Influx of Calcium through a Redox-sensitive Plasma Membrane Channel in Thymocytes Causes Early Necrotic Cell Death Induced by the Epipolythiodioxopiperazine Toxins

Abstract

Gliotoxin, a member of the epipolythiodioxopiperazine (ETP) class of toxins, induces both apoptotic and necrotic cell death in a concentration-dependent manner. Whereas the specific trigger for apoptotic death caused by these toxins is unclear, the reactive disulfide bond in the ETP toxins is required for biological activity. Thus it is likely that it is the interaction of this disulfide moiety with macromolecules in cells that was responsible for activity of ETP toxins. Here we present evidence that necrosis induced by gliotoxin and a simple synthetic ETP toxin is largely because of an influx of extracellular calcium through a redox-sensitive calcium channel in the plasma membrane of murine thymocytes. The calcium rises are strongly dependent on the pH of the external medium and the presence of external calcium and are abrogated and/or reversed by the presence of dithiothreitol, cell impermeant glutathione, and the calcium channel blocker Ni 2؉ . Comparisons with thapsigargin, which indirectly causes release of calcium from internal stores, indicates that ETP toxins do not provoke calcium rises by store depletion. A mechanism of oxidation by ETP toxins of cell surface thiol groups resulting in direct entry of calcium through a redox active channel in the plasma membrane is proposed. Necrotic but not apoptotic cell death was abrogated by inhibition of calcium entry.Gliotoxin is a member of the epipolythiodioxopiperazine (ETP) 1 of toxic fungal metabolites that display antibacterial, antiviral, and immunosuppressive properties (1). The ETP toxins contribute to pathological damage in fungal infections in animals (2) including humans and their known immunosuppressive activity could contribute to the establishment of disseminated infections by ETP producing fungi (3). The presence of a reactive disulfide bridge accounts for much of the biological activity of these toxins (4, 5). The ETP toxins induce apoptotic cell death in a variety of cells across a window of concentrations typically 0.1-3 M (1, 6 -8). Typical of many toxins, the mode of cell death switches from apoptotic to necrotic at relatively higher concentrations (9). Although the exact cellular targets of the ETP toxins have yet to be defined, they have been shown to inhibit a variety of enzymes including alcohol dehydrogenase (10), creatine kinase (11), the transcription factor NFB (12), farnesyl transferase (13), and RNA polymerase (14). In particular the ETP toxins target proteins with accessible thiol groups and gliotoxin has been shown to induce intramolecular disulfide formation in creatine kinase (11). Apoptosis induced by gliotoxin in thymocytes has been strongly linked to increased cAMP levels and histone H3 phosphorylation (15).Because gliotoxin inhibits NFB in intact cells, gliotoxin has been used as a tool to probe the requirement for this transcription factor. However, gliotoxin and other ETP toxins can potentially target multiple sites in cells resulting in a variety of effects, including altering intracellular calcium levels (9). In this pape...

Activation and Inhibition of Cellular Calcium and Tyrosine Kinase Signaling Pathways Identify Targets of the HBx Protein Involved in Hepatitis B Virus Replication

“…These data are consistent in demonstrating that through a variety of pathways, compounds that stimulate cytoplasmic calcium accumulation or mobilization replace the requirement for HBx in specifically promoting HBV DNA replication in this system. Thapsigargin increases intracellular calcium by acting on the endoplasmic reticulum (18), valinomycin does so by acting on mitochondrial membrane potential (5), gliotoxin does so by acting on the plasma membrane (9), and glibenclamide does so by disrupting cation homeostasis (10). Despite the disparate mechanisms by which these agents stimulate an increase in cytosolic calcium, all four recover HBV DNA replication in the absence of HBx.…”

“…Gliotoxin is a fungal toxin that increases the level of calcium in cells, predominantly by acting on the plasma membrane and causing calcium influx (9). HepG2 cells transfected with wildtype HBV or HBV HBx(Ϫ) genomic DNA were treated for 4 days with 0.5 M gliotoxin, and viral DNA replication was assessed.…”

Redox-Directed Cancer Therapeutics: Molecular Mechanisms and Opportunities

“…Gliotoxin inactivates alcohol dehydrogenase and NFkB by either covalent modification or free radical damage mediated by redox cycling (365). Necrosis of thymocytes caused by gliotoxin has been attributed to increased cellular calcium levels thought to result from interaction of gliotoxin with a redox-sensitive thiol residue in the plasma membrane calcium channel (158). Calcium dysregulation may then cause subsequent oxidative damage.…”

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones,

“…Sporidesmin and other ETPs exhibit a variety of biological activities, including antibacterial, antiviral, immunosuppressive and antineoplastic actions (12,13). The mode of action of the ETP compounds is unknown but multiple effects have been reported including altered calcium flux (14), interference with NFκB action (15), inhibition of farnesyl and geranylgeranyl transferases (16) and formation of mixed disulfides between the ETP moiety and cysteine residues on vital proteins, as reported for creatine kinase (17). Also implicated is the ability of the ETPs to undergo redox cycling in the presence of oxygen, thereby generating reactive oxygen species (18).…”