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Volume 271, Number 43, Issue of October 25, 1996 pp. 26954-26961
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Synergistic Activation of Interleukin-8 Gene Transcription by All-trans-retinoic Acid and Tumor Necrosis Factor-alpha Involves the Transcription Factor NF-kappa B

(Received for publication, March 14, 1996, and in revised form, June 14, 1996)

Hanna Harant Dagger § , Rainer de Martin par , Penelope J. Andrew Dagger , Elisabeth Foglar Dagger , Christian Dittrich § and Ivan J. D. Lindley Dagger

From the Dagger  Sandoz Research Institute, A-1235 Vienna, Austria, the § Ludwig Boltzmann Institute for Applied Cancer Research, Kaiser Franz Josef-Hospital, Vienna, A-1100 Austria and the par  Department of Vascular Biology and Thrombosis Research, Vienna International Research Cooperation Center, A-1235, Vienna, Austria

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-kappa B. 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-kappa B 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-kappa B 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-kappa B complexes bound to the IL-8 NF-kappa B 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-kappa B to its cognate site.


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