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A novel role for the apoptosis inhibitor ARC in suppressing death receptor-mediated necrosis.

机译：凋亡抑制剂ARC在抑制死亡受体介导的坏死中的新作用。

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摘要

Apoptosis and necrosis are two morphologically distinct forms of cell death that have been described. Apoptosis has long been recognized as a form of regulated cell death. In contrast, necrosis has traditionally been viewed as cell death that is unregulated and passive that occurs due to excessive trauma or stress. Yet, increasing evidence has emerged to indicate that necrotic cell death can be actively controlled. Regulated necrosis can be stimulated by the same death ligands that activate apoptosis, such as tumor necrosis factor α (TNFα) and Fas ligand. Programmed necrosis initiated by the ligation of TNF receptor 1 (TNFR1) has been most extensively characterized. Activation of TNFR1 can promote cell survival mediated by Nuclear Factor-κB (NF-κB) activation, apoptosis, or necrosis depending on cell type and the environment. Although the mechanism that mediates the selection amongst these outcomes is not well understood, a basic tenet of this decision-making is that inhibition of apoptosis obligates the cell to undergo necrosis. ARC (Apoptosis Repressor with a CARD (Caspase Recruitment Domain)) is an endogenous apoptosis inhibitor that antagonizes both the mitochondrial and death receptor apoptosis pathways through protein-protein interactions involving its CARD, a death-fold motif. In keeping with the existing paradigm, we hypothesized that ARC would inhibit apoptosis and thereby promote necrosis induced by the death receptor pathway.;To test this hypothesis, we employed a well-defined system utilizing L929 murine fibrosarcoma cells, in which TNFα can induce either apoptosis or necrosis. In these cells, necrosis is elicited through the administration of TNFα alone or in conjunction with pan-caspase inhibitor z-VAD-fmk. Conversely, the application of TNFα along with the protein synthesis inhibitor cycloheximide results in apoptosis. Assays that detect cellular release of high mobility group box protein 1 and lactate dehydrogenase and entry of propidium iodide were used to monitor cellular loss of plasma membrane integrity, a defining feature of necrosis. Poly ADP-ribose polymerase cleavage and caspase-3 cleavage are markers of apoptosis that were used to assess apoptotic cell death. Surprisingly, we found that overexpression of ARC suppresses, rather than promotes, TNFα-induced programmed necrosis, while retaining its ability to block apoptosis. Overexpression of the CARD-defective L31F; G69R double-point mutant form of ARC that is deficient in inhibition of apoptosis, abrogates the ability of ARC to suppress TNFα-induced necrosis. In line with this finding, the knockdown of ARC exacerbates TNFα-induced necrosis, an effect that can be rescued by reconstitution of knockdown cells with wild type, but not the CARD-defective mutant, ARC. Moreover, using fractionation and NE-κB p65 ELISA binding assays, we found that ARC inhibits NF-κB activation, another event downstream of TNFα stimulation, in a CARD-dependent manner. Together, these data indicate that the CARD of ARC is required in suppressing downstream TNFα-TNFR1 signaling. Furthermore, through immunoprecipitations, we discovered an interaction between ARC and TNFR that inhibited the recruitment of RIP1, a critical mediator of TNFα-induced necrosis.;To test the relevance of the effect of ARC in TNFα-induced necrosis pathway in vivo, we infected wild type mice and mice lacking ARC with vaccinia virus, which has been demonstrated previously to induce necrosis through a pathway mediated by TNFα. We found that mice lacking ARC exhibited markedly increased necrosis/inflammation in the adipose tissue compared with wild type controls, paralleling our results that indicate the role of ARC in limiting necrosis.;These studies demonstrate that ARC suppresses TNFα-induced necrosis and apoptosis by interacting with TNFR1 and disrupting TNFα signaling. Given that both apoptosis and necrosis play important roles in multiple diseases, such as myocardial infarction and stroke, ARC may provide pharmacological strategies to maintain tissue viability in these ischemic syndromes.
机译：凋亡和坏死是已描述的两种形态学上不同的细胞死亡形式。长期以来，凋亡被认为是受调节的细胞死亡的一种形式。相反，坏死传统上被认为是由于过度的创伤或压力而发生的细胞死亡，它是不受控制的和被动的。但是，越来越多的证据表明可以积极控制坏死细胞的死亡。可以通过激活凋亡的相同死亡配体（例如肿瘤坏死因子α（TNFα）和Fas配体）刺激调节性坏死。由TNF受体1（TNFR1）的连接引发的程序性坏死已得到最广泛的表征。 TNFR1的激活可促进由细胞核因子-κB（NF-κB）激活，凋亡或坏死介导的细胞存活，具体取决于细胞类型和环境。尽管尚未很好地理解介导这些结果之间选择的机制，但该决策的基本原则是抑制细胞凋亡使细胞必须发生坏死。 ARC（具有CARD（胱天蛋白酶募集结构域）的细胞凋亡阻遏物）是一种内源性凋亡抑制剂，可通过涉及其CARD（一种死亡折叠基序）的蛋白质-蛋白质相互作用来拮抗线粒体和死亡受体的凋亡途径。根据现有的范例，我们假设ARC会抑制凋亡，从而促进死亡受体途径诱导的坏死。为了验证这一假设，我们采用了定义明确的系统，利用L929鼠纤维肉瘤细胞，其中TNFα可以诱导细胞凋亡或坏死。在这些细胞中，通过单独或与泛半胱天冬酶抑制剂z-VAD-fmk一起施用TNFα引起坏死。相反，TNFα与蛋白质合成抑制剂环己酰亚胺一起使用会导致细胞凋亡。检测高迁移率族盒蛋白1和乳酸脱氢酶的细胞释放以及碘化丙锭进入细胞的检测方法可用于监测细胞质膜完整性的丧失，这是坏死的一个重要特征。聚ADP-核糖聚合酶裂解和caspase-3裂解是凋亡的标志物，用于评估凋亡细胞的死亡。令人惊讶地，我们发现ARC的过度表达抑制而不是促进TNFα诱导的程序性坏死，同时保留其阻断细胞凋亡的能力。 CARD缺陷L31F的过表达；缺乏抑制凋亡的G69R双点突变形式的ARC，消除了ARC抑制TNFα诱导的坏死的能力。与这一发现一致的是，敲除ARC会加剧TNFα诱导的坏死，这种作用可以通过重组具有野生型而不是CARD缺陷型突变体ARC的敲除细胞来挽救。此外，使用分级分离和NE-κBp65 ELISA结合试验，我们发现ARC以CARD依赖性方式抑制NF-κB活化，这是TNFα刺激下游的另一事件。总之，这些数据表明在抑制下游TNFα-TNFR1信号传导中需要ARC的CARD。此外，通过免疫沉淀，我们发现ARC与TNFR之间的相互作用抑制了TNFα诱导的坏死的关键介体RIP1的募集。为了检测ARC在体内由TNFα诱导的坏死途径中的相关性，我们进行了感染野生型小鼠和缺乏带有痘苗病毒的ARC的小鼠，先前已证明可通过TNFα介导的途径诱导坏死。我们发现缺乏ARC的小鼠与野生型对照组相比脂肪组织中的坏死/炎症明显增加，这与我们的研究结果表明ARC在限制坏死中的作用相似;这些研究表明ARC通过相互作用抑制TNFα诱导的坏死和细胞凋亡。 TNFR1并破坏TNFα信号传导。鉴于凋亡和坏死在多种疾病（例如心肌梗塞和中风）中都起着重要作用，ARC可以提供药理策略来维持这些缺血性综合征的组织活力。

著录项

  • 作者

    Kung, Gloria.;

  • 作者单位

    Yeshiva University.;

  • 授予单位 Yeshiva University.;
  • 学科 Biology Cell.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 202 p.
  • 总页数 202
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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