Calmodulin Binding to Death Receptor 5-mediated Death-Inducing Signaling Complex in Breast Cancer Cells.
Calmodulin Binding to Death Receptor 5-mediated Death-Inducing Signaling Complex in Breast Cancer Cells.
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DOI：
10.1002/jcb.25882
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发表时间：
2017-08
影响因子：
4
通讯作者：
Song Y
中科院分区：
文献类型：
--
作者：
Fancy RM;Kim H;Zhou T;Zinn KR;Buchsbaum DJ;Song Y
死亡受体-5(DR5)的激活导致死亡诱导信号复合体(DISC)的形成。TrA-8是一种DR5特异性激动型抗体，在体内外均显示出显著的细胞毒活性，且无肝毒性。钙调蛋白(CaM)在乳腺癌中过表达，在调控DR5介导的细胞凋亡中起着关键作用。然而，CaM调节DR5介导的细胞凋亡信号的机制仍不清楚。在这项研究中，我们用免疫共沉淀、荧光显微镜成像、caspase信号分析和细胞存活率分析等方法，研究了CaM与DR5介导的DISC结合在TRA-8敏感的乳腺癌细胞系中诱导细胞凋亡的作用。结果表明，在DR5激活后，CaM以钙依赖的方式被募集到DR5介导的视盘中。CaM拮抗剂三氟拉嗪(TFP)可抑制CaM募集进入视盘并减弱视盘的形成。DR5寡聚在细胞凋亡盘形成过程中起关键作用。TFP抑制TRA-8激活的DR5寡聚，这与TFP对DR5介导的视盘形成的影响一致。TFP和钙离子螯合剂EGTA可阻断TRA-8激活的caspase依赖的凋亡信号，TFP可降低TRA-8诱导的细胞毒作用。这些结果表明，CaM以钙依赖的方式与DR5介导的视盘结合，并可能确认CaM是DR5介导的乳腺癌细胞凋亡视盘形成的关键调节因子。
Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. TRA-8, a DR5 specific agonistic antibody, has demonstrated significant cytotoxic activity in vitro and in vivo without inducing hepatotoxicity. Calmodulin (CaM) that is overexpressed in breast cancer plays a critical role in regulating DR5-mediated apoptosis. However, the mechanism of CaM in regulating DR5-mediated apoptotic signaling remains unknown. In this study, we characterized CaM binding to DR5-mediated DISC for apoptosis in TRA-8 sensitive breast cancer cell lines using co-immunoprecipitation, fluorescence microscopic imaging, caspase signaling analysis and cell viability assay. Results show that upon DR5 activation, CaM was recruited into DR5-mediated DISC in a calcium dependent manner. CaM antagonist, trifluoperazine (TFP), inhibited CaM recruitment into the DISC and attenuated DISC formation. DR5 oligomerization is critical for DISC formation for apoptosis. TFP decreased TRA-8 activated DR5 oligomerization, which was consistent with TFP’s effect on DR5-mediated DISC formation. TFP and Ca2+ chelator, EGTA, impeded TRA-8 activated caspase-dependent apoptotic signaling, and TFP decreased TRA-8 induced cell cytotoxicity. These results demonstrated CaM binding to DR5-mediated DISC in a calcium dependent manner and may identify CaM as a key regulator of DR5-mediated DISC formation for apoptosis in breast cancer.