*Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
†Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Clin Sci (Lond). 2014 Aug;127(3):171-83. doi: 10.1042/CS20130676.
Up-regulation of ICAM-1 (intercellular adhesion molecule-1) is frequently implicated in lung inflammation and lung diseases, such as IPF (idiopathic pulmonary fibrosis). Thrombin has been shown to play a key role in inflammation via the induction of adhesion molecules, which then causes lung injury. However, the mechanisms underlying thrombin-induced ICAM-1 expression in HPAEpiCs (human pulmonary alveolar epithelial cells) remain unclear. In the present study, we have shown that thrombin induced ICAM-1 expression in HPAEpiCs. Pre-treatment with the inhibitor of thrombin [PPACK (D-Phe-Pro-Arg-chloromethyl ketone)], c-Src (PP1), PDGFR (platelet-derived growth factor receptor) (AG1296), PI3K (phosohinositide 3-kinase) (LY294002), NF-κB (nuclear factor κB) (Bay11-7082) or p300 (GR343) and transfection with siRNAs of c-Src, PDGFR, Akt, p65 and p300 markedly reduced thrombin-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with thrombin. In addition, we established that thrombin stimulated the phosphorylation of c-Src, PDGFR, Akt and p65, which were inhibited by pre-treatment with their respective inhibitors PP1, AG1296, LY294002 or Bay11-7082. In addition, thrombin also enhanced Akt and NF-κB translocation from the cytosol to the nucleus, which was reduced by PP1, AG1296 or LY294002. Thrombin induced NF-κB promoter activity and the formation of the p65-Akt-p300 complex, which were inhibited by AG1296, LY294002 or PP1. Finally, we have shown that thrombin stimulated in vivo binding of p300, Akt and p65 to the ICAM-1 promoter, which was reduced by AG1296, LY294002, SH-5 or PP1. These results show that thrombin induced ICAM-1 expression and monocyte adherence via a c-Src/PDGFR/PI3K/Akt/NF-κB-dependent pathway in HPAEpiCs. Increased understanding of the signalling mechanisms underlying ICAM-1 gene regulation will create opportunities for the development of anti-inflammatory therapeutic strategies.
细胞间黏附分子-1（ICAM-1）的上调通常与肺部炎症和肺部疾病有关，如特发性肺纤维化（IPF）。已有研究表明，凝血酶通过诱导黏附分子的产生在炎症中发挥关键作用，进而导致肺损伤。然而，凝血酶诱导 HPAEpiCs（人肺肺泡上皮细胞）中 ICAM-1 表达的机制尚不清楚。在本研究中，我们已经表明凝血酶可诱导 HPAEpiCs 中 ICAM-1 的表达。用凝血酶抑制剂[PPACK（D-Phe-Pro-Arg-氯甲基酮）]、c-Src（PP1）、血小板衍生生长因子受体（PDGFR）（AG1296）、PI3K（磷酸肌醇 3-激酶）（LY294002）、核因子κB（NF-κB）（Bay11-7082）或 p300（GR343）预处理，或用 c-Src、PDGFR、Akt、p65 和 p300 的 siRNA 转染，可显著降低凝血酶诱导的 ICAM-1 表达和凝血酶刺激的 HPAEpiCs 上单核细胞的黏附。此外，我们发现凝血酶刺激 c-Src、PDGFR、Akt 和 p65 的磷酸化，而用其各自的抑制剂 PP1、AG1296、LY294002 或 Bay11-7082 预处理则可抑制磷酸化。此外，凝血酶还增强了 Akt 和 NF-κB 从细胞质向细胞核的易位，而用 PP1、AG1296 或 LY294002 则可减少易位。凝血酶诱导 NF-κB 启动子活性和 p65-Akt-p300 复合物的形成，而用 AG1296、LY294002 或 PP1 则可抑制该活性和复合物的形成。最后，我们发现凝血酶刺激 p300、Akt 和 p65 与 ICAM-1 启动子在体内的结合，而用 AG1296、LY294002、SH-5 或 PP1 处理则可减少结合。这些结果表明，凝血酶通过 HPAEpiCs 中的 c-Src/PDGFR/PI3K/Akt/NF-κB 依赖性途径诱导 ICAM-1 表达和单核细胞黏附。深入了解 ICAM-1 基因调控的信号机制将为抗炎治疗策略的发展创造机会。
