Thrombin-induced NF-κB activation and IL-8/CXCL8 release is mediated by c-Src-dependent Shc, Raf-1, and ERK pathways in lung epithelial cells

In addition to its functions in thrombosis and hemostasis, thrombin also plays an important role in lung inflammation. Our previous report showed that thrombin activates the protein kinase C (PKC)alpha/c-Src and G beta gamma/Rac1/PI3K/Akt signaling pathways to induce I kappa B kinase alpha/beta (IKK alpha/beta) activation, NF-kappa B transactivation, and IL-8/CXCL8 expressions in human lung epithelial cells (ECs). In this study, we further investigated the mechanism of c-Src-dependent Shc, Raf-1, and extracellular signal-regulated kinase (ERK) signaling pathways involved in thrombin-induced NF-kappa B activation and IL-8/CXCL8 release. Thrombin-induced increases in IL-8/CXCL8 release and kappa B-luciferase activity were inhibited by the Shc small interfering RNA (siRNA), p66Shc siRNA, GW 5074 (a Raf-1 inhibitor), and PD98059 (a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor). Treatment of A549 cells with thrombin increased p66Shc and p46/p52Shc phosphorylation at Tyr239/240 and Tyr317, which was inhibited by cell transfection with the dominant negative mutant of c-Src (c-Src DN). Thrombin caused time-dependent phosphorylation of Raf-1 and ERK, which was attenuated by the c-Src DN. Thrombin-induced IKK alpha/beta phosphorylation was inhibited by GW 5074 and PD98059. Treatment of cells with thrombin induced G beta gamma, c-Src, and p66Shc complex formation in a time-dependent manner. Taken together, these results show for the first time that thrombin activates Shc, Raf-1, and ERK through G beta gamma, c-Src, and Shc complex formation to induce IKK alpha/beta phosphorylation, NF-kappa B activation, and IL-8/CXCL8 release in human lung ECs. (C) 2013 Elsevier Inc. All rights reserved.