Department of Dermatology and Venereology, Laboratory for Experimental Dermatology, Otto-von-Guericke-University Magdeburg, Magdeburg 39120, Germany.
J Biol Chem. 2011 May 13;286(19):16631-46. doi: 10.1074/jbc.M110.148585. Epub 2011 Mar 22.
Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.
死亡受体（DRs）诱导细胞凋亡，但也会刺激促炎的“非凋亡”信号（如 NF-κB 和丝裂原活化蛋白激酶（MAPK）的激活），并抑制 DR 激活前半胱天冬酶-8 成熟的不同步骤。为了研究细胞型 FLIP（cFLIP）的同种型或其切割产物是否差异调节 DR 信号，我们建立了表达 cFLIP(S)、cFLIP(L)或 cFLIP(L)的突变体（cFLIP(D376N)和 cFLIP(p43)）的 HaCaT 细胞。cFLIP 变体阻断 TRAIL 和 CD95L 诱导的细胞凋亡，但半胱天冬酶-8 在诱导凋亡信号复合物中的切割模式不同：cFLIP(L)诱导 caspase-8 加工为 p43/41 片段，而与 cFLIP 切割无关。cFLIP(S)或 cFLIP(p43)阻断了前半胱天冬酶-8 的切割。分析非凋亡信号通路时，我们发现 TRAIL 和 CD95L 在 15 分钟内激活 JNK 和 p38。cFLIP 变体和不同的半胱天冬酶抑制剂阻断了晚期死亡配体诱导的 JNK 或 p38 MAPK 激活，表明这些反应是细胞死亡的继发事件。cFLIP 同种型/突变体也阻断了死亡配体介导的 CXCL-8（IL-8）基因诱导。半胱天冬酶-8 的敲低完全抑制了凋亡和非凋亡信号。原代人角质形成细胞中 cFLIP 同种型的敲低增强了 CD95L 和 TRAIL 诱导的 NF-κB 激活以及 JNK 和 p38 的激活，强调了 cFLIP 对这些 DR 介导信号的调节作用。虽然半胱天冬酶-8 的存在对于凋亡和非凋亡信号是至关重要的，但 cFLIP 同种型是 TRAIL 和 CD95L 诱导的细胞凋亡、NF-κB 激活以及晚期 JNK 和 p38 MAPK 激活的有效抑制剂。cFLIP 介导的对 CD95 和 TRAIL DR 的抑制作用在角质形成细胞皮肤癌变期间以及在皮肤中 DR 激活触发的固有和/或适应性免疫反应的激活中可能具有至关重要的意义。
