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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:2056.)
© 2008 American Heart Association, Inc.

Cell Biology/Signaling

Lysosomal Targeting and Trafficking of Acid Sphingomyelinase to Lipid Raft Platforms in Coronary Endothelial Cells

Si Jin; Fan Yi; Fan Zhang; Justin L. Poklis; Pin-Lan Li

From the Department of Pharmacology & Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University.

Correspondence to Pin-Lan Li, MD, PhD, Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, 410 N 12th, Richmond, VA 23298. E-mail pli{at}vcu.edu

Objective— The purpose of this study was to determine whether lysosome trafficking and targeting of acid sphingomyelinase (ASMase) to this organelle contribute to the formation of lipid raft (LR) signaling platforms in the membrane of coronary arterial endothelial cells (CAECs).

Methods and Results— By measurement of fluorescent resonance energy transfer (FRET), it was found that in FasL-stimulated CAECs, membrane lamp1 (a lysosome marker protein) or Fas and GM1 (a LR marker) were trafficking together. Cofocal colocalization assay showed that ceramide was enriched in these LR platforms. Further studies demonstrated that these ceramide molecules in LR platforms were colocalized with ASMase, a ceramide producing enzyme. Fluorescence imaging of living CAECs loaded with lysosomal specific dyes demonstrated that lysosomes fused with membrane on FasL stimulation. In the presence of lysosome function inhibitors, bafilomycin (Baf) or glycyl-L-phenylalanine-β-naphthylamide (GPN), these FasL-induced changes were abolished. Moreover, this FasL-induced formation of LR platforms was also blocked in ECs transfected with siRNA of sortilin, an intracellular transporter for targeting of ASMase to lysosomes. Functionally, FasL-induced impairment of vasodilator response was reversed by lysosomal inhibitors or sortilin gene silencing.

Conclusions— Lysosomal trafficking and targeting of ASMase are importantly involved in LRs clustering in ECs membrane, leading to the formation of signaling platforms or signalosomes.

Key Words: Fas ligand • lysosome • coronary circulation • vascular endothelium • sphingolipid

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