Abstract
Induction of interleukin-8 (IL-8) by IL-1 or tumor necrosis factor (TNF), and repression by interferons or glucocorticoids have been shown to involve sequences between nucleotides -94 and -71 of the 5'-flanking region, and the transcription factors NF-IL-6 and NF-kappaB. The A3 cell line was derived from the human melanoma cell line G-361 by stable transfection with part of the IL-8 promoter (nucleotides -101 to +40 from transcription start) fused to the luciferase coding region. These regulatory sequences were sufficient for transcriptional activation by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, IL-1beta, or TNF-alpha. Simultaneous treatment of A3 cells with ATRA and TNF-alpha resulted in a dose- and time-dependent synergistic increase in luciferase expression and IL-8 mRNA levels. Transient transfections of the parental cell line demonstrated that the NF-kappaB binding site is essential for this synergistic transactivation. Electrophoretic mobility shift assays with nuclear extracts of A3 cells showed that stimulation with ATRA and TNF-alpha for more than 16 h resulted in enhanced NF-kappaB binding compared to that induced by TNF-alpha alone. The simultaneous treatment with ATRA and TNF-alpha also resulted in changes in the composition of NF-kappaB complexes bound to the IL-8 NF-kappaB site, preventing the formation of two TNF-alpha-inducible binding activities. We suggest that these complexes consist of repressive factors which, when removed, allow enhanced binding of NF-kappaB to its cognate site.
Highlights
Retinoic acid (RA)1 exerts profound effects on proliferation and differentiation, and has immunomodulatory properties
We have established the cell line A3, which was derived from the human melanoma cell line G-361 by stable transfection with a reporter gene construct containing a portion of the IL-8 promoter fused to the firefly luciferase coding region, and show that the promoter can be activated by all-trans-retinoic acid (ATRA)
Combination of ATRA with tumor necrosis factor (TNF)-␣ induced synergistic activation of the IL-8 promoter in a dose- and time-dependent manner. These results are reflected by IL-8 mRNA levels, which are synergistically induced by ATRA and TNF-␣ in this cell line
Summary
Reagents—All-trans-retinoic acid was obtained from Sigma. 9-cisRetinoic acid and 4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB) were provided by J. The cell line A3 was generated by transfection with the NAPs promoter-luciferase construct, and selection and subsequent growth in a medium containing 350 g/ml of the antibiotic G418 (Geneticin; Life Technologies, Inc., Scotland). Both cell lines were cultivated in minimal essential medium supplemented with 5% fetal calf serum. After 30 s centrifugation in a microcentrifuge, the buffer was completely aspirated and the pelleted nuclei suspended in 25 l of buffer C (20 mM HEPES, 20% glycerol, 0.420 M NaCl, 0.150 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice with shaking for 15 min. Luciferase values were obtained from cell lysates as described under “Experimental Procedures.” Relative luciferase activities compared to untreated control cells from one representative experiment from at least four individual experiments are shown
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