Ceramide 1-Phosphate, a Mediator of Phagocytosis

Abstract

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the Fc␥RIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERKtransfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERKtransfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.The second messengers produced by membrane lipids through agonist stimulation include not only glycerolipids but also sphingolipids. Sphingolipids are comprised of lipids that contain a long chain sphingoid base. Sphingolipids, in addition to being structural components of membranes, regulate cell-cell and cell-substrate interactions, proliferation, and differentiation. Members of this diverse group of lipids have emerged as a novel class of signaling molecules that also regulate phagocytosis. The mechanisms by which sphingolipids exert these effects remain incompletely defined. More than a decade ago, it was found that ceramide can be phosphorylated to ceramide 1-phosphate (C1P) 1 (1-3).C1P is found in brain synaptic vesicles, and it is thought to play a role in regulating the secretion of neurotransmitters by promoting the fusion of vesicle membranes (4). Ceramide kinase activity exists in HL-60 cells where C1P is derived from ceramide released from sphingomyelin (5). Human ceramide kinase (hCERK) was recently cloned based on the homology to two isoforms of mice and human sphingosine kinase (6). The expressed kinase displayed specific ceramide phosphorylating activity. BLAST search analyses using the hCERK sequence revealed that a series of putative CERKs exist in a variety of cellular organisms, including plants, nematodes, insects, and vertebrates. CERKs represent a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases (6, 7).C1P shares struc...

The interaction between the pleckstrin homology domain of ceramide kinase and phosphatidylinositol 4,5-bisphosphate regulates the plasma membrane targeting and ceramide 1-phosphate levels

“…Shayman and co-workers reported that C1P is formed during phagocytosis and that it promotes the fusion of complex liposomes [14,15]. We recently demonstrated that the calcium-dependent activation of CERK is involved in the degranulation 17 pathway, possibly through the fusion of the plasma membrane with the secretory granules in the mast cells [18].…”

“…Therefore, we concluded that the plasma membrane targeting of CERK is induced by the interaction of the PH domain and PI(4,5)P 2 , and, consequently, the translocation of CERK enhances the formation of C1P in the vicinity of the plasma membrane. The translocation might facilitate CERK to interact with their substrates.Shayman and co-workers reported that C1P is formed during phagocytosis and that it promotes the fusion of complex liposomes [14,15]. We recently demonstrated that the calcium-dependent activation of CERK is involved in the degranulation 17 pathway, possibly through the fusion of the plasma membrane with the secretory granules in the mast cells [18].…”

“…A decade later, CERK was cloned based on its sequence homology to sphingosine kinase (SPHK) type 1 [10]. Both C1P and CERK have been implicated in the regulation of vital cellular processes, such as cell proliferation [11,12], apoptosis [13], phagocytosis [14,15] and inflammation [16,17]. Recently, we demonstrated that CERK and C1P are required for the degranulation process in mast cells [18], and that this process is dependent on calmodulin [19].…”

Sphingolipid targets in cancer therapy

“…Ceramide may be phosphorylated to ceramide-1-PO 4 in the plasma membrane (52). Ceramide kinase activity has been isolated from several cell lines and tissues and is Ca 2+ stimulatable, in contrast to diacylglycerol kinase, which is Mg 2+ dependent.…”

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).…”