Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602, USA.
Am J Respir Cell Mol Biol. 2011 Aug;45(2):411-8. doi: 10.1165/rcmb.2010-0231OC. Epub 2010 Dec 3.
We previously demonstrated up-regulation of the receptor for advanced glycation end-products (RAGE) and its ligands by cigarette smoke extract (CSE) in rat R3/1 cells, a type I-like alveolar epithelial cell line. However, RAGE-mediated intracellular signaling pathways that lead to pulmonary inflammation remained unclear. Using ELISAs, we demonstrate that alveolar epithelial cell lines exposed to 25% CSE for 2 hours induce the activation of Ras, a small GTPase that functions as a molecular switch in the control of several intracellular signaling networks. Conversely, cells treated with siRNA for RAGE (siRAGE) resulted in decreased Ras activation. Furthermore, Ras was significantly diminished in lungs from RAGE null mice exposed to chronic tobacco smoke when compared with smoke-exposed wild-type mice. The use of a luciferase reporter containing NF-κB binding sites also demonstrated elevated NF-κB activation in R3/1 cells after CSE stimulation and decreased NF-κB activation in cells transfected with siRAGE before CSE exposure. ELISA revealed an increase in the secretion of IL-1β and CCL5 by R3/1 cells, two cytokines induced by NF-κB and associated with leukocyte chemotaxis. Furthermore, real-time RT-PCR and ELISAs revealed decreased cytokine secretion in RAGE null mouse lung exposed to tobacco smoke compared with lungs from smoke-exposed wild-type animals. These results support the conclusion that CSE-induced RAGE expression functions in pathways that involve Ras-mediated NF-κB activation and cytokine elaboration. This RAGE-Ras-NF-κB axis likely contributes to inflammation associated with several smoking-related inflammatory lung diseases.
我们之前的研究表明，香烟烟雾提取物（CSE）可上调大鼠 R3/1 细胞（一种Ⅰ型肺泡上皮细胞系）中晚期糖基化终产物受体（RAGE）及其配体的表达。然而，RAGE 介导的导致肺部炎症的细胞内信号通路仍不清楚。通过 ELISA，我们证明暴露于 25% CSE 2 小时的肺泡上皮细胞系会诱导 Ras 的激活，Ras 是一种小 GTP 酶，作为控制几种细胞内信号网络的分子开关。相反，用 RAGE 的 siRNA（siRAGE）处理的细胞导致 Ras 激活减少。此外，与暴露于烟雾的野生型小鼠相比，暴露于慢性烟草烟雾的 RAGE 缺失小鼠的肺部 Ras 明显减少。使用含有 NF-κB 结合位点的荧光素酶报告基因也表明，CSE 刺激后 R3/1 细胞中 NF-κB 的激活增加，而在 CSE 暴露前转染 siRAGE 的细胞中 NF-κB 的激活减少。ELISA 显示，R3/1 细胞分泌的白细胞介素 1β（IL-1β）和趋化因子 5（CCL5）增加，这两种细胞因子均由 NF-κB 诱导，并与白细胞趋化作用有关。此外，实时 RT-PCR 和 ELISA 显示，与暴露于烟雾的野生型动物的肺部相比，暴露于烟草烟雾的 RAGE 缺失小鼠的肺部细胞因子分泌减少。这些结果支持这样的结论，即 CSE 诱导的 RAGE 表达在涉及 Ras 介导的 NF-κB 激活和细胞因子表达的途径中起作用。这个 RAGE-Ras-NF-κB 轴可能与几种与吸烟相关的炎症性肺部疾病相关的炎症有关。
