Department of Pulmonary and Critical Care Medicine, Gachon University, Korea.
J Toxicol Sci. 2010 Feb;35(1):33-9. doi: 10.2131/jts.35.33.
Cigarette smoke (CS), a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrated cigarette smoke extract (CSE) induced DISC formation in human lung fibroblasts (MRC-5). The aim of this study was to investigate the involvement of extracellular signal-regulated kinase (ERK) MAPK activation in CSE induced DISC formation. Immunoprecipitation (IP) for Fas and Western Immunoblot (IB) analysis for caspase 8 were then performed to show DISC. Lactate dehydrogenase (LDH) release was measured using a cytotoxicity detection kit. MTT assay was used as a measure of cell viability. We demonstrated that CSE induces DISC formation in MRC-5 using IP for Fas and IB for caspase 8. ERK was expressed in MRC-5 exposed to CSE. MEK-1 inhibitor (PD98059) decreased DISC formation in MRC-5 exposed to 20% CSE at 1 hr, and cell viability, as assessed by colorimetric MTT assay, was increased in MEK-1 inhibitor treated MRC-5 cells after 24 hr CSE exposure compared to the control. Inhibiting ERK significantly decreased the caspase-3,-8 activity in MEK-1 inhibitor treated MRC-5 cells compared to the control.The DISC formation, initial event of extrinsic apoptotic pathway, is a primary component of CSE- induced death in MRC-5, and ERK activation plays an active role in the DISC formation and downstream pathway. These results suggest that modulation of ERK may have therapeutic potential in the prevention of smoke-related lung injury.
香烟烟雾（CS）是肺气肿的主要危险因素，其通过尚未完全阐明的机制导致细胞死亡。死亡诱导信号复合物（DISC）的形成是 Fas 介导致凋亡的初始事件。我们证明香烟烟雾提取物（CSE）可诱导人肺成纤维细胞（MRC-5）中 DISC 的形成。本研究的目的是研究细胞外信号调节激酶（ERK）MAPK 激活在 CSE 诱导的 DISC 形成中的作用。然后进行 Fas 的免疫沉淀（IP）和 caspase 8 的 Western 免疫印迹（IB）分析以显示 DISC。使用细胞毒性检测试剂盒测量乳酸脱氢酶（LDH）释放。MTT 测定法用于测量细胞活力。我们通过 Fas 的 IP 和 caspase 8 的 IB 证明了 CSE 在 MRC-5 中诱导 DISC 的形成。ERK 在暴露于 CSE 的 MRC-5 中表达。MEK-1 抑制剂（PD98059）降低了在 1 小时暴露于 20％CSE 的 MRC-5 中的 DISC 形成，并且在用 MEK-1 抑制剂处理的 MRC-5 细胞中，与对照相比，在 24 小时 CSE 暴露后通过比色 MTT 测定法评估的细胞活力增加。与对照相比，在 MEK-1 抑制剂处理的 MRC-5 细胞中，ERK 的抑制显著降低了 caspase-3、-8 的活性。DISC 的形成是外源性凋亡途径的初始事件，是 CSE 诱导的 MRC-5 细胞死亡的主要组成部分，ERK 激活在 DISC 形成及其下游途径中发挥积极作用。这些结果表明，ERK 的调节可能在预防与吸烟有关的肺损伤方面具有治疗潜力。
