Lens S M, den Drijver B F, Pötgens A J, Tesselaar K, van Oers M H, van Lier R A
Department of Clinical Viro-Immunology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
J Immunol. 1998 Jun 15;160(12):6083-92.
To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1beta-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.
为剖析参与B细胞抗原受体（BCR）介导的以及Fas诱导的人类B细胞死亡的细胞内信号通路，我们分离了具有不同凋亡敏感性的伯基特淋巴瘤细胞系Ramos的克隆。对Fas诱导凋亡敏感的克隆筛选同时也筛选出了对BCR介导的细胞死亡敏感性增强的克隆，反之亦然。相比之下，对Fas介导的凋亡具有抗性的克隆仍可发生BCR诱导的细胞死亡。基于这些克隆的功能表型，我们推测这两条受体诱导的凋亡信号通路最初是不同的，但最终可能会汇聚。事实上，Fas和BCR的连接均导致白细胞介素-1β转化酶/Ced-3样蛋白酶caspase 3及其底物N-乙酰天冬氨酰-谷氨酰-缬氨酰-天冬氨酸醛和聚（ADP-核糖）聚合酶的裂解。值得注意的是，观察到Fas或BCR连接诱导的caspase 3裂解模式存在质的差异；Fas连接产生19/20 kDa和17 kDa的caspase 3裂解产物，而BCR交联后仅发现后者的裂解产物。caspase抑制剂缬氨酰-丙氨酰-天冬氨酸-氟甲基酮可阻断Fas和BCR介导的凋亡，但对两种刺激诱导的caspase 3裂解有不同影响。最后，发现Fas相关死亡结构域（FADD）显性负性突变蛋白的过表达可抑制Fas诱导的凋亡，但不抑制BCR诱导的凋亡。我们的研究结果共同表明，Fas和BCR分别通过依赖FADD和不依赖FADD的机制，与caspase 3上游的不同蛋白酶偶联。
