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.

FIG. 1.

Confocal microscopic analysis of colocalization of LR clusters and ASM or NOX subunits in CAECs. The cells were stained with Texas red–labeled anti-ASM (TR-ASM) or anti-gp91^phox (TR-gp91) antibody and LRs marker, Al488-CTXB. siRNA and Scra indicate sortilin siRNA and scramble sRNA transfection group, respectively. *p < 0.05 vs. control group. ^#p < 0.05 vs. FasL group; n = 6 batches of cells.

FIG. 2.

Effects of sortilin siRNA transfection on FasL-induced O₂^·− production in CAECs measured with ESR spectrometry. Veh, vehicle group. *p < 0.05 vs. control group. ^#p < 0.05 vs. FasL group; n = 5 batches of cells.

FIG. 3.

Detection of sortilin, Lamp-1, and ASM on cell membrane in CAECs with flow cytometry. Control or FasL-stimulated (10 ng/ml for 15 min) cells were stained with FITC-labeled anti-gp91^phox, sortilin, Lamp-1, and ASM and analyzed with flow cytometry. Al488-CTXB was also used to detect LRs level on the cell membrane. *p < 0.05 vs. control group; n = 4 batches of cells.

FIG. 4.

Representative images of FRET analysis among sortilin, Lamp-1, and ASM in CAECs. The left group of images was obtained from control CAECs, and the right group of images, from FasL-stimulated (10 ng/ml for 15 min) cells. FRET was detected by using an acceptor-bleaching protocol. The blue images (representing FRET) on the bottom were obtained by subtracting a prebleaching image from a postbleaching image. Arrows, where the FRET happened. (A) FRET between Lamp-1 and sortilin. (B) FRET between ASM and sortilin. (C) FRET between ASM and Lamp-1.

FIG. 5.

Summarized results of detected FRET efficiency among sortilin, Lamp-1, and ASM. *p < 0.05 vs. control group; n = 5 batches of cells.

FIG. 6.

Interaction of sortilin and ASM in lysosomes of CAECs detected with co-immunoprecipitation. *p < 0.05 vs. control group; n = 5 batches of cells. Ctrl, control; FasL, FasL stimulation; No-IP, no immunoprecipitation conducted; IP-ASM, immunoprecipitation with beads conjugated with anti-ASM antibody.

FIG. 7.

Effects of FasL on BK-induced Ca²⁺ release (A) and NO production (B) in intact endothelium of bovine coronary artery before and after sortilin gene silencing, lysosome-function inhibition, LRs disruption, or NOX inactivation. BK (1 μM) was used to induce Ca²⁺ release and NO production in arteries with or without 20-min pretreatments with FasL (10 ng/ml) alone or FasL accompanied with sotilin siRNA or scramble sRNA transfection, Baf (100 nM), MCD (100 μM), or gp91ds-tat (50 μM). *p < 0.05 vs. BK-only group; ^#p < 0.05 vs. FasL + BK group; n = 5 bovine hearts.