In Situ Analysis of Interleukin-1-Induced Transcription of cox-2 and il-8 in Cultured Human Myometrial Cells

Abstract

The specific binding of transcription factors to DNA has been shown to be inhibited by chromatin structure and increased by cooperative interactions with other proteins. Consequently, in situ analysis using chromatin immunoprecipitation offers the most accurate view of transcriptional control. Transient transfection studies and in vitro analyses of IL-1-induced cox-2 transcription in a number of cell types have indicated regulation by either nuclear factor kappa B (NF-kappa B) or CCAAT/enhancer binding protein (C/EBP beta), or both acting cooperatively. To determine the mechanisms of COX-2 (cyclooxygenase or prostaglandin endoperoxide synthase) induction in cultured human myometrial cells in situ, we examined the cross-linking of the RelA subunit of NF-kappa B and C/EBP beta to the cox-2 promoter and flanking sequences. As a control, we inspected the interaction of these transcription factors with the IL-8 gene, which has been shown in other cell types to be activated by the cooperative interaction of NF-kappa B and C/EBP beta. Indeed, both transcription factors were cross-linked to the il-8 promoter after IL-1 treatment, but only RelA was cross-linked to cox-2 DNA. The il-8 promoter was also found to physically interact with proteins cross-linked to sites further upstream. IL-1 treatment also increased polymerase II cross-linking to both promoters and increased histone H4 acetylation at specific sites. These results indicate that modification of chromatin structure is part of the response to IL-1 stimulation. Chromatin immunoprecipitation thus provides critical insight into the mechanisms of COX-2 and IL-8 expression in human myometrial cells.

Identification of interleukin-1β regulated genes in uterine smooth muscle cells

“…It is well established that IL-1b is a major activator of the crucial and rate-determining enzyme in prostaglandin biosynthesis (PTGS2, also called COX-2; Rauk & Chiao 2000). This action is likely mediated by the nuclear factor NFkB (Ackerman et al 2004, Soloff et al 2004. In agreement with these data, expression of PTGS2 and NFkB genes is strongly induced by IL-1b (7.9-and 10.9-fold increase for PTGS2 and NFkB respectively).…”

The role of nuclear factor kappa B in human labour

“…NF-kB inhibition by SASP does, however, repress secretory type II phospholipase A 2 (an enzyme upstream of COX-2 in the PG biosynthetic pathway) in gestational tissues (Lappas et al 2004), as does NF-kB inhibition by NAC (Lappas et al 2003). The in situ binding of the p65 NF-kB subunit to the COX-2 promoter has been demonstrated in IL-1b-stimulated myometrial cells by chromatin immunoprecipitation (Soloff et al 2004). Treatment of myometrial cells with proteasome inhibitors, a COX-2-derived PG metabolite, or a more selective IKKb inhibitor, all of which block NF-kB activity, inhibits IL-1b-induced COX-2 expression and PG synthesis (Belt et al 1999, Lindstrom & Bennett 2005.…”

“…Repression of NF-kB DNAbinding by the anti-inflammatory agent sulfasalazine (SASP) or the anti-oxidant N-acetyl-cysteine (NAC) inhibits the release of IL-6, IL-8 and TNF-a from LPS-treated placental, choriodecidual and amnion explants (Lappas et al 2002, Lappas et al 2003. Soloff et al (2004) recently reported the in situ binding of the p65 NF-kB subunit to the endogenous IL-8 promoter in IL-1b-stimulated myometrial cells. Reporter studies in amnion and cervical cells demonstrated that the NF-kB response element is required for transcription of the IL-8 gene in these cells (Elliott et al 2001).…”

Role of the progesterone receptor (PR) in the regulation of inflammatory response pathways and aromatase in the breast

“…The human COX-2 gene contains two well-characterized NF-κB response elements in its promoter that bind NF-κB in vitro and in vivo [75][76][77]. In studies using human myometrial cells in culture, we found using chromatin immunoprecipitation (ChIP) that the effects of IL-1β to stimulate recruitment of NF-κB p65 to both proximal and distal NF-κB elements of the COX-2 promoter were markedly diminished by co-incubation with progesterone [72].…”