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. 2010 Oct 14;467(7317):863-7.
doi: 10.1038/nature09413.

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

Faraaz B Chekeni  1 , Michael R Elliott, Joanna K Sandilos, Scott F Walk, Jason M Kinchen, Eduardo R Lazarowski, Allison J Armstrong, Silvia Penuela, Dale W Laird, Guy S Salvesen, Brant E Isakson, Douglas A Bayliss, Kodi S Ravichandran
Affiliations

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

Faraaz B Chekeni et al. Nature. .

Abstract

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, 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 overexpression 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 dyes. 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.

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Figures

Figure 1
Figure 1. Release of find-me signals by apoptotic cells is pannexin-1-dependent
a, Time course of ATP release from apoptotic Jurkat cells in the presence of 500 μM carbenoxolone (CBX) or 2 mM probenecid (Prob). n = 3. No Tx, no treatment. b, mRNA levels for PANX1, PANX2 and PANX3 in Jurkat cells determined by qPCR, normalized to PANX1. n = 2. c, PANX1 protein expression in Jurkat cells transfected with control or PANX1 siRNA. Glycosylated PANX1 (upper band; arrow) was quantified. d, e, PANX1 knockdown does not affect the progression of apoptosis (assessed by apoptotic caspase activity) (d, n = 3), but decreases ATP (e, n = 10) and UTP (e, n = 3) release 4 h after apoptosis induction. *, P < 10−5; **, P < 0.01. f, Transwell migration of THP-1 monocytes towards apoptotic cell supernatants from PANX1-siRNA-treated cells (4 h after ultraviolet (UV) treatment). *, P < 0.05. Representative of four independent experiments. g, Left, Schematic of mouse air pouch model for monitoring chemotactic activity of apoptotic cell supernatants in vivo. Right, CD45+ leukocytes migrating into the pouch were determined after injecting apoptotic cell supernatants from siRNA-transfected cells. n = 9–10 mice per group. *, P < 0.05, by ANOVA with Bonferroni post-analysis. Error bars, s.e.m., except in b, f, where they represent s.d.
Figure 2
Figure 2. Pannexin 1 expression level correlates with find-me signal release and membrane permeability
a, Immunoblotting of lysates from control or PANX1–Flag Jurkat cells. b, ATP release by Jurkat cells after induction of Fas-mediated apoptosis, and 4 h after UV-induced apoptosis. n = 2. c, UTP levels in supernatants of Jurkat cells 2 h after apoptosis induction. n = 4. d, Migration of THP-1 monocytes to supernatants from control and PANX1–Flag expressing cells, collected 2 h after induction of apoptosis. *, P < 0.05. Representative of four experiments. e, Left, flow cytometry histograms showing YO-PRO-1 dye uptake by control versus PANX1-siRNA-transfected Jurkat cells with or without apoptosis induction. Right, uptake of YO-PRO-1 presented as mean fluorescence intensity (MFI) of the entire cell population. *, P < 0.0005, n = 3. f, Left, YO-PRO-1 uptake by primary murine thymocytes undergoing anti-Fas-induced apoptosis, with zVAD or CBX treatments. Right, percentage of apoptotic cells (annexin V positive and propidium iodide negative). n = 2. Error bars represent s.d., except in c (s.e.m.).
Figure 3
Figure 3. Carbenoxolone-sensitive current induced during apoptosis is pannexin-1-dependent
a, Morphology of anti-Fas treated Jurkat cells used to identify ‘live’ and ‘dying’ cells (based on blebbing). b, Patch-clamp recordings from Jurkat cells, receiving indicated treatments. Peak whole-cell current (at +90 mV) is shown under conditions when bath solution was perfused with FFA (100 μM, blue shading) or CBX (100 μM, pink shading). Exemplar traces are representative of 5–15 cells per group. c, CBX-sensitive current density in indicated Jurkat cells, normalized to whole-cell capacitance (pA/pF). *, P < 0.01. n ≥ 7 per group. d, Current–voltage relationships of apoptosis-induced current in dying Jurkat cells (from b), with current measured over a range of voltages (only dying cells shown, as live cells have very little current). Note different y axes. Traces representative of 4–15 cells. e, CBX-sensitive current density in PANX1-siRNA-transfected cells induced to undergo apoptosis. *, P < 0.05. n ≥ 8 per group. All error bars represent s.e.m.
Figure 4
Figure 4. Pannexin 1 is a target of effector caspase cleavage during apoptosis
a, b, Fas-mediated apoptosis results in loss of PANX1 detection with antibody targeted to PANX1 C terminus in Jurkat cells (a) and primary murine thymocytes (b). c, Schematic of PANX1–Flag protein indicating the predicted caspase cleavage sites (sites A and B) and the epitopes recognized by the EL2 (extracellular loop 2) and C-terminal antibodies. The predicted products of caspase cleavage at sites A and B are also shown. d, In vitro cleavage of immunoprecipitated PANX1–Flag incubated with the indicated purified active caspases. Loss of immunoreactivity to Flag assessed. Representative of two independent experiments. e, Jurkat cells stably expressing a C-terminally GFP-tagged PANX1 were induced to undergo apoptosis and assessed for the cleaved C-terminal fragment (which runs at ~33 kDa). Representative of two independent experiments. f, In vitro cleavage of indicated PANX1–Flag proteins (wild type and caspase cleavage site mutants), incubated with the indicated caspases.
Figure 5
Figure 5. Caspase-mediated cleavage of pannexin 1 results in channel activation during apoptosis
a, b, Jurkat cells transiently transfected with indicated constructs were assessed for YO-PRO-1 uptake (a) and current density (b) 48 h post-transfection. The MFI for YO-PRO-1 uptake by the gated population is shown (a) and the analysis of dying cells in b was performed as in Fig. 3. *, P < 0.01, n ≥ 5 for each group. c,d, Jurkat cells stably transfected with the indicated plasmids were assessed for ATP release (c, n = 3), TO-PRO-3 uptake (d, left) and annexin V staining (d, right) 2 h after induction of apoptosis. MFI for the TO-PRO-3 positive peaks and percentage of annexin V positive cells are shown. n = 3. e, TO-PRO-3 uptake by Jurkat cells 16 h after transient transfection with full-length or truncated PANX1. Schematic of full-length and truncated PANX1 (amino acids 1–426 and 1–371, respectively) are shown. n = 3. f, CBX-sensitive currents in Jurkat cells transiently transfected with indicated PANX1 proteins (full length or truncation mutant). Patch-clamp was performed on cells that appeared morphologically healthy. n ≥ 5 per group. Error bars and ± represent s.e.m.
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References

    1. Lauber K, Blumanthal SC, Waibel M, Wesselborg S. Clearance of apoptotic cells: getting rid of the corpses. Mol Cell. 2004;14:277–287. - PubMed
    1. Elliott MR, et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature. 2009;461:282–286. - PMC - PubMed
    1. Idziorek T, Estaquier J, De Bels F, Ameisen JC. YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J Immunol Methods. 1995;185:249–258. - PubMed
    1. Ghiringhelli F, et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β-dependent adaptive immunity against tumors. Nature Med. 2009;15:1170–1178. - PubMed
    1. Fujiwara T, Oda K, Yokota S, Takatsuki A, Ikehara Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem. 1988;263:18545–18552. - PubMed

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