The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne, Victoria, Australia.
Cell Death Dis. 2013 Jan 17;4(1):e465. doi: 10.1038/cddis.2012.201.
Ligation of tumor necrosis factor receptor 1 (TNFR1) can cause cell death by caspase 8 or receptor-interacting protein kinase 1 (RIPK1)- and RIPK3-dependent mechanisms. It has been assumed that because RIPK1 bears a death domain (DD), but RIPK3 does not, RIPK1 is necessary for recruitment of RIPK3 into signaling and death-inducing complexes. To test this assumption, we expressed elevated levels of RIPK3 in murine embryonic fibroblasts (MEFs) from wild-type (WT) and gene-deleted mice, and exposed them to TNF. Neither treatment with TNF nor overexpression of RIPK3 alone caused MEFs to die, but when levels of RIPK3 were increased, addition of TNF killed WT, Ripk1(-/-), caspase 8(-/-), and Bax(-/-)/Bak(-/-) MEFs, even in the presence of the broad-spectrum caspase inhibitor Q-VD-OPh. In contrast, Tnfr1(-/-) and Tradd(-/-) MEFs did not die. These results show for the first time that in the absence of RIPK1, TNF can activate RIPK3 to induce cell death both by a caspase 8-dependent mechanism and by a separate Bax/Bak- and caspase-independent mechanism. RIPK1 is therefore not essential for TNF to activate RIPK3 to induce necroptosis nor for the formation of a functional ripoptosome/necrosome.
肿瘤坏死因子受体 1（TNFR1）的交联可通过半胱天冬酶 8 或受体相互作用蛋白激酶 1（RIPK1）和 RIPK3 依赖性机制导致细胞死亡。人们认为，由于 RIPK1 具有死亡结构域（DD），而 RIPK3 没有，因此 RIPK1 对于将 RIPK3 募集到信号和死亡诱导复合物中是必需的。为了验证这一假设，我们在来自野生型（WT）和基因缺失小鼠的鼠胚胎成纤维细胞（MEFs）中表达了高水平的 RIPK3，并将其暴露于 TNF 中。TNF 的处理或单独过表达 RIPK3 均不会导致 MEFs 死亡，但当 RIPK3 水平增加时，TNF 的添加会杀死 WT、Ripk1(-/-)、caspase 8(-/-)和 Bax(-/-)/Bak(-/-) MEFs，即使存在广谱半胱天冬酶抑制剂 Q-VD-OPh 也是如此。相比之下，Tnfr1(-/-)和 Tradd(-/-) MEFs 不会死亡。这些结果首次表明，在没有 RIPK1 的情况下，TNF 可以激活 RIPK3，通过半胱天冬酶 8 依赖性机制和独立的 Bax/Bak 和 caspase 非依赖性机制诱导细胞死亡。因此，RIPK1 对于 TNF 激活 RIPK3 以诱导坏死性凋亡或形成功能性 ripoptosome/necrosome 不是必需的。
