Caspase activation regulates the extracellular export of autophagic vacuoles.
The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy. Nutrient deficiency induced a significant and rapid increase in autophagic features, as imaged by electron microscopy and immunoblotting analysis of LC3-II/LC3-I ratios. Increased autophagic flux was confirmed by exposing serum-starved cells to bafilomycin A 1. Induction of autophagy was followed by indices of an apoptotic response, as assessed by microscopy and poly (ADP-ribose) polymerase cleavage in absence of cell membrane permeabilization indicative of necrosis. Pan-caspase inhibition with ZVAD-FMK did not prevent the development of autophagy but negatively impacted autophagic vacuole (AV) maturation. Adopting a multidimensional proteomics approach with validation by immunoblotting, we determined that nutrient-deprived EC released AV components (LC3I, LC3-II, ATG16L1 and LAMP2) whereas pan-caspase inhibition with ZVAD-FMK blocked AV release. Similarly, nutrient deprivation in aortic murine EC isolated from CASP3/caspase 3-deficient mice induced an autophagic response in absence of apoptosis and failed to prompt LC3 release. Collectively, the present results demonstrate the release of autophagic components by nutrient-deprived apoptotic human cells in absence of cell membrane permeabilization. These results also identify caspase-3 as a novel regulator of AV release.
Research Centre, Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Canada.
Isabelle
I
Sirois
Jessika
J
Groleau
Nicolas
N
Pallet
Nathalie
N
Brassard
Katia
K
Hamelin
Irène
I
Londono
Alexey V
AV
Pshezhetsky
Moise
M
Bendayan
Marie-Josée
MJ
Hébert
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D053148:Caspase 3 / Q000172:deficiency / Q000378:metabolism; D061945:Caspase Inhibitors / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D002853:Chromatography, Liquid; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D061945:Caspase Inhibitors; D016895:Culture Media, Serum-Free; D053148:Caspase 3; D020169:Caspases