title: Activation of membrane NADPH oxidase associated with lysosome-targeted acid sphingomyelinase in coronary endothelial cells.
abstract: This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O(2)(.-) production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, 410 North 12th, Richmond, VA 23298, USA.
authors.forename: Jun-Xiang
authors.initials: JX
authors.lastname: Bao
authors.forename: Si
authors.initials: S
authors.lastname: Jin
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Zheng-Chao
authors.initials: ZC
authors.lastname: Wang
authors.forename: Ningjun
authors.initials: N
authors.lastname: Li
authors.forename: Pin-Lan
authors.initials: PL
authors.lastname: Li
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology* / Q000201:enzymology; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D053222:Fas Ligand Protein / Q000378:metabolism; D020868:Gene Silencing; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D000099183:Sortilin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D053222:Fas Ligand Protein; D052118:Lysosomal-Associated Membrane Protein 1; D019255:NADPH Oxidases; D034741:RNA, Small Interfering; D013108:Sphingomyelin Phosphodiesterase; D000099183:Sortilin