University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.
Waldkliniken Eisenberg, Eisenberg, Germany.
Arthritis Rheumatol. 2019 Jan;71(1):63-72. doi: 10.1002/art.40667. Epub 2018 Nov 20.
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).
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.
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 Ca release-activated Ca /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.
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.
研究 ADAM15 将 FasL 介导的死亡诱导信号转化为类风湿关节炎滑膜成纤维细胞（RASFs）中Src 和粘着斑激酶（FAK）的促生存激活的机制。
使用酶测定法和 Annexin V 染色法，在 Fas/CD95 触发后，在 RASFs 和转染 ADAM15 的 T/C28a4 细胞中测定 caspase 3/7 活性和凋亡率。通过免疫印迹分析磷酸化 Src 和 FAK。使用 CaM-Sepharose 进行下拉实验和用特异性抗体进行共沉淀实验，分析 ADAM15 和 CD95 与钙调蛋白（CaM）、Src 或 FAK 的相互作用。使用重组 CaM 和 ADAM15 进行蛋白结合测定。通过免疫荧光实验研究 ADAM15、Fas/CD95 和 CaM 的亚细胞共定位。
与转染全长 ADAM15 的细胞相比，转染缺乏细胞质结构域的 ADAM15 构建体的细胞的凋亡增加，或者在敲低 ADAM15 后 RASFs 的凋亡抵抗能力降低，证明 ADAM15 在 FasL 刺激的细胞中具有抗凋亡作用。Fas 配体触发钙释放激活钙/钙释放激活钙通道蛋白 1（CRAC/Orai1）通道依赖性 CaM 募集到 Fas/CD95 和细胞膜上的 ADAM15。同时，与 CaM 结合的 Src 被证明参与含有细胞质结合的 FAK 的 ADAM15 复合物。因此，Fas 配体在 RASFs 中导致 ADAM15 依赖性 Src 和 FAK 的磷酸化，这与存活增加有关。同时应用钙调蛋白抑制剂三氟拉嗪或 CRAC/Orai 抑制剂 BTP-2 对 FasL 的药理学干扰在 Fas 介导的 RASFs 凋亡中协同增强。
ADAM15 为 FasL 诱导的死亡信号通过 CRAC/Orai 共激活形成依赖 CaM 的促生存信号复合物提供了一个支架，是打破 RASFs 凋亡抵抗的潜在治疗靶点。
