Reduction of coactivator expression by antisense oligodeoxynucleotides inhibits ERα transcriptional activity and MCF-7 proliferation

Ilaria T R Cavarretta, Ratna Mukopadhyay, David M. Lonard, Lex M. Cowsert, C. Frank Bennett, Bert W. O'Malley, Carolyn L. Smith

Research output: Contribution to journalArticle

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 ERα 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 ERα 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. ERα-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 ERα transcriptional activity to an extent greater than individual asODN treatment alone, suggesting that these coactivators cooperate, in at least an additive fashion, to activate ERα-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 ERα biological action and avoids potential artifacts arising from overexpression of coactivators in transient transfection assays.

Original languageEnglish
Pages (from-to)253-270
Number of pages18
JournalMolecular Endocrinology
Volume16
Issue number2
DOIs
Publication statusPublished - 2002

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

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    Cavarretta, I. T. R., Mukopadhyay, R., Lonard, D. M., Cowsert, L. M., Bennett, C. F., O'Malley, B. W., & Smith, C. L. (2002). Reduction of coactivator expression by antisense oligodeoxynucleotides inhibits ERα transcriptional activity and MCF-7 proliferation. Molecular Endocrinology, 16(2), 253-270. https://doi.org/10.1210/me.16.2.253