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Neuroendocrine Unit Massachusetts General Hospital and Department of Medicine Harvard Medical School Boston, Massachusetts 02114
This study examines the cooperative effects of a
human estrogen receptor-
(ER) isoform on estrogen
(E2)-mediated gene activation in U2-OS
osteosarcoma cells. 
5ER, an alternatively spliced ER variant
lacking exon 5, is coexpressed with normal ER in several
E2-responsive neoplastic tissues. However, the
potential interactions of 5ER with normal ER have not been
functionally characterized. 5ER 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 5ER protein is
prematurely truncated and lacks the majority of the hormone-binding
and activating function-2 (AF-2) domains. When 5ER mammalian
expression vector was transfected alone in human ER/ERß-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 ER
increased EREtk81Luc activity up to 20-fold in response to 10
nM E2. However, when
5ER was cotransfected with normal ER, both basal and
E2-stimulated EREtk81Luc reporter activation
were increased approximately 500% over levels observed when cells were
transfected with ER alone. Similar effects of 5ER and normal
ER coexpression were observed using an
E2-responsive human C3 promoter/luciferase
reporter construct. The effects of 5ER on normal ER were
further assessed in U2-OS cells stably transfected with normal ER.
Transfection of increasing amounts of 5ER expression vector into
[ER+]OS cells resulted in potentiation of
E2-stimulated ERELuc activity in a synergistic,
dose-dependent manner. Moreover, coexpression of 5ER in
[ER+]OS cells improved E2 sensitivity
100-fold over cells expressing ER alone. Proliferation rates of
stable U2-OS cell lines expressing 5ER were significantly
increased (P < 0.05), with cell doubling times
reduced from 35 h in control parental U2-OS cells to 28 h in
[5ER]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 ER and 5ER confirmed the
constitutive binding of 5ER and ER protein to
estrogen-response element (ERE) sequence independent of
E2 and also showed an increase in
5ER/ER-ERE complexes with E2
treatment. These data are consistent with interactive effects of normal
ER and 5ER on transcription from classic ERE gene promoters.
5ER appears to therefore act as a dominant positive receptor that
increases both basal and E2-stimulated gene
transactivation of normal ER.
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