Kim Hong Pyo, Wang Xue, Chen Zhi-Hua, Lee Seon-Jin, Huang Min-Hsin, Wang Yong, Ryter Stefan W, Choi Augustine M K
Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, MUH 628NW, Pittsburgh, Pennsylvania, USA.
Autophagy. 2008 Oct;4(7):887-95. doi: 10.4161/auto.6767. Epub 2008 Oct 12.
Cigarette smoke-induced cell death contributes to the pathogenesis of chronic obstructive pulmonary disease, though the relative roles of apoptosis and autophagy remain unclear. The inducible stress protein heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. We examined the relationships between these processes in human bronchial epithelial cells (Beas-2b) exposed to cigarette smoke extract (CSE). CSE induced morphological and biochemical markers of autophagy in Beas-2b cells and induced autophagosome formation as evidenced by formation of GFP-LC3 puncta and electron microscopic analysis. Furthermore, CSE increased the processing of microtubule-associated protein-1 light chain-3 (LC3B-I) to LC3B-II, within 1 hr of exposure. Increased LC3B-II was associated with increased autophagy, since inhibitors of lysosomal proteases and of autophagosome-lysosome fusion further increased LC3B-II levels during CSE exposure. CSE concurrently induced extrinsic apoptosis in Beas-2b cells involving early activation of death-inducing-signaling-complex (DISC) formation and downstream activation of caspases (-8,-9,-3). The induction of extrinsic apoptosis by CSE was dependent in part on autophagic proteins. Reduction of Beclin 1 levels with beclin 1 siRNA inhibited DISC formation and caspase-3/8 activation in response to CSE. LC3B siRNA also inhibited caspase-3/8 activation. The stress protein HO-1 protected against CSE-induced cell death by concurrently downregulating apoptosis and autophagy-related signaling. Adenoviral mediated expression of HO-1 inhibited DISC formation and caspase-3/9 activation in CSE-treated epithelial cells, diminished the expression of Beclin 1, and partially inhibited the processing of LC3B-I to LC3B-II. Conversely, transfection of Beas-2b with ho-1 siRNA augmented CSE-induced DISC formation and increased intracellular reactive oxygen species formation. HO-1 expression augmented CSE-induced phosphorylation of NFkappaB p65 in Beas-2b cells. Consistently, expression of IkappaB, the inhibitor of NFkappaB, increased CSE-induced DISC formation. LC3B siRNA also enhanced p65 phosphorylation. In fibroblasts from beclin 1 heterozygous knockout mice, p65 phosphorylation was dramatically upregulated, while CSE-induced DISC formation was inhibited, consistent with an anti-apoptotic role for NFkappaB and a pro-apoptotic role for Beclin 1. These studies demonstrated an interdependence of autophagic and apoptogenic signaling in CSE-induced cell death, and their coordinated downregulation by HO-1. An understanding of the regulation of cell death pathways during smoke exposure may provide therapeutic strategies in smoke-related illness.
香烟烟雾诱导的细胞死亡参与慢性阻塞性肺疾病的发病机制，尽管凋亡和自噬的相对作用仍不清楚。诱导性应激蛋白血红素加氧酶-1（HO-1）赋予细胞对氧化应激的保护作用。我们研究了暴露于香烟烟雾提取物（CSE）的人支气管上皮细胞（Beas-2b）中这些过程之间的关系。CSE诱导Beas-2b细胞中自噬的形态学和生化标志物，并诱导自噬体形成，绿色荧光蛋白-微管相关蛋白1轻链3（GFP-LC3）斑点的形成和电子显微镜分析证明了这一点。此外，CSE在暴露1小时内增加了微管相关蛋白1轻链3（LC3B-I）向LC3B-II的加工。LC3B-II的增加与自噬增加有关，因为溶酶体蛋白酶抑制剂和自噬体-溶酶体融合抑制剂在CSE暴露期间进一步增加了LC3B-II水平。CSE同时诱导Beas-2b细胞中的外源性凋亡，涉及死亡诱导信号复合物（DISC）形成的早期激活和半胱天冬酶（-8、-9、-3）的下游激活。CSE诱导的外源性凋亡部分依赖于自噬蛋白。用Beclin 1小干扰RNA降低Beclin 1水平可抑制DISC形成和对CSE的半胱天冬酶-3/8激活。LC3B小干扰RNA也抑制半胱天冬酶-3/8激活。应激蛋白HO-1通过同时下调凋亡和自噬相关信号来保护细胞免受CSE诱导的细胞死亡。腺病毒介导的HO-1表达抑制CSE处理的上皮细胞中的DISC形成和半胱天冬酶-3/9激活，减少Beclin 1的表达，并部分抑制LC3B-I向LC3B-II的加工。相反，用HO-1小干扰RNA转染Beas-2b可增强CSE诱导的DISC形成并增加细胞内活性氧的形成。HO-1表达增强了CSE诱导的Beas-2b细胞中核因子κB p65的磷酸化。同样，核因子κB抑制剂IkappaB的表达增加了CSE诱导的DISC形成。LC3B小干扰RNA也增强了p65磷酸化。在Beclin 1杂合敲除小鼠的成纤维细胞中，p65磷酸化显著上调，而CSE诱导的DISC形成受到抑制，这与核因子κB的抗凋亡作用和Beclin 1的促凋亡作用一致。这些研究证明了CSE诱导的细胞死亡中自噬和凋亡信号的相互依赖性，以及HO-1对它们的协同下调作用。了解烟雾暴露期间细胞死亡途径的调节可能为烟雾相关疾病提供治疗策略。
