Lysosomal targeting and trafficking of acid sphingomyelinase to lipid raft platforms in coronary endothelial cells.
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).


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-beta-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.
Jin
Si
S
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
Yi
Fan
F
Zhang
Fan
F
Poklis
Justin L
JL
Li
Pin-Lan
PL
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002417:Cattle; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D003331:Coronary Vessels / Q000187:drug effects / Q000201:enzymology*; D004151:Dipeptides / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D053222:Fas Ligand Protein / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005677:G(M1) Ganglioside / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D018942:Macrolides / Q000494:pharmacology; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology*; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014664:Vasodilation; D019014:fas Receptor / Q000378:metabolism; D000099183:Sortilin
D033942:Adaptor Proteins, Vesicular Transport; D001920:Bradykinin; D051242:Caveolin 1; D002518:Ceramides; D004151:Dipeptides; D053222:Fas Ligand Protein; D005456:Fluorescent Dyes; D005677:G(M1) Ganglioside; D052118:Lysosomal-Associated Membrane Protein 1; D018942:Macrolides; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D013108:Sphingomyelin Phosphodiesterase; D019014:fas Receptor; D000099183:Sortilin; C073804:FM1 43; C041865:glycylphenylalanine 2-naphthylamide
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