ADAM15 in Apoptosis Resistance of Synovial Fibroblasts: Converting Fas/CD95 Death Signals Into the Activation of Prosurvival Pathways by Calmodulin Recruitment
ADAM15 in Apoptosis Resistance of Synovial Fibroblasts: Converting Fas/CD95 Death Signals Into the Activation of Prosurvival Pathways by Calmodulin Recruitment
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DOI：
10.1002/art.40667
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发表时间：
2019-01-01
影响因子：
13.3
通讯作者：
Burkhardt, Harald
中科院分区：
文献类型：
--
作者：
Janczi, Tomasz;Boehm, Beate B.;Burkhardt, Harald
目的 探讨 ADAM15 将 FasL 介导的死亡诱导信号转化为类风湿性关节炎滑膜成纤维细胞 (RASF) 中 Src 和粘着斑激酶 (FAK) 的促生存激活的机制。方法 使用酶测定和膜联蛋白 V 染色测定 Fas/CD95 触发后 RASF 和 ADAM15 转染的 T/C28a4 细胞中的 Caspase 3/7 活性和凋亡率。通过免疫印迹分析磷酸化的Src和FAK。 ADAM15 和 CD95 与钙调蛋白 (CaM)、Src 或 FAK 的相互作用通过使用 CaM-Sepharose 的下拉和与特异性抗体的免疫共沉淀进行分析。使用重组 CaM 和 ADAM15 进行蛋白质结合测定。进行免疫荧光研究 ADAM15、Fas/CD95 和 CaM 的亚细胞共定位。结果 ADAM15 在 FasL 刺激的细胞中的抗凋亡作用通过与转染全长 ADAM15 的细胞相比，转染缺乏胞质结构域的 ADAM15 构建体的细胞凋亡增加或通过 ADAM15 RNA 干扰沉默后 RASF 的凋亡抗性降低来证明。 Fas 连接触发了 Ca2+ 释放激活的 Ca2+/钙释放激活的钙通道蛋白 1 (CRAC/Orai1) 通道依赖性 CaM 募集至细胞膜中的 Fas/CD95 和 ADAM15。同时，与 CaM 相关的 Src 被证明参与到也含有细胞质结合 FAK 的 ADAM15 复合物中。因此，RASF 中的 Fas 连接导致 Src 和 FAK 发生 ADAM15 依赖性磷酸化，这与存活率增加相关。 CaM 抑制剂三氟拉嗪或 CRAC/Orai 抑制剂 BTP-2 的药理学干扰与 FasL 同时应用，可协同增强 Fas 介导的 RASF 细胞凋亡。结论 ADAM15 为 FasL 诱导的死亡信号共同激活 CRAC/Orai 时形成 CaM 依赖性促生存信号复合物提供了支架，并且是打破 RASF 中细胞凋亡抵抗的潜在治疗靶点。
Objective To investigate mechanisms underlying the capability of ADAM15 to transform FasL-mediated death-inducing signals into prosurvival activation of Src and focal adhesion kinase (FAK) in rheumatoid arthritis synovial fibroblasts (RASFs). Methods Caspase 3/7 activity and apoptosis rate were determined in RASFs and ADAM15-transfected T/C28a4 cells upon Fas/CD95 triggering using enzyme assays and annexin V staining. Phosphorylated Src and FAK were analyzed by immunoblotting. Interactions of ADAM15 and CD95 with calmodulin (CaM), Src, or FAK were analyzed by pull-downs using CaM-Sepharose and coimmunoprecipitations with specific antibodies. Protein binding assays were performed using recombinant CaM and ADAM15. Immunofluorescence was performed to investigate subcellular colocalization of ADAM15, Fas/CD95, and CaM. Results The antiapoptotic effect of ADAM15 in FasL-stimulated cells was demonstrated either by increased apoptosis of cells transfected with an ADAM15 construct lacking the cytoplasmic domain compared to cells transfected with full-length ADAM15 or by reduced apoptosis resistance of RASFs upon RNA interference silencing of ADAM15. Fas ligation triggered a Ca2+ release-activated Ca2+/calcium release-activated calcium channel protein 1 (CRAC/Orai1) channel-dependent CaM recruitment to Fas/CD95 and ADAM15 in the cell membrane. Simultaneously, Src associated with CaM was shown to become engaged in the ADAM15 complex also containing cytoplasmic-bound FAK. Accordingly, Fas ligation in RASFs led to ADAM15-dependent phosphorylation of Src and FAK, which was associated with increased survival. Pharmacologic interference with either the CaM inhibitor trifluoperazine or the CRAC/Orai inhibitor BTP-2 simultaneously applied with FasL synergistically enhanced Fas-mediated apoptosis in RASFs. Conclusion ADAM15 provides a scaffold for formation of CaM-dependent prosurvival signaling complexes upon CRAC/Orai coactivation by FasL-induced death signals and a potential therapeutic target to break apoptosis resistance in RASFs.