title: Reduction of coactivator expression by antisense oligodeoxynucleotides inhibits ERalpha transcriptional activity and MCF-7 proliferation.
abstract: Steroid receptor RNA activator (SRA) is a novel coactivator for steroid receptors that acts as an RNA molecule, whereas steroid receptor coactivator (SRC) family members, such as steroid receptor coactivator-1 (SRC-1) and transcriptional intermediary factor 2 (TIF2) exert their biological effects as proteins. Individual overexpression of each of these coactivators, which can form multimeric complexes in vivo, results in stimulated ERalpha transcriptional activity in transient transfection assays. However there is no information on the consequences of reducing SRC-1, TIF2, or SRA expression, singly or in combination, on ERalpha transcriptional activity. We therefore developed antisense oligodeoxynucleotides (asODNs) to SRA, SRC-1, and TIF2 mRNAs, which rapidly and specifically reduced the expression of each of these coactivators. ERalpha-dependent gene expression was reduced in a dose-dependent fashion by up to 80% in cells transfected with these oligonucleotides. Furthermore, treatment of cells with combinations of SRA, SRC-1, and TIF2 asODNs reduced ERalpha transcriptional activity to an extent greater than individual asODN treatment alone, suggesting that these coactivators cooperate, in at least an additive fashion, to activate ERalpha-dependent target gene expression. Finally, treatment of MCF-7 cells with asODN against SRC-1 and TIF2 revealed a requirement of these coactivators, but not SRA, for hormone-dependent DNA synthesis and induction of estrogen-dependent pS2 gene expression, indicating that SRA and SRC family coactivators can fulfill specific functional roles. Taken together, we have developed a rapid method to reduce endogenous coactivator expression that enables an assessment of the in vivo role of specific coactivators on ERalpha biological action and avoids potential artifacts arising from overexpression of coactivators in transient transfection assays.
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3498, USA.
authors.forename: Ilaria T R
authors.initials: IT
authors.lastname: Cavarretta
authors.forename: Ratna
authors.initials: R
authors.lastname: Mukopadhyay
authors.forename: David M
authors.initials: DM
authors.lastname: Lonard
authors.forename: Lex M
authors.initials: LM
authors.lastname: Cowsert
authors.forename: C Frank
authors.initials: CF
authors.lastname: Bennett
authors.forename: Bert W
authors.initials: BW
authors.lastname: O'Malley
authors.forename: Carolyn L
authors.initials: CL
authors.lastname: Smith
mesh_terms: D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D051548:Histone Acetyltransferases; D006801:Humans; D056920:Nuclear Receptor Coactivator 1; D051958:Nuclear Receptor Coactivator 2; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D062085:RNA, Long Noncoding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D047628:Estrogen Receptor alpha; D004967:Estrogens; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D016376:Oligonucleotides, Antisense; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D022661:RNA, Untranslated; D011960:Receptors, Estrogen; D014157:Transcription Factors; C405423:steroid receptor RNA activator; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1