Abstract
Extracellular vesicles (EVs) are shed from the plasma membrane, but the regulation and function of these EVs remain unclear. We found that oxidative stress induced by H 2 O 2 in Hela cells stimulated filopodia formation and the secretion of EVs. EVs were small (150 nm) and labeled for CD44, indicating that they were derived from filopodia. Filopodia-derived small EVs (sEVs) were enriched with the sphingolipid ceramide, consistent with increased ceramide in the plasma membrane of filopo- dia. Ceramide was colocalized with neutral sphingomyelinase 2 (nSMase2) and acid sphingomyelinase (ASM), two sphingomyelinases generating ceramide at the plasma membrane. Inhibition of nSMase2 and ASM prevented oxidative stress-induced sEV shedding but only nSMase2 inhibition prevented filopodia formation. nSMase2 was S-palmitoylated and interacted with ASM in filopodia to generate ceramide for sEV shedding. sEVs contained nSMase2 and ASM and decreased the level of these two enzymes in oxidatively stressed Hela cells. A novel metabolic labeling technique for EVs showed that oxidative stress induced secretion of fluorescent sEVs labeled with NBD-ceramide. NBD-ceramide-labeled sEVs transported ceramide to mitochondria, ultimately inducing cell death in a proportion of neuronal (N2a) cells. In conclu- sion, using Hela cells we provide evidence that oxidative stress induces interaction of nSMase2 and ASM at filopodia, which leads to shedding of ceramide-rich sEVs that target mitochondria and propagate cell death.
Document Type
Article
Publication Date
7-2024
Digital Object Identifier (DOI)
https://doi.org/10.1002/jev2.12477
Funding Information
We are grateful for the support of the Department of Physiology (Chair Dr. Alan Daugherty) at the University of Kentucky College of Medicine. This work was supported by grants to EB from the National Institutes of Health, R01AG064234, R21AG078601, RF1AG078338; and the U.S. Department of Veterans Affairs, I01BX003643.
Repository Citation
Quadri, Zainuddin; Elsherbini, Ahmed; Crivelli, Simone M.; El-Amouri, Salim S.; Tripathi, Priyanka; Zhu, Zhihui; Ren, Xiaojia; Zhang, Liping; Spassieva, Stefka D.; Nikolova‑Karakashian, Mariana N.; and Bieberich, Erhard, "Ceramide-mediated orchestration of oxidative stress response through filopodia-derived small extracellular vesicles" (2024). Markey Cancer Center Faculty Publications. 274.
https://uknowledge.uky.edu/markey_facpub/274
Notes/Citation Information
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.