The Formation of Ceramide-1-phosphate during Neutrophil Phagocytosis and Its Role in Liposome Fusion

Abstract

Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32 PO 4 -labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.

Distinct receptor-mediated activities in macrophages for natural ceramide-1-phosphate (C1P) and for phospho-ceramide analogue-1 (PCERA-1)

“…In addition, C1P regulates apoptosis (GomezMunoz, 1998;Gomez-Munoz, 2004), is implicated in the inflammatory response (Chalfant and Spiegel, 2005;Lamour and Chalfant, 2005), and is important in the regulation of phagocytosis (Hinkovska-Galcheva et al, 2005;Hinkovska-Galcheva et al, 1998).…”

Ceramide Kinase, a Novel Lipid Kinase

“…Recently, it was shown that ceramide thus formed can be converted to ceramide-1-phosphate through activation of ceramide kinase. Hence, it was suggested that ceramide-1-phosphate may promote phagolysosome formation (25). On the other hand, ceramide-1-phosphate has also been reported to have mitogenic effects.…”

Ceramide‐1‐phosphate promotes cell survival through activation of the phosphatidylinositol 3‐kinase/protein kinase B pathway

“…A major mechanism whereby growth factors promote cell survival is the activation of the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) (Akt) pathway [17]. Activation of PI3-K leads to phosphatidylinositol (3,4,5) trisphosphate (PI (3,4,5) P3) formation, and this results in the recruitment of pleckstrin homology domain containing proteins such as PKB. Recruitment of PKB to the cell membrane results in its phosphorylation and activation, and this allows it to phosphorylate downstream effectors.…”

“…Abbreviations: A-SMase, acid sphingomyelinase; BMDM, bone marrow-derived macrophages; C1P, ceramide-1-phosphate; C 2 -, acetyl; C 8 -, octanoyl; CAPE, caffeic acid phenylethyl ester; EMSA, electromobility shift assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M-CSF, macrophage colony stimulating factor; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; MEK, MAPK/ERK kinase; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]; PI, phosphatidylinositol; PI3-K, phosphatidylinositol 3-kinase; PI (3,4,5) P3, phosphatidylinositol (3,4,5) trisphosphate; PKB, protein kinase B; PLD, phospholipase D; cPLA 2 , calcium-dependent cytosolic phospholipase A 2 ; PMS, phenazine methosulfate; PTX, pertussis toxin; S1P, sphingosine-1-phosphate; TBS, Tris-buffered saline; TLC, thinlayer chromatography A target of PKB that is known to regulate apoptosis is the transcription factor NF-jB. The latter is an inducible heterodimeric factor responsible for the transcription of many important antiapoptotic molecules including Bcl-X L .…”

“…Ceramide-1-phosphate (C1P) is emerging as a new bioactive molecule [1] capable of regulating vital pathophysiological functions including cell proliferation [2,3], apoptosis [4], phagocytosis [5], and inflammation [6,7]. Although the existence of C1P and its metabolizing enzymes, ceramide kinase and C1P phosphatase, in mammalian tissues have been known for over a decade [8][9][10], current understanding of the metabolic and signaling pathways that are affected by this bioactive sphingolipid is incomplete.…”