Related Experiment Videos
Intracellular mediators of granulysin-induced cell death
Satoshi Okada1, Qing Li, John C Whitin
1Division of Immunology and Transplantation Biology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
Abstract:
Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.
Insights
Granulysin triggers cell death by disrupting calcium and potassium channels, leading to mitochondrial damage and reactive oxygen species generation. Glutathione protects cells, highlighting the role of redox balance in granulysin-mediated apoptosis.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Granulysin, a cytotoxic protein from cytotoxic T lymphocytes (CTL) and NK cells, targets microbes and tumors.
- Granulysin induces apoptosis by damaging mitochondria, releasing cytochrome c and apoptosis-inducing factor, leading to DNA fragmentation.
Purpose of the Study:
- To elucidate the specific ion channels and cellular mechanisms involved in granulysin-mediated Jurkat cell death.
- To investigate the role of intracellular calcium (Ca2+), potassium (K+), and reactive oxygen species (ROS) in granulysin-induced apoptosis.
Main Methods:
- Utilized Ca2+ and K+ channel blockers (nickel, econazole, tetraethylammonium chloride, apamin, charybdotoxin) to assess their impact on granulysin-induced cell death.
- Employed thapsigargin to investigate the source of intracellular Ca2+ increase.
- Measured intracellular Ca2+ and K+ levels, ROS generation, and glutathione levels in granulysin-treated Jurkat cells.
Main Results:
- Granulysin-induced Jurkat cell death involves an increase in intracellular Ca2+ ([Ca2+](i)) originating from intracellular stores, followed by a decrease in intracellular K+.
- Ca2+ and K+ channel blockers, as well as elevated extracellular K+, inhibited granulysin-induced apoptosis.
- Granulysin treatment led to uncoupled electron transport, ROS generation, and increased susceptibility to cell death, which was mitigated by increased intracellular glutathione.
Conclusions:
- Granulysin-mediated cell death is critically dependent on the influx of Ca2+ and efflux of K+ through specific ion channels.
- The generation of reactive oxygen species and the cellular redox state play significant roles in granulysin's cytotoxic mechanism.
- Targeting these ion channels or modulating the redox state may offer therapeutic strategies against granulysin-mediated cytotoxicity.