A novel mechanism of retinoic acid-enhanced interleukin-8 gene expression in airway epithelium

A 3- to 8-fold stimulation of interleukin (IL)-8 gene expression by all-tra ns-retinoic acid (ATRA) was demonstrated in primary cultures of human and m onkey tracheobronchial epithelial cells and BEAS-2B serum-sensitive cell li ne. The effect of ATRA on IL-8 gene expression Is dose- and time-dependent. Using cycloheximide, it was observed that new protein synthesis was requir ed for the stimulation. ATRA had no effect on IL-8 messenger RNA stability. A difference in nuclear run-on activity suggests that a transcriptional me chanism is involved in ATRA-enhanced IL-8 gene expression. Promoter-reporte r gene transfection studies demonstrated ATRA enhanced IL-8 promoter activi ty, especially when cells were cotransfected with retinoic acid nuclear rec eptor-alpha expression vector. Deletion and site-directed mutagenesis analy sis revealed the involvement of nuclear factor (NF)-kappa B binding site of the IL-8 gene in ATRA-enhanced promoter activity. Electrophoretic mobility shift assay (EMSA) demonstrated that ATRA enhanced DNA-NF-kappa B complex formation, especially with the p65 subunit. Western blot analysis demonstra ted that ATRA did not enhance the protein amount of both the p50 and the p6 5 subunits in the nuclei. Because ATRA also enhances thioredoxin (TRX) gene expression, the effect of TRX on IL-8 gene expression was examined. IL-8 p romoter activity was enhanced in transfected cells by the addition of TRX p rotein. Treatment of nuclear extracts with TRX also enhanced DNA-NF-kappa B complex formation as observed by EMSA, particularly the p65 subunit, Takin g these data together, a novel mechanism is proposed in which ATRA activate s promoter activity of IL-8 gene through TRX-dependent NF-kappa B activatio n.