Lipopolysaccharide induces ICAM-1 expression via a c-Src/NADPH oxidase/ROS-dependent NF-κB pathway in human pulmonary alveolar epithelial cells
Lipopolysaccharide induces ICAM-1 expression via a c-Src/NADPH oxidase/ROS-dependent NF-κB pathway in human pulmonary alveolar epithelial cells
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DOI：
10.1152/ajplung.00109.2014
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发表时间：
2016-04-01
影响因子：
4.9
通讯作者：
Yang, Chuen-Mao
中科院分区：
文献类型：
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作者：
Cho, Rou-Ling;Yang, Chien-Chung;Yang, Chuen-Mao
细胞间粘附分子 1 (ICAM-1) 的上调常常与肺部炎症有关。脂多糖（LPS）已被证明通过粘附分子诱导在炎症中发挥关键作用，然后导致肺损伤。然而，LPS 诱导人肺泡上皮细胞 (HPAEpiCs) 中 ICAM-1 表达的机制仍不清楚。通过蛋白质印迹、RT-PCR、实时 PCR 和启动子测定，我们发现 LPS 诱导 HPAEpiC 中 ICAM-1 的表达。用 c-Src（蛋白磷酸酶-1，PP1）、活性氧（ROS）（依达拉奉）、NADPH 氧化酶（罗布麻宁和二亚苯基碘鎓氯化物）、EGFR (AG1478)、PDGFR (AG1296)、磷脂酰肌醇-3-激酶 (PI3K) (LY294002)、MEK1/2 (U0126) 抑制剂进行预处理，或 NF-kappa B (Bay11-7082) 并用 c-Src、EGFR、PDGFR、Akt、p47(phox)、Nox2、Nox4、p42 和 p65 的 siRNA 转染，显着降低 LPS 诱导的 ICAM-1 表达和单核细胞对 LPS 攻击的 HPAEpiC 的粘附。此外，我们还确定 LPS 会刺激 c-Src、EGFR、PDGFR、Akt 或 p65 的磷酸化，而这些磷酸化可通过使用各自的抑制剂进行预处理来抑制。 LPS 诱导 Toll 样受体 4 (TLR4)、MyD88、TNF 受体相关因子 6 (TRAF6)、c-Src、p47(phox) 和 Rac1 复合物形成 2，通过 c-Src 或 TRAF6 siRNA 转染可减弱这种作用。此外，LPS 显着增强了 NADPH 氧化酶的激活和细胞内 ROS 的生成，而这些都被 PP1 抑制。我们确定 LPS 通过这些细胞中的 c-Src/NADPH 氧化酶/ROS/EGFR、PDGFR/PI3K/Akt 依赖性途径诱导 p42/p44 MAPK 激活。最后，我们观察到 LPS 显着增强 NF-kappa B 和 I kappa B α 磷酸化、NF-kappa B 易位和 NF-kappa B 启动子活性，而这些活性可被 PP1、依达拉奉、罗布麻素、二亚苯基碘鎓、AG1478、AG1296、LY294002 或 U0126 抑制。这些结果表明，LPS 通过 TLR4/MyD88/TRAF6/c-Src/NADPH 氧化酶/ROS/EGFR、PDGFR/PI3K/Akt 途径诱导 p42/p44 MAPK 激活，进而启动 NF-kappa B 的激活，最终诱导 HPAEpiC 中 ICAM-1 的表达。
Upregulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via adhesion molecule induction and then causes lung injury. However, the mechanisms underlying LPS-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. We showed that LPS induced ICAM-1 expression in HPAEpiCs, revealed by Western blotting, RT-PCR, real-time PCR, and promoter assay. Pretreatment with the inhibitor of c-Src (protein phosphatase-1, PP1), reactive oxygen species (ROS) (Edaravone), NADPH oxidase (apocynin and diphenyleneiodonium chloride), EGFR (AG1478), PDGFR (AG1296), phosphatidylinositol-3-kinase (PI3K) (LY294002), MEK1/2 (U0126), or NF-kappa B (Bay11-7082) and transfection with siRNAs of c-Src, EGFR, PDGFR, Akt, p47(phox), Nox2, Nox4, p42, and p65 markedly reduced LPS-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with LPS. In addition, we established that LPS stimulated phosphorylation of c-Src, EGFR, PDGFR, Akt, or p65, which was inhibited by pretreatment with their respective inhibitors. LPS induced Toll-like receptor 4 (TLR4), MyD88, TNF receptor-associated factor 6 (TRAF6), c-Src, p47(phox), and Rac1 complex formation 2, which was attenuated by transfection with c-Src or TRAF6 siRNA. Furthermore, LPS markedly enhanced NADPH oxidase activation and intracellular ROS generation, which were inhibited by PP1. We established that LPS induced p42/p44 MAPK activation via a c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt-dependent pathway in these cells. Finally, we observed that LPS significantly enhanced NF-kappa B and I kappa B alpha phosphorylation, NF-kappa B translocation, and NF-kappa B promoter activity, which were inhibited by PP1, Edaravone, apocynin, diphenyleneiodonium chloride, AG1478, AG1296, LY294002, or U0126. These results demonstrated that LPS induces p42/p44 MAPK activation mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt pathway, which in turn initiates the activation of NF-kappa B and ultimately induces ICAM-1 expression in HPAEpiCs.