title: 15-deoxy-Delta12,14-prostaglandin J2 inhibits transcriptional activity of estrogen receptor-alpha via covalent modification of DNA-binding domain.
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.
authors.affiliation: Cancer Stem Cell Section, Laboratory of Cancer Prevention, Science Applications International Corporation-Frederick, Maryland, USA.
authors.forename: Han-Jong
authors.initials: HJ
authors.lastname: Kim
authors.forename: Joon-Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Zhaojing
authors.initials: Z
authors.lastname: Meng
authors.forename: Li Hua
authors.initials: LH
authors.lastname: Wang
authors.forename: Fa
authors.initials: F
authors.lastname: Liu
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Burke
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: William L
authors.initials: WL
authors.lastname: Farrar
mesh_terms: D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003545:Cysteine / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D047495:PPAR gamma; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D016335:Zinc Fingers
chemical_list: C097240:15-deoxy-delta(12,14)-prostaglandin J2; D004273:DNA, Neoplasm; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D047495:PPAR gamma; D004958:Estradiol; D003545:Cysteine; D015230:Prostaglandin D2