Neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane

Abstract

Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g (“microvesicles” (MV)) and 100,000 g (“exosomes”) are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that – despite the different vesicle biogenesis – the routes of vesicular export are adaptable.

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion

“…Ceramide is involved in MVE formation, and as originally reported by Trajkovic et al, blocking of ceramide synthesis by inhibition of neutral sphingomyelinase‐2 (nSmase2) has been widely used to interfere with exosome production. By contrast, nSmase2 inhibition does not prevent and can instead enhance the production of ectosomes …”

“…By contrast, nSmase2 inhibition does not prevent and can instead enhance the production of ectosomes. 40 In order to characterize the origin of the EVs involved in Hck-activated vesicle secretion of TACE we generated knock-down derivatives of HEK293 cells in which nSmase2 expression had been suppressed by lentivirally delivered shRNAs. We found that similar to the spontaneous EV secretion of LAMP-1, Hck-activated packaging of TACE into EVs was almost completely blocked as a consequence of the Smase2 knock-down ( Figure 6).…”

Ceramide and Exosomes: A Novel Target in Cancer Biology and Therapy

“…Wang et al, 2012). To date, inhibition or genetic deficiency of nSMase2 is a method widely used to prevent exosome secretion in various cell types and tissues (Chairoungdua, Smith, Pochard, Hull, & Caplan, 2010; Dinkins et al, 2014; Dinkins, Enasko, et al, 2016; Dinkins, Wang, & Bieberich, 2016; Goldkorn, Chung, & Filosto, 2013; Guo, Bellingham, & Hill, 2015; Kong, He, et al, 2015; Kosaka et al, 2010; Marsh & van Meer, 2008; Menck et al, 2017; Shamseddine, Airola, & Hannun, 2015; Tan et al, 2013; Trajkovic et al, 2008; Yuyama, Sun, Mitsutake, & Igarashi, 2012). nSMase2 converts sphingomyelin into ceramide, a reaction shown to be instrumental for exosome secretion in vitro (Trajkovic et al, 2008) (Fig.…”

Senescent stromal cells promote cancer resistance through SIRT1 loss-potentiated overproduction of small extracellular vesicles

“…1L). Further analysis with successive differential ultracentrifugation of pellets from conditioned media, a strategy that effectively separates sEVs from MVs (14,000 g and 100,000 g of sedimentation, respectively) 28 , indicated enhanced secretion of sEVs by SEN cells, while MV release remained largely unchanged, implying engagement of a mechanism specifically responsible for production of sEVs, but not all EV subtypes (Fig. 1M).…”