Freiberg R A, Spencer D M, Choate K A, Peng P D, Schreiber S L, Crabtree G R, Khavari P A
Veterans Administration Palo Alto Health Care System, Palo Alto, California 94304, USA.
J Biol Chem. 1996 Dec 6;271(49):31666-9. doi: 10.1074/jbc.271.49.31666.
The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.
表皮持续暴露于基因毒性损伤，需要一种有效的机制来清除基因改变的细胞。膜受体Fas在包括角质形成细胞在内的多种细胞类型中引发细胞凋亡。受体交联是Fas信号转导中配体结合后的关键步骤，我们利用能够使工程化包含FK506结合蛋白（FKBP）的蛋白质寡聚化的FK1012，通过人角质形成细胞中与FKBP连接的受体胞质结构域触发Fas。含有靶向质膜的Fas胞质结构域的FKBP嵌合体，响应FK1012，导致角质形成细胞活力下降高达89%，这由组成型启动子的活性反映出来。通过FK1012使工程化的Fas.FKBP寡聚化或通过全长Fas的抗体交联诱导的细胞变化与细胞凋亡一致。lpr Fas点突变消除了这种效应。在此试验中，与膜未连接的Fas.FKBP构建体具有完全活性。细胞的早期发育阶段或用GM-CSF、TGF-β、EGF、KGF、IFN-γ或佛波酯预处理均不能保护细胞免受Fas效应的影响。这些发现表明，Fas信号转导途径在角质形成细胞中具有活性，无需诱导，且能显著超越生长刺激。
