Pannexin-1 Is Required for ATP Release during Apoptosis but Not for Inflammasome Activation

Abstract

Apoptotic cell death is important for embryonic development, immune cell homeostasis, and pathogen elimination. Innate immune cells also undergo a very rapid form of cell death termed pyroptosis after activating the protease caspase-1. The hemichannel pannexin-1 has been implicated in both processes. In this study, we describe the characterization of pannexin-1–deficient mice. LPS-primed bone marrow-derived macrophages lacking pannexin-1 activated caspase-1 and secreted its substrates IL-1β and IL-18 normally after stimulation with ATP, nigericin, alum, silica, flagellin, or cytoplasmic DNA, indicating that pannexin-1 is dispensable for assembly of caspase-1–activating inflammasome complexes. Instead, thymocytes lacking pannexin-1, but not the P2X7R purinergic receptor, were defective in their uptake of the nucleic acid dye YO-PRO-1 during early apoptosis. Cell death was not delayed but, unlike their wild-type counterparts, Panx1−/− thymocytes failed to recruit wild-type peritoneal macrophages in a Transwell migration assay. These data are consistent with pannexin-1 liberating ATP and other yet to be defined “find me” signals necessary for macrophage recruitment to apoptotic cells.

Molecular mechanisms of ATP secretion during immunogenic cell death

“…45 The administration of cumate to tPANX1-transfected, but not to control, MCA205 cells rendered them permeable to the small fluorescent dye YO-PRO-1, but not to the vital dye DAPI (Figure 3c), in line with the previously reported capacity of tPANX1 to promote a selective permeabilization of the plasma membrane. [32][33][34] The expression of tPANX1 drove a YO-PRO-1 influx coupled to an ATP efflux that could be inhibited in a dose-dependent manner by monensin and DIDS but not by Y-27632, blebbistatin and Z-VAD-fmk (Figures 3d and e), confirming the specificity of this panel of inhibitors.…”

“…[32][33][34] Surprisingly, however, the Figure 7 Lysosomes and autophago(lyso)somes participate in the trafficking and/or in the maintenance of the intracellular ATP pool. (a-c) Human osteosarcoma U2OS cells were transfected with a non-targeting siRNA (siUNR) or with the indicated siRNAs for 48 h, and then maintained in control conditions (Co) or treated with 4 mM mitoxantrone (MTX) or 300 mM oxaliplatin (OXA) for additional 18 h. Finally, cells were processed for the fluorescence microscopy-assisted visualization of nuclei (with Hoechst 33342, blue fluorescence), ATP-containing vesicles (with quinacrine, green fluorescence) or LAMP1 (revealed with an secondary antibody emitting in red) (a and b), and ATP levels in culture supernatants assessed by a luciferase-based test (c).…”

“…31 In line with this notion, the pharmacological inhibition of caspases, the knockout of Panx1, the depletion of PANX1 as well as the substitution of PANX1 by a non-cleavable variant all abolish apoptosis-associated ATP release. [32][33][34] Moreover, caspase activation and PANX1 expression are both required for the partial, apoptotic increase in the permeability of the plasma membrane to small solutes that precedes the complete, post-apoptotic (necrotic) plasma membrane breakdown. [32][33][34] In a cell death-unrelated setting, astrocytes have been reported to secrete ATP by lysosomal exocytosis, a process in which lysosomes fuse with the plasma membrane to release their luminal content into the extracellular space.…”

“…[32][33][34] Moreover, caspase activation and PANX1 expression are both required for the partial, apoptotic increase in the permeability of the plasma membrane to small solutes that precedes the complete, post-apoptotic (necrotic) plasma membrane breakdown. [32][33][34] In a cell death-unrelated setting, astrocytes have been reported to secrete ATP by lysosomal exocytosis, a process in which lysosomes fuse with the plasma membrane to release their luminal content into the extracellular space. 35 Driven by these premises and incognita, we decided to characterize the mechanisms through which cancer cells extrude ATP in response to chemotherapeutic agents.…”

Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

“…This is most likely an indication of advanced injury and irreversible apoptosis due to the ablation treatment Xiao et al 2012). Coordinated activation of P2X receptors and Pannexin1 results in diverse arrays of membrane trafficking events, including opening of pores in the membrane, allowing large molecules, such as YP1 to enter cells (Iglesias et al 2008;Qu et al 2011;Xiao et al 2012). This observation may explain the discordance between cell death marker TUNEL and cell-permeability marker YP1 in the current study.…”