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. 2018 Feb 20;9(3):300.
doi: 10.1038/s41419-018-0373-8.

Contribution of TMEM16F to Pyroptotic Cell Death

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Free PMC article

Contribution of TMEM16F to Pyroptotic Cell Death

Jiraporn Ousingsawat et al. Cell Death Dis. .
Free PMC article

Abstract

Pyroptosis is a highly inflammatory form of programmed cell death that is caused by infection with intracellular pathogens and activation of canonical or noncanonical inflammasomes. The purinergic receptor P2X7 is activated by the noncanonical inflammasome and contributes essentially to pyroptotic cell death. The Ca2+ activated phospholipid scramblase and ion channel TMEM16F has been shown earlier to control cellular effects downstream of purinergic P2X7 receptors that ultimately lead to cell death. As pyroptotic cell death is accompanied by an increases in intracellular Ca2+, we asked whether TMEM16F is activated during pyroptosis. The N-terminal cleavage product of gasdermin D (GD-N) is an executioner of pyroptosis by forming large plasma membrane pores. Expression of GD-N enhanced basal Ca2+ levels and induced cell death. We observed that GD-N induced cell death in HEK293 and HAP1 cells, which was depending on expression of endogenous TMEM16F. GD-N activated large whole cell currents that were suppressed by knockdown or inhibition of TMEM16F. The results suggest that whole cell currents induced by the pore forming domain of gasdermin-D, are at least in part due to activation of TMEM16F. Knockdown of other TMEM16 paralogues expressed in HAP1 cells suggest TMEM16F as a crucial element during pyroptosis and excluded a role of other TMEM16 proteins. Thus TMEM16F supports pyroptosis and other forms of inflammatory cell death such as ferroptosis. Its potent inhibition by tannic acid may be part of the anti-inflammatory effects of flavonoids.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. TMEM16F supports gasdermin D-induced cell death.
a, b Dot blot diagram of cell death analysis by flow cytometry. Cell death (7-AAD and AnnexinV-FITC double staining) was significantly enhanced in HEK293 cells expressing the N-terminal pore–forming domain of gasdermin D (GD-N), but not in mock transfected cells. c LDH release in HEK293 cells transfected with empty plasmid (mock), full length gasdermin D (GD), or GD-N. Flow cytometry and LDH assays were performed 24 h after transfection. LDH release was inhibited by CaCCinhAO1 (AO1; 20 µM), tannic acid (TA; 10 µM) or niflumic acid (NFA; 100 µM). d LDH release measured in cells expressing empty plasmids, GD, or GD-N, which were treated with siRNA for TMEM16F or with scrambled RNA. e Immunocytochemistry of GD and GD-N expressed in HEK293 cells. f Western blot indicating knockdown of TMEM16F by siRNA. g LDH release in GD-N transfected human B-lymphocytes (wt-BL) and Scott-lymphocytes lacking expression of TMEM16F (Scott-BL). h Western blot of TMEM16F indicating lack of expression in Scott-B-lymphocytes. Mean ± SEM (number of FACS and LDH assays). #significant increase when compared to mock (p < 0.05, ANOVA). §significant inhibition by inhibitors or si-TMEM16F (p < 0.05, ANOVA)
Fig. 2
Fig. 2. Increase of intracellular Ca2+ and activation of whole cell Cl- currents by pore forming gasdermin D.
a, b Original recordings from each 200 experiments (a) and summary of the baseline 340/380 fluorescence ratio in Fura-2 loaded cells expressing the N-terminal pore–forming domain of gasdermin D (GD-N) or empty plasmid (mock). Tannic acid (TA; 10 µM) inhibited the baseline Ca2+ increase in GD-N expressing cells. c Cell morphology of cells transfected with empty plasmid (mock), full length gasdermin D (GD), or GD-N. d, e Whole cell currents and corresponding current/voltage relationships obtained in non-stimulated mock-transfected HEK293 cells and cells expressing GD, or GD-N. The whole cell current in GD-N expressing cells was enhanced, which was inhibited by removal of extracellular chloride (5Cl) from the bath solution. f Current densities for all individual cells examined by patch clamping, obtained at Vc = + 100 mV. g Summaries for the 5Cl--inhibited whole cell current indicating activation of Cl- permeable currents in cells expressing GD-N. Cells were voltage clamped ± 100 mV (1 s) in steps of 20 mV. Mean + /− SEM (number of experiments). Significant difference when compared to mock or inhibition by tannic acid, respectively (unpaired t-test). *Significant inhibition by 5 Cl (p < 0.05; paired t-test)
Fig. 3
Fig. 3. Gasdermin D-induced whole cell currents are inhibited by blockers of TMEM16F.
a Whole cell currents obtained in non-stimulated HEK293 cells expressing the N-terminal pore-forming domain of gasdermin D (GD-N). The enhanced whole cell currents detected in these cells were significantly inhibited by CaCCinhAO1 (AO1; 20 µM) or tannic acid (TA; 10 µM). b Corresponding current/voltage relationships for the experiments shown in a. c whole cell currents measured in mock transfected HEK293 cells and cells expressing GD-N in the presence of scrambled RNA (scrbld) or after siRNA-knockdown of TMEM16F. d Corresponding current/voltage relationships. Cells were voltage clamped ± 100 mV (1 s) in steps of 20 mV. Mean±SEM (number of experiments). *Significant inhibition by AO1 and TA, respectively (p < 0.05; paired t-test)
Fig. 4
Fig. 4. Knockdown of TMEM16F in HAP1 cells eliminates Ca2+-activated PS exposure.
a RT-PCR analysis of HAP1 cells (horizon, Cambridge, UK) detected expression of TMEM16D,F,H,K. b Western blots indicating expression of TMEM16D,F,H,K in HAP1 parental cells (Parental) and knockout of expression of TMEM16F (KO_16F) or knockout of TMEM16D,F,H,K (KO_T16all) by gene editing. Very right panel shows loading controls in Parental, KO_16F, and KO-all (β-actin). c, d Dot blot diagram from flow cytometry and summary of annexin V positive cells, indicating Ca2+-dependent activation of phospholipid scrambling (exposure of phosphatidylserine, PS) by ionomycin in parental HAP1 cells but not in cells lacking expression of TMEM16F (KO_16F) or TMEM16F and additional TMEM16 proteins. e Proliferation of HAP1 cells lacking expression of TMEM16F or lacking expression of all endogenous TMEM16 proteins was significantly reduced when compared with parental cells. Mean±SEM (number of experiments). #Significant activation by ionomycin, or inhibition of proliferation (p < 0.05; unpaired t-test)
Fig. 5
Fig. 5. Knockdown of TMEM16F attenuates different cell death pathways.
a Summary of basal intracellular Ca2+ levels, which was not affected by knockout of TMEM16 proteins. b Increase in intracellular Ca2+ levels by stimulation with ATP (100 µM) or cyclopiazonic acid (CPA, 100 µM) in the presence of a Ca2+ free extracellular bath solution. Increase in intracellular Ca2+ by ATP was significantly reduced in KO_T16F and KO_T16all cells. c Summary of cell death (7-AAD and annexinV-FITC double staining) induced by expression of GD-N in parental, KO-T16F, and KO_T16all cells. d Baseline Ca2+ levels measured in mock transfected cells and cells expressing GD-N. Increase in intracellular Ca2+ by expression of GD-N was completely inhibited by tannic acid (TA; 10 µM). e, f Summary of cell death induced by ferroptosis (incubation with 1 µM RSL3 and 10 µM erastin for 24 h) or apoptosis (incubation with 100 ng/ml TNFα for 24 h). g Cell morphology assessed by quantitative holographic phase microscopy. Scale bar indicates cell height. Mean + /− SEM (number of cells). #Significant inhibition when compared to parental cells (p < 0.05; unpaired t-test)

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