Lysosomal Targeting of Acid Sphingomyelinase in the Formation of Lipid Raft‐Redox Signaling Platforms of Coronary Arterial Endothelial Cells

Abstract

Lipid rafts (LRs) are emerging as an important cellular signaling mechanism currently. Recent studies have demonstrated that LRs are able to cluster NADPH oxidase subunits to form a LR‐redox signaling platform in response to death receptor activation. However, the mechanisms are still poorly understood. In the present study, we examined the role of a sortilin‐mediated lysosomal targeting and trafficking of acid sphingomyelinase (ASM) in bovine coronary arterial endothelial cells (CAECs). By confocal microscopy and detection of fluorescence resonance energy transfer (FRET), ASM was found to be targeted on lysosomal membrane via sortilin in response to FasL stimulation as shown by detection of ASM with sortilin or Lamp‐1, a lysosomal membrane marker. The FRET efficiency between these molecules was over 20% compared to 5% in control cells, indicating a FasL‐stimulated lysosomal targeting of ASM via sortilin. After CAECs were transfected by an siRNA of sortilin, this FasL stimulated targeting of ASM was abolished and therefore LRs clustering could not be formed, resulting in failure of LR‐redox signaling platform formation, as shown by substantial reduction of LR clusters containing NADPH oxidase subunits gp91phox or p47phox. This was also confirmed by Western blot analysis of isolated LRs membrane fractions, showing that sortilin siRNA efficiently prevented FasL‐induced translocation of gp91phox or p47phox into LR microdomains. Consequently, FasL‐induced superoxide production via NADPH oxidase in LRs clusters was also abolished by sortilin siRNA, as measured by electron spin resonance (ESR) spectrometry. It is concluded that lysosomal targeting of ASM through sortilin is present in CAECs and this targeting mechanism is attributed to the formation of LR‐redox signaling platforms in these cells in response to FasL.