Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis

Abstract

Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes1. The nucleotides ATP and UTP represent one class of find-me signals2, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 over-expression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes3. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

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

“…The mechanism involved incorporates the release of ATP and UTP molecules into the extracellular space, leading to the activation of P2X7 receptors (Virginio et al 1999). The opening of cation channels follows, allowing entry of YP1, as well as other large molecules (Chekeni et al 2010;Michel et al 2000;Virginio et al 1999). Functional activity of P2X7 receptors can influence apoptotic pathways (Chow et al 1997), and activation of these receptors has been used to promote cell death in cancer cells (Gorodeski 2009).…”