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Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides.

ABSTRACT: Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd)-dependent lytic cell death called pyroptosis that promotes antimicrobial host defense but may contribute to sepsis. The nature of the caspase-1-dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium(2+) (Pro(2+)) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDMs) as an indicator of this PM permeabilization. BMDMs were characterized by rapid Pro(2+) influx after initiation of NLRP3 or Pyrin inflammasomes by nigericin (NG) or Clostridium difficile toxin B (TcdB), respectively. No Pro(2+) uptake in response to NG or TcdB was observed in Casp1(-/-) or Asc(-/-) BMDMs. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro(2+) influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro(2+) influx and pyroptotic lysis. Extracellular La(3+) and Gd(3+) rapidly and reversibly blocked the induced Pro(2+) influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome assembly and caspase-1 activation. Thus, caspase-1-driven pyroptosis requires induction of initial prelytic pores in the PM that are dependent on Gsdmd expression. These PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca(2+) Although lanthanides and Gsdmd deletion both suppressed PM pore activity and pyroptotic lysis, robust IL-1? release was observed in lanthanide-treated BMDMs but not in Gsdmd-deficient cells. This suggests roles for Gsdmd in both passive IL-1? release secondary to pyroptotic lysis and in nonlytic/nonclassical IL-1? export.

SUBMITTER: Russo HM

PROVIDER: S-EPMC4976007 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides.

Russo Hana M HM Rathkey Joseph J Boyd-Tressler Andrea A Katsnelson Michael A MA Abbott Derek W DW Dubyak George R GR

Journal of immunology (Baltimore, Md. : 1950) 20160706 4

Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd)-dependent lytic cell death called pyroptosis that promotes antimicrobial host defense but may contribute to sepsis. The nature of the caspase-1-dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium(2+) (Pro(2+)) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDMs) as an indicator of th ...[more]

PMID: 27385778

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Project description:BACKGROUND:Pyroptosis belongs to a novel inflammatory programmed cell death pathway, with the possible prognosis of endometrial cancer related to the terminal protein GSDMD. Hydrogen exerts a biphasic effect on cancer by promoting tumor cell death and protecting normal cells, which might initiate GSDMD pathway-mediated pyroptosis. METHODS:We performed immunohistochemical staining and western immunoblotting analysis to observe expression of NLRP3, caspase-1, and GSDMD in human and xenograft mice endometrial cancer tissue and cell lines. We investigated treatment with hydrogen could boost ROS accumulation in endometrial cancer cells by intracellular and mitochondrial sources. GSDMD shRNA lentivirus was used to transfect endometrial cancer cells to investigate the function of GSDMD protein in pyroptosis. Propidium iodide (PI) staining, TUNEL assay, measurement of lactate dehydrogenase (LDH) release and IL-1β ELISA were used to analysis pyroptosis between hydrogen-supplemented or normal culture medium. We conducted in vivo human endometrial tumor xenograft mice model to observe anti-tumor effect in hydrogen supplementation. RESULTS:We observed overexpression of NLRP3, caspase-1, and GSDMD in human endometrial cancer and cell lines by IHC and western immunoblotting. Hydrogen pretreatment upregulated ROS and the expression of pyroptosis-related proteins, and increased the number of PI- and TUNEL-positive cells, as well as the release of LDH and IL-1β, however, GSDMD depletion reduced their release. We further demonstrated that hydrogen supplementation in mice was sufficient for the anti-tumor effect to inhibit xenograft volume and weight of endometrial tumors, as mice subjected to hydrogen-rich water displayed decreased radiance. Tumor tissue sections in the HRW groups presented moderate-to-strong positive expression of NLRP3, caspase-1 and GSDMD. Hydrogen attenuated tumor volume and weight in a xenograft mouse model though the pyroptotic pathway. CONCLUSIONS:This study extended our original analysis of the ability of hydrogen to stimulate NLRP3 inflammasome/GSDMD activation in pyroptosis and revealed possible mechanism (s) for improvement of anti-tumor effects in the clinical management of endometrial cancer.

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Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides.

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Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides.

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