title: Both internalization and AIP1 association are required for tumor necrosis factor receptor 2-mediated JNK signaling.
abstract: OBJECTIVE
  The proinflammtory cytokine tumor necrosis factor (TNF), primarily via TNF receptor 1 (TNFR1), induces nuclear factor-κB (NF-κB)-dependent cell survival, and c-Jun N-terminal kinase (JNK) and caspase-dependent cell death, regulating vascular endothelial cell (EC) activation and apoptosis. However, signaling by the second receptor, TNFR2, is poorly understood. The goal of this study was to dissect how TNFR2 mediates NF-κB and JNK signaling in vascular EC, and its relevance to in vivo EC function.
  
  
  RESULTS
  We show that TNFR2 contributes to TNF-induced NF-κB and JNK signaling in EC as TNFR2 deletion or knockdown reduces the TNF responses. To dissect the critical domains of TNFR2 that mediate the TNF responses, we examine the activity of TNFR2 mutant with a specific deletion of the TNFR2 intracellular region, which contains conserved domain I, domain II, domain III, and 2 TNFR-associated factor-2-binding sites. Deletion analyses indicate that different sequences on TNFR2 have distinct roles in NF-κB and JNK activation. Specifically, deletion of the TNFR-associated factor-2-binding sites (TNFR2-59) diminishes the TNFR2-mediated NF-κB, but not JNK activation; whereas, deletion of domain II or domain III blunts TNFR2-mediated JNK but not NF-κB activation. Interestingly, we find that the TNFR-associated factor-2-binding sites ensure TNFR2 on the plasma membrane, but the di-leucine LL motif within the domain II and aa338-355 within the domain III are required for TNFR2 internalization as well as TNFR2-dependent JNK signaling. Moreover, domain III of TNFR2 is responsible for association with ASK1-interacting protein-1, a signaling adaptor critical for TNF-induced JNK signaling. While TNFR2 containing the TNFR-associated factor-2-binding sites prevents EC cell death, a specific activation of JNK without NF-κB activation by TNFR2-59 strongly induces caspase activation and EC apoptosis.
  
  
  CONCLUSIONS
  Our data reveal that both internalization and ASK1-interacting protein-1 association are required for TNFR2-dependent JNK and apoptotic signaling. Controlling TNFR2-mediated JNK and apoptotic signaling in EC may provide a novel strategy for the treatment of vascular diseases.
authors: Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, 10 Amistad St., 401B, New Haven, CT 06520, USA.
authors: Weidong
authors: W
authors: Ji
authors: Yonghao
authors: Y
authors: Li
authors: Ting
authors: T
authors: Wan
authors: Jing
authors: J
authors: Wang
authors: Haifeng
authors: H
authors: Zhang
authors: Hong
authors: H
authors: Chen
authors: Wang
authors: W
authors: Min
mesh_terms: D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D004789:Enzyme Activation; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*
chemical_list: C462952:DAB2IP protein, human; C561450:Dab2ip protein, mouse; D016328:NF-kappa B; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D047992:TNF Receptor-Associated Factor 2; C415902:TNFRSF1B protein, human; C506236:Tnfrsf1a protein, mouse; D014409:Tumor Necrosis Factor-alpha; D020703:ras GTPase-Activating Proteins; D048031:JNK Mitogen-Activated Protein Kinases