Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
J Leukoc Biol. 2011 Jun;89(6):917-26. doi: 10.1189/jlb.1209812. Epub 2011 Mar 14.
Actin cytoskeleton has been shown to play a regulating role in several signaling pathways, and disruption of actin filament has been reported to increase sTNF-α-induced cell death. However, whether actin is involved in tmTNF-α-mediated cytotoxicity remains unclear. Here, we demonstrated that pretreatment of HL-60 with CytD or LatA to depolymerize actin significantly suppressed tmTNF-α-mediated apoptosis. Interestingly, tmTNF-α increased the actin immunoprecipitated by anti-TNFR2 but not anti-TNFR1 antibody, and disruption of the actin filament totally blocked this effect. In addition, TNFR1 knockdown by siRNA did not affect tmTNF-α-mediated cytotoxicity and the inhibitory effect of CytD, suggesting that the involvement of actin in the tmTNF-α-induced apoptosis is linked to the TNFR2 pathway. Our results revealed further that tmTNF-α signaled the inhibition of IκB degradation and NF-κB activity by recruiting RIP1 to and uncoupling TRAF2 from the TNFR2 complex. Nevertheless, CytD totally reversed the tmTNF-α signaling and activated NF-κB by recruiting TRAF2 to and dissociating RIP1 from the TNFR2 complex. Furthermore, tmTNF-α led to activation of caspase-8 by dissociation of cFLIP from TNFR2 and inhibition of the cFLIP expression. Activated caspase-8 cleft RIP1 to suppress NF-κB activity and also mediated tmTNF-α-induced apoptosis. However, CytD blocked the tmTNF-α-induced uncoupling of cFLIP from TNFR2 and prevented caspase-8 activation and the resulting cleavage of RIP1, converting the signaling for tmTNF-α-mediated apoptosis into one for activating NF-κB to survive. These results suggest that the actin cytoskeleton functions in transmitting signals via TNFR2 to mediate tmTNF-α-induced apoptosis.
肌动蛋白细胞骨架已被证明在几种信号通路中发挥调节作用，并且肌动蛋白丝的破坏已被报道会增加 sTNF-α诱导的细胞死亡。然而，肌动蛋白是否参与 tmTNF-α介导的细胞毒性仍不清楚。在这里，我们证明了用 CytD 或 LatA 预处理 HL-60 以解聚肌动蛋白会显著抑制 tmTNF-α介导的细胞凋亡。有趣的是，tmTNF-α增加了抗 TNFR2 抗体免疫沉淀的肌动蛋白，但不增加抗 TNFR1 抗体免疫沉淀的肌动蛋白，并且肌动蛋白丝的破坏完全阻断了这种效应。此外，siRNA 敲低 TNFR1 并不影响 tmTNF-α介导的细胞毒性和 CytD 的抑制作用，这表明肌动蛋白在 tmTNF-α诱导的细胞凋亡中的参与与 TNFR2 途径有关。我们的结果进一步揭示，tmTNF-α通过招募 RIP1 并使 TRAF2 与 TNFR2 复合物解偶联，从而抑制 IκB 降解和 NF-κB 活性来发出信号。然而，CytD 通过招募 TRAF2 并使 RIP1 与 TNFR2 复合物解离，完全逆转了 tmTNF-α信号并激活了 NF-κB。此外，tmTNF-α通过使 cFLIP 与 TNFR2 解离来激活 caspase-8，并抑制 cFLIP 的表达。激活的 caspase-8 切割 RIP1 以抑制 NF-κB 活性，也介导了 tmTNF-α诱导的细胞凋亡。然而，CytD 阻断了 tmTNF-α诱导的 cFLIP 与 TNFR2 的解偶联，阻止了 caspase-8 的激活和由此产生的 RIP1 切割，将 tmTNF-α介导的细胞凋亡信号转导转化为激活 NF-κB 以存活的信号转导。这些结果表明，肌动蛋白细胞骨架通过 TNFR2 传递信号以介导 tmTNF-α诱导的细胞凋亡。
