Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells

Vania Hinkovska-Galcheva, Andrea Clark, Susan VanWay, Ji Biao Huang, Miki Hiraoka, Akira Abe, Michael Borofsky, Robin G. Kunkel, Thomas Shanley, James A. Shayman, Frederick Lanni, Howard R. Petty, Laurence A. Boxer

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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 FcγRIIA or both FcγRIIA/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 FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA 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.

Original languageEnglish (US)
Pages (from-to)531-542
Number of pages12
JournalJournal of Lipid Research
Volume49
Issue number3
DOIs
StatePublished - Mar 1 2008

Fingerprint

COS Cells
Phagocytosis
Fusion reactions
Immunoglobulin G
Phagosomes
Cells
Bearings (structural)
Transient Receptor Potential Channels
Cell Fractionation
Caveolae
Sphingolipids
Phosphorylation
Ceramides
Fluorescence microscopy
Fractionation
Electric sparks
Fluorescence Microscopy
ceramide kinase
Pharmacology
ceramide 1-phosphate

Keywords

  • Ceramide-1-phosphate
  • Immunoglobulin G
  • Phagocytosis
  • Store operated channels
  • Transient potential channel

Cite this

Hinkovska-Galcheva, V., Clark, A., VanWay, S., Huang, J. B., Hiraoka, M., Abe, A., ... Boxer, L. A. (2008). Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells. Journal of Lipid Research, 49(3), 531-542. https://doi.org/10.1194/jlr.M700442-JLR200

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells. / Hinkovska-Galcheva, Vania; Clark, Andrea; VanWay, Susan; Huang, Ji Biao; Hiraoka, Miki; Abe, Akira; Borofsky, Michael; Kunkel, Robin G.; Shanley, Thomas; Shayman, James A.; Lanni, Frederick; Petty, Howard R.; Boxer, Laurence A.

In: Journal of Lipid Research, Vol. 49, No. 3, 01.03.2008, p. 531-542.

Research output: Contribution to journalArticle

Hinkovska-Galcheva, V, Clark, A, VanWay, S, Huang, JB, Hiraoka, M, Abe, A, Borofsky, M, Kunkel, RG, Shanley, T, Shayman, JA, Lanni, F, Petty, HR & Boxer, LA 2008, 'Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells', Journal of Lipid Research, vol. 49, no. 3, pp. 531-542. https://doi.org/10.1194/jlr.M700442-JLR200
Hinkovska-Galcheva, Vania ; Clark, Andrea ; VanWay, Susan ; Huang, Ji Biao ; Hiraoka, Miki ; Abe, Akira ; Borofsky, Michael ; Kunkel, Robin G. ; Shanley, Thomas ; Shayman, James A. ; Lanni, Frederick ; Petty, Howard R. ; Boxer, Laurence A. / Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells. In: Journal of Lipid Research. 2008 ; Vol. 49, No. 3. pp. 531-542.
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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 FcγRIIA or both FcγRIIA/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 FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA 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.",
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AU - Hinkovska-Galcheva, Vania

AU - Clark, Andrea

AU - VanWay, Susan

AU - Huang, Ji Biao

AU - Hiraoka, Miki

AU - Abe, Akira

AU - Borofsky, Michael

AU - Kunkel, Robin G.

AU - Shanley, Thomas

AU - Shayman, James A.

AU - Lanni, Frederick

AU - Petty, Howard R.

AU - Boxer, Laurence A.

PY - 2008/3/1

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N2 - 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 FcγRIIA or both FcγRIIA/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 FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA 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.

AB - 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 FcγRIIA or both FcγRIIA/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 FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA 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.

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KW - Store operated channels

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