cPLA2α Activity Research Articles

C2-di-ethyl-ceramide-1-phosphate as an inhibitor of group IVA cytosolic phospholipase A2

Ceramide-1-phosphate (C1P) has been shown to bind with C2 domain in group IVA cytosolic phospholipase A2 (cPLA2α, PLA2G4A) and activate the enzyme activity directly. In cells, C1P causes translocation of cPLA2α to perinuclear regions including the Golgi complex by interacting with C2 domain in the enzyme, and then cPLA2α releases arachidonic acid from substrate phospholipids in the regions. In this study, we synthesized new di-ethyl (DE) phosphate ester analogs of C1P with N-acyl chains of different lengths, and examined their effects on cPLA2α. A DE-C1P analog with a C2-N-acyl chain (C2-DE-C1P), but not DE-C1P analogs with longer N-acyl chain, such as C6- and C16-DE-C1P, inhibited release of arachidonic acid via cPLA2α activation in CHO-W11A cells expressing platelet-activating factor (PAF) receptors without changing secretory phospholipase A2-induced release. Treatment with C2-DE-C1P did not modify phosphorylation of extracellular signal-regulated kinase 1/2 and cPLA2α and increase of intracellular Ca2+ level induced by PAF, but inhibited Ca2+- and PAF-induced accumulation of cPLA2α in the Golgi complex. Phosphatidylcholine vesicles containing C2-DE-C1P reduced cPLA2α activity in vitro. C2-DE-C1P disturbed the binding of the enzyme to glycerophospholipids in the lipid–protein overlay assay, and the reagent alone did not bind to the enzyme. Interestingly, C2-DE-C1P inhibited neither Ca2+- and PAF-induced accumulation of C2 domain of cPLA2α in the Golgi complex nor binding of cPLA2α to C16-C1P. These results suggest that C2-DE-C1P appeared to inhibit cPLA2α, probably by interaction with a site in the catalytic domain of the enzyme, not with the site in C2 domain responsible for native C1P.

Inhibition of cytosolic phospholipase A2 alpha protects against focal ischemic brain damage in mice

It is postulated that inhibition of cytosolic phospholipase A2 alpha (cPLA2α) can reduce severity of stroke injury. This is supported by the finding that cPLA2α-deficient mice are partially protected from transient, focal cerebral ischemia. The object of this study was to determine the effect of cPLA2α inhibition with arachidonyl trifluoromethyl ketone (ATK) on stroke injury in mice. Male C57BL/6 mice were subjected to 1h of focal cerebral ischemia followed by 24 or 72h of reperfusion. Mice were treated with ATK or vehicle by intermittent intraperitoneal injection or continuous infusion via an implanted infusion pump. ATK injections 1h before and then 1 and 6h after the start of reperfusion significantly reduced infarction volumes in striatum and hemisphere after 24h of reperfusion. ATK did not reduce injury if it was not administered before onset of ischemia or was not administered after 6h of reperfusion. Intermittent doses of ATK failed to reduce infarct volume after 72h of reperfusion. Continuous infusion with ATK throughout 72h of reperfusion significantly reduced cortical and whole hemispheric infarct volume compared to vehicle treatment. Following ischemia and reperfusion, ATK treatment significantly reduced brain PLA2 activity. These results are the first to demonstrate a therapeutic effect of cPLA2α inhibition on ischemia and reperfusion injury and define a therapeutic time window. cPLA2α activity augments injury in the acute and delayed phases of cerebral ischemia and reperfusion injury. We conclude that cPLA2α inhibition may be clinically useful if started before initiation of cerebral ischemia.

A novel C(28)-hydroxylated lupeolic acid suppresses the biosynthesis of eicosanoids through inhibition of cytosolic phospholipase A2

Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4β-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC50 values of 2.3–6.9μM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA2α, we show that Ac-OH-LA may directly interfere with cPLA2α activity (IC50=3.6μM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA2α and consequently suppresses eicosanoid biosynthesis in intact cells.

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A2α (cPLA2α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA2α which coincided with a significant increase in cell proliferation. The inhibition of cPLA2α activity by pyrrophenone or by antisense oligonucleotide against cPLA2α (AS) or inhibition of prostaglandin E2 (PGE2) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE2. The secreted PGE2 activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE2. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE2. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA2α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA2α-dependent PGE2 production. PGE2via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.

The Characterization and Identification of Ceramide‐1‐Phosphate Binding Proteins

The activation of cytosolic phospholipase A2 α (cPLA2α) has been implicated in atherosclerosis and cerebral ischemia in previous animal models. The sphingolipid metabolite ceramide‐1‐phosphate (C1P) is known to specifically regulate the activation of cPLA2α and has been shown to increase enzymatic activity 5–10 fold through increased membrane residence. We have recently determined the amino acids that are responsible for C1P binding to cPLA2α in vitro and in a cellular system. In addition to heart disease, the conversation of ceramide to C1P has been implicated in cancer and phagocytosis signaling, suggesting that there are unidentified proteins that are regulated by C1P binding in membranes. Using the novel cPLA2α consensus sequence to search the proteome, we have predicted and are currently investigating new C1P binding proteins. Through the characterization of the C1P binding site, we have constructed a novel model for C1P recognition in membranes that deviates from the traditional lipid binding mechanism. This molecular characterization provides great insight into the molecular basis of C1P recognition and selectivity in biological membranes and will significantly increase the molecular basis of C1P binding by proteins. The understanding of this binding site in particular would lay the groundwork for a promising therapy for heart disease.AHA 11PRE7640028 (KEW)NIH T32GM075762 (KEW)

Disruption of group IVA cytosolic phospholipase A2attenuates myocardial ischemia-reperfusion injury partly through inhibition of TNF-α-mediated pathway

Group IVA cytosolic phospholipase A(2) (cPLA(2)α), which preferentially cleaves arachidonic acid from phospholipids, plays a role in apoptosis and tissue injury. Downstream signals in response to tumor necrosis factor (TNF)-α, a mediator of myocardial ischemia-reperfusion (I/R) injury, involve cPLA(2)α activation. This study examined the potential role of cPLA(2)α and its mechanistic link with TNF-α in myocardial I/R injury using cPLA(2)α knockout (cPLA(2)α(-/-)) mice. Myocardial I/R was created with 10-wk-old male mice by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. As a result, compared with wild-type (cPLA(2)α(+/+)) mice, cPLA(2)α(-/-) mice had a 47% decrease in myocardial infarct size, preservation of echocardiographic left ventricle (LV) function (%fractional shortening: 14 vs. 21%, respectively), and lower content of leukotriene B(4) and thromboxane B(2) (62 and 50% lower, respectively) in the ischemic myocardium after I/R. Treatment with the TNF-α inhibitor (soluble TNF receptor II/IgG1 Fc fusion protein, sTNFR:Fc) decreased myocardial I/R injury and LV dysfunction in cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) mice. sTNFR:Fc also suppressed cPLA(2)α phosphorylation in the ischemic myocardium after I/R of cPLA(2)α(+/+) mice. Similarly, sTNFR:Fc exerted protective effects against hypoxia-reoxygenation (H/R)-induced injury in the cultured cardiomyocytes from cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) cardiomyocytes. H/R and TNF-α induced cPLA(2)α phosphorylation in cPLA(2)α(+/+) cardiomyocytes, which was reversible by sTNFR:Fc. In cPLA(2)α(-/-) cardiomyocytes, TNF-α induced apoptosis and release of arachidonic acid to a lesser extent than in cPLA(2)α(+/+) cardiomyocytes. In conclusion, disruption of cPLA(2)α attenuates myocardial I/R injury partly through inhibition of TNF-α-mediated pathways.

CPLA2α gene activation by IL-1β is dependent on an upstream kinase pathway, enzymatic activation and downstream 15-lipoxygenase activity: A positive feedback loop

Cytosolic phospholipase A2α (cPLA2α) is the most widely studied member of the Group IV PLA2 family. The enzyme is Ca2+-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA2α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA2α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca2+ levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA2α is phosphorylated within 10min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA2α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA2α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA2α→15-LOX2-dependent, positive feedback loop where a protein's enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus.

Signaling via Macrophage G2A Enhances Efferocytosis of Dying Neutrophils by Augmentation of Rac Activity

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.

Ceramide and Ceramide‐1‐phosphate: Enigmatic Lipids Generating New Signaling Paradigms

In 1884, Johann Thudichum first described sphingolipids naming these lipids due to their enigmatic nature. Over 100 years later, new roles for sphingolipids such as ceramide, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate continue to emerge. My research, for example, has implicated C1P as a major regulator of eicosanoid synthesis, and despite the importance of eicosanoids in the inflammatory process, the regulation of eicosanoid synthesis proximal to the activation of Group IVA phospholipase A2 (cPLA2α) is still an enigma. In this regard, my laboratory demonstrated that C1P is a direct and required lipid cofactor for cPLA2α activation. Specifically, we demonstrated that the interaction of C1P with cPLA2α was required for this enzyme to translocate to membranes in response to A23187 and ATP. Furthermore, mutation of the C1P interaction site blocked the ability of re-expressed cPLA2α to “rescue” eicosanoid synthesis in the cPLA2α −/− cells. These findings solidified a role for C1P, and the enzyme that produces C1P from ceramide, ceramide kinase (CERK), in eicosanoid synthesis. Recently, the role for CERK in eicosanoid synthesis has recently become controversial due to studies with the CERK −/− mouse. Our laboratory explains this conundrum showing that the conventional knock-out of CERK has developmentally adapted by increasing novel C1P subspecies. To overcome this adaptation and examine the role of the C1P/cPLA2α interaction site in eicosanoid synthesis in vivo, our laboratory generated a cPLA2α knock-in mouse. In this lecture, the progress in the initial characterization and inflammatory phenotypes of this intriguing mouse model will be overviewed. Furthermore, biological mechanisms implicated by the identification of the C1P binding site in cPLA2α will be described. Overall, data will be presented that strongly implicate C1P in modulating eicosanoid synthesis.

Cytosolic Phospholipase A 2 α Contributes to Blood Pressure Increases and Endothelial Dysfunction Under Chronic NO Inhibition

Nitric oxide (NO) is an important modulator of cardiovascular function. In this study, we examined whether cytosolic phospholipase A2α (cPLA2α), an initial enzyme in the arachidonic acid pathway, is involved in blood pressure (BP) elevation in a murine model of chronic NO inhibition. cPLA2α gene-deficient mice (cPLA2α-/-) and wild-type mice (WT) were administered the NO synthesis inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) for 4 weeks. Before treatment, BP was comparable in both groups; it increased significantly in the WT but not in the cPLA2α-/- after treatment. Bone marrow transplantation experiments showed that cPLA2α in blood cells and plasma eicosanoid concentrations were not involved in BP elevation by L-NAME treatment. Activation of cPLA2α and subsequent production of eicosanoids in the aortic endothelium but not in aortic smooth muscle cell, heart, or kidney was observed after L-NAME treatment. Aortic ring assays revealed that endothelial function was comparable in both groups of mice before treatment. L-NAME treatment disturbed endothelial function in WT but not in cPLA2α-/-. These results suggest that endothelial cPLA2α may play a principal role in L-NAME-induced hypertension and may be a target molecule for maintaining endothelial function under NO inhibition.

Endothelial ICAM-1 Protein Induction Is Regulated by Cytosolic Phospholipase A2α via Both NF-κB and CREB Transcription Factors

The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A(2)α (cPLA(2)α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA(2)α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA(2)α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA(2)α (AS) that reduced cPLA(2)α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA(2)α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA(2)α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA(2)α activation, suggesting that NADPH oxidase acts upstream to cPLA(2)α. Attenuating cPLA(2)α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE(2) that were essential for ICAM-1 upregulation. Inhibition of cPLA(2)α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser(536) and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA(2)α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE(2)-mediated protein kinase A.

Protease-Activated Receptor Signaling in Platelets Activates Cytosolic Phospholipase A 2α Differently for Cyclooxygenase-1 and 12-Lipoxygenase Catalysis

The rate-limiting step in the biosynthesis of thromboxane A(2) (TxA(2)) and 12-hydroxyeicosatetraenoic acid (12-HETE) by platelets is activation of cytosolic phospholipase A(2α) (cPLA(2α)), which releases arachidonic acid, which is the substrate for cyclooxygenase-1 (COX-1) and 12-lipoxygenase. We evaluated signaling via the human platelet thrombin receptors, protease-activated receptor (PAR) 1 and PAR4, to the activation of cPLA(2α), which provides a substrate for the biosynthesis of TxA(2) and 12-HETE. Stimulating washed human platelets resulted in delayed biosynthesis of 12-HETE, which continues after maximal formation of TxA(2) is completed, suggesting that 12-HETE is not formed by the same pool of arachidonic acid that provides a substrate to COX-1. PAR1-induced formation of TxA(2) was inhibited by the phosphatidylinositol kinase inhibitor LY294002, whereas this inhibitor did not block 12-HETE biosynthesis. Both 1-butanol and propranolol also blocked TxA(2) biosynthesis but did not inhibit 12-HETE formation. The concerted evidence indicates that the platelet thrombin receptors signal activation of cPLA(2α) coupled to COX-1 by a pathway different from that signaling activation of the cPLA(2α) coupled to 12-lipoxygenase.

Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours

Background:The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A2α (cPLA2α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA2α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines.Methods:The importance of differential cPLA2α activity in clinical breast cancer was established by relating the expression of cPLA2α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters.Results:High cPLA2α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA2α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA2α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up.Conclusion:This study shows a role of cPLA2α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.

1-(5-Carboxyindol-1-yl)propan-2-one Inhibitors of Human Cytosolic Phospholipase A2α: Effect of Substituents in Position 3 of the Indole Scaffold on Inhibitory Potency, Metabolic Stability, Solubility, and Bioavailability

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 have been found to be potent inhibitors of human cytosolic phospholipase A2α (cPLA2α). In the course of structure-activity relationship studies, we investigated the effect of a substitution of indole 3 position with acyl, alkyl, and oxadiazole residues. The highest increase of inhibitory potency could be achieved by a 3-methyl-1,2,4-oxadiazol-5-yl-moiety. Appropriate compound 40 revealed an IC50 of 0.0021 μM against isolated cPLA2α. In a cellular assay applying human platelets 40 blocked cPLA2α activity even with an IC50 of 0.0006 μM. Metabolic stability and aqueous solubility of the target compounds were also determined. Furthermore, one selected compound was tested for peroral bioavailability in mice.

A ceramide analog inhibits cPLA 2 activity and consequent PGE 2 formation in LPS-stimulated macrophages

Prostaglandin E 2 (PGE 2) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE 2 production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE 2 production in macrophages. Inhibition of PGE 2 production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA 2α in an in vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA 2α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE 2 production in LPS-stimulated macrophages by direct interaction with cPLA 2, and suggest that ceramide may similarly counteract C1P effect on cPLA 2 activity in cells. The suppression of PGE 2 production is suggested to contribute to the anti-inflammatory action of PCERA-1.

Pathways Regulating Cytosolic Phospholipase A2 Activation and Eicosanoid Production in Macrophages by Candida albicans

Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A2 (cPLA2α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the β-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88−/− but not Dectin-1−/− macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA2α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA2α phosphorylation, and COX2 expression in response to particulate β-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA2α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both β-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA2α and eicosanoid production.

Newly synthetic ceramide-1-phosphate analogs; their uptake, intracellular localization, and roles as an inhibitor of cytosolic phospholipase A 2α and inducer of cell toxicity

Ceramide-1-phosphate (C1P) regulates cellular functions including arachidonic acid (AA) metabolism and modulates cell fate. The mechanism by which C1P is taken up is unclear, and the development of lipophilic analogs may be useful for regulating C1P's actions. We synthesized new mono- and di-methyl-ester (MM and DM, respectively) analogs of C1P with N-acyl chains of different lengths, and examined their effects on AA release and cell toxicity. Short- N-acyl-DM-C1P analogs including C5- and C6-DM-C1P, but not long- N-acyl-DM-C1P analogs, inhibited the release of AA mediated by α type cytosolic phospholipase A 2 (cPLA 2α) in Chinese hamster ovary (CHO) cells and the enzymatic activity. Short- N-acyl-DM-C1P analogs including C6-DM-C1P caused morphological changes with cell toxicity 24 h after the treatment in three cells lines (CHO, L929, and RLC-18 cells), although the role of AA in the toxicity was not clear. Neither long- N-acyl-DM-C1P analogs nor MM-C1P analogs including C6-MM-C1P affected cPLA 2α activity and cell toxicity. Similar to C6-ceramide having a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) group on a C6- N-acyl chain (NBD-C6-ceramide), NBD-C6-DM-C1P and C6-DM-C1P-NBD (with a C6- N-acyl chain and an NBD-labeled C14-alkyl chain) were accumulated in the Golgi complex, although less C6-DM-C1P-NBD than NBD-C6-DM-C1P was taken up. NBD-C6-ceramide was converted to various metabolites including NBD-C6-sphingomyelin, but both NBD-C6-DM-C1P and C6-DM-C1P-NBD were stable in cells within 2 h. The short- N-acyl-DM-C1P analogs acted directly as an inhibitor of cPLA 2α and an inducer of cell toxicity, and may be useful for the regulation of ceramide/C1P-regulated responses.

Advanced glycation end products inhibit Na + K + ATPase in proximal tubule epithelial cells: Role of cytosolic phospholipase A 2α and phosphatidylinositol 4-phosphate 5-kinase γ

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. In diabetes, renal Na + K + ATPase (NKA) activity is downregulated and phosphoinositide metabolism is upregulated. We examined the effects of AGEs on NKA activity in porcine LLC-PK1 and human HK2 proximal tubule epithelial cells. AGE-BSA increased cellular phosphoinositol 4,5 bisphosphate (PIP2) production as determined by immunofluorescence microscopy and thin layer chromatography. AGE-BSA (40 μM) induced 3H-arachidonic acid release and reactive oxygen species (ROS) production via cytosolic phospholipase A 2 (cPLA 2) activation. Within minutes, AGE-BSA significantly inhibited NKA surface expression and activity in a dose- and time-dependent manner as determined by immunofluorescence staining and [ 86Rb +] uptake, respectively, suggesting AGEs inhibit NKA by stimulating its endocytosis. The AGE-BSA-induced decrease in cell surface NKA was reversed by a cPLA 2α inhibitor, neomycin, a PIP2 inhibitor, and PP2, a Src inhibitor. AGE-BSA increased binding of NKA to the α-adaptin but not β2- or μ2-adaptin subunits of the AP-2 clathrin pit adaptor complex. Transfection of HK2 cells with PIP5Kγ siRNA prevented AGE-BSA inhibition of NKA activity. AGEs may stimulate PIP5Kγ to increase PIP2 production, which may enhance AP-2 localisation to clathrin pits, increase clathrin pit formation, enhance NKA cargo recognition by AP-2 and/or stimulate cPLA 2α activity. These results suggest AGEs modulate arachidonic acid and phosphoinositide metabolism to inhibit NKA via clathrin-mediated endocytosis. Elucidation of new intracellular AGE signaling pathways may lead to improved therapies for diabetic nephropathy.

Critical role of cytosolic phospholipase A2α in bronchial mucus hypersecretion in CFTR-deficient mice

Cystic fibrosis (CF) is due to mutations in the CF transmembrane conductance regulator gene CFTR. CF is characterised by mucus dehydration, chronic bacterial infection and inflammation, and increased levels of cytosolic phospholipase A2α (cPLA2α) products in airways. We aimed to examine the role of cPLA2α in the modulation of mucus production and inflammation in CFTR-deficient mice and epithelial cells. Mucus production was assessed using histological analyses, immuno-histochemistry and MUC5AC ELISA. cPLA2α activation was measured using an enzymatic assay and lung inflammation determined by histological analyses and polymorphonuclear neutrophil counts in bronchoalveolar lavages. In lungs from Cftr(-/-) mice, lipopolysaccharide induced mucus overproduction and MUC5AC expression associated with an increased cPLA2α activity. Mucus overproduction was mimicked by instillation of the cPLA2α product arachidonic acid, and abolished by either a cPLA2α null mutation or pharmacological inhibition. An increased cPLA2α activity was observed in bronchial explants from CF patients. CFTR silencing induced cPLA2α activation and MUC5AC expression in bronchial human epithelial cells. This expression was enhanced by arachidonic acid and reduced by cPLA2α inhibition. However, inhibition of CFTR chloride transport function had no effect on MUC5AC expression. Reduction of CFTR expression increased cPLA2α activity. This led to an enhanced mucus production in airway epithelia independent of CFTR chloride transport function. cPLA2α represents a suitable new target for therapeutic intervention in CF.

Modulation of the activity of cytosolic phospholipase A2α (cPLA2α) by cellular sphingolipids and inhibition of cPLA2α by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2alpha (cPLA2alpha) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca(2+) ionophore A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2alpha inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2alpha. Phosphatidylcholine vesicles containing SM reduced cPLA2alpha activity. Furthermore, SM disturbed the binding of cPLA2alpha to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2alpha-dependent release of AA by inhibiting the binding of cPLA2alpha to glycerophospholipids.