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Synergistic Activation of Interleukin-8 Gene Transcription by All-trans-retinoic Acid and Tumor Necrosis Factor-α Involves the Transcription Factor NF-κB*

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-κB. 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-1β, or TNF-α. Simultaneous treatment of A3 cells with ATRA and TNF-α 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-κB 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-α for more than 16 h resulted in enhanced NF-κB binding compared to that induced by TNF-α alone. The simultaneous treatment with ATRA and TNF-α also resulted in changes in the composition of NF-κB complexes bound to the IL-8 NF-κB site, preventing the formation of two TNF-α-inducible binding activities. We suggest that these complexes consist of repressive factors which, when removed, allow enhanced binding of NF-κB to its cognate site.