RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation.
RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation.
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DOI：
10.1371/journal.pone.0023209
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Chan FK
中科院分区：
文献类型：
--
作者：
Cho Y;McQuade T;Zhang H;Zhang J;Chan FK
程序性坏死/坏死性凋亡是一种新兴的细胞死亡形式，在哺乳动物发育和免疫系统中发挥着重要作用。受体相互作用蛋白 (RIP) 家族中的促坏死激酶是程序性坏死的重要介质。阻止程序性坏死的化学抑制剂的鉴定极大地促进了坏死研究的最新进展。 Necrostatin-1 (Nec-1) 及其衍生物先前已被证明可靶向促坏死激酶 RIP1/RIPK1。 Nec-1 及其衍生物在许多实验模型系统中所赋予的保护作用通常归因于 RIP1 功能的抑制。我们比较了 Nec-1 和 siRNA 介导的 RIP1 沉默在小鼠纤维肉瘤细胞系 L929 中的效果。用泛半胱天冬酶抑制剂 zVAD-fmk 或外源 TNF 处理 L929 细胞会诱导坏死。引人注目的是，我们发现 siRNA 介导的 RIP1 沉默可抑制 zVAD-fmk 诱导的坏死，但不能抑制 TNF 诱导的坏死。在 RIP1 敲低的 L929 细胞中，TNF 诱导的细胞死亡被 Nec-1 抑制，但 caspase 抑制剂 zVAD-fmk 则不受抑制。我们发现 Nec-1 中度抑制 PKA-C§ 表达，但不抑制 Jnk 或 Erk 激活。此外，我们发现 Nec-1 独立于 RIP1 抑制近端 T 细胞受体信号传导，从而抑制 T 细胞增殖。我们的结果表明，除了 RIP1 之外，Nec-1 还针对 L929 细胞中对诱导坏死至关重要的其他因子。此外，高剂量的 Nec-1 会抑制其他信号转导途径，例如 T 细胞受体激活的信号转导途径。这些结果凸显了独立验证使用 Nec-1 与其他方法（例如 siRNA 介导的基因沉默）获得的结果的重要性。我们建议根据我们的发现重新评估之前发表的使用 Nec-1 获得的一些结果。
Programmed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis. Necrostatin-1 (Nec-1) and its derivatives were previously shown to target the pro-necrotic kinase RIP1/RIPK1. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function. We compared the effect of Nec-1 and siRNA-mediated silencing of RIP1 in the murine fibrosarcoma cell line L929. Treatment of L929 cells with the pan-caspase inhibitor zVAD-fmk or exogenous TNF induces necrosis. Strikingly, we found that siRNA-mediated silencing of RIP1 inhibited zVAD-fmk induced necrosis, but not TNF-induced necrosis. TNF-induced cell death in RIP1 knocked down L929 cells was inhibited by Nec-1, but not the caspase inhibitor zVAD-fmk. We found that PKA-C§ expression, but not Jnk or Erk activation, was moderately inhibited by Nec-1. Moreover, we found that Nec-1 inhibits proximal T cell receptor signaling independent of RIP1, leading to inhibition of T cell proliferation. Our results reveal that besides RIP1, Nec-1 also targets other factors crucial for necrosis induction in L929 cells. In addition, high doses of Nec-1 inhibit other signal transduction pathways such as that for T cell receptor activation. These results highlight the importance to independently validate results obtained using Nec-1 with other approaches such as siRNA-mediated gene silencing. We propose that some of the previous published results obtained using Nec-1 should be re-evaluated in light of our findings.
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