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• W1996870265 abstract "Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A2α (cPLA2α), the initial rate-limiting step. The current view is that cPLA2α 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 cPLA2α in vitro via a novel site in the cationic β-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467–204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA2α to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA2α interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA2α−/−) coupled to “rescue” studies. Therefore, this study provides a paradigm shift in how cPLA2α is activated during inflammation. Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A2α (cPLA2α), the initial rate-limiting step. The current view is that cPLA2α 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 cPLA2α in vitro via a novel site in the cationic β-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467–204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA2α to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA2α interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA2α−/−) coupled to “rescue” studies. Therefore, this study provides a paradigm shift in how cPLA2α is activated during inflammation. Eicosanoids are a class of bioactive lipids derived from the 20-carbon fatty acid, arachidonic acid (AA), 2The abbreviations used are: AAarachidonic acidC1Pceramide 1-phosphateCERKceramide kinaseIL-1βinterleukin-1βPCphosphatidylcholinePGE2prostaglandin E2PLA2phospholipase A2cPLA2cytosolic PLA2siRNAsmall interfering RNATNFαtumor necrosis factor αWTwild typeGFPgreen fluorescent proteinCFPcyan fluorescent proteinYFPyellow fluorescent proteinCaLB domainCa2+-dependent lipid binding domainPIPphosphoinositideMEFmouse embryonic fibroblastPBSphosphate-buffered salinePAPC1-palmitoyl-2-arachidonyl-sn-phosphatidylcholineCAMKIIcalcium/calmodulin-dependent protein kinase. 2The abbreviations used are: AAarachidonic acidC1Pceramide 1-phosphateCERKceramide kinaseIL-1βinterleukin-1βPCphosphatidylcholinePGE2prostaglandin E2PLA2phospholipase A2cPLA2cytosolic PLA2siRNAsmall interfering RNATNFαtumor necrosis factor αWTwild typeGFPgreen fluorescent proteinCFPcyan fluorescent proteinYFPyellow fluorescent proteinCaLB domainCa2+-dependent lipid binding domainPIPphosphoinositideMEFmouse embryonic fibroblastPBSphosphate-buffered salinePAPC1-palmitoyl-2-arachidonyl-sn-phosphatidylcholineCAMKIIcalcium/calmodulin-dependent protein kinase. 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 A2 (cPLA2α) (2Clark J.D. Schievella A.R. Nalefski E.A. Lin L.L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). Activation of cPLA2 in cells requires the association of the enzyme with intracellular membranes in a Ca2+-dependent manner. This translocation of cPLA2α from the cytosol to intracellular membranes is mediated by a Ca2+-dependent lipid binding domain (CaLB domain) located at the N terminus of the enzyme (4Reynolds L.J. Hughes L.L. Louis A.I. Kramer R.M. Dennis E.A. Biochim. Biophys. Acta. 1993; 1167: 272-280Crossref PubMed Scopus (151) Google Scholar, 5Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar, 6Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar, 7Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar). The CaLB domain is ∼60 amino acids and binds phosphatidylcholine (PC) in a Ca2+-dependent manner (3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 8Tay A. Simon J.S. Squire J. Hamel K. Jacob H.J. Skorecki K. Genomics. 1995; 26: 138-141Crossref PubMed Scopus (81) Google Scholar, 9Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. et al.J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar, 10Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar). However, it is not known if physiologic calcium is sufficient to activate and translocate cPLA2α 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 (11Casas J. Gijón M.A. Vigo A.G. Crespo M.S. Balsinde J. Balboa M.A. Mol. Biol. Cell. 2006; 17: 155-162Crossref PubMed Scopus (59) Google Scholar) 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 cPLA2α after the enzyme translocated to the membrane (11Casas J. Gijón M.A. Vigo A.G. Crespo M.S. Balsinde J. Balboa M.A. Mol. Biol. Cell. 2006; 17: 155-162Crossref PubMed Scopus (59) Google Scholar). Another recent report by Leslie and co-workers (12Tucker D.E. Ghosh M. Ghomashchi F. Loper R. Suram S. St. John B. Girotti M. Bollinger J.G. Gelb M.H. Leslie C.C. J. Biol. Chem. 2009; 284: 9596-9611Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) confirmed these findings, and a recent study by our laboratory corroborated these findings utilizing biophysical approaches (1Stahelin R.V. Subramanian P. Vora M. Cho W. Chalfant C.E. J. Biol. Chem. 2007; 282: 20467-20474Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Specifically, we showed that C1P induced a dramatic increase of cPLA2α activity strictly by increasing the residence time of cPLA2α to membranes, whereas phosphatidylinositol 4,5-diphosphate enhanced the enzymes catalytic activity and membrane penetration (13Subramanian P. Stahelin R.V. Szulc Z. Bielawska A. Cho W. Chalfant C.E. J. Biol. Chem. 2005; 280: 17601-17607Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14Subramanian P. Vora M. Gentile L.B. Stahelin R.V. Chalfant C.E. J. Lipid Res. 2007; 48: 2701-2708Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).Recent studies from our laboratory have also demonstrated that C1P enhances the association of cPLA2α with membranes in vitro via a novel interactions site adjacent to the calcium binding region II of the C2 domain. Mutations of specific amino acids of this region significantly reduced the affinity for C1P (>65%) without an effect on basal enzyme activity, calcium-dependent PC affinity (supplemental Table 1), and phosphatidylinositol 4,5-diphosphate activation/affinity (1Stahelin R.V. Subramanian P. Vora M. Cho W. Chalfant C.E. J. Biol. Chem. 2007; 282: 20467-20474Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Subramanian P. Vora M. Gentile L.B. Stahelin R.V. Chalfant C.E. J. Lipid Res. 2007; 48: 2701-2708Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The identification and characterization of the C1P interaction site in cPLA2α allowed our laboratory to determine whether C1P played a role in regulating cPLA2α translocation and, thus, eicosanoid synthesis in response to inflammatory agonists.DISCUSSIONIn this study we demonstrate that C1P is a proximal and required bioactive lipid for the activation of cPLA2α. Specifically, the interaction of C1P with cPLA2α was necessary for the translocation of the enzyme to internal membranes and for the production of the eicosanoid subspecies, PGE2, in response to various inflammatory agonists (e.g. calcium ionophore, ATP, TNFα, and IL-1β). Previously activation/translocation of cPLA2α in cells was shown to require the association of cPLA2α with membranes via the Ca2+-dependent lipid binding domain (CaLB domain) (3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 5Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar, 8Tay A. Simon J.S. Squire J. Hamel K. Jacob H.J. Skorecki K. Genomics. 1995; 26: 138-141Crossref PubMed Scopus (81) Google Scholar, 9Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. et al.J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar, 10Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar). For two decades, the theory of cPLA2α activation/translocation held that the enzyme interacted with PC-rich membranes in response to an intracellular calcium increase via charge-negation and hydrophobic interactions. Whereas this theory could not explain the micromolar kca for the interaction of the enzyme with PC vesicles, the CaLB domain was indeed required for the enzyme to bind substrate PC in a Ca2+-dependent manner as well as required for agonist-induced activation of the enzyme in cells (3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 5Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar, 6Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar, 8Tay A. Simon J.S. Squire J. Hamel K. Jacob H.J. Skorecki K. Genomics. 1995; 26: 138-141Crossref PubMed Scopus (81) Google Scholar, 9Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. et al.J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar, 31Chatterjee S. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1523-1533Crossref PubMed Scopus (219) Google Scholar). However, this study explicitly demonstrates that Ca2+ alone is not sufficient to activate and translocate the enzyme to internal membranes in cells (3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 32Balsinde J. Balboa M.A. Li W.H. Llopis J. Dennis E.A. J. Immunol. 2000; 164: 5398-5402Crossref PubMed Scopus (66) Google Scholar). We demonstrate that the interaction of C1P with β-groove of the CaLB domain of cPLA2α is a requirement for efficient recruitment of the enzyme to internal PC-rich membranes in response to calcium and inflammatory agonists, dispelling the dogmatic theory of two decades while corroborating the requirement of the CaLB domain. Furthermore, substrate PC is not sufficient to effect the translocation of cPLA2α under physiologic conditions as the enzyme is cytosolic before activation even though PC is present in membranes in great abundance (33Evans J.H. Spencer D.M. Zweifach A. Leslie C.C. J. Biol. Chem. 2001; 276: 30150-30160Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Therefore, this study demonstrates the absolute requirement of the C1P/cPLA2α interaction for the activation of cPLA2α during the inflammatory response. Because C1P has been shown to be essential in the translocation of cPLA2α, we can also now deduce a link between the production of C1P and inflammatory responses.The requirement of C1P in the translocation/activation of cPLA2α adds another level of complexity to the activation of this enzyme. Multiple levels of regulation are not uncommon for many rate-limiting steps in biosynthetic pathways, and both lipid co-factors and post-translational modifications such as phosphorylation at serine 505, 515, and 727 have been reported in regulating the activation of cPLA2α. Currently, the role of phosphorylation of cPLA2α in the induction of translocation is controversial due to conflicting reports, but the requirement of the phosphorylation of cPLA2α at serine 505, 515, and 727 for full activity of the enzyme in vitro and in vivo is not questioned based on studies from a number of laboratories (34Thomas W. Coen N. Faherty S. Flatharta C.O. Harvey B.J. Steroids. 2006; 71: 256-265Crossref PubMed Scopus (42) Google Scholar, 35Pavicevic Z. Leslie C.C. Malik K.U. J. Lipid Res. 2008; 49: 724-737Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 36Hefner Y. Borsch-Haubold A.G. Murakami M. Wilde J.I. Pasquet S. Schieltz D. Ghomashchi F. Yates 3rd, J.R. Armstrong C.G. Paterson A. Cohen P. Fukunaga R. Hunter T. Kudo I. Watson S.P. Gelb M.H. J. Biol. Chem. 2000; 275: 37542-37551Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Specifically, recent findings suggest that in response to an inflammatory agonist, cPLA2α is phosphorylated on serine 515 by CAMKII followed by serines 505 and 727 by serine/threonine kinases of the mitogen-activated protein kinase pathway (34Thomas W. Coen N. Faherty S. Flatharta C.O. Harvey B.J. Steroids. 2006; 71: 256-265Crossref PubMed Scopus (42) Google Scholar, 35Pavicevic Z. Leslie C.C. Malik K.U. J. Lipid Res. 2008; 49: 724-737Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 36Hefner Y. Borsch-Haubold A.G. Murakami M. Wilde J.I. Pasquet S. Schieltz D. Ghomashchi F. Yates 3rd, J.R. Armstrong C.G. Paterson A. Cohen P. Fukunaga R. Hunter T. Kudo I. Watson S.P. Gelb M.H. J. Biol. Chem. 2000; 275: 37542-37551Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). As with the lipid co-factor, phosphatidylinositol 4,5-diphosphate, the bulk of the data suggests that phosphorylation plays more of a role in enzyme activity once the enzyme is associated with endogenous membranes. In regard to the translocation of cPLA2α, the only other report as to a required mechanism for the translocation of cPLA2α suggested that ceramide played a role in the association of cPLA2α to membranes via the CaLB domain (37Huwiler A. Johansen B. Skarstad A. Pfeilschifter J. FASEB J. 2001; 15: 7-9Crossref PubMed Scopus (114) Google Scholar). These findings have met with skepticism in recent years because of the similarity of ceramide effects to diacylglycerol on cPLA2α activity in vitro and the inability of other laboratories to repeat the findings on the ceramide/cPLA2α interaction at near physiologic salt concentrations. Thus, ceramide 1-phosphate is currently the only lipid known to strongly interact with cPLA2α and dramatically regulate the association of enzyme with PC-rich vesicles and, now, because of this study, internal membranes in “live” cells.Based on the findings in this study and the reports in the literature, one can now theorize the scenario of this complex activation of cPLA2α. Essentially, most inflammatory agonists induce an increase in intracellular calcium. Upon the increase in intracellular calcium, CAMKII becomes activated, leading to the phosphorylation of serine 515 followed by phosphorylation of serine 505 and serine 727. Simultaneously, calcium and possibly CAMKII (there is a consensus CAMKII phospho-site in ceramide kinase) lead to the induction of ceramide 1-phosphate in PC-rich membranes. A fully active cPLA2α interacts with C1P increasing residence time at internal membranes followed by phosphoinositide (PIP) association and membrane penetration of the catalytic domain of cPLA2α (Fig. 5). In support of this theory of cPLA2α activation by PIPs after C1P association, PIPs would be localized near “pools” of C1P in the trans-Golgi due to 1) ceramide kinase localizing to PIPs via its PH-domain and 2) ceramide transport protein, the protein that delivers substrate ceramide to the trans-Golgi for ceramide kinase, which requires PIP association to insert ceramide into membranes. Thus, future studies are warranted to explore this hypothesis in detail.One inconsistency with our findings of a role for CERK-derived C1P as a major player in cPLA2α activation and eicosanoid synthesis in cells and in vivo are recent studies by Bornancin and co-workers (26Graf C. Niwa S. Müller M. Kinzel B. Bornancin F. Biochem. Biophys. Res. Commun. 2008; 373: 159-163Crossref PubMed Scopus (41) Google Scholar, 27Boath A. Graf C. Lidome E. Ullrich T. Nussbaumer P. Bornancin F. J. Biol. Chem. 2008; 283: 8517-8526Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). These researchers found that cells derived from mice with the genetic ablation of CERK had no effect on AA release and eicosanoid synthesis in response to inflammatory agonists. There are two plausible explanations for the incongruent findings of this study and the Bornancin study. First, at least one other biosynthetic pathway for the generation of C1P exists (see Fig. 1, panel D), which may provide the C1P for activation of cPLA2α in the cell models used by the Bornancin group (e.g. peritoneal macrophages) (26Graf C. Niwa S. Müller M. Kinzel B. Bornancin F. Biochem. Biophys. Res. Commun. 2008; 373: 159-163Crossref PubMed Scopus (41) Google Scholar). Our presented study utilizes a mutant cPLA2α, which specifically focuses on the interaction of C1P and cPLA2α, negating the unknown specificity of CERK for eicosanoid generation in other cell models. A second explanation could simply be adaptation in the CERK knock-out mouse. To date, only one chain length of C1P has been examined in the CERK knock-out mouse. It is plausible that increases (or changes in subcellular localization) in other chain lengths of C1P via up-regulation of separate anabolic pathways could lead to adaptation, making conclusions for a non-role of CERK in eicosanoid synthesis premature at this time. Regardless, the presented study demonstrates an absolute requirement for the C1P/cPLA2α interaction for the induction of cPLA2α translocation and eicosanoid synthesis in response to inflammatory agonists. Further studies will hopefully determine whether these unknown pathways of C1P generation can “cross”-signal or whether they play roles in different cellular mechanisms.This plausible link between C1P generation and inflammation coupled with this study demonstrating that C1P is upstream of cPLA2α activation may lead to a much-needed and new generation of therapeutics for inflammatory disorders and cancer. For example, a chemical inhibitor of cPLA2α as well as genetic ablation of cPLA2α was also shown to prevent the development of airway inflammation in the AHR mouse model of asthma (38Malaviya R. Ansell J. Hall L. Fahmy M. Argentieri R.L. Olini Jr., G.C. Pereira D.W. Sur R. Cavender D. Eur. J. Pharmacol. 2006; 539: 195-204Crossref PubMed Scopus (26) Google Scholar). Ablation of the cPLA2α gene also significantly reduced pulmonary edema, and cPLA2α is also found to be a mediator of acute lung injury induced by sepsis via production of inflammatory mediators, such as eicosanoids (38Malaviya R. Ansell J. Hall L. Fahmy M. Argentieri R.L. Olini Jr., G.C. Pereira D.W. Sur R. Cavender D. Eur. J. Pharmacol. 2006; 539: 195-204Crossref PubMed Scopus (26) Google Scholar). In regard to cancer phenotypes, COX-2-derived eicosanoids, such as PGE2, contribute to tumorigenesis (39Jakobsson P.J. Thorén S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 7220-7225Crossref PubMed Scopus (890) Google Scholar). Indeed, COX-2 has been shown to be up-regulated in colon cancer, breast, and lung cancers (40Kutchera W. Jones D.A. Matsunami N. Groden J. McIntyre T.M. Zimmerman G.A. White R.L. Prescott S.M. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 4816-4820Crossref PubMed Scopus (445) Google Scholar, 41Hida T. Yatabe Y. Achiwa H. Muramatsu H. Kozaki K. Nakamura S. Ogawa M. Mitsudomi T. Sugiura T. Takahashi T. Cancer Res. 1998; 58: 3761-3764PubMed Google Scholar, 42Wolff H. Saukkonen K. Anttila S. Karjalainen A. Vainio H. Ristimäki A. Cancer Res. 1998; 58: 4997-5001PubMed Google Scholar), and research studies have shown that the levels of PGE2 is higher in cancer tissue and than in normal tissue (43McLemore T.L. Hubbard W.C. Litterst C.L. Liu M.C. Miller S. McMahon N.A. Eggleston J.C. Boyd M.R. Cancer Res. 1988; 48: 3140-3147PubMed Google Scholar, 44Hubbard W.C. Alley M.C. Gray G.N. Green K.C. McLemore T.L. Boyd M.R. Cancer Res. 1989; 49: 826-832PubMed Google Scholar). Recent studies by Nemenoff and co-workers (45Blaine S.A. Meyer A.M. Hurteau G. Wick M. Hankin J.A. Murphy R.C. Dannenberg A.J. Geraci M.W. Subbaramaiah K. Nemenoff R.A. Carcinogenesis. 2005; 26: 209-217Crossref PubMed Scopus (33) Google Scholar) demonstrated that increased PGE2 levels are necessary for the production of lung tumors in mice. Furthermore, breeding the knock-out mice for cPLA2α or COX-2 with a mouse model of adenomatous polyposis dramatically reduced the multiplicity and size of colon tumors (46Fosslien E. Crit. Rev. Clin. Lab. Sci. 2000; 37: 431-502Crossref PubMed Scopus (233) Google Scholar, 47Williams J.L. Borgo S. Hasan I. Castillo E. Traganos F. Rigas B. Cancer Res. 2001; 61: 3285-3289PubMed Google Scholar) as well as in the urethane mouse model of lung tumorigenesis (45Blaine S.A. Meyer A.M. Hurteau G. Wick M. Hankin J.A. Murphy R.C. Dannenberg A.J. Geraci M.W. Subbaramaiah K. Nemenoff R.A. Carcinogenesis. 2005; 26: 209-217Crossref PubMed Scopus (33) Google Scholar). Taken together, these observations suggested that ablation of this pathway regulated by the direct target of C1P, cPLA2α, has potential as a drug target for lung inflammation, osteoarthritis, and cancer.For specific targeting of eicosanoid synthesis, inhibitors of the cPLA2α/C1P interaction would provide high specificity for this mechanism while keeping the potential therapeutic value for treating the mentioned disorders. The production of this type of inhibitor may not even be a challenging undertaking because C1P interacts with cPLA2α with a stoichiometry of >4.8 molecules of C1P (13Subramanian P. Stahelin R.V. Szulc Z. Bielawska A. Cho W. Chalfant C.E. J. Biol. Chem. 2005; 280: 17601-17607Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 20Pettus B.J. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. Chalfant C.E. J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Furthermore, published findings from Goñi and Alonso suggest that a multimeric lipid structure of ≥6 molecules of C1P is formed in cellular membranes (48Goñi F.M. Alonso A. Biochim. Biophys. Acta. 2009; 1788: 169-177Crossref PubMed Scopus (161) Google Scholar). Based on the calcium dependence of the C1P/cPLA2α interaction, it is not inconceivable that calcium may regulate the conformation of this lipid structure modulating the specificity of protein/lipid interactions. Compounds synthesized to specifically block the formation of this structure may indeed provide a new generation of specific anti-inflammatory therapeutics while limiting unwanted side effects.In conclusion, this study shows that C1P fulfills all five requirements of a regulated and bioactive lipid. First, both this study and previous studies have shown that C1P levels are regulated in response to inflammatory agonists. Second, our previous work has also shown that exogenous C1P induces a specific biochemical and cellular response (release of AA), and this action of C1P demonstrated high lipid specificity in the induction of cPLA2α interaction/activation, AA release, and eicosanoid synthesis (1Stahelin R.V. Subramanian P. Vora M. Cho W. Chalfant C.E. J. Biol. Chem. 2007; 282: 20467-20474Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Subramanian P. Stahelin R.V. Szulc Z. Bielawska A. Cho W. Chalfant C.E. J. Biol. Chem. 2005; 280: 17601-17607Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14Subramanian P. Vora M. Gentile L.B. Stahelin R.V. Chalfant C.E. J. Lipid Res. 2007; 48: 2701-2708Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 20Pettus B.J. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. Chalfant C.E. J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 49Wijesinghe D.S. Subramanian P. Lamour N.F. Gentile L.B. Granado M.H. Szulc Z. Bielawska A. Gomez-Munoz A. Chalfant C.E. J. Lipid Res. 2009; (in press)Google Scholar). Third, endogenous C1P generated in cells by sphingomyelinase D reproduced these effects (19Pettus B.J. Bielawska A. Spiegel S. Roddy P. Hannun Y.A. Chalfant C.E. J. Biol. Chem. 2003; 278: 38206-38213Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Fourth, a direct target (cPLA2α) that binds/interacts with and is activated by C1P has been identified and validated (1Stahelin R.V. Subramanian P. Vora M. Cho W. Chalfant C.E. J. Biol. Chem. 2007; 282: 20467-20474Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Subramanian P. Vora M. Gentile L.B. Stahelin R.V. Chalfant C.E. J. Lipid Res. 2007; 48: 2701-2708Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 20Pettus B.J. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. Chalfant C.E. J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 49Wijesinghe D.S. Subramanian P. Lamour N.F. Gentile L.B. Granado M.H. Szulc Z. Bielawska A. Gomez-Munoz A. Chalfant C.E. J. Lipid Res. 2009; (in press)Google Scholar). Last, the interaction of C1P and cPLA2α is explicitly required for activation and translocation of the enzyme as well as subsequent prostanoid synthesis. Therefore, the presented study provides the last “missing link” in cPLA2α translocation and activation, validating C1P as a new lipid messenger in biological systems. This study should generate significant insight into novel aspects of signal transduction by lipids, activation of cPLA2α, and the regulation of prostaglandin synthesis. It also suggests C1P/cPLA2α interaction as a novel target for the development of anti-inflammatory therapeutics for inflammatory disorders, cancer, and sepsis. Eicosanoids are a class of bioactive lipids derived from the 20-carbon fatty acid, arachidonic acid (AA), 2The abbreviations used are: AAarachidonic acidC1Pceramide 1-phosphateCERKceramide kinaseIL-1βinterleukin-1βPCphosphatidylcholinePGE2prostaglandin E2PLA2phospholipase A2cPLA2cytosolic PLA2siRNAsmall interfering RNATNFαtumor necrosis factor αWTwild typeGFPgreen fluorescent proteinCFPcyan fluorescent proteinYFPyellow fluorescent proteinCaLB domainCa2+-dependent lipid binding domainPIPphosphoinositideMEFmouse embryonic fibroblastPBSphosphate-buffered salinePAPC1-palmitoyl-2-arachidonyl-sn-phosphatidylcholineCAMKIIcalcium/calmodulin-dependent protein kinase. 2The abbreviations used are: AAarachidonic acidC1Pceramide 1-phosphateCERKceramide kinaseIL-1βinterleukin-1βPCphosphatidylcholinePGE2prostaglandin E2PLA2phospholipase A2cPLA2cytosolic PLA2siRNAsmall interfering RNATNFαtumor necrosis factor αWTwild typeGFPgreen fluorescent proteinCFPcyan fluorescent proteinYFPyellow fluorescent proteinCaLB domainCa2+-dependent lipid binding domainPIPphosphoinositideMEFmouse embryonic fibroblastPBSphosphate-buffered salinePAPC1-palmitoyl-2-arachidonyl-sn-phosphatidylcholineCAMKIIcalcium/calmodulin-dependent protein kinase. 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 A2 (cPLA2α) (2Clark J.D. Schievella A.R. Nalefski E.A. Lin L.L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). Activation of cPLA2 in cells requires the association of the enzyme with intracellular membranes in a Ca2+-dependent manner. This translocation of cPLA2α from the cytosol to intracellular membranes is mediated by a Ca2+-dependent lipid binding domain (CaLB domain) located at the N terminus of the enzyme (4Reynolds L.J. Hughes L.L. Louis A.I. Kramer R.M. Dennis E.A. Biochim. Biophys. Acta. 1993; 1167: 272-280Crossref PubMed Scopus (151) Google Scholar, 5Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-" @default.
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