c-Src mediates thrombin-induced NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells.
In this study, we examined the regulation of NF-kappaB activation and IL-8/CXCL8 expression by thrombin in human lung epithelial cells (EC). Thrombin caused a concentration-dependent increase in IL-8/CXCL8 release in a human lung EC line (A549) and primary normal human bronchial EC. In A549 cells, thrombin, SFLLRN-NH2 (a protease-activated receptor 1 (PAR1) agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide), but not TFRGAP-NH2 (a PAR3 agonist peptide), induced an increase in IL-8/CXCL8-luciferase (Luc) activity. The thrombin-induced IL-8/CXCL8 release was attenuated by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (a thrombin inhibitor), U73122 (a phosphoinositide-phospholipase C inhibitor), Ro-32-0432 (a protein kinsase C alpha (PKC alpha) inhibitor), an NF-kappaB inhibitor peptide, and Bay 117082 (an IkappaB phosphorylation inhibitor). Thrombin-induced increase in IL-8/CXCL8-Luc activity was inhibited by the dominant-negative mutant of c-Src and the cells transfected with the kappaB site mutation of the IL-8/CXCL8 construct. Thrombin caused time-dependent increases in phosphorylation of c-Src at tyrosine 416 and c-Src activity. Thrombin-elicited c-Src activity was inhibited by Ro-32-0432. Stimulation of cells with thrombin activated IkappaB kinase alphabeta (IKK alphabeta), IkappaB alpha phosphorylation, IkappaB alpha degradation, p50 and p65 translocation from the cytosol to the nucleus, NF-kappaB-specific DNA-protein complex formation, and kappaB-Luc activity. Pretreatment of A549 cells with Ro-32-4032 and the dominant-negative mutant of c-Src DN inhibited thrombin-induced IKK alphabeta activity, kappaB-Luc activity, and NF-kappaB-specific DNA-protein complex formation. Further studies revealed that thrombin induced PKC alpha, c-Src, and IKK alphabeta complex formation. These results show for the first time that thrombin, acting through PAR1 and PAR4, activates the phosphoinositide-phospholipase C/PKC alpha/c-Src/IKK alphabeta signaling pathway to induce NF-kappaB activation, which in turn induces IL-8/CXCL8 expression and release in human lung EC.
Lin
Chien-Huang
CH
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taiwan.
Cheng
Hui-Wen
HW
Hsu
Ming-Jen
MJ
Chen
Mei-Chieh
MC
Lin
Chia-Chin
CC
Chen
Bing-Chang
BC
D000818:Animals; D045744:Cell Line, Tumor; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*
C508606:CXCL8 protein, human; D019743:Chemokines, CXC; D016209:Interleukin-8; D016328:NF-kappa B; D010716:Phosphatidylinositols; D016392:Proto-Oncogene Proteins pp60(c-src); D051550:I-kappa B Kinase; D051571:Protein Kinase C-alpha; D013917:Thrombin