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-1␤ 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 M) 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 M. 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.

Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A 2 ␣ (cPLA 2 ␣) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic ␤-groove (Arg 57 , Lys 58 , Arg 59 ) of the C2 domain of cPLA 2 ␣. In this regard, mutants of this region of cPLA 2 ␣ were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/ R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA 2 ␣. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA 2 ␣ also showed a significant reduction in the ability of C1P to: 1) increase the V max of the reaction; and 2) significantly decrease the dissociation constant (K s A ) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA 2 ␣ demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA 2 ␣. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic ␤-groove of the C2 domain of cPLA 2 ␣.

Previous findings from our laboratory have demonstrated that cPLA 2 ␣ is directly activated by the emerging bioactive sphingolipid, ceramide 1-phosphate (C-1-P) (1). In this study, a Triton X-100/phosphatidylcholine (PC) mixed micelle assay was utilized to determine the kinetics and specificity of this lipid-enzyme interaction. Using this assay, the addition of C-1-P induced a dramatic increase in the activity of cPLA 2 ␣ (>15-fold) with a K a of 2.4 mol % C-1-P/Triton X-100 micelle. This activation was highly specific as the addition of other lipids had insignificant effects on cPLA 2 ␣ activity. Studies using surface-dilution kinetics revealed that C-1-P had no effect on the MichaelisMenten constant, K m B , but decreased the dissociation constant (K s A ) value by 87%. Thus, C-1-P not only increases the membrane affinity of cPLA 2 ␣ but also may act as an allosteric activator of the enzyme. Surface plasmon resonance analysis of the C-1-P/cPLA 2 ␣ interaction verified a decrease in the dissociation constant, demonstrating that cPLA 2 ␣ bound PC vesicles containing C-1-P with increased affinity (5-fold) compared with PC vesicles alone. The effect on the dissociation rate of cPLA 2 ␣ was also found to be lipid-specific with the exception of phosphatidylinositol 4,5-bisphosphate, which caused a modest increase in vesicle affinity (2-fold). Lastly, the binding site for C-1-P was determined to be within the C2-domain of cPLA 2 ␣, unlike phosphatidylinositol 4,5-bisphosphate. These data demonstrate a novel interaction site for C-1-P and suggest that C-1-P may function to recruit cPLA 2 ␣ to intracellular membranes as well as allosterically activate the membrane-associated enzyme.Group IVA cytosolic phospholipase A 2 (cPLA 2 ␣) 1 was first characterized in platelets and macrophage cells and subsequently cloned from a macrophage cDNA library (2-8). The cDNA of cPLA 2 ␣ encodes a 85-kDa protein, and the mRNA for cPLA 2 is widely expressed in brain, lung, kidney, heart, and spleen (2-8). cPLA 2 ␣ is the major phospholipase that regulates eicosanoid synthesis in response to inflammatory agonists (2, 3). In vitro, cPLA 2 ␣ is activated by Ca 2ϩ ; however, the addition of salt at physiologic concentrations will also induce enzyme activation and thus the catalytic activity of cPLA 2 ␣ is not dependent on Ca 2ϩ (2, 9, 10). The cellular activation of cPLA 2 ␣ requires Ca 2ϩ -dependent membrane translocation of the enzyme, which is mediated by the N-terminal C2 domain (2-5). Cell-specific and agonist-dependent events coordinate translocation of cPLA 2 ␣ to the nuclear envelope, endoplasmic reticulum, and Golgi apparatus via this domain (2-7, 9, 10). At these membranes, cPLA 2 ␣ hydrolyzes membrane phospholipids to produce arachidonic acid, initiating the eicosanoid synthetic pathways (2-7, 9, 10). However, the specific membrane lipids that regulate the association of this domain with membranes, especially at low cellular calcium concentration (e.g. submicromolar), have yet to be defined (4).One possible candidate for an activa...

FTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD 2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A 2 (PLA 2 )-mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA 2 or ERK1/2 activation, suggesting that it does not interfere with Fc⑀RI-mediated events leading to cPLA 2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA 2 ␣ activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA 2 ␣ and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis. IntroductionFTY720 is a novel immunosuppressive agent that was derived from myriocin, a sphingosine-like fungal metabolite. 1 FTY720 potently inhibits a variety of experimental autoimmune disorders, such as type I diabetes 2 and arthritis, 3 but clinically, its greatest potential for humans appears to be in the prevention of renal graft rejection and treatment of multiple sclerosis (MS) where it is currently in phase 3 clinical trials. 4,5 The immunosuppressive properties of FTY720 stem from its ability to prevent T cells' egress from secondary lymphoid organs and back into circulation, sequestering them away from the transplanted graft, [6][7][8] or preventing their entry into the central nervous system. 9 FTY720 is phosphorylated by sphingosine kinase 2 (SphK2), [10][11][12] and a large body of evidence suggests that the phosphorylated drug (FTY720-P), an analog of sphingosine-1-phosphate (S1P), is the biologically active form. FTY720-P can bind to all of the known S1P receptors, except S1P 2 , 6,13,14 and has been shown to regulate S1P 1 actions that are crucial for lymphocyte migration and trafficking. [15][16][17][18] Nevertheless, the exact mechanism of action of this prodrug is still a matter of debate. [19][20][21] S1P is also an important chemoattractant in the immune system, modulating T-cell responses to chemokines (reviewed in Cyster 13 and Rosen and Goetzl 18 ).FTY720 inhibits airway inflammation, induction of bronchial hyperresponsiveness, and lymphocyte and eosinophil infiltration in an actively Ag-sensitized murine asthma model, 22 suggesting that it might have therapeutic benefits in asthma.Given that S1P and its receptors may pla...

Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A 2 ␣ (cPLA 2 ␣), the initial rate-limiting step. The current view is that cPLA 2 ␣ associates with intracellular/phosphatidylcholine-rich membranes strictly via hydrophobic interactions in response to an increase of intracellular calcium. In opposition to this accepted mechanism of two decades, ceramide 1-phosphate (C1P) has been shown to increase the membrane association of cPLA 2 ␣ in vitro via a novel site in the cationic ␤-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA 2 ␣ to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/ cPLA 2 ␣ interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA 2 ␣ ؊/؊ ) coupled to "rescue" studies. Therefore, this study provides a paradigm shift in how cPLA 2 ␣ is activated during inflammation.Eicosanoids are a class of bioactive lipids derived from the 20-carbon fatty acid, arachidonic acid (AA), 2 including prostaglandins, prostacyclins, thromboxanes, and leukotrienes. The production of AA is the initial rate-limiting step in the production of eicosanoids, and the major phospholipase that regulates eicosanoids synthesis in response to agonists is group IVA cytosolic phospholipase A 2 (cPLA 2 ␣) (2, 3). Activation of cPLA 2 in cells requires the association of the enzyme with intracellular membranes in a Ca 2ϩ -dependent manner. This translocation of cPLA 2 ␣ from the cytosol to intracellular membranes is mediated by a Ca 2ϩ -dependent lipid binding domain (CaLB domain) located at the N terminus of the enzyme (4 -7). The CaLB domain is ϳ60 amino acids and binds phosphatidylcholine (PC) in a Ca 2ϩ -dependent manner (3, 8 -10). However, it is not known if physiologic calcium is sufficient to activate and translocate cPLA 2 ␣ to membranes in cells or if activation also requires the generation of other activating lipids, such as the focus of this study, ceramide 1-phosphate (C1P).One possible activating lipid, phosphatidylinositol 4,5-diphosphate, was ruled out by Balboa and co-workers (11) as a lipid co-factor required for the translocation of the enzyme. This group showed that the interaction with this lipid (via its catalytic domain) was required for full activity of cPLA 2 ␣ after the enzyme translocated to the membrane (11). Another recent report by Leslie and co-workers (12) confirmed these findings, and a recent study by our laboratory corroborated these findings utilizing biophysical approaches (1). Specifically, we showed that C1P induced a dramatic increase of cPLA 2 ␣ activity strictly by increasing the residence time of cPLA 2 ␣ to membranes, whereas phosphatidylinositol 4,5-diphosphate enhanced the enzymes catalytic activity and membrane penetration (13,14).Recent...

Background: PEDF has neurotrophic activity and interacts with PEDF-R, a membrane-linked lipase. Results: A PEDF-binding region of PEDF-R is required for PEDF-R enzymatic stimulation, and peptides derived from this region block PEDF⅐PEDF-R-mediated retinal survival activities. Conclusion: A ligand binding domain is identified in PEDF-R, a critical receptor for the survival activity of PEDF. Significance: The findings provide mechanistic insight into the survival activity of PEDF.

Across neurodegenerative diseases, common mechanisms may reveal novel therapeutic targets based on neuronal protection, repair, or regeneration, independent of etiology or site of disease pathology. To address these mechanisms and discuss emerging treatments, in April, 2021, Glaucoma Research Foundation, BrightFocus Foundation, and the Melza M. and Frank Theodore Barr Foundation collaborated to bring together key opinion leaders and experts in the field of neurodegenerative disease for a virtual meeting titled “Solving Neurodegeneration”. This “think-tank” style meeting focused on uncovering common mechanistic roots of neurodegenerative disease and promising targets for new treatments, catalyzed by the goal of finding new treatments for glaucoma, the world’s leading cause of irreversible blindness and the common interest of the three hosting foundations. Glaucoma, which causes vision loss through degeneration of the optic nerve, likely shares early cellular and molecular events with other neurodegenerative diseases of the central nervous system. Here we discuss major areas of mechanistic overlap between neurodegenerative diseases of the central nervous system: neuroinflammation, bioenergetics and metabolism, genetic contributions, and neurovascular interactions. We summarize important discussion points with emphasis on the research areas that are most innovative and promising in the treatment of neurodegeneration yet require further development. The research that is highlighted provides unique opportunities for collaboration that will lead to efforts in preventing neurodegeneration and ultimately vision loss.

Background: Pigment epithelium-derived factor (PEDF) interacts with its receptor PEDF-R to exert cytoprotection. Results: Alanine scanning of a small fragment (17-mer) of PEDF reveals key interacting residues for binding PEDF-R and alternative retinoprotective peptide versions with higher efficacy. Conclusion: The 17-mer contains a novel PEDF-R binding region important for retinoprotection. Significance: Altered PEDF peptides could be exploited pharmacologically to improve protection of photoreceptors from degeneration.