4.7 Article

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

Journal

ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
Volume 28, Issue 11, Pages 2056-U275

Publisher

LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/ATVBAHA.108.172478

Keywords

Fas ligand; lysosome; coronary circulation; vascular endothelium; sphingolipid

Funding

  1. NHLBI NIH HHS [HL-57244, R29 HL057244, R01 HL057244-12, HL-75316, R01 HL057244, R01 HL075316, R01 HL057244-11, R01 HL075316-03, R01 HL075316-04] Funding Source: Medline
  2. NIDDK NIH HHS [R01 DK054927-11, R01 DK054927, R01 DK054927-10, DK54927] Funding Source: Medline

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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-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. (Arterioscler Thromb Vasc Biol. 2008; 28: 2056-2062)

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