title: Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells.
abstract: Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcgammaRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcgammaRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcgammaRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. tzgalch@umich.edu
authors.forename: Vania
authors.initials: V
authors.lastname: Hinkovska-Galcheva
authors.forename: Andrea
authors.initials: A
authors.lastname: Clark
authors.forename: Susan
authors.initials: S
authors.lastname: VanWay
authors.forename: Ji-Biao
authors.initials: JB
authors.lastname: Huang
authors.forename: Miki
authors.initials: M
authors.lastname: Hiraoka
authors.forename: Akira
authors.initials: A
authors.lastname: Abe
authors.forename: Michael
authors.initials: M
authors.lastname: Borofsky
authors.forename: Robin G
authors.initials: RG
authors.lastname: Kunkel
authors.forename: Thomas
authors.initials: T
authors.lastname: Shanley
authors.forename: James A
authors.initials: JA
authors.lastname: Shayman
authors.forename: Frederick
authors.initials: F
authors.lastname: Lanni
authors.forename: Howard R
authors.initials: HR
authors.lastname: Petty
authors.forename: Laurence A
authors.initials: LA
authors.lastname: Boxer
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D020013:Calcium Signaling; D021941:Caveolae / Q000737:chemistry; D002458:Cell Fractionation; D002522:Chlorocebus aethiops; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D008247:Lysosomes; D009895:Opsonin Proteins; D010587:Phagocytosis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000032:analysis / Q000378:metabolism; D014162:Transfection
chemical_list: D015703:Antigens, CD; C080568:Fc gamma receptor IIA; D007074:Immunoglobulin G; D009895:Opsonin Proteins; D017452:Receptors, IgG; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase