Results ASK 1 and Trx 2 Form a Complex in Mitochondria That Is Dissociated in Response to TNF / ROS

Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)–induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondriaspecific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities. (Circ Res. 2004;94:1483-1491.)

[1]  W. Min,et al.  AIP1 mediates TNF-alpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. , 2003, The Journal of clinical investigation.

[2]  G. Powis,et al.  The Absence of Mitochondrial Thioredoxin 2 Causes Massive Apoptosis, Exencephaly, and Early Embryonic Lethality in Homozygous Mice , 2003, Molecular and Cellular Biology.

[3]  Yahong Lin,et al.  A JNK-dependent pathway is required for TNFalpha-induced apoptosis. , 2003, Cell.

[4]  Dean P. Jones,et al.  Overexpressed Human Mitochondrial Thioredoxin Confers Resistance to Oxidant-induced Apoptosis in Human Osteosarcoma Cells* , 2002, The Journal of Biological Chemistry.

[5]  J. Gustafsson,et al.  Human Mitochondrial Thioredoxin , 2002, The Journal of Biological Chemistry.

[6]  W. Min,et al.  Thioredoxin Promotes ASK1 Ubiquitination and Degradation to Inhibit ASK1-Mediated Apoptosis in a Redox Activity-Independent Manner , 2002, Circulation research.

[7]  Kiyoshi Inoue,et al.  ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. , 2002, Genes & development.

[8]  田中 亨.  Thioredoxin-2(TRX-2)is an essential gene regulating mitochondria-dependent apoptosis , 2002 .

[9]  E Vicaut,et al.  Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide. , 2001, American journal of respiratory cell and molecular biology.

[10]  G. Powis,et al.  Properties and biological activities of thioredoxins. , 2001, Annual review of pharmacology and toxicology.

[11]  T Takahashi,et al.  ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis , 2001, EMBO reports.

[12]  畑井 多喜子.  Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation , 2001 .

[13]  D. Green.  Apoptotic Pathways Paper Wraps Stone Blunts Scissors , 2000, Cell.

[14]  D. Bar-Sagi,et al.  Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. , 2000, Science.

[15]  Guido Kroemer,et al.  Mitochondrial control of cell death , 2000, Nature Medicine.

[16]  Hong Liu,et al.  Activation of Apoptosis Signal-Regulating Kinase 1 (ASK1) by Tumor Necrosis Factor Receptor-Associated Factor 2 Requires Prior Dissociation of the ASK1 Inhibitor Thioredoxin , 2000, Molecular and Cellular Biology.

[17]  F. Urano,et al.  Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. , 2000, Science.

[18]  Zhi-Min Yuan,et al.  Translocation of SAPK/JNK to Mitochondria and Interaction with Bcl-xL in Response to DNA Damage* , 2000, The Journal of Biological Chemistry.

[19]  Masashi Narita,et al.  Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC , 1999, Nature.

[20]  S. O’Rahilly,et al.  Tumor necrosis factor is delivered to mitochondria where a tumor necrosis factor-binding protein is localized. , 1998, Laboratory investigation; a journal of technical methods and pathology.

[21]  Kohei Miyazono,et al.  Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1 , 1998, The EMBO journal.

[22]  Minoru Takagi,et al.  Induction of Apoptosis by ASK1, a Mammalian MAPKKK That Activates SAPK/JNK and p38 Signaling Pathways , 1997, Science.

[23]  M. Matsui,et al.  Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. , 1996, Developmental biology.