Association with actin mediates the EGTA-resistant binding of cytosolic phospholipase A2-alpha to the plasma membrane of activated platelets

The association of cytosolic phospholipase A₂-α (cPLA₂α) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A₂ in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA₂α was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA₂α relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA₂α was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA₂α co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA₂α and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA₂α with its membrane substrate via interaction with actin.     

C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes

Group IVA cytosolic phospholipase A₂ (cPLA₂α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA₂α binds zwitterionic lipids such as phosphatidylcholine in a Ca²⁺-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA₂α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA₂α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA₂α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA₂α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA₂α.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-α

Cytosolic phospholipase A₂-α (cPLA₂) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA₂ is dependent on a rise in cytosolic Ca²⁺ concentration, membrane association via the Ca²⁺-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA₂ via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), including the role of a “pleckstrin homology (PH)-like” region of cPLA₂ (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate + the Ca²⁺ ionophore, A23187, or epidermal growth factor + A23187, expression of the PH domain of phospholipase C-δ1 (which sequesters membrane PIP₂) attenuated cPLA₂ activity. Stimulated cPLA₂ activity was also attenuated by the expression of cPLA₂ 135-366, or cPLA₂ 2-366, and expression of a PIP₂-specific 5′-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP₂, with high affinity, only cPLA₂ 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA₂s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA₂ activity can be modulated by sequestration or depletion of cellular PIP₂, although the interaction of cPLA₂ with membrane PIP₂ appears to be indirect, or of weak affinity.     

Thermodynamic characterization of cytosolic phospholipase A2 alpha inhibitors

Cytosolic phospholipase A₂ alpha (cPLA₂α, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA₂α inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA₂α and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA₂α inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 °C and pH 7.4. The compounds tested bound cPLA₂α with a 1:1 stoichiometry, and the dissociation constant K_d of the inhibitors calculated from the ITC experiments correlated well with the IC₅₀ values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA₂α was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA₂α. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA₂α was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.     

Characterization of arachidonic acid-induced apoptosis

Tumor necrosis factor (TNF) can induce apoptosis in a number of different cell types. This response often depends on the activity of cytosolic phospholipase A₂ (cPLA₂), which catalyzes the release of arachidonic acid from the sn-2 position of membrane phospholipids. In this study, we investigate the ability of arachidonic acid itself to cause cell death. We show that in assays with 10% fetal bovine serum (FBS) arachidonic acid will not kill, nor does act synergistically with TNF. In contrast, by lowering the concentration of FBS to 2% it is possible to use arachidonic acid to induce cell death. Arachidonic acid-induced cell death was judged to be apoptotic based on morphology and the cleavage of poly (ADP) ribose polymerase. Arachidonic acid was able to kill all cell lines tested including two human melanoma-derived cell lines, and susceptibility to arachidonic acid was not influenced by adenovirus gene products that control susceptibility to TNF. Finally, we show that arachidonic acid is unique among 20 carbon fatty acids for its ability to induce apoptosis and that several other unsaturated, but not saturated fatty acids can also induce apoptosis.     

Hyperforin induces Ca-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions

Here, we investigated the modulation of cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA₂α-inhibition, and induced translocation of cPLA₂ to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca²⁺ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA₂ at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA₂ phosphorylation. However, neither AA release nor translocation of cPLA₂ was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA₂ in the presence of Ca²⁺. However, when Ca²⁺ was omitted, hyperforin caused a prominent increase in cPLA₂ activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional ¹H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA₂ activation by enabling its access towards select lipid membranes independent of Ca²⁺ ions. Such Ca²⁺- and phosphorylation-independent mechanism of cPLA₂ activation may apply also to other membrane-interfering molecules.     

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death

The purpose of this study was to determine the roles of calcium-dependent phospholipase A₂ (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) in thapsigargin-induced membrane susceptibility to secretory phospholipase A₂ (sPLA₂) and programmed cell death. ³H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA₂ (BEL) and completely by an inhibitor of both cPLA₂ and iPLA₂ (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA₂ in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA₂. In conclusion, the results suggested that cPLA₂ and iPLA₂ are active during thapsigargin-induced apoptosis in S49 cells and that cPLA₂ tempers the tendency of the cells to become susceptible to sPLA₂ during apoptosis.     

RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA<Subscript>2</Subscript> Activation

Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A₂ (cPLA₂), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA₂. Inhibition of RhoA, Rho kinase and cPLA₂ significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA₂ activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA₂. The immunofluorescence staining showed that ROCK₁ or ROCK₂, two isoforms of Rho kinase, was co-localized with cPLA₂ in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK₁ or ROCK₂ bonded directly with cPLA₂ and phospho-cPLA₂. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA₂ activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA₂ activation.     

Role of Cytosolic Phospholipase A2 in Oxidative and Inflammatory Signaling Pathways in Different Cell Types in the Central Nervous System

Phospholipases A₂ (PLA₂s) are important enzymes for the metabolism of fatty acids in membrane phospholipids. Among the three major classes of PLA₂s in the mammalian system, the group IV calcium-dependent cytosolic PLA₂ alpha (cPLA₂α) has received the most attention because it is widely expressed in nearly all mammalian cells and its active participation in cell metabolism. Besides Ca²⁺ binding to its C2 domain, this enzyme can undergo a number of cell-specific post-translational modifications, including phosphorylation by protein kinases, S-nitrosylation through interaction with nitric oxide (NO), as well as interaction with other proteins and lipid molecules. Hydrolysis of phospholipids by cPLA₂ yields two important lipid mediators, arachidonic acid (AA) and lysophospholipids. While AA is known to serve as a substrate for cyclooxygenases and lipoxygenases, which are enzymes for the synthesis of eicosanoids and leukotrienes, lysophospholipids are known to possess detergent-like properties capable of altering microdomains of cell membranes. An important feature of cPLA₂ is its link to cell surface receptors that stimulate signaling pathways associated with activation of protein kinases and production of reactive oxygen species (ROS). In the central nervous system (CNS), cPLA₂ activation has been implicated in neuronal excitation, synaptic secretion, apoptosis, cell-cell interaction, cognitive and behavioral function, oxidative-nitrosative stress, and inflammatory responses that underline the pathogenesis of a number of neurodegenerative diseases. However, the types of extracellular agonists that target intracellular signaling pathways leading to cPLA₂ activation among different cell types and under different physiological and pathological conditions have not been investigated in detail. In this review, special emphasis is given to metabolic events linking cPLA₂ to activation in neurons, astrocytes, microglial cells, and cerebrovascular cells. Understanding the molecular mechanism(s) for regulation of this enzyme is deemed important in the development of new therapeutic targets for the treatment and prevention of neurodegenerative diseases.     

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA₂) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA₂ are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA₂ and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA₂ by 1.5-fold (p < 0.01), which was blocked by the cPLA₂ inhibitor MAFP (10 μM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 μM; 15 min) and ERK1/2 inhibitor PD98059 (20 μM; 15 min) abolished the INS-mediated increase in cPLA₂ phosphorylation by 59% (p < 0.001), and by 75% (p < 0.001), respectively. Further, inhibition of cPLA₂ with cPLA₂ inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% (p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% (p < 0.001). The effect of INS on VSMCs proliferation was significantly (p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA₂ and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA₂ activity.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

Phospholipases A2 in Ischemic and Toxic Brain Injury

Phospholipases A₂ (PLA₂s) regulate hydrolysis of fatty acids, including arachidonic acid, from the sn-2 position of phospholipid membranes. PLA₂ activity has been implicated in neurotoxicity and neurodegenerative processes secondary to ischemia and reperfusion and other oxidative stresses. The PLA₂s constitute a superfamily whose members have diverse functions and patterns of expression. A large number of PLA₂s have been identified within the central nervous systems of rodents and humans. We postulated that group IV large molecular weight, cytosolic phospholipase A₂ (cPLA₂) has a unique role in neurotoxicity associated with ischemic or toxin stress. We created mice deficient in cPLA₂ and tested this hypothesis in two injury models, ischemia/reperfusion and MPTP neurotoxicity. In each model cPLA₂ deficient mice are protected against neuronal injury when compared to their wild type littermate controls. These experiments support the hypothesis that cPLA₂ is an important mediator of ischemic and oxidative injuries in the brain.     

Oxidative stress mediates synthesis of cytosolic phospholipase A2 after UVB injury

UVB irradiation has previously been shown to significantly increase phospholipase activity and prostaglandin synthesis. Because UVB irradiation is a potent oxidative stress, the role of active oxygen species in regulating UV-induced cPLA₂ synthesis and phosphorylation was examined. In the present study, irradiation produced a 3-fold increase in synthesis within 6 h following irradiation. Phosphorylation of cPLA₂ was also increased to a similar extent. UVB-induced synthesis and phosphorylation of cPLA₂ could be inhibited by pretreatment with the antioxidants 2,2,5,7,8-pentamethyl-6-hydroxychromane (50 μM) or N-acetylcysteine (10 mM). Treatment of unirradiated cultures with the potent oxidant tert-butyl hydroperoxide (500 μM) also increased cPLA₂ synthesis and phosphorylation, suggesting that oxidative injury is an important regulator of cPLA₂ synthesis. Increased synthesis of cPLA₂ correlated well with increased [³H]arachidonic acid release, PGE₂ synthesis and lipid peroxidation in epidermis after oxidant or UVB treatment. The results indicate that UVB-induced upregulation of cPLA₂ synthesis is mediated by UVB-induced formation of free radicals.     

Toll-like receptor 9-mediated cytosolic phospholipase A2 activation regulates expression of inducible nitric oxide synthase

Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A₂ (cPLA₂) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA₂-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA₂ activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA₂ protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA₂ activity via p38 or Akt pathways and mediates iNOS expression.     

2-Oxoamide inhibitors of phospholipase A2 activity and cellular arachidonate release based on dipeptides and pseudodipeptides

A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A₂ (GIVA cPLA₂ and GVIA iPLA₂) and one human secretory phospholipase A₂ (GV sPLA₂) were evaluated. Derivatives containing a free carboxyl group are selective GIVA cPLA₂ inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-oxoamide, which preferentially inhibits GVIA iPLA₂. The effect of 2-oxoamides on the generation of arachidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA₂ inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC₅₀ value of 2 μM.     

Expression of phospholipases A2 in primary human lung macrophages: Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA₂) and secreted phospholipases A₂ (sPLA₂) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA₂s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA₂ (iPLA₂). Two structurally-divergent inhibitors of group IV cPLA₂ completely block arachidonic acid release by macrophages in response to non-physiological (Ca²⁺ ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA₂s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA₂s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA₂ inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA₂-alpha, iPLA₂ and several sPLA₂s. Cytosolic PLA₂-alpha is the major enzyme responsible for lipid mediator production in human macrophages.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.