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c-Src Mediates Thrombin-Induced NF-B Activation and IL-8/CXCL8 Expression in Lung Epithelial Cells¹

Chien-Huang Lin^*, Hui-Wen Cheng, Ming-Jen Hsu^*, Mei-Chieh Chen, Chia-Chin Lin^¶ and Bing-Chang Chen^2,

^* Graduate Institute of Medical Sciences, Graduate Institute of Biomedical Technology, Department of Microbiology and Immunology, School of Respiratory Therapy, College of Medicine, and ^¶ Graduate Institute of Nursing, College of Nursing, Taipei Medical University, Taipei, Taiwan

In this study, we examined the regulation of NF-B 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 (PKC) inhibitor), an NF-B inhibitor peptide, and Bay 117082 (an IB 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 B 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 IB kinase (IKK), IB phosphorylation, IB degradation, p50 and p65 translocation from the cytosol to the nucleus, NF-B-specific DNA-protein complex formation, and B-Luc activity. Pretreatment of A549 cells with Ro-32-4032 and the dominant-negative mutant of c-Src DN inhibited thrombin-induced IKK activity, B-Luc activity, and NF-B-specific DNA-protein complex formation. Further studies revealed that thrombin induced PKC, c-Src, and IKK complex formation. These results show for the first time that thrombin, acting through PAR1 and PAR4, activates the phosphoinositide-phospholipase C/PKC/c-Src/IKK signaling pathway to induce NF-B activation, which in turn induces IL-8/CXCL8 expression and release in human lung EC.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the National Science Council of Taiwan (NSC91-2320-B-038-048, NSC92-2314-B-038-059, and NSC94-2320-B-038-047).

² Address correspondence and reprint requests to Dr. Bing-Chang Chen, School of Respiratory Therapy, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. E-mail address: bcchen{at}tmu.edu.tw

³ Abbreviations used in this paper used in this paper: EC, epithelial cell; BAL, bronchoalveolar lavage, COX-2, cyclooxygenase-2; GPCR, G protein-coupled receptor; IKK, IB kinase; PAR1, protease-activated receptor 1; PI, phosphoinositide; PLC, phospholipase C; PKC-, protein kinase C-, PPACK, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone; c-Src DN, domainant-negative mutant of c-Src; NHBEC, normal human bronchial EC; KC, keratinocyte-derived chemokine; Ser, serine; Tyr, tyrosine; Luc, luciferase; wt, wild type.

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