A tumor-specific truncated estrogen receptor splice variant enhances estrogen-stimulated gene expression.

Abstract

This study examines the cooperative effects of a human estrogen receptor-alpha (ERalpha) isoform on estrogen (E2)-mediated gene activation in U2-OS osteosarcoma cells. Delta5ERalpha, an alternatively spliced ERalpha variant lacking exon 5, is coexpressed with normal ERalpha in several E2-responsive neoplastic tissues. However, the potential interactions of delta5ERalpha with normal ERalpha have not been functionally characterized. Delta5ERalpha encodes the hormone-independent trans-activating function (AF-1), as well as the constitutive receptor dimerization and DNA-binding domains. It is generated by an alternate splice event that omits exon 5 and alters the reading frame of the resulting mRNA. The delta5ERalpha protein is prematurely truncated and lacks the majority of the hormone-binding and activating function-2 (AF-2) domains. When delta5ERalpha mammalian expression vector was transfected alone in human ERalpha/ERbeta-negative osteosarcoma U2-OS cells, it had no effect on either basal or E2-mediated EREtk81Luc reporter transcriptional activity, while transfected cells expressing control normal ERalpha increased EREtk81 Luc activity up to 20-fold in response to 10 nM E2. However, when delta5ERalpha was cotransfected with normal ERalpha, both basal and E2-stimulated EREtk81Luc reporter activation were increased approximately 500% over levels observed when cells were transfected with ERalpha alone. Similar effects of delta5ERalpha and normal ERa coexpression were observed using an E2-responsive human C3 promoter/luciferase reporter construct. The effects of delta5ERalpha on normal ERalpha were further assessed in U2-OS cells stably transfected with normal ERalpha. Transfection of increasing amounts of delta5ERalpha expression vector into [ERalpha+]OS cells resulted in potentiation of E2-stimulated ERELuc activity in a synergistic, dose-dependent manner. Moreover, coexpression of delta5ERalpha in [ERalpha+]OS cells improved E2 sensitivity 100-fold over cells expressing ERalpha alone. Proliferation rates of stable U2-OS cell lines expressing delta5ERalpha were significantly increased (P < 0.05), with cell doubling times reduced from 35 h in control parental U2-OS cells to 28 h in [delta5ERalpha]OS cells. However, growth rates were not affected by either E2 or tamoxifen treatment. Electromobility shift/supershift assays using nuclear extracts of U2-OS cells stably transfected with ERalpha and delta5ERalpha confirmed the constitutive binding of delta5ERalpha and ERalpha protein to estrogen-response element (ERE) sequence independent of E2 and also showed an increase in delta5ERalpha/ERalpha-ERE complexes with E2 treatment. These data are consistent with interactive effects of normal ERalpha and delta5ERalpha on transcription from classic ERE gene promoters. Delta5ERalpha appears to therefore act as a dominant positive receptor that increases both basal and E2-stimulated gene transactivation of normal ERalpha.