100 treatments followed by purification on sucrose gradient at 4 degrees C were necessary to obtain DRM with a maximum of alkaline phosphatase activity and a typical protein pattern. A third Triton treatment did not alter this DRM composition. Among the enriched protein, we identified, by mass spectrometry, a microsomal dipeptidase, which was GPI membrane-anchored. Protein-kinase activities, mainly serine-kinase, were enriched during the DRM purification. Using the typical FTIR olefinic =C-H bands of the acyl chains, a global decrease in the unsaturation level of DRM lipids was observed as compared with total membranes. Three main phospholipids were identified in DRM. Their fatty acid compositions were determined by gas chromatography and compared with those of total membranes. The most enriched saturated fatty acid was palmitic acid (+44% for phosphatidylethanolamine, +52% for phosphatidylcholine and +49% for sphingomyelin), agreeing with a selection of specific phospholipids among the saturated ones during the DRM purification.

The mechanism of docosahexaenoic acid-induced phospholipase D activation in human lymphocytes involves exclusion of the enzyme from lipid rafts.

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid that inhibits T lymphocyte activation, has been shown to stimulate phospholipase D (PLD) activity in stimulated human peripheral blood mononuclear cells (PBMC). To elucidate the mechanisms underlying the DHA-induced PLD activation, we first characterized the PLD expression pattern of PBMC. We show that these cells express PLD1 and PLD2 at the protein and mRNA level and are devoid of oleate-dependent PLD activity. DHA enrichment of PBMC increased the DHA content of cell phospholipids, which was directly correlated with the extent of PLD activation. The DHA-induced PLD activation was independent of conventional protein kinase C but inhibited by brefeldin A, which suggests ADP-ribosylation factor (ARF)-dependent mechanism. Furthermore, DHA enrichment dose-dependently stimulated ARF translocation to cell membranes. Whereas 50% of the guanosine 5'-3-O-(thio)triphosphate plus ARF-dependent PLD activity and a substantial part of PLD1 protein were located to the detergent-insoluble membranes, so-called rafts, of non-enriched PBMC, DHA treatment strongly displaced them toward detergent-soluble membranes where ARF is present. Collectively, these results suggest that the exclusion of PLD1 from lipid rafts, due to their partial disorganization by DHA, and its relocalization in the vicinity of ARF, is responsible for its activation. This PLD activation might be responsible for the immunosuppressive effect of DHA because it is known to transmit antiproliferative signals in lymphoid cells.

Role of calcium and membrane organization on phospholipase D localization and activity. Competition between a soluble and insoluble substrate.

The phospholipase D (PLD) from Streptomyces chromofuscus is a soluble enzyme known to be activated by the phosphatidic acid-calcium complexes. PLD-catalyzed hydrolysis of phospholipids in aqueous medium leads to the formation of phosphatidic acid (PA). Previous studies concluded on an allosteric activation of PLD by the PA-calcium complexes. In this work, the role of PA and calcium was investigated in terms of membrane structure and dynamics. The role of calcium in PLD partitioning between the soluble phase and the water-lipid interface was tested. The monomolecular film technique was used to measure both membrane dynamics and PLD activity. These experiments provided information on PLD activity at a water-lipid interface. Moreover, the ability of PA to enhance PLD activity toward phosphatidylcholine was correlated to the physical properties of PA itself, affecting the rheology of the membrane. The effect of calcium was investigated on PLD binding to lipids and on the catalytic process by competition experiments between a soluble and a vesicular substrate. These experiments confirmed the absolute PLD requirement for calcium and pointed out the importance of calcium for PLD catalytic process and for the enzyme location at the water-lipid interface.