ABSTRACT
The ability of steroid receptor RNA activator (SRA), an AF-1 coactivator, to contribute to differences in estrogen receptor (ER)-alpha and ERbeta transcriptional activity was tested. In transient transfections, SRA expression increased ERalpha- and ERbeta-dependent gene expression. However, when the receptors' amino-terminal A/B regions were examined as GAL4 DNA binding domain fusions, SRA enhanced the activity of GAL-ABalpha but not GAL-ABbeta. Exogenous SRA also enhanced AF-2 activity for both receptors, indicating that SRA effects are not limited to AF-1. Simultaneously mutating three phosphorylation sites within GAL-ABalpha domain only modestly reduced SRA coactivation of GAL-ABalpha, suggesting that phosphorylation does not play a major role in SRA function relative to this domain. SRA enhanced ERalpha activity stimulated by 4-hydroxytamoxifen, but was unable to convert this mixed antiestrogen to an ERbeta agonist. Thus, SRA is an ERalpha AF-1-specific coactivator that enhances the agonist activity of tamoxifen-bound ERalpha and may contribute to tamoxifen resistance.
Subject(s)
Drug Resistance, Neoplasm , Estrogen Receptor alpha/metabolism , RNA, Untranslated/physiology , Tamoxifen/analogs & derivatives , Tamoxifen/agonists , Tamoxifen/pharmacology , Blotting, Western , Cell Line , DNA/metabolism , Estrogen Receptor beta/metabolism , Gene Deletion , Gene Expression Regulation , HeLa Cells , Humans , Mutation , Phosphorylation , Plasmids/metabolism , Protein Structure, Tertiary , RNA, Long Noncoding , RNA, Untranslated/metabolism , Transcription, Genetic , TransfectionABSTRACT
Although increases in intracellular cAMP can stimulate estrogen receptor-alpha (ER alpha) activity in the absence of exogenous hormone, no studies have addressed whether ER beta can be similarly regulated. In transient transfections, forskolin plus 3-isobutyl-1-methylxanthine (IBMX), which increases intracellular cAMP, stimulated the transcriptional activities of both ER alpha and ER beta. This effect was blocked by the protein kinase A inhibitor H89 (N-(2-(p-bromocinnamylamino)-ethyl)-5-isoquinolinesulfonamide) and was dependent on an estrogen response element. A 12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 5' to the estrogen response element was necessary for cAMP-dependent activation of gene expression by ER beta but not ER alpha, indicating that the former subtype requires a functional interaction with TRE-interacting factor(s) to stimulate transcription. Both p160 and CREB-binding protein coactivators stimulated cAMP-induced ER alpha and ER beta transcriptional activity. However, mutation of the two cAMP-inducible SRC-1 phosphorylation sites important for cAMP activation of chicken progesterone receptor or all seven known SRC-1 phosphorylation sites did not specifically impair cAMP activation of ER alpha. The E/F domains of ER alpha are sufficient for activation by forskolin/IBMX, and this is accompanied by an increase in receptor phosphorylation. In contrast, cAMP signaling reduces the phosphorylation of the corresponding region of ER beta, and this correlates with the lack of forskolin/IBMX stimulated transcriptional activity. Our data suggest that cAMP activation of ER alpha transcriptional activity is associated with receptor instead of SRC-1 phosphorylation. Moreover, differences in the cofactor requirements, domains of ER alpha and ER beta sufficient for forskolin/IBMX activation, and the effect of cAMP on receptor phosphorylation indicate that this signaling pathway utilizes distinct mechanisms to stimulate ER alpha and ER beta transcriptional activity.
