title: Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate.
abstract: We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A(2) and protein kinase C-α, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-α and cPLA(2)-α in this pathway.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain.
authors.forename: Lide
authors.initials: L
authors.lastname: Arana
authors.forename: Patricia
authors.initials: P
authors.lastname: Gangoiti
authors.forename: Alberto
authors.initials: A
authors.lastname: Ouro
authors.forename: Io-Guané
authors.initials: IG
authors.lastname: Rivera
authors.forename: Marta
authors.initials: M
authors.lastname: Ordoñez
authors.forename: Miguel
authors.initials: M
authors.lastname: Trueba
authors.forename: Ravi S
authors.initials: RS
authors.lastname: Lankalapalli
authors.forename: Robert
authors.initials: R
authors.lastname: Bittman
authors.forename: Antonio
authors.initials: A
authors.lastname: Gomez-Muñoz
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology*; D005260:Female; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D019255:NADPH Oxidases / Q000378:metabolism / Q000502:physiology; D054510:Phospholipases A2, Cytosolic / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000378:metabolism / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D002518:Ceramides; D017382:Reactive Oxygen Species; C065576:ceramide 1-phosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051571:Protein Kinase C-alpha; D054510:Phospholipases A2, Cytosolic