Reactive Oxygen Species-Induced Necrotic Cell Death

Mechanisms of cell death extend beyond the simple apoptosis/necrosis relationship to include regulated modes of cell death that do not readily fit either of the classic descriptors. One such mechanism of cell death involves poly(ADP-ribose)polymerase-1 (PARP-1)-mediated cell death. 2,3,5-Tris(Glutathion-S-yl)-hydroquinone (TGHQ), a reactive oxygen species (ROS) generating nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes necrotic renal cell death, the basis for which is unclear. We therefore investigated TGHQ-mediated cell death in human renal proximal tubule epithelial HK-2 cells. TGHQ induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca2+ concentrations, loss of mitochondrial membrane potential, and subsequent necrotic cell death. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Inhibition of PARP-1 with PJ34 blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers, NAD consumption, and the consequent necrotic cell death. However, HK-2 cell death was only delayed by PJ34, and cell death remained necrotic in nature. In contrast, chelation of intracellular Ca2+ with BAPTA-AM completely abrogated TGHQ-induced necrotic cell death. Ca2+ chelation not only prevented the collapse in the mitochondrial potential but also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca2+ homeostasis. Moreover, TGHQ caused a sequential oxidation of peroxiredoxin III (PrxIII), a protein considered the primary antioxidant defense within mitochondria. Thus, TGHQ induced two acidic shifts in PrxIII, with both pI shifted spots representing oxidized forms of PrxIII. Transient expression of a dominant negative version of PrxIII resulted in a significant increase in TGHQ-induced cytotoxicity, whereas overexpression of wild-type PrxIII significantly attenuated cytotoxicity. Our studies provide new insights into PARP-1-mediated necrotic cell death. Changes in intracellular Ca2+ concentrations appear to couple PARP-1-hyperactivation to subsequent cell death, but in the absence of AIF release from mitochondria. NAD depletion, mitochondrial membrane depolarization, Ca2+-mediated calpain activation, and PrxIII oxidation, all contribute to TGHQ-driven ROS-mediated necrotic cell death.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Pharmacology & Toxicology

Grantor dc:publisher

The University of Arizona.

Year dc:date.issued

2009

Author dc:creator

• Xie, Ruiyu

Advisor dc:contributor.advisor

• Monks, Terrence J

Committee members dc:contributor.committeemember

• Jacobson, Myron K
• Lau, Serrine S
• Chen, Qin M
• Futsher, Bernard W

• calcium
• cell death
• mechanism
• necrosis
• PARP
• reactive oxygen species

Statement dc:rights

• Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Language dc:language.iso

EN

Handle dc:identifier.uri

http://hdl.handle.net/10150/195215

OAI identifier oai:identifier

oai:repository.arizona.edu:10150/195215