ATP release depends on autophagic stimulation and rab21 in hela cells

Abstract

ATP exocytosis has emerged as an important autocrine/paracrine signal to trigger responses regarding platelet aggregation, inter-astrocytes communication, cell migration, differentiation, etc. The molecular mechanisms underlying this process have not been well defined so far. In addition, autophagy-dependent ATP release was shown to be an important process involved in immunogenic cell death and inflammation. Autophagic exocytosis has been also implicated in IL-1β, IL-18, galectin-3 secretion and antigens presentation to the major histocompatibility complex. The regulation of this process is poorly understood. Our group has previously shown that there is an important role of the v-SNARE protein VAMP7 in the autophagic ATP exocytosis. Since there is a close connection between VAMP7, the small GTP-ase Rab21 and autophagy we decided to investigate the role of this Rab protein, its guanine-nucleotide exchange factor VARP and another Rab related to VARP (Rab32) in the autophagic exocytosis of ATP. HeLa cells were transiently transfected with GFP-Vector, YFP-Rab32wt, YFP-Rab32T39N (negative dominant mutant), GFP-Rab21wt, GFP-Rab21T33N (negative dominant mutant) and/or RFP-LC3. Then we incubated this cells in full medium, resveratrol 50nM and rapamycin 50nM for 4 hours or starvation medium for 2 hours. We used LC3B or VARP antibodies to detect endogenous proteins. Lysotracker red was put 30 minutes before the incubation finished. For ATP release assays, we used the luciferin-luciferase kit to measure the amounts of ATP in the extracellular medium. We took samples at 0, 30, 60, 90, 120, 180 and 240 minutes after the autophagic stimulus began. We found that autophagy leads to a redistribution of Rab32 or Rab21 molecules to the cell periphery, where they colocalized with LC3 and VARP. ATP release was significantly increased in starvation and in less content in resveratrol condition. Our results suggest a role for Rab21, Rab32 and VARP in the autophagic exocytosis of ATP.