15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Transcriptional Activity of Estrogen Receptor-α via Covalent Modification of DNA-Binding Domain

Han-Jong Kim,Fa Liu,Joon-Young Kim,Thomas P Conrads,Timothy D Veenstra,Zhaojing Meng,Terrence R Burke,Li Hua Wang,William L Farrar

doi:10.1158/0008-5472.can-06-3043

Abstract

The cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-gamma (PPARgamma)-dependent and PPARgamma-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-alpha (ERalpha) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ(2) inhibits both 17beta-estradiol (E(2))-dependent and E(2)-independent ERalpha transcriptional activity by PPARgamma-independent mechanism. In addition, 15d-PGJ(2) directly modifies ERalpha protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ(2) into ERalpha, both in vitro and in vivo. Nanoflow reverse-phase liquid chromatography tandem mass spectrometry analysis identifies two cysteines (Cys(227) and Cys(240)) within the COOH-terminal zinc finger of ERalpha DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ(2). Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ(2) inhibits DNA binding of ERalpha and subsequent repression of ERalpha target gene expression, such as pS2 and c-Myc. Therefore, our results suggest that 15d-PGJ(2) can block ERalpha function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERalpha DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ERalpha transcriptional activity.

Highlights

15-Deoxy-D12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone PG, is a naturally occurring derivative of PGD2 and acts as an endogenous ligand for the nuclear receptor peroxisome proliferator-activated receptor-g (PPARg; refs. 1, 2)
We show that 15d-PGJ2 inhibits 17h-estradiol (E2)– dependent and E2-independent ERa transcriptional activity by PPARg-independent mechanism
Our result shows that 15d-PGJ2 at low concentration (0.5–2.5 Amol/L) strongly decreases proliferation of ERa-positive MCF-7 cells in the absence or presence of E2 but not that of ERa-negative MDA-MB-231 cells (Fig. 1D)

Summary

Introduction

15-Deoxy-D12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone PG, is a naturally occurring derivative of PGD2 and acts as an endogenous ligand for the nuclear receptor peroxisome proliferator-activated receptor-g (PPARg; refs. 1, 2). J series of PGs, including 15d-PGJ2, unlike other classes of PGs are characterized by the presence of a reactive a,h-unsaturated carbonyl group in the cyclopentenone ring This moiety confers 15d-PGJ2 the capability to form covalent adducts with thiols of cysteine residues in target proteins by Michael’s addition, resulting in an alteration of protein function [12,13,14,15]. The middle DBD contains two nonequivalent Cys zinc fingers critical for binding to short palindromic nucleotide sequences called estrogen response element (ERE) in the target gene promoters These two zinc fingers in ERa DBD function cooperatively in ERa dimerization and DNA binding [25, 26]. In addition to defining the molecular mechanism underlying ERa inhibition by 15d-PGJ2, our results validate targeting the vulnerable cysteines in the COOHterminal zinc finger of ERa DBD by covalent modification or electrophilic attack as a means of disrupting hormone- and growth factor–mediated transactivation of ERa

Materials and Methods

Results

Findings

Discussion