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Institute of Pharmacological Sciences (P.C., G.D.L., S.B.,
G.P., E.V., A.M.) University of Milan 20133 Milan,
Italy
Regina Elena Institute (L.T., C.T.) 00158 Rome,
Italy
In addition to their well known control of
reproductive functions, estrogens modulate important physiological
processes. The identification of compounds with tissue-selective
activity will lead to new drugs mimicking the beneficial effects of
estrogen on the prevention of osteoporosis and cardiovascular or
neurodegenerative diseases, while avoiding its detrimental
proliferative effects. As an innovative model for the in
vivo identification of new selective estrogen receptor modulators
(SERMs), we engineered a mouse genome to express a luciferase reporter
gene ubiquitously. The constructs for transgenesis consist of the
reporter gene driven by a dimerized estrogen-responsive element (ERE)
and a minimal promoter. Insulator sequences, either matrix attachment
region (MAR) or ß-globin hypersensitive site 4 (HS4), flank the
construct to achieve a generalized, hormoneresponsive luciferase
expression. In the mouse we generated, the reporter expression is
detectable in all 26 tissues examined, but is induced by
17ß-estradiol (E2) only in 15 of them, all
expressing estrogen receptors (ERs). Immunohistochemical studies show
that in the mouse uterus, luciferase and ERs colocalize. In primary
cultures of bone marrow cells explanted from the transgenic mice and
in vivo, luciferase activity accumulates with increasing
E2 concentration. E2
activity is blocked by the ER full antagonist ICI 182,780. Tamoxifen
shows partial agonist activity in liver and bone when administered to
the animals. In the mouse system here illustrated, by biochemical,
immunohistochemical, and pharmacological criteria, luciferase content
reflects ER transcriptional activity and thus represents a novel system
for the study of ER dynamics during physiological fluctuations of
estrogen and for the identification of SERMs or endocrine disruptors.
. At the present time,
molecules active through estrogen receptors (ERs) are used in fertility
control, endocrine dysfunction, and cancer therapy. In postmenopausal
women, estrogen replacement therapy (2) was proven efficacious for the
prevention of osteoporosis (3), and several lines of study suggested
that 17ß-estradiol (E2) has beneficial effects
in cardiovascular (4, 5) and selected neurodegenerative diseases (6).
Unfortunately, the prolonged use of this hormone has been associated
with increased risk of breast and uterine cancer (7). The discovery
that synthetic ligands of the ER may exhibit tissue-specific agonist or
antagonist activity raised a new interest in the use of these compounds
for estrogen replacement therapy (8, 9). These selective estrogen
receptor modulators or SERMs are identified by comparative screening in
cells of different origin to characterize their tissue-specific profile
(agonist/antagonist). Generally, the study is carried out in
transformed cell lines stably or transiently transfected with ER
or
-ß and a reporter of the receptor’s activated state. In addition to
limiting the analysis to a selected number of cells, this method may
also provide erroneous or defective results. In fact, the
tissue-specific agonist/antagonist activity of SERMs has been
attributed to the presence of cell-specific proteins capable of
interacting with the hormone receptor complex (10), and these proteins
may be aberrantly expressed in cancer cells (11). Thus, the major
shortcoming of this screening procedure is associated with the
requirement of further in vivo analysis for the
identification of the pharmacodynamic properties of the molecule to be
developed. The availability of an engineered mouse carrying an ER
reporter expressed ubiquitously as a transgene would represent a
remarkable advancement for the identification and profiling of new
SERMs. In addition, such a model would be invaluable for the
spatio-temporal localization of ER activity and could provide data of
major impact for the full comprehension of estrogens and ER functions
from development to aging. Such an experimental system can hardly be
generated by classical transgenesis because of the difficulty in
obtaining a regulated expression of the transgene (12). To overcome
this limitation, we made use of insulator sequences previously
described to oppose the interference of the host genome on the
expression of the ectopic genes (13, 14).
In this study we describe a construct that led to ubiquitous and estrogen-regulated expression of a reporter transgene. The transgenic mouse we generated represents an innovative model for the study of the in vivo dynamics of intracellular receptor activity.
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