BACKGROUND: NF-κB is the most important transcriptional factor in Il-8 gene expression. Triptolide is a new compound that recently has been shown to inhibit NF-κB activation. The purpose of this study is to investigate how triptolide inhibits NF-κB-dependent IL-8 gene transcription in lung epithelial cells and to pilot the potential for the clinical application of triptolide in inflammatory lung diseases. METHODS: A549 cells were used and triptolide was provided from Pharmagenesis Company (Palo Alto, CA). In order to examine NF-κB-dependent IL-8 transcriptional activity, we established stable A549 IL-8-NF-κB-luc. cells and performed luciferase assays. IL-8 gene expression was measured by RT-PCR and ELISA. A Western blot was done for the study of IκBα degradation and as electromobility shift assay was done to analyze NF-κB DNA binding. p65 specific transactivation was analyzed by a cotransfection study using a Gal4-p65 fusion protein expression system. To investigate the involvement of transcriptional coactivators, we perfomed a transfection study with CBP and SRC-1 expression vectors. RESULTS: We observed that triptolide significantly suppresses NF-κB-dependent IL-8 transcriptional activity induced by IL-1β and PMA. RT-PCR showed that triptolide represses both IL-1β- and pMA-induced IL-8 mRNA expression and ELISA confirmed this triptolide-mediated IL-8 suppression at the protein level. However, triptolide did not affect IκBα degradation and NF-κB DNA binding. In a p65-specific transactivation study, triptolide significantly suppressed Gal4-p65TA1 and Gal4-p65TA2 activity suggesting that triptolide inhibits NF-κB activation by inhibiting p65 transactivation. However, this triptolide-mediated inhibition of p65 transactivation was not rescued by the overexpression of CBP or SRC-1, thereby excluding the role of transcriptional coactivators. CONCLUSIONS: Triptolide is a new compound that inhibits NF-κB-dependent IL-8 transcriptional activation by inhibiting p65 transactivation, but not by an IκBα-dependent mechanism. This suggests that triptolide may have a therapeutic potential for inflammatory lung diseases.
Blotting, Western ; DNA ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells* ; Gene Expression ; Interleukin-8* ; Luciferases ; Lung Diseases ; Lung* ; NF-kappa B* ; RNA, Messenger ; Transcriptional Activation ; Transfection