Yang Jun-Hai, Le Wei-Dong, Basinger Scott F, Wu Samuel M, Yang Chao-yuh
Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA.
Invest Ophthalmol Vis Sci. 2005 Mar;46(3):1039-46. doi: 10.1167/iovs.04-0325.
To investigate the mechanism underlying apoptosis in retinal pigment epithelium (RPE) induced by TNF-alpha in conditions of heavy metal ion deficiency.
Apoptotic morphology was assessed with Hoechst 33342. FITC-VAD-fmk or antibody specific to cleaved caspase 3 was used to detect in situ activated caspases or cleaved caspase 3, respectively. JC-1 and carboxylated dichlorodihydrofluorescein diacetate were used as probes to measure mitochondrial membrane potential (Deltapsi(m)) and intracellular reactive oxygen species (rOx).
The apoptotic response of RPE cells was markedly enhanced when TNF-alpha plus actinomycin D (act-D) was coapplied with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a heavy metal ion chelator. The apoptosis was caspase dependent, and a blockade with cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition (MPT), but not FK506, a calcineurin inhibitor, abolished caspase activation and subsequent apoptosis, suggesting that apoptosis requires the MPT, and that caspase activation is downstream to the MPT. MPT, observed in situ as Deltapsi(m) loss, was prevented when cells were pretreated with either CsA or the pan-caspase inhibitor z-VAD-fmk. This result suggests that apoptotic signaling is initiated by the MPT and further amplified by downstream caspases, probably through a feedback loop. An increase in rOx was observed in cells exposed to TNF-alpha+act-D+TPEN, and rOx production did not require MPT or caspase activation. In addition, application of antioxidants significantly inhibited rOx production, Deltapsi(m) loss, and apoptosis. These data suggest that the rOx may play a role as a proapoptotic signal.
The data suggest that intracellular heavy metal ions play a role in determining the apoptosis induction threshold of the inflammatory response to TNF-alpha in RPE.
研究在重金属离子缺乏条件下，肿瘤坏死因子-α（TNF-α）诱导视网膜色素上皮（RPE）细胞凋亡的机制。
用Hoechst 33342评估凋亡形态。分别用FITC-VAD-fmk或特异性针对裂解型半胱天冬酶-3的抗体检测原位活化的半胱天冬酶或裂解型半胱天冬酶-3。用JC-1和羧基二氯二氢荧光素二乙酸酯作为探针测量线粒体膜电位（Δψm）和细胞内活性氧（ROS）。
当TNF-α加放线菌素D（act-D）与重金属离子螯合剂N,N,N',N'-四（2-吡啶甲基）乙二胺（TPEN）共同应用时，RPE细胞的凋亡反应明显增强。凋亡是半胱天冬酶依赖性的，线粒体通透性转换（MPT）抑制剂环孢素A（CsA）可阻断凋亡，但钙调神经磷酸酶抑制剂FK506不能，这表明凋亡需要MPT，且半胱天冬酶激活在MPT下游。原位观察到的MPT表现为Δψm丧失，当细胞用CsA或泛半胱天冬酶抑制剂z-VAD-fmk预处理时可被阻止。这一结果表明凋亡信号由MPT启动，并可能通过反馈环由下游半胱天冬酶进一步放大。在暴露于TNF-α+act-D+TPEN的细胞中观察到ROS增加，且ROS产生不需要MPT或半胱天冬酶激活。此外，应用抗氧化剂可显著抑制ROS产生、Δψm丧失和凋亡。这些数据表明ROS可能作为促凋亡信号发挥作用。
数据表明细胞内重金属离子在决定RPE对TNF-α炎症反应的凋亡诱导阈值中起作用。
