Activation of the interleukin-6 promoter by a dominant negative mutant of c-Jun

Abstract

The human IL-6 promoter contains multiple regulatory elements such as those binding transcription factors belonging to the NF-kappaB (-75/-63), C/EBP (-158/-145 and -87/-76) and AP-1 (-283/-277) families. Herein, we report that ectopic expression of c-Jun, C/EBPdelta, and the p65 subunit of NF-kappaB synergistically activates an IL-6 promoter construct containing only a TATA box and a kappaB binding site. These results suggest that interactions among NF-kappaB, C/EBP, and AP-1, which are all activated by the most powerful physiological inducers of the IL-6 gene, namely TNF-alpha and IL-1, may be crucial for maximal activation of the IL-6 promoter in response to the two cytokines. Furthermore, we show that a mutated form of c-Jun lacking the transactivation domain (TAM-67) was a much stronger activator of the IL-6 promoter than c-Jun. In combination with p65 and/or C/EBPdelta, TAM-67 also synergistically activated the IL-6 promoter, while it inhibited TNF-alpha induced AP-1 activity directing an AP-1-responsive reporter plasmid. Lastly, electrophoretic mobility shift assay (EMSA) results strongly suggest the formation of complexes between p65, C/EBPdelta, and/or c-Jun or TAM-67 on the kappaB site, supporting the idea that the functional synergism is determined by a physical interaction. These data provide new insight into the molecular mechanisms regulating the formation of the transcription complex responsible for IL-6 promoter activation.

Homeobox Transcription Factor VentX Regulates Differentiation and Maturation of Human Dendritic Cells

“…To determine whether VentX inhibits IL6 expression at the transcriptional level, we performed luciferase reporter assays with IL6 promoter. The effect of VentX on the activity of 592 bp IL6 promoter luciferase reporter and several deletion mutant constructs was assessed (26). As shown in Fig.…”

Greater Than Additive Suppression of TLR3-Induced IL-6 Responses by Administration of Dieldrin and Atrazine

“…The results from mathematical modeling support the feasibility that the greater than additive suppression by these compounds of IL-6 expression is mediated by reduced formation of an NF-κB-AP-1 complex that synergistically enhances IL-6 gene expression (Faggioli et al, 2004). This can be examined experimentally, and the model can be modified after concentrations of the complex are measured experimentally by immunoprecipitation followed by Western blot.…”

“…If it is then assumed that the rate of IL-6 production per mole associated with a cell having both the ARE and the NRE sites occupied by NF-κ B is r , then the production of IL-6 per mole of cells will be rq 1 q 2 . The rate of IL-6 per mole production associated with a cell having both the ARE site occupied and the NRE site occupied by NF-κB-AP-1 is about five times the amount produced by cells in which the NRE site is occupied only by NF-κB (Faggioli et al, 2004). Thus, the total IL-6 production per mole of cells will be rq 1 (q 2 + 5q 3 ).…”

“…Such behavior is consistent with results indicating that a physical interaction between NF-κB and AP-1 is associated with a synergistic increase in transcription. Presumably decreasing this interaction by decreasing the concentration of both transcription factors would synergistically decrease gene expression (Faggioli et al, 2004).…”

“…In particular, the transcription factors AP-1 and NF-κB are required for interleukin (IL)-6 transcription, and a recent report indicates that these factors can bind to each other and form a novel transcription factor which synergistically increases IL-6 transcription by binding to the NF-κB response element in the IL-6 promoter (Faggioli et al, 2004). This mechanism was used to devise a mathematical model that predicts the greater than additive effects of dieldrin and atrazine on IL-6 production.…”