Tuberc Respir Dis.  2001 Jan;50(1):52-66. 10.4046/trd.2001.50.1.52.

Triptolide-induced Transrepression of IL-8 NF-kappaB in Lung Epithelial Cells

• Jee YK
• Kim YS
• Yun SY
• Kim YH
• Choi EK
• Park JS
• Kim KY
• Chea GN
• Kwak SJ
• Lee KY

• KMID: 2160056
• DOI: http://doi.org/10.4046/trd.2001.50.1.52

Abstract

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.

Keyword

IL-8; NF-κB; Triptolide; Transrepression; p65 transactivation; CBP; SCR-1

MeSH Terms

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
DNA
Interleukin-8
Luciferases
NF-kappa B
RNA, Messenger