Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, 10 Amistad St., 401B, New Haven, CT 06520, USA.
Arterioscler Thromb Vasc Biol. 2012 Sep;32(9):2271-9. doi: 10.1161/ATVBAHA.112.253666. Epub 2012 Jun 28.
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.
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.
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.
促炎细胞因子肿瘤坏死因子（TNF）主要通过 TNF 受体 1（TNFR1）诱导核因子-κB（NF-κB）依赖性细胞存活，以及 c-Jun N 端激酶（JNK）和半胱天冬酶依赖性细胞死亡，调节血管内皮细胞（EC）的激活和凋亡。然而，第二个受体 TNFR2 的信号转导知之甚少。本研究的目的是剖析 TNFR2 如何介导血管 EC 中的 NF-κB 和 JNK 信号转导，以及其与体内 EC 功能的相关性。
我们表明，TNFR2 有助于 TNF 诱导的 EC 中 NF-κB 和 JNK 信号转导，因为 TNFR2 缺失或敲低会降低 TNF 的反应。为了剖析介导 TNF 反应的 TNFR2 的关键结构域，我们检查了具有 TNFR2 细胞内区特定缺失的 TNFR2 突变体的活性，该缺失区包含保守的 I 结构域、II 结构域、III 结构域和 2 个 TNFR 相关因子-2 结合位点。缺失分析表明，TNFR2 上的不同序列在 NF-κB 和 JNK 激活中具有不同的作用。具体来说，TNFR 相关因子-2 结合位点（TNFR2-59）的缺失会减弱 TNFR2 介导的 NF-κB，但不会减弱 JNK 激活；而 II 结构域或 III 结构域的缺失会减弱 TNFR2 介导的 JNK，但不会减弱 NF-κB 激活。有趣的是，我们发现，TNFR 相关因子-2 结合位点确保了 TNFR2 位于质膜上，但 II 结构域内的双亮氨酸 LL 基序和 III 结构域内的 aa338-355 是 TNFR2 内化以及 TNFR2 依赖性 JNK 信号所必需的。此外，TNFR2 的 III 结构域负责与 ASK1 相互作用蛋白-1 结合，后者是 TNF 诱导的 JNK 信号所必需的信号衔接蛋白。虽然含有 TNFR 相关因子-2 结合位点的 TNFR2 可防止 EC 细胞死亡，但 TNFR2-59 特异性激活 JNK 而不激活 NF-κB 会强烈诱导半胱天冬酶激活和 EC 凋亡。
我们的数据表明，TNFR2 依赖的 JNK 和凋亡信号转导需要内化和 ASK1 相互作用蛋白-1 结合。控制 EC 中 TNFR2 介导的 JNK 和凋亡信号转导可能为血管疾病的治疗提供新策略。
