Stimulation of TLR3 Triggers Release of Lysosomal ATP in Astrocytes and Epithelial Cells that Requires TRPML1 Channels

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Departmental Papers (Dental)
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Adenosine Triphosphate
Animals
Astrocytes
Autophagy
Biomarkers
Calcium
Cells
Cultured
Epithelial Cells
Hydrogen-Ion Concentration
Lysosomes
Mice
RNA
Small Interfering
Toll-Like Receptor 3
Transient Receptor Potential Channels
adenosine triphosphate
biological marker
calcium
Mcoln1 protein
mouse
small interfering RNA
toll like receptor 3
transient receptor potential channel
animal
astrocyte
autophagy
cell culture
epithelium cell
genetics
lysosome
metabolism
mouse
pH
Dentistry
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Beckel, Jonahan M.
Gómez, Néstor Más
Lu, Wennan
Campagno, Keith E.
Nabet, Bardia
Albalawi, Farraj
Lim, Jason C.
Boesze-Battaglia, Kathleen
Mitchell, Claire H.
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Abstract

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and "non-targeting" scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1-/- cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways. © 2018 The Author(s).

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2018-12-01
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Scientific Reports
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