15-deoxy-Delta(12,14)-prostaglandin J(2) inhibits transcriptional activity of estrogen receptor-alpha via covalent modification of DNA-binding domain

The cyclopentenone 15-deoxy-Delta(12,14)-prostagiandin J(2) (15d-PGJ(2)) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-gamma (PPAR gamma)-dependent and PPAR gamma-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-alpha (ER alpha) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ2 inhibits both 17 beta-estradiol (E-2)-dependent and E-2-independent ER alpha transcriptional activity by PPAR gamma-independent mechanism. In addition, 15d-PGJ(2) directly modifies ER alpha protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ(2) into ER alpha, 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 ER alpha DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ2. Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ(2) inhibits DNA binding of ER alpha and subsequent repression of ER alpha target gene expression, such as pS2 and c-Myc. Therefore, our results suggest that 15d-PGJ(2) can block ER alpha function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ER alpha DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ER alpha transcriptional activity.

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