Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2

Recently, we demonstrated that ceramide kinase, and its product, ceramide 1-phosphate (Cer-1-P), were mediators of arachidonic acid released in cells in response to interleukin-1beta and calcium ionophore (Pettus, B. J., Bielawska, A., Spiegel, S., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2003) J. Biol. Chem. 278, 38206-38213). In this study, we demonstrate that down-regulation of cytosolic phospholipase A(2) (cPLA(2)) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA(2) is the key phospholipase A(2) downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 microm) induced the translocation of full-length cPLA(2) from the cytosol to the Golgi apparatus/perinuclear regions, which are known sites of translocation in response to agonists. Cer-1-P also induced the translocation of the CaLB/C2 domain of cPLA(2) in the same manner, suggesting that this domain is responsive to Cer-1-P either directly or indirectly. In vitro studies were then conducted to distinguish these two possibilities. In vitro binding studies disclosed that Cer-1-P interacts directly with full-length cPLA(2) and with the CaLB domain in a calcium- and lipid-specific manner with a K(Ca) of 1.54 microm. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA(2) enzymatic activity as well as lowering the EC(50) of calcium for the enzyme from 191 to 31 nm. This study identifies Cer-1-P as an anionic lipid that translocates and directly activates cPLA(2), demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.     

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

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.     

Sphingolipids and cholesterol modulate membrane susceptibility to cytosolic phospholipase A(2)

Modulation of cytosolic phospholipase A(2) (cPLA(2)) activity by sphingomyelin (SPH), ceramide (Cer), and cholesterol (Chol) was investigated in CHO-2B cells activated by the calcium ionophore A23187 and epinephrine. Chol depletion of CHO-2B cells by treatment with methyl-beta-cyclodextrin (5 mm) resulted in the inhibition of the release of arachidonic acid whereas the restoration of the level by Chol-loaded cyclodextrin relieved inhibition. Conversion of CHO-2B cellular SPH to Cer by Staphylococcus aureus sphingomyelinase enhanced endogenous cPLA(2) activation as well as uptake by cells of C2- and C6-ceramide analogs. These results were confirmed in vitro with purified human recombinant cPLA(2) acting on a model phospholipid substrate. The enzyme activity was inhibited by SPH but reactivated by Cer as well as by Chol added to glycerophospholipid liposomal substrates containing SPH. The results of this study, which combine in situ and in vivo experimental approaches, indicate that membrane microdomains enriched in SPH and Chol play a role in the modulation of the activity of cPLA2 and in arachidonic acid-derived mediator production.     

Acceleration by ceramide of calcium-dependent translocation of phospholipase A2 from cytosol to membranes in platelets

The effect of ceramide on Ca2+-dependent translocation of cytosolic phospholipase A2 (cPLA2) to membranes was studied. Pretreatment of platelets with sphingomyelinase or C6-ceramide (N-hexanoylsphingosine) led to apparent enhancement of Ca2+-ionophore A23187-stimulated arachidonic acid release but did not affect the cytosolic phospholipase A2 (cPLA2) activity. Under these conditions, the cPLA2 proteins in membranes increased significantly, compared with those by A23187 alone. Sphingomyelinase and C6-ceramide, but not C6-dihydroceramide, a control analog of C6-ceramide, also facilitated the Ca2+-dependent increase in the cPLA2 protein, as well as the activity, in membranes induced by addition of Ca2+ into platelet lysate. Protein kinase Calpha, which possesses a Ca2+-dependent lipid binding domain, was increased in membranes in a Ca2+-dependent manner, but the increase was not accelerated by sphingomyelinase or C6-ceramide. These findings suggest that ceramide in membranes potentiates Ca2+-dependent cPLA2 translocation from cytosol to membranes, probably through modification of membrane phospholipid organization.     

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.     

Regulation of the specific release of arachidonic acid by cytosolic phospholipase A2

Cytosolic phospholipase A(2) alpha (cPLA(2)alpha) is the only PLA(2) that exhibits specificity for sn-2 arachidonic acid consistent with its primary role in mediating the agonist-induced release of arachidonic acid for eicosanoid production. It is subject to complex mechanisms of regulation that ensure that levels of free arachidonic acid are tightly controlled. The calcium-induced translocation of cPLA(2)alpha from the cytosol to membrane regulates its interaction with phospholipid substrate. cPLA(2)alpha is additionally regulated by phosphorylation on sites in the catalytic domain. Because of its central position as the upstream regulatory enzyme for initiating production of several classes of bioactive lipid mediators (leukotrienes, prostaglandins and platelet-activating factor), it is a potentially important pharmacological target for the control of inflammatory diseases.     

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.     

Stimulation-dependent recruitment of cytosolic phospholipase A2-alpha to EA.hy.926 endothelial cell membranes leads to calcium-independent association.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme involved in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid is predominantly converted into prostacyclin, a potent vasodilator and inhibitor of platelet activation. As the rate-limiting step in prostacyclin production is the generation of free arachidonic acid by cPLA2-alpha, this enzyme has become an attractive pharmacological target and the focus of many studies. Following stimulation with calcium-mobilizing agonists, cPLA2-alpha translocates to intracellular phospholipid membranes via its C2 domain. In this study, the calcium-induced association of cPLA2-alpha with EA.hy.926 endothelial cell membranes was investigated. Subcellular fractionation and immunofluorescence studies showed that following stimulation with histamine, thrombin or the calcium ionophore A23187, cPLA2-alpha relocated to intracellular membranes. Treatment of A23187-stimulated cells with EGTA or BAPTA-AM demonstrated that a substantial pool of cPLA2-alpha remained associated with membrane fractions in a calcium-independent manner. Furthermore, immunofluorescence microscopy studies revealed that cells stimulated for periods of greater than 10 min showed a high proportion of calcium-independent membrane-associated cPLA2-alpha. Calcium-independent membrane association of cPLA2-alpha was not due to hydrophobic or cytoskeletal interactions. Finally, the recombinant C2 domain of cPLA2-alpha exhibited calcium-independent membrane binding to membranes isolated from A23187-stimulated cells but not those isolated from nonstimulated cells. These findings suggest that novel mechanisms involving accessory proteins at the target membrane play a role in the regulation of cPLA2-alpha. Such regulatory associations could enable the cell to discriminate between the varying levels of cytosolic calcium induced by different stimuli.     

Enzymatic properties of human cytosolic phospholipase A(2)gamma

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.     

Structure and mechanism of human cytosolic phospholipase A(2)

cPLA(2) is an 85-kDa enzyme whose primary function, the release of arachidonic acid from phospholipid membranes, is a crucial reaction in the metabolism of lipid mediators of inflammation. cPLA(2) consists of two domains: an N-terminal, C2-type unit analogous to those present in other membrane-targeting molecules, and a catalytic domain harboring an active site dyad at the bottom of a deep, mostly hydrophobic catalytic funnel. The absence of a third active site residue in the cPLA(2) cleft, as observed in other lipases, suggests that the enzyme proceeds through a novel catalytic mechanism. Crystallographic and biochemical studies of cPLA(2) will provide essential information for the development of small molecule inhibitors which may be employed in the control of inflammatory and other highly regulated processes.     

Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate levels

Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.     

NO induced apoptosis of vascular smooth muscle cells accompanied by ceramide increase

We have shown previously that nitric-oxide (NO) can induce apoptosis of vascular smooth muscle cells (VSMCs) and that the NO-induced apoptosis is accompanied by an increase in arachidonic acid release via cytoplasmic Ca(2+)-dependent phospholipase A(2) (cPLA(2)). We have evidence that during NO-induced apoptosis there is an increase in ceramide synthesis. The use of inhibitors of ceramide synthesis, namely, fumonisin B1 and desipramine, which block ceramide synthase and sphingomyelinase, respectively revealed that the ceramide was produced via the sphingomyelinase pathway. Inhibition of acid sphingomyelinase by desipramine was shown to inhibit NO-induced apoptosis while fumonisin B1 failed to inhibit this process. C(2)-ceramide could induce apoptosis in cultured VSMCs. Apoptosis in smooth muscle cells was accompanied by the increased activity of DNA fragmentation factor-40 and the secretion of cathepsin D from the cells. In this study, ceramide appears to function as a mediator of apoptosis.     

Cytosolic phospholipase A2 is an effector of Jak/STAT signaling and is involved in platelet-derived growth factor BB-induced growth in vascular smooth muscle cells

Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). To understand its mitogenic and chemotactic signaling events, we studied the role of cytosolic phospholipase A(2) (cPLA(2)) and the Jak/STAT pathway. PDGF-BB induced the expression and activity of cPLA(2) in a time-dependent manner in VSMC. Arachidonyl trifluoromethyl ketone, a potent and specific inhibitor of cPLA(2), significantly reduced PDGF-BB-induced arachidonic acid release and DNA synthesis. PDGF-BB stimulated tyrosine phosphorylation of Jak-2 in a time-dependent manner. In addition, PDGF-BB activated STAT-3 as determined by its tyrosine phosphorylation, DNA-binding activity, and reporter gene expression, and these responses were suppressed by AG490, a selective inhibitor of Jak-2. AG490 and a dominant-negative mutant of STAT-3 also attenuated PDGF-BB-induced expression of cPLA(2,) arachidonic acid release, and DNA synthesis in VSMC. Together, these results suggest that induction of expression of cPLA(2) and arachidonic acid release are involved in VSMC growth in response to PDGF-BB and that these events are mediated by Jak-2-dependent STAT-3 activation.     

CTP:phosphocholine cytidylyltransferase inhibition by ceramide via PKC-alpha, p38 MAPK, cPLA2, and 5-lipoxygenase

In a companion paper (Vivekananda J, Smith D, and King RJ. Am J Physiol Lung Cell Mol Physiol 281: L98-L107, 2001), we demonstrated that tumor necrosis factor (TNF)-alpha inhibited the activity of CTP:phosphocholine cytidylyltransferase (CT), the rate-limiting enzyme in the de novo synthesis of phosphatidylcholine (PC), and that its actions were likely exerted through a metabolite of sphingomyelin. In this paper, we explore the signaling pathway employed by TNF-alpha using C2 ceramide as a cell-penetrating sphingolipid representative of the metabolites induced by TNF-alpha. We found that in H441 cells, as reported in other cell types, cytosolic phospholipase A2 (cPLA2) is activated by TNF-alpha. We also observed that the inhibiting action of C2 ceramide on CT requires protein kinase C-alpha, p38 mitogen-activated protein kinase, and cPLA2. The actions of C2 ceramide on CT activity can be duplicated by adding 2 microM lysoPC to these cells. Furthermore, we found that the effects of C2 ceramide are dependent on 5-lipoxygenase but that cyclooxygenase II is unimportant. We hypothesize that CT activity is inhibited by the lysoPC generated as a consequence of the activation of cPLA2 by protein kinase C-alpha and p38 mitogen-activated protein kinase. The other product of the activation of cPLA2, arachidonic acid, is a substrate for the synthesis of leukotrienes, which raise intracellular Ca2+ levels and complete the activation of cPLA2.     

cPLA2 activator peptide, PLAP, increases arachidonic acid release and apoptosis of vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (VSMCs) has recently drawn a lot of interest in various laboratories due to its importance in atherogenesis. We have shown previously that nitric-oxide (NO) can induce apoptosis of VSMCs and that the NO-induced apoptosis is accompanied by an increase in arachidonic acid release via cytoplasmic Ca(2+)-dependent phospholipase A(2) (cPLA(2)). We have demonstrated here that NO-induced activation of cPLA(2) leading to increased arachidonic acid release can be mimicked via direct activation of cPLA(2) with a cPLA(2) activator peptide, PLAP. The PLAP induced arachidonic acid release and apoptosis is inhibitable by a cPLA(2)-specific inhibitor, AACOCF(3), indicating the direct involvement of cPLA(2). In this study, activation of cPLA(2) appears to be preceded by activation and binding by PLAP indicating that the cPLA(2) functions are mediated via PLAP.     

Modulation of the activity and arachidonic acid selectivity of group X secretory phospholipase A2 by sphingolipids

To investigate the role of sphingomyelin (SM) in the regulation of inflammatory reactions, we studied its effect on the activity and fatty acid specificity of group X secretory phospholipase A(2) (sPLA(2)X). Compared with other phospholipases, recombinant sPLA(2)X released more arachidonate from HDL. Pretreatment of HDL with sphingomyelinase (SMase) C activated the sPLA(2)X activity, but the release of arachidonate was stimulated less than that of linoleate. In liposomes containing synthetic phosphatidylcholines (PCs), sPLA(2)X showed no clear selectivity among the various sn-2 unsaturated fatty acids. However, when SM was incorporated into liposomes at 30 mol%, the enzyme exhibited strong preference for arachidonate, although its overall activity was inhibited. Degradation of liposomal SM by SMase C resulted in sPLA(2)X activation and loss of its arachidonate preference. Incorporation of ceramide into HDL or PC liposomes activated the enzyme activity, the release of arachidonate being stimulated more than that of linoleate. SM-deficient cells released more arachidonate than normal cells in response to exogenous sPLA(2)X. SMase pretreatment of normal cells stimulated the release of arachidonate by the exogenous sPLA(2)X. These results show that SM not only inhibits sPLA(2)X activity but also contributes to its selectivity for arachidonate, whereas ceramide stimulates the hydrolysis of arachidonate-containing PCs.     

Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.     

Cytosolic phospholipase A(2) activity during the ongoing cell cycle

Cytosolic phospholipase A(2) (cPLA(2)) is of special interest because it selectively releases arachidonic acid from membrane phospholipids. Arachidonic acid has been implicated to play an important role in various cellular responses. Recently arachidonic acid release and prostaglandin synthesis have been shown to be cell cycle dependent and therefore the activity of cPLA(2) during the ongoing cell cycle was investigated, using the mitotic shake off method for cell synchronisation. cPLA(2) activity was high in mitotic cells and decreased rapidly in the early G1 phase. A strong increase in activity was measured following the G1/S transition in both neuroblastoma and Chinese hamster ovary cells. The changes in activity were not due to a difference in cPLA(2) expression but due to phosphorylation of cPLA(2). Phosphorylation of cPLA(2) occurs through MAPK since the use of a specific MAPK kinase inhibitor and serum depletion of synchronised cells inhibited cPLA(2) activity.