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.
Highlights
Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK)
As CERK and transient receptor potential channel (TRPC)-1 have been associated with caveolin and/or lipid rafts [27,28,29], we speculate that TRPC-1 and its store-operated channels (SOCs)-like activity may be relevant to calcium signaling in these transfectants
After challenging cells with EIgG, TRPC-1 was found in lipid rafts of all transfectants, the strongest signal was found in human ceramide kinase (hCERK) transfected cells
Summary
Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). We evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK colocalizes with EIgG in FcgRIIA/hCERKbearing COS-1 cells. The addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca21 signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca21 signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.— Hinkovska-Galcheva, V., A. Ceramide kinase promotes Ca21 signaling near IgGopsonized targets and enhances phagolysosomal fusion in COS-1 cells.
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