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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
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ABSTRACT |
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 TOP ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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RESULTS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The efficiency of these boundary
elements was tested by stable cotransfection of the constructs
generated, and the pSV2Neo vector in the ER-positive MCF-7. 48
clones for each construct were isolated, expanded, and tested for
luciferase expression in the presence or absence of 1
nM E2 (Fig. 1B). In the
absence of hormone, luciferase activity could be measured in 77%
(37/48) and 40% (19/48) of the cells transfected with pHS4 and pMAR,
respectively. In about 80% of these (inducible clones), 16 h of
E2 treatment caused a significant increase in the
reporter activity (at least 3-fold over basal levels). When pERE was
transfected, basal luciferase activity could be detected only in 19%
(9/48) of the clones, and in 44% of these the enzymatic activity was
E2 inducible. Next, we evaluated the relationship
between the number of copies integrated and luciferase expression in
the absence or presence of E2. In the absence of
E2, linear correlation analysis of the two
variables produced lines of best fit with an r coefficient
of 0.66 for pHS4- and 0.79 for pMAR-transfected clones. After 16 h
of E2 induction, the r calculated was
0.47 and 0.54 for the two groups of clones. These values of
r indicate a positive correlation between the two variables
analyzed, even though these data most likely underestimate the
insulator activity because the clones in which transgene rearrangements
had occurred were not eliminated from the analysis.
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Effect of Insulators on Estrogen-Dependent Transcription of the
Reporter Gene in the Mouse
Linearized pMAR and pHS4 vectors deleted of plasmid sequences were
microinjected into oocytes explanted from C57Bl/6xDBA/2
F2 of mice zygotes. This outbred strain was
chosen to ensure a high efficiency of transgenesis (17); furthermore,
the presence of C57BL/6 in the genetic background confers a good
responsiveness to estrogens (18, 19). Seventeen independent lines were
obtained, but only 12 of these were fertile, 9 carrying the pMAR and 3
carrying the pHS4 construct. An initial screening for assessing basal
and estrogen-inducible expression of the luciferase reporter was done
by measuring the reporter enzymatic activity in tissue homogenates from
ovariectomized mice of the F1 generation. Five
organs were initially taken into consideration: uterus, liver and brain
as well known targets for the hormone, and lung and heart as negative
controls. Table 1 shows that among the
lines that integrated the MAR transgene, three showed an
estrogen-inducible expression of the reporter in uterus, brain, liver,
and lung. In line 31 the hormone-inducible expression of the reporter
was found in uterus, liver, and brain, while in lines 56 and 59 it was
restricted to brain. We did not detect any basal or estrogen-inducible
luciferase activity in the heart. In lines 13 and 77, basal expression
of the reporter is low; however, treatment with
E2 did not result in its increase. In transgenic
mice carrying the HS4 construct, we observed very little expression of
the reporter in the organs investigated; only line 61 showed low basal
and E2-induced expression of luciferase.
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Characterization of Estrogen-Dependent Luciferase Expression in
Transgenic Mice
A further characterization of the activity of the transgene
was carried out in line 2. Luciferase activity was measured in 26
different tissues from 2-month-old female mice, which had been
ovariectomized 2 weeks before the experiment. To verify the capability
of E2 to induce the transgene transcription, mice
were treated for 16 h with either vehicle or
E2 subcutaneously. Figure 2A shows that in the absence of hormonal
stimulation a considerable level of luciferase expression was found in
tissues such as bone marrow, brain, pituitary, liver, tongue, and
mammary gland, while in others the enzymatic activity found was low, at
the limit of detection. The hormonal treatment induced an increase of
the enzyme content higher than 5-fold with respect to controls in
liver, lung, spleen, bone marrow, brain, and thymus. In eye, uterus,
bladder, skin, adipocyte, and spinal cord, the hormonal treatment
resulted in an accumulation of luciferase less remarkable (between 2.5-
and 4.9-fold over controls), but still clearly visible. Finally, the
treatment did not result in any change in pancreas, tail, aorta,
esophagus, thyroid, stomach, blood, tongue, skeletal muscle, or heart
(Fig. 2B). When compared with the distribution of ER and -ß, the
distribution of luciferase activity indicated a strict correlation
between E2 responsiveness and presence of the
hormone receptors. Interestingly, the lung, which was originally taken
as a control ER-negative organ, showed a high responsiveness to the
hormonal treatment. This finding is in line with the recent
report on the high content of ERß in lung (20).
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or
luciferase (Fig. 3). ER
immunoreactivity was clearly detected in nuclei of cells in stroma,
endometrium, and glandular epithelium. Cytoplasmic staining of
luciferase was clearly visible in the same cell types. In both
cases, no staining was detected when preimmune serum was used.
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). The cells were treated for 16 h
with increasing concentrations of E2 (0.01–10
nM) or with 100 nM of two ER antagonists:
4-hydroxytamoxifen (T) and ICI 182,780 (ICI) alone or in the presence
of 1 nM E2. E2
induced a dose-dependent increase of luciferase accumulation blocked by
the presence of ICI 182,780. ICI 182,780 by itself did not
produce any effect. Conversely, 4-hydroxytamoxifen induced a
significant increase of luciferase levels even though lower than
E2 at the same concentration. In coadministration
with E2, 4-hydroxytamoxifen induced higher
luciferase accumulation, yet the level reached was still lower than
with E2 alone. This is compatible with the
partial agonist activity of 4-hydroxytamoxifen and with the fact that
it is present in the solution at a concentration 100-fold higher than
E2. As control, we also tested progesterone and
dexamethasone (10 nM). Neither ligand had any effect on the
ER reporter (Fig. 4).
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Pharmacological Modulation of Luciferase Expression in
Vivo
Two-month-old male mice were injected s.c. with 50 µg
E2/kg and killed after 3, 6, or 16 h. As
shown in Fig. 5 (upper panel)
the maximal luciferase accumulation was observed at 6 h after
treatment both in liver and bone tissues. When mice were treated for
6 h with increasing concentrations of the hormone (Fig. 5, middle panel), the maximal effect on luciferase activity was
detected at 50 µg/kg. Interestingly, the administration of 250 µg
E2 /kg induced, in the bone, a luciferase
accumulation lower then with 50 µg/kg. Thus, the luciferase
accumulation is time and dose dependent. Next, the effect of in
vivo administration of the two ER antagonists was investigated.
Figure 5 (lower panel) shows that the s.c administration of
250 µg tamoxifen/kg for 6 h increased the level of luciferase in
liver and bone 12 times and 7 times, respectively, confirming also
in vivo the partial agonist activity of tamoxifen in these
tissues. The injection of 250 µg tamoxifen/kg or ICI 182,780, 1
h before the administration of 50 µg E2/kg,
inhibited the E2-dependent activation of
luciferase expression as expected from the antagonist effect of these
compounds with respect to E2.
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DISCUSSION |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We believe that the key to the realization of our model was the use of insulators. It is, in fact, well known that the expression of transgenes driven by weak promoters is heavily influenced by enhancers/silencers surrounding the regions of insertion; in addition, methylation may gradually extinguish their transcriptional activity. In the past, insulators have been successfully used to counteract these effects in specific tissues. Here, we demonstrate that their use can be extended to the achievement of the ubiquitous and regulated expression of a given gene. Sixty percent of the transgenic lines obtained expressed luciferase in an estrogen-dependent fashion at least in some organ. In-depth analysis of one positive line showed that in 26 target tissues, the expression of the transgene is correctly regulated after in vivo administration of E2. Yet, in 40% of the mouse lines developed, the expression of luciferase was either undetectable or not modulated by E2.
This could be due to the use of a weak promoter, which might have slightly restricted the possibility of reaching detectable levels of reporter expression. The E2-independent expression of luciferase may be ascribed to rearrangements of the vector during the integration in the mouse genome. Indeed, also the study of stably transfected MCF-7 cells showed that in about 20% of the clones the expression of luciferase was insensitive to the presence of E2.
The system generated represents a major advancement for the understanding of the physiology of compounds active through the ERs. In the last decade, the ability to transfect cells in culture with reporters of ER transcriptional activity granted a novel insight into the complexity of estrogen action. It was shown that the binding of the hormone-receptor complex to the specific sequences of the promoter is not sufficient to ensure the hormone-regulated transcription of the target genes. The ER must, in fact, interact with a series of proteins modulating its transcriptional activity (22, 23). These findings were supported by crystallographic studies showing the structural conformation of ER bound to natural or synthetic ligands (24, 25). These and other investigations on steroid receptors demonstrated how synthetic ligands, by inducing specific structural conformations that modify the possibilities of the receptor to interact with its coregulators, may change its transcriptional activity in a tissuespecific fashion (10, 23, 26). In addition, several studies underlined that the binding of the specific hormone is not indispensable to ER transcriptional activation. Unliganded ER was shown to regulate the transcription of target genes after activation of specific kinases (27, 28). Finally, ER dosage may also constitute an important element in the control of ER tissue-specific activities.
A major challenge at present is to demonstrate how these mechanisms are relevant in physiological systems and how ER activity is regulated in its numerous target cells. The model generated will facilitate these studies by providing a system in which the activity of the receptor on ERE-containing genes can be assessed in a very restricted time frame. To this aim, we purposely made use of the natural firefly luciferase gene, the turnover of which in mammalian cells is about 3 h (29). By measuring the levels of luciferase accumulation, therefore, we will be able to monitor the state of activity of the receptor in response to the fluctuating hormone levels during the estrous cycle or after administration of ER ligands. In addition, these mice will allow identification of novel tissues and cell types targeted for the hormone in vivo in both sexes.
Further investigation is necessary to understand whether the high
content of luciferase in bone marrow, brain, tail, tongue, or liver
observed in this study should be ascribed to the tissue characteristics
facilitating the recovery/measurement of the luciferase enzymatic
activity, to the low catabolism of the exogenous protein, or to the
activation of the unliganded resident receptor via cross-coupling with
membrane receptors. We would rule out the possibility of luciferase
induction by ERR (ER-related receptor) orphan receptors based on the
observation that tissues such as kidney and heart, known to express
very high concentrations of ERR and 
, display a very low basal
activity of the reporter (30, 31, 32). Similarly, a more accurate
evaluation of the time course of E2 induction is
necessary before drawing any conclusion on the potency of the hormonal
treatment in the various organs. The present study was carried out at
16 h of hormonal treatment. It is likely that the relatively low
E2-dependent accumulation of luciferase that we
observed in certain organs (e.g. uterus, mammary glands) is
due to ER down-regulation, which in these organs occurs in a few hours
after E2 administration. Appropriate time-course
studies will better clarify the kinetics of ER activity in the various
tissues.
From the pharmacological point of view, the system generated is very interesting, particularly for the identification of novel SERMs because it will identify in which organs the molecule of interest displays full, partial agonist, or antagonist activity. The preliminary assessment of the activity of 4-hydroxytamoxifen in bone marrow cells and in vivo, supports the validity of this model in this type of studies. In previous studies, reporter-based systems for the in vivo identification of ligands for intracellular receptors were generated using fusion proteins between the RXR and RAR ligand binding domain and the DNA binding domain of the yeast protein GAL4 (33, 34). These systems were proved to be useful for the detection of endogenous ligands; however, because of the relevance of protein/protein interaction in the activity of intracellular receptors, GAL4-receptors fusion products might be unable to undergo conformational changes indispensable for the action of tissue selective synthetic ligands (10, 23, 26).
In spite of the fact that the system generated will not provide an
insight on the exact nature of the ER activated [ER, ERß, or
other proteins not known active through estrogen response elements
(EREs)], appropriate breeding of the ERE transgenic mice with
selective ER and -ß knockout (K.O.) mice will erase this
limitation.
The transgenic mouse generated in this study can be used to produce models for the physiological, pharmacological, and toxicological analysis of other intracellular receptors. In addition, because of the intrinsic characteristics of the reporter above specified, the model could be particularly suited for studying the pharmacokinetic profile of natural and synthetic ER ligands. Finally, these transgenic mice can be used as biosensors to investigate whether environmental or food pollutants act as endocrine disruptors by interfering with the physiological state of ER activity.
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MATERIALS AND METHODS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). This construct was
assembled with the following components: 1) the 2,731-bp DNA fragment
encoding the luciferase excised from the pGl2basic vector
(Promega Corp., Madison, WI) with the SalI
restriction enzyme blunted and ligated into the blunted
HindIII site of pBluescript; 2) the 168-bp
BamHI/XhoI fragment containing the tk
promoter from pBLCAT2 (37), blunted and ligated into the blunted
PstI site of the pBluescript; 3) the 82-bp
XhoI/ClaI fragment containing the ERE2X excised
from pGL2basic vector (Promega Corp.) in which it has been
previously cloned (see below), blunted, and ligated into the blunted
SalI site of the pBluescript. Two tandem copies of the insulators HS4 (2.4-kb DNA fragment) from chicken ß-globin gene were obtained by digesting the vector pBS(II)HS4, generously provided by S. Y. Tsai (38), with SalI restriction enzyme; a single copy of MAR (3-kb DNA fragment) from chicken lysozyme gene was excised with digestion of the pBSKMAR, kindly provided by L. Hennighausen (39), by XbaI/BamHI restriction enzymes. The insulator fragments were blunted and inserted in the blunted KpnI and NotI sites located at the 5'- and 3'-end of pERE, giving the pHS4 (EMBL accession no. AJ2777959) and pMAR (EMBL accession no. AJ2777959) constructs.
Generation of ERE2X
The two oligonucleotides, 5'-GATCCGCAGGTCACAGTGAC CTA-3' and
5'-GATCTAGGTCACTGTGACCTGCG-3', were annealed, the resulting double
strand oligo was ligated and digested with BamHI, and the
bands corresponding to monomers or multimers were extracted from an
acrylamide gel as described previously (40) and ligated into the
BglII site of pGL2basic vector.
Cell Cultures and Transfections
Breast carcinoma MCF-7, neuroblastoma SK-NBE, and cervix
carcinoma HeLa cell lines were routinely grown in RPMI 1640 medium
supplemented with 10% FBS. Stable transfections of MCF-7 cells were
performed with the calcium phosphate procedure as previously described
(41). Twenty four hours before transfection, 1.5 x
106 cells were seeded in Petri dishes with RPMI
1640 supplemented with 10% FCS; 6 h before addition of 1 ml
CaPO4/DNA mixture the medium was replaced with
DMEM supplemented with 10% FCS. The CaPO4/DNA
mixture used for transfection contained 1 ng of pSV2Neo plasmid
expressing G418 resistance gene (CLONTECH Laboratories, Inc., Palo Alto, CA; GenBank accession no. U02434) together with
10 µg of the pERE, pHS4, or pMAR vectors and 9 µg salmon sperm DNA.
Forty eight hours after transfection, 300 µg/ml G418 (Life Technologies, Inc.) were added to the culture medium. Medium and
selective agents were replaced three times a week. After 21 days
selection, 48 clones for each transfection were isolated with cloning
rings and expanded. To test the expression of luciferase, each clone
was grown in RPMI 1640 without phenol red and supplemented with 10%
dextran charcoal-stripped FBS (DCC-FBS) (41) for at least 1 week
before 16 h induction with E2 (1
nM in 0.00001% ethanol). Control cells were treated for
24 h with 0.00001% ethanol. Protein extracts were obtained as
previously described (27), and the enzymatic assay was carried out as
described in detail below.
Transgenic Mice
For microinjection, linearized pMAR and pHS4 constructs depleted
of plasmid sequences were obtained with BsshII restriction
enzyme digestion. With these vectors two different types of transgenic
mice were produced by pronuclear DNA injection of zygotes
C57Bl/6xDBA/2, F2 generation, using standard
procedures (17). Injected zygotes were reimplanted into pseudopregnant
B6D2F1 (C57Bl/6xDBA/2) foster mothers to complete their development.
Genomic DNA was extracted as previously described (42) from tail
biopsies and used for genotyping. Briefly, tissues were lysed by
addition of 1% SDS, 50 mM Tris-HCl, pH 8, and
200 µg/ml Proteinase K and incubation overnight at 37 C; DNA was then
purified by phenol extraction and ethanol precipitation. DNAs from the
founders and their littermates were screened by PCR analysis. PCR
amplification was carried out in a buffer containing 10
mM Tris-HCl (pH 8.0), 50 mM
KCl, 1.5 mM MgCl2, 0.2
mM deoxynucleotide triphosphates, 0.25
µM of each primer, and 2 U of TAQ polymerase
for 1 µg genomic DNA template. The primers used were
5'-GGCAGAAGCTATGAAACGAT-3' and 5'-CGACTGAAATCCCTGGTAAT-3'; after 30
cycles (30 sec at 95 C, 30 sec at 55 C, and 30 sec at 72 C) the
products were analyzed on 2.5% agarose gels stained with ethidium
bromide. At the third week of age, all the potential founders obtained
from pHS4 and pMAR microinjection were screened by PCR. From the pMAR
and pHS4 groups, 10 and 7 individuals, respectively, were identified as
positives for the presence of the transgene. For the experiments we
used heterozygous littermates obtained by mating our founders with
B6D2F1 wild-type mice.
Heterozygous female mice (2 months old) were ovariectomized and after 2 weeks injected s.c. with 50 µg E2/kg or with vehicle (vegetable oil) as control. Sixteen hours later the animals were killed, and the tissues were dissected and immediately frozen on dry ice. For the in vivo pharmacological studies with ER antagonists, 2-month-old heterozygous male mice were treated by s.c. injections of the different compounds dissolved in vegetable oil. Tissue extracts were prepared by homogenization in 500 µl of 100 mM KPO4 lysis buffer (pH 7.8) containing 1 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, 0.7 mM phenylmethylsulfonyl fluoride, three cycles of freezing-thawing, and 30 min of microfuge centrifugation at maximum speed. Supernatants, containing luciferase, were collected and protein concentration was determined by Bradford’s assay (43).
Luciferase Enzymatic Assay
Luciferase enzymatic activity in the cell and tissue extracts
was measured by a commercial kit (luciferase assay system,
Promega Corp.) according to the supplier indications. The
light intensity was measured with a luminometer (Lumat LB 9501/16,
Berthold, Wildbad, Germany) over 10 sec and expressed as relative light
units (RLU) over 10 sec/µg proteins.
Immunohistochemistry
Uteri of ovariectomized mice, treated as before, were dissected
and fixed through immersion in 4% paraformaldehyde in 0.1
M phosphate buffer pH 7.2 (PB), for 5 h. Tissues were
dehydrated with an ascending ethanol scale, clarified with xylene, and
processed for paraffin embedding. Serial 4 µm microtome sections were
cut and collected onto slides coated with poly-L-lysine
(Sigma, St. Louis, MO). After 16 h drying at 37 C,
sections were hydrated through a descending ethanol scale and boiled in
10 mM citrate buffer (pH 6.0) for 15 min in a microwave
oven, washed for 10 min with PBS, and then processed for luciferase and
ER immunodetection at room temperature. Sections were first
incubated for 30 min with 0.3%
H2O2 to quench endogenous
peroxidase activity and subsequently washed three times with PBS for 10
min. After saturation with 10% preimmune goat serum
supplemented with 0.3% Tween 20 (Sigma), sections
were incubated with the antiluciferase (Sigma, 1:1800
dilution in PBS with 10% goat serum and 0.3% Tween 20) or anti-ER
(kindly provided by J. Green, 5 µg/ml in 10% goat serum and 0.3%
Tween 20) polyclonal antibodies for 16 h, washed with PBS (six
times, 10 min each), incubated for 60 min with an antirabbit secondary
antibody (raised in goat, 1:200 dilution in PBS supplemented by 1%
goat serum and 0.3% Tween 20; Vector Laboratories, Inc.,
Burlingame, CA) and then washed again (six times with PBS, 10 min
each). Antibody-antigen detection was obtained by 40 min incubation
with avidin-biotin-horseradish peroxidase (HRP) from an ABC kit
(Vector Laboratories, Inc.). Immunostaining was visualized
by exposure to HRP substrate 3,3'-diaminobenzidine (DAB Fast, Tablet
Set, Sigma). After one wash in PBS and few tap water
changes, sections were allowed to air dry and then covered. Pictures
were taken with a digital camera (Coolpix 990; Nikon,
Melville, NY) applied to a Axioscope microscope (Carl Zeiss, Thornwood, NY).
Primary Bone Marrow Culture
After mice were killed, bone marrow cells were flushed out from
femur and tibia of ovariectomized animals, using a syringe filled with
PBS. Cells were collected in a 15 ml Falcon tube (Becton Dickinson and Co., Meylan Cedex, France) and washed once with
PBS. After centrifugation the cell pellet was resuspended in RPMI 1640
supplemented with 10% DCC-FBS; cells were counted and plated in a
six-well dish (2 x 106cells per well). For
the treatment, all compounds were dissolved in ethanol and added to the
medium at the indicated concentration. After 16 h the cells were
collected in Eppendorf tubes, washed once with PBS,
re-suspended in 100 µl KPO4 lysis buffer (as
above), and frozen and thawed three times. After 30 min microfuge
centrifugation at maximum speed, supernatants were collected for the
determination of protein concentration and luciferase activity.
Experimental Animals
Animal experiments performed in this study were conducted
according to the "Guidelines for Care and Use of Experimental
Animals."
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by the European Community Program BIOMED (Grant BMH4-CT97–2286); Telethon (Grant E.600); Italian Association for Cancer Research (AIRC); and CNR Targeted Project Biotechnology, Murst 40%.
Received for publication December 4, 2000. Revision received February 28, 2001. Accepted for publication March 19, 2001.
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER) and estrogen receptor-ß (ERß) messenger ribonucleic acid in
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K. Toda, Y. Okada, M. Zubair, K.-i. Morohashi, T. Saibara, and |
,
P < 0.05 as compared with the T-treated);
P values were calculated with ANOVA followed by
Scheffé test.
,
P < 0.01 as compared with the
E2-treated); P values were calculated with
ANOVA followed by the Scheffé test.