NFκB and AP-1 drive human myometrial IL8 expression

Abstract

The uterine expression of the chemokine IL8 increases dramatically with the onset of labour both at term and preterm. The IL8 promoter contains binding sites for the transcription factors nuclear factor-kappa B (NFκB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein (CEBP). In this study we investigated the roles of these transcription factors in IL1B regulation of the IL8 gene in human myometrium. Using chromatin immune precipitation (ChIP) assay, we showed that each of NFκB, CEBP, and AP-1 binds to the IL8 promoter upon IL1B stimulation. To examine the relative importance of each site in IL8 gene expression, site-directed mutagenesis of each of these sites was performed. We found that the NFκB site was essential for basal and IL1B-stimulated gene expression. Mutation of the AP-1 site reduced both basal and IL1B-stimulated expression but to a lesser extent. Mutation of the CEBP site had no effect upon basal expression but eliminated the IL1B response. Small interfering RNA (siRNA) silencing of NFκB abolished the IL8 response to IL1B significantly; siRNA against AP-1 reduced it to a lesser extent whilst knockdown of CEBP enhanced the response. Our data confirms a central and essential role for NFκB in regulation of IL8 in human myometrium.

Figure 1

(a) Schematic of the IL8 promoter enhanceosome region. (b) ChIP analysis demonstrates the in vivo binding of NFκBp65, CEBPβ and d, c-Jun, c-Fos, and H4 to the transcriptional enhanceosome of the IL8 promoter. ChIP assay using antibodies against NFκBp65 and CEBPβ and -d, c-Jun, and c-Fos was performed in nonstimulated (N/s) and IL1B stimulated conditions. The immunoprecipitates were subjected to PCR analysis using primer pairs spanning the IL8 promoter transcriptional enhanceosome. The first two lanes are “input” lanes where no immunoprecipitation was performed prior to PCR. The second two lanes contained IgG antibody for immunoprecipitation (negative controls).

Figure 2

Representation of the IL8 promoter region and the mutations used in this study, as indicated by the underlined nucleotides. mAP-1: mutation of AP-1. mNFκB: mutation of NFκB and mCEBP: mutation of CEBP binding site. Functional effect of site-directed mutagenesis of transcription factor binding sites in the IL8 promoter in primary human myometrial cells. Data are presented as mean ± SEM (n = 4). An Anova test was performed with a Dunnett's multiple comparison post-test. **indicates a significant difference of P less than 0.01 compared to WT promoter.

Figure 3

(a) siRNA knockdown of p65 (a), CEBPβ (b), and c-Jun (c). (b) The effects of siRNA against NFκBp65, CEBPβ, c-Jun, and c-Fos on IL8 protein production in a nonstimulated and IL1B-stimulated state. Data is normalised to the nontargeting control. Data are presented as mean ± SEM (n = 4). An ANOVA was performed with a Dunnett's multiple comparison post-test. *Indicates a significant difference of P less than 0.05. (c) The effects of siRNA against NFκBp65, CEBPβ, and CEBPD on IL8 protein production in a nonstimulated and IL1B-stimulated state. Data is normalised to the non-targeting control. Data are presented as mean ± SEM (n = 4). An ANOVA was performed with a Dunnett's multiple comparison post-test. *Indicates a significant difference of P less than 0.05.