Regulation of human microsomal prostaglandin E synthase-1 by IL-1β requires a distal enhancer element with a unique role for C/EBPβ

Abstract

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1β (interleukin 1β) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPβ (CCAAT/enhancer-binding protein β) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPβ binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPβ in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPβ highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

Krüppel-like Factor 5 Transcription Factor Promotes Microsomal Prostaglandin E2 Synthase 1 Gene Transcription in Breast Cancer

“…Interestingly, EGR1 was previously suggested to induce KLF5 expression (42,43). Two other mPGES1 transcription factors, C/EBP␤ (19) and NF-B (18,23), were shown to be KLF5 cofactors (40,44). However, how these transcription factors coordinately regulate mPGES1 gene transcription needs further investigation.…”

“…Previous studies showed that inflammatory factors, such as TNF␣ (17), IL-1␤ (17)(18)(19), LPS (20), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (18), induce mPGES1 expression. Several transcription factors, such as Sp1/3 (21), EGR1 (10,22), CREB (cAMP-responsive element-binding protein) (20), C/EBP␤ (CCAAT/enhancer-binding protein ␤) (19), and NF-B (18,23), have also been shown to regulate mPGES1 gene transcription. However, the transcriptional regulatory mechanism of mPGES1 in breast cancer has not been explored to date.…”

Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders

“…Various binding sites for transcription factors have been proposed and were partially experimentally confirmed. Specificity protein (SP)1 and 3, which bind to the proximal promoter, and CCAAT/enhancer-binding protein β, which binds to a distal enhancer element, are required for basal expression of mPGES-1 [41,42]. Diverse pro-inflammatory stimuli such as IL-1β, TNF-α and lipopolysaccharide (LPS) as well as growth factors such as epidermal growth factor induce the expression of mPGES-1 in a variety of tissues and cell-types [1,4].…”

A distal enhancer controls cytokine-dependent human cPLA2α gene expression

“…We also evaluated C/EBP ␤ by ChIP analysis because of its involvement in transcriptional activation of other members of the eicosanoid pathway, specifi cally cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES), based on our studies and others ( 38,52 ). We found that both c-Jun and C/EBP ␤ were constitutively associated with the enhancer in control cells and that the association of C/EBP ␤ increased more than 2-fold following 8 h of IL-1 ␤ treatment ( Fig.…”

“…Cell permeabilization, DNase I treatment, and Southern analysis were performed as previously described ( 37,38 ). Following DNase I treatment, cell lysates were incubated overnight at 50°C, and genomic DNA was isolated by organic extraction, alcohol precipitation, and resuspension in TE.…”