Abstract
The bioactive sphingolipid, ceramide 1-phosphate (C-1-P), has been implicated as an extracellular chemotactic agent directing cellular migration in hematopoietic stem/progenitor cells and macrophages. However, interacting proteins that could mediate these actions of C-1-P have, thus far, eluded identification. We have now identified and characterized interactions between ceramide 1-phosphate and the annexin a2-p11 heterotetramer constituents. This C-1-P-receptor complex is capable of facilitating cellular invasion. Herein, we demonstrate in both coronary artery macrovascular endothelial cells and retinal microvascular endothelial cells that C-1-P induces invasion through an extracellular matrix barrier. By employing surface plasmon resonance, lipid-binding ELISA, and mass spectrometry technologies, we have demonstrated that the heterotetramer constituents bind to C-1-P. Although the annexin a2-p11 heterotetramer constituents do not bind the lipid C-1-P exclusively, other structurally similar lipids, such as phosphatidylserine, sphingosine 1-phosphate, and phosphatidic acid, could not elicit the potent chemotactic stimulation observed with C-1-P. Further, we show that siRNA-mediated knockdown of either annexin a2 or p11 protein significantly inhibits C-1-P-directed invasion, indicating that the heterotetrameric complex is required for C-1-P-mediated chemotaxis. These results imply that extracellular C-1-P, acting through the extracellular annexin a2-p11 heterotetrameric protein, can mediate vascular endothelial cell invasion.
Highlights
Extracellular ceramide 1-phosphate is presumed to interact with extracellular proteins to mediate cellular invasion
We found that exogenous ceramide 1-phosphate (C-1-P) dramatically enhanced the capacity of both cell lines to invade through MatrigelTM in a dose-dependent manner, up to 10 M in human retinal microvascular endothelial cells (HREC) and 3 M in human coronary artery endothelial cells (HCAEC) (Fig. 1, A and B)
To explore the specificity of C-1-P-mediated invasion, we tested whether other lipids, such as the structurally similar sphingolipids ceramide and sphingosine 1-phosphate (S-1-P), or the anionic phosphoglycerolipids PA and DOPS, which are known to interact with A2t [17, 23], could direct invasion in endothelial cells
Summary
Extracellular ceramide 1-phosphate is presumed to interact with extracellular proteins to mediate cellular invasion. We have identified and characterized interactions between ceramide 1-phosphate and the annexin a2-p11 heterotetramer constituents This C-1-P-receptor complex is capable of facilitating cellular invasion. We show that siRNA-mediated knockdown of either annexin a2 or p11 protein significantly inhibits C-1-P-directed invasion, indicating that the heterotetrameric complex is required for C-1-P-mediated chemotaxis. These results imply that extracellular C-1-P, acting through the extracellular. Recent evidence documents the direct inhibition of TNF␣-converting enzyme by C-1-P binding [11] These data supported previous research demonstrating that C-1-P, produced via ceramide generated from acid sphingomyelinase activity, could negatively modulate TNF-␣ release [12, 13]
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