title: Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner.
abstract: 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. Mitochondria-specific 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.
authors.affiliation: Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, New Haven, Conn 06510, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhang
authors.forename: Rafia
authors.initials: R
authors.lastname: Al-Lamki
authors.forename: Lanfang
authors.initials: L
authors.lastname: Bai
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Streb
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Miano
authors.forename: John
authors.initials: J
authors.lastname: Bradley
authors.forename: Wang
authors.initials: W
authors.lastname: Min
mesh_terms: D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D042783:Endothelial Cells / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D048848:MAP Kinase Kinase Kinase 5 / Q000037:antagonists & inhibitors* / Q000737:chemistry; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D008928:Mitochondria / Q000201:enzymology*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
chemical_list: D000873:Anthracenes; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; C432165:pyrazolanthrone; D013879:Thioredoxins; D045304:Cytochromes c; D048031:JNK Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5