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)α/c-Src and Gβγ/Rac1/PI3K/Akt signaling pathways to induce IκB kinase α/β (IKKα/β) activation, NF-κB 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-κB activation and IL-8/CXCL8 release. Thrombin-induced increases in IL-8/CXCL8 release and κB-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 IKKα/β phosphorylation was inhibited by GW 5074 and PD98059. Treatment of cells with thrombin induced Gβγ, 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 Gβγ, c-Src, and Shc complex formation to induce IKKα/β phosphorylation, NF-κB activation, and IL-8/CXCL8 release in human lung ECs.
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Chien-Huang
CH
Lin
Ming-Chih
MC
Yu
Chia-Chieh
CC
Chiang
Mauo-Ying
MY
Bien
Ming-Hsien
MH
Chien
Bing-Chang
BC
Chen
D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D016209:Interleukin-8 / Q000378:metabolism*; D008168:Lung / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013917:Thrombin / Q000494:pharmacology*; D019061:src-Family Kinases / Q000378:metabolism
D005419:Flavonoids; D007211:Indoles; D016209:Interleukin-8; D016328:NF-kappa B; D010636:Phenols; D034741:RNA, Small Interfering; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D019908:Proto-Oncogene Proteins c-raf; D051550:I-kappa B Kinase; D048049:Extracellular Signal-Regulated MAP Kinases; D013917:Thrombin; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one