Cho Rou-Ling, Yang Chien-Chung, Lee I-Ta, Lin Chih-Chung, Chi Pei-Ling, Hsiao Li-Der, Yang Chuen-Mao
Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan;
Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan; Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Lin-Kou, Kwei-San, Tao-Yuan, Taiwan;
Am J Physiol Lung Cell Mol Physiol. 2016 Apr 1;310(7):L639-57. doi: 10.1152/ajplung.00109.2014. Epub 2016 Jan 8.
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-κB (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-κB and IκBα phosphorylation, NF-κB translocation, and NF-κB 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-κB and ultimately induces ICAM-1 expression in HPAEpiCs.
细胞间黏附分子-1（ICAM-1）的上调常与肺部炎症有关。脂多糖（LPS）已被证明通过诱导黏附分子在炎症中起关键作用，进而导致肺损伤。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。我们发现，通过蛋白质印迹法、逆转录-聚合酶链反应（RT-PCR）、实时定量PCR和启动子分析显示，LPS可诱导HPAEpiCs中ICAM-1的表达。用c-Src抑制剂（蛋白磷酸酶-1，PP1）、活性氧（ROS）（依达拉奉）、NADPH氧化酶（阿朴吗啡和二苯基碘鎓氯化物）、表皮生长因子受体（EGFR）（AG1478）、血小板衍生生长因子受体（PDGFR）（AG1296）、磷脂酰肌醇-3-激酶（PI3K）（LY294002）、丝裂原活化蛋白激酶1/2（MEK1/2）（U0126）或核因子κB（NF-κB）（Bay11-7082）预处理，以及用c-Src、EGFR、PDGFR、Akt、p47（phox）、Nox2、Nox4、p42和p65的小干扰RNA（siRNA）转染，均可显著降低LPS诱导的ICAM-1表达以及单核细胞对受LPS刺激的HPAEpiCs的黏附。此外，我们证实LPS刺激了c-Src、EGFR、PDGFR、Akt或p65的磷酸化，而用它们各自的抑制剂预处理可抑制这种磷酸化。LPS诱导Toll样受体4（TLR4）、髓样分化因子88（MyD88）、肿瘤坏死因子受体相关因子6（TRAF6）、c-Src､p47（phox）和Rac1复合物形成2，用c-Src或TRAF6 siRNA转染可使其减弱。此外，LPS显著增强NADPH氧化酶的活化和细胞内ROS的产生，而PP1可抑制这些作用。我们证实LPS通过c-Src/NADPH氧化酶/ROS/EGFR、PDGFR/PI3K/Akt依赖的途径诱导p42/p44丝裂原活化蛋白激酶（MAPK）的活化。最后，我们观察到LPS显著增强NF-κB和IκBα的磷酸化、NF-κB的易位以及NF-κB启动子活性，而PP1、依达拉奉、阿朴吗啡、二苯基碘鎓氯化物、AG1478、AG1296、LY294002或U0126可抑制这些作用。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的活化，进而启动NF-κB的活化并最终诱导HPAEpiCs中ICAM-1的表达。
