Received for publication, November 30, 1999, and in revised form, April 26, 2000
Although it is well established that estrogen
deficiency causes osteoporosis among the postmenopausal women, the
involvement of estrogen receptor (ER) in its pathogenesis still remains
uncertain. In the present study, we have generated rats harboring a
dominant negative ER
, which inhibits the actions of not only ER
but also recently identified ER
. Contrary to our expectation, the
bone mineral density (BMD) of the resulting transgenic female rats was
maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was
observed almost equally in both groups. Strikingly, however, the BMD of
the transgenic female rats, after ovariectomized, remained decreased
even if 17-estradiol (E2) was administrated,
whereas, in contrast, the decrease of littermate BMD was completely
prevented by E2. Moreover, bone histomorphometrical
analysis of ovariectomized transgenic rats revealed that the higher
rates of bone turnover still remained after treatment with
E2. These results demonstrate that the prevention from the
ovariectomy-induced bone loss by estrogen is mediated by ER
pathways and that the maintenance of BMD before ovariectomy might
be compensated by other mechanisms distinct from ER and ER pathways.
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INTRODUCTION |
Postmenopausal osteoporosis is characterized by increased bone
resorption due to estrogen deficiency (1, 2). The supplement of
estrogen markedly prevents the disease (3). Action of estrogen is
generally supposed to be mediated by estrogen receptor
(ER),1 a member of the
nuclear receptor superfamily (4-6). However, the precise mechanism of
ER responsible for the bone metabolism remains to be understood.
Both classical ER and recently identified ER (7) were
differentially expressed during the osteoblast differentiation, implying their functional roles in the bone metabolism (8, 9). Several
studies have suggested that the estrogen action mediated by ER cause
the mineralization of bone-forming cells, the osteoblasts (10-12). On
the other hand, it is reported that the estrogen response element
(ERE)-mediated transcription is not necessarily observed in the
mineralizing process, suggesting the existence of the estrogen-induced
but ER-independent signaling pathways (13).
In contrast to the report of a man homozygous for ER gene mutation
who suffered from severe osteoporosis based on the increased bone
turnover (14), ER knockout (ERKO) mice (15) were shown to
decrease in their skeletal mineralization compared with wild-type mice
to a small extent (16, 17). The unclear phenotypes of ERKO mice in
the bone may be partly because of the possible compensation of
ER-mediated cascade by ER, as well as the involvement of the
so-called non-genomic action of estrogen. These possibilities are also
suggested in the vascular walls of ERKO mice, which show a response
to estrogen similar to that of wild-type mice when they were injured
(18, 19).
We have recently demonstrated that a dominant negative mutant of human
ER constructed by C-terminal truncation, namely ER-(1-530), blocked both ER and ER signaling pathways (20). In order to clarify the roles of ERs in exerting the physiological effects of
estrogen, we introduced the expression vector encoding the corresponding mutant of rat ER, namely rER-(1-535), into rats, thereby inhibiting both ER and ER signaling pathways in
vivo. Taking advantage of the analytical merits of rat as a
species for the physiological and the histomorphometrical studies over that of mice (21, 22), we studied effects of estrogen on the bone
metabolism using these transgenic rats. As a result, we have established a transgenic animal model with the impaired estrogen sensitivity in the bone, exhibiting the decreased mineralizing response
after ovariectomy followed by 17-estradiol (E2) administration.
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EXPERIMENTAL PROCEDURES |
Production of Transgenic Rats--
The PCR-amplified products of
rat ER-(1-535) (cDNA fragment encoding amino acids from 1 to
535) (23) were cloned into pCXN2 vector (24) that has chicken -actin
promoter to construct pCXN2-ER-(1-535). The obtained DNA sequence
was compiled and confirmed using DNASIS computer programs (Hitachi Co.,
Tokyo, Japan). The construction of transgenic rats were described
elsewhere (22). Briefly, insert of pCXN2-ER-(1-535) digested with
BamHI was microinjected into the male pronuclei of
fertilized eggs from Wistar rats. Injected eggs were transferred to
oviducts of pseudopregnant rats. Three independent founders, each with
intact copies of transgene, were identified by Southern blot analysis.
Two lines, designated line 3 and line 5, were able to transmit the
transgenes to their offspring and were bred out into permanent lines.
The copy numbers of the integrated transgenes were 10 and 8, respectively.
Study Protocol--
Sixty 6-month-old Wistar rats, weighing
approximately 300 g each, were used in this study. The rats were
acclimated to the local vivarium conditions (24 °C; 12-h light/12-h
dark cycle), allowed free access to water and a pelleted commercial
diet (PMI Feeds, Inc.) containing 0.60% calcium, 0.40% phosphorus,
and 2.2 IU/g vitamin D3. All the experimental protocols
were approved by the Animal Research Committee of Saitama Medical
School. Two weeks prior to the operation, all rats were changed to be
fed a pelleted low calcium diet (PMI Feeds, Inc.) containing 0.02% calcium instead of 0.60%.
The rats were sham-operated or bilaterally ovariectomized, and they
were received subcutaneous daily injection of E2 (10 µg/kg body weight, Sigma) suspended in corn oil as vehicle or of only vehicle for 4 weeks. Double bone labelings were performed by the injection with demeclocyclin (Sigma) and calcein (Sigma) both at a dose
of 15 mg/kg body weight, given intraperitoneally as described (25, 26).
After 4 weeks of treatment, the rats were sacrificed by cardiac
puncture under ether anesthesia. Blood was obtained by cardiac
puncture, allowed to clot at 4 °C, and then centrifuged at 2000 × g for 10 min. In all experiments, uteri were isolated and
wet uterine weights were measured to confirm the effects of treatment.
Reverse Transcription-PCR (RT-PCR)--
cDNA was synthesized
from 0.1 µg of rat poly(A)+ RNA of primary
osteoblast-like cells using random 9-mers and avian myeloblastosis virus reverse transcriptase (TaKaRa, Kyoto, Japan). Subsequent PCR
amplification was carried out by the RNA PCR kit (TaKaRa) for 30 cycles
using an annealing temperature of 55 °C in a Perkin Elmer
thermalcycler (Perkin Elmer-Cetus, Norwalk, CT). The oligonucleotides 5'-CTCTTGGACAGGAATCAAGG-3' and 5'-TAGAGAGGCACGACATTCTT-3' were used for
amplification of 386-bp fragment of ER mRNA (23). The
ER-(1-535) should not be amplified by this set of primers. The
oligonucleotides 5'-CATCAGTAACAAGGGCATGG-3' and
5'-CACTGAGACTGTAGGTTCTG-3' were used for amplification of 192-bp
fragment of ER mRNA (7). The oligonucleotides
5'-CTCTTGGACAGGAATCAAGG-3' and 5'-CAGTGGTATTTGTGAGCCAG-3' were used for
amplification of 455-bp fragment of ER-(1-535) mRNA
specifically. The plasmids containing rat ER (23) and ER (7) were
used for positive controls of PCR reactions.
Northern Blot Analysis--
Total RNA was prepared from the
tissues and primary osteoblast-like cells using acid guanidinium
thiocyanate-phenol-chloroform extraction as described previously (27).
For each sample, 10 µg of total RNA was separated in 1% agarose.
Northern blot analysis was performed as described previously (28). The
32P-labeled, 500-bp,
EcoRI-NotI-digested fragment of
pCXN2-rER-(1-535) was used as a probe. Autoradiography was carried
out at 
80 °C with an intensifying screen for 1 day.
Cell Transfection and Whole Cell Extract
Preparation--
Osteoblast-like cells were isolated by three
sequential enzymatic digestion as described previously (29). Briefly,
21-day-old rat embryo calvariae were incubated at room temperature for
20 min with gentle shaking of an enzyme solution containing 0.1% collagenase, 0.05% trypsin, and 4 mM EDTA in
phosphate-buffered saline. Only the cells released from the fourth to
sixth consecutive digests were cultured separately with -modified
minimum essential medium supplemented with 10% fetal calf serum and
with antibiotics (100 µg/ml streptomycin and 100 IU/ml penicillin G),
and the second passage of the cells were used for experimental
determinations. COS-7 cells were maintained in Dulbecco's modified
Eagle's medium without phenol red, supplemented with 10%
dextran-coated charcoal-stripped fetal calf serum. The 5 × 105 cells in 10-cm Petri dishes were transfected with a
total of 15 µg of plasmids using DOTAP (Roche Molecular
Biochemicals). Cells were harvested 36 h after transfection and
whole cell extracts were prepared using 1× Passive lysis buffer
(Promega, Madison, WI).
Luciferase Assays--
Luciferase assay was performed using Dual
Luciferase Reporter Assay System (Promega) as described (30). Briefly,
5 × 105 cells were transfected with a total of 15 µg of DNA. Two µg of ERE-TK-LUC reporter plasmid was co-transfected
with indicated amount of receptor expression vectors. All assays were
performed in the presence of 2 µg of pRL-TK, a Renilla
luciferase reporter plasmid, as an internal control (Promega). The
total amount of DNA and expression vectors for transfection was
adjusted using pGEM3Zf (Promega) and pCXN2, respectively. After a 12-h
incubation, the cells were washed with fresh medium and incubated for
an additional 24 h in the absence or presence of
10
7 M E2. Cell
extracts were assayed for luciferase activity by Luminoskan (Labsystems
Inc., Beverly, MA).
Western Blot Analysis--
Twenty µg of proteins were
subjected to Western blot analysis using the 6F11 mouse monoclonal
antibody against ER (Novocastra Laboratories Ltd.) and rabbit
polyclonal antibody against ER as described (28, 31), using the
chemiluminescence-based ECL detection system (Amersham Pharmacia
Biotech) according to the manufacturer's instruction.
Bone Size and Bone Mineral Measurements--
The left femur from
each rat was removed at autopsy and scanned using dual energy x-ray
absorptiometry (QDR 1000/W, Aloka, Japan) equipped with Regional High
Resolution Scan software, with antero-posterior application of the
radiation beams to the specimens using two energy x-ray beams (27 KeV
and 53 KeV) with 1-mm beam width. The scan speed was 2 mm/s. The
femoral scan images were analyzed, and the value of the BMD of distal
one-third region of the femur was determined.
Histology--
The proximal right tibia specimens were refixed
with 70% ethanol and embedded in methyl methacrylate after Goldner's
staining. With a microtome (model 2050 Supercut; Reichert-Jung,
Heidelberg, Germany), 8-µm-thick midfrontal sections of
proximal tibia were obtained.
Bone Histomorphometry--
Histomorphometry was performed as
described (26), using a semiautomatic image analyzing system linked to
a light microscope (Cosmozone 1S, Nikon, Tokyo, Japan). In the tibial
specimen, the metaphyseal cancellous bone area located within 4.0 mm
distal to the growth plate-metaphyseal junction was measured. The
parameters of trabecular bone volume (BV/TV, %) and trabecular bone
surface (BS, mm) were measured. For indices of bone formation, double- and single-labeled perimeters (dLS/BS, sLS/BS, %) and the interlabel distance (µm) were measured on the trabecular perimeter. The
mineralizing surface (MS/BS, %) was calculated as the sum of the
double-labeled surface and half of the single-labeled surface. The
mineral apposition rate (MAR, µm/day) was calculated as the mean
distance between double labels divided by the interval labeling time
and multiplied by 
/4, and the bone formation rate (BFR/BS,
µm3/µm2/day) was expressed per unit of bone
surface. The parameter of trabecular osteoclast surface (OcS/BS, %)
was measured.
Serum Parameters--
Serum calcium, phosphorus levels were
determined with an autoanalyzer (Hitachi 7170). Plasma osteocalcin
levels were determined by a two-site sandwich enzyme immunoassay system
using anti-rat osteocalcin. Serum E2 levels were determined
by an radioimmunoassay kit (Diagnostic Products Co.).
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RESULTS |
Construction of the Transgenic Rats--
We introduced the
expression vector encoding rER-(1-535), the corresponding rat
mutant of human ER-(1-530), into rats (Fig. 1A). Chicken -actin
promoter (32) was adopted for expressing rER-(1-535) in various
kinds of tissues. Three independent founders were identified by
Southern blot analysis. Two lines, designated line 3 and line 5, respectively, were able to transmit the transgenes to their offspring
and were bred out into permanent lines. The F1 offspring obtained by
the back-cross of the founder rats with normal rats were used for
subsequent studies. The fertility and lactation in offspring of these
founders were not markedly different from the wild-type rats. The copy
numbers of the integrated transgenes, determined by comparison with the
endogenous ER, were 10 and 8 in line 3 and line 5, respectively
(Fig. 1B). RT-PCR analysis detected the rER-(1-535)
mRNA in various tissues including bone of the F1 transgenic female
rat (line 3), as well as the endogenous expression of ER and ER
(Fig. 2A). The similar
expression pattern was detected in the F1 transgenic female rat of line
5 (data not shown). Especially in primary osteoblast-like cells of the
transgenic embryos, we demonstrated the expression of the
rER-(1-535) at the mRNA level by Northern blotting (Fig.
2B), as well as the protein level as a 59-kDa band by
Western blotting (Fig. 2D). Endogenous ER and ER
mRNA was demonstrated in primary osteoblast-like cells of both
transgenic rats and their littermates by RT-PCR (Fig. 2C),
whose protein could not detected by Western blotting (Fig.
2D).
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Fig. 1.
Structure of the transgene and its expression
pattern. A, schematic structure of the transgene is
presented. The transgene is under control of the chicken -actin
promoter with cytomegalovirus immediate-early (CMV-IE)
enhancer, and includes the dominant negative ER (rER-(1-535))
cDNA, and rabbit -globin polyadenylation site. The box represent
gene or cDNA fragments. B, Southern blot analysis of
tail DNA from heterozygous offspring of the two founders that were
successfully bred. The copy number was indicated for each line of
transgenic rats. The EcoRI-NotI-digested 500-bp
fragment of the rat ER cDNA was used as a hybridization probe.
The 2.9-kilobase pair signal observed in the wild-type and transgenic
rats represent the endogenous ER.
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Fig. 2.
Expression of the
rER-(1-535) transcript in various rat
tissues. A, RT-PCR analysis with poly(A)+
RNA from various rat tissues using specific primers for ER-(1-535),
endogenous ER, and ER, respectively. Expression of
ER-(1-535), ER, and ER transcripts (455, 386, and 192 bp,
respectively) was analyzed by RT-PCR with 0.1 µg of
poly(A)+ RNA as described. The RT-PCR products were
analyzed by agarose gel electrophoresis. B, Northern blot
analysis using primary osteoblast-like cells derived from wild-type and
transgenic embryos. Total RNA was hybridized with
32P-labeled, EcoRI-NotI-digested
fragment of the rat ER cDNA probe. Each lane contained 10 µg
of total RNA and g;yceraldehyde-3-phosphate dehydrogenase was used for
the internal control. C, RT-PCR analysis from
poly(A)+ RNA of rat primary osteoblast-like cells using
specific primers for endogenous ER and ER. Expression of ER
and ER transcripts (386 and 192 bp, respectively) was analyzed by
RT-PCR with 0.1 µg of poly(A)+ RNA as described. The
RT-PCR products were analyzed by agarose gel electrophoresis.
D, determination of rER-(1-535) by immunoblot analysis
of primary osteoblast-like cells. Twenty µg of whole cell extracts
were resolved by SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose filter, and blots were probed with the ER-specific
monoclonal antibody (6F11, 1:100) and ER-specific polyclonal
antibody (1:500), respectively.
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In Vivo Dominant Negative Activity of rER-(1-535)--
First,
the inhibitory effect of rER-(1-535) was confirmed against
wild-type ER and ER transactivation in COS-7 cells. In the
presence of E2, equivalent co-expression ratio of the
former leads to nearly the basal level of transactivation by ER and ER, respectively (Fig. 3, A
and B). Next, when the primary osteoblast-like cells
isolated from the transgenic embryo calvariae and those of the
littermate were transfected with ER or ER expression vector
exogenously, an inhibitory effect of rER-(1-535) was also demonstrated (Fig. 3C). Endogenous E2-induced
transactivation was shown as about 1.8-fold induction in the
littermate-derived osteoblast-like cells, but it was lost in the
transgenic osteoblast-like cells, indicating the dominant negative
effect of transgene against ER and ER transactivation in
vivo (Fig. 3D).
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Fig. 3.
Dominant negative activities of
rER-(1-535) in the ER
and ER signaling pathways.
A, dose-dependent inhibition of the
rER-(1-535) against ER transactivation. The COS-7 cells were
transfected with 0.03 µg of the ER expression vector and the
rER-(1-535) expression vector at the indicated amounts (0.03-0.3
µg) in the presence (+) or absence () of
107 M E2. Luciferase
activities are indicated as means ± standard deviations,
calculated from three independent experiments. B, inhibitory
effect of rER-(1-535) against the transactivation by ER. The
experimental conditions were the same as A, except that the
COS-7 cells were transfected with 0.03 µg of the ER expression
vector instead of ER expression vector. ER, ER, and
ER-(1-535) are the abbreviations of pCXN2-rER, pCXN2-rER, and
pCXN2-rER-(1-535), respectively. C, the inhibition of
rER-(1-535) in the primary calvarial osteoblast-like cells
transfected with 0.1 µg of the ER and ER expression vectors,
respectively. D, transactivation of the ERE-TK-LUC reporter
in the primary osteoblastic-like cells. The primary calvarial
osteoblastic-like cells were transfected with 10 µg of ERE-TK-LUC
reporter plasmid. The transfected cells were incubated for 24 h in
the presence (+) or absence () of 107
M E2, and luciferase activities were measured
and are shown as means ± standard deviations (-fold inductions),
calculated from three independent experiments.
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Radiographic and Histological Evaluation of Impaired Estrogen
Sensitivity in Bone--
We then examined the dominant negative
effects of the transgene product against both ER and ER signaling
pathways with respect to the bone mineral properties in
vivo, one of the most important targets of estrogen. The distal
femur BMD of the sexually mature 7-month-old female transgenic rats and
their littermates were investigated by dual energy x-ray
absorptiometry. As shown in Fig.
4A, the BMD of sham-operated
rats exhibited no significant difference among transgenic 2 lines and
littermate rats. Ovariectomy caused the decrease of BMD in transgenic
rats to the same extent as the littermates. The decrease of BMD of the
control littermate rats due to ovariectomy was completely prevented in
response to E2 administration. Strikingly however, the BMD
of the transgenic rats of both line 3 and line 5 remained decreased
even after treatment with E2. When intact
non-ovariectomized transgenic rats (n = 5) and their
littermates (n = 5) were treated with E2,
the distal femur BMD exhibited no significant difference (data not
shown). The representative radiographic analyses of the femurs were
compared in Fig. 4B, which supported the dual energy x-ray
absorptiometry results that the transgenic femur failed to be prevented
from the ovariectomy-induced bone loss in response to E2
administration. Representative fluorescent micrographs of the
calcein-labeled mineralization fronts (green) were also
shown in Fig. 4C. Seven-month-old transgenic rats, compared
with their littermates, showed the marked decrease in calcified
cancellous bone of proximal tibia under the
ovariectomized+E2 conditions, whereas no apparent
histological difference was observed when they were sham-operated or
ovariectomized.
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Fig. 4.
Bone mineral property of transgenic
rats. A, distal femur BMD (g/cm2) measured
in 7-month-old sham-operated (sham, bright
shaded box), ovariectomized (OVX,
dark shaded box), and ovariectomized
and E2 treatment (OVX+E2,
black box) rats. Values of measurements are
means ± standard deviations (n = 5-6).
Significance of difference between groups was evaluated with Student's
t test; *, p < 0.05, significantly
different from the sham-operated rats within the each strain; **,
p < 0.05, significantly different from the
ovariectomized rats within each strain. B, radiological
analysis of the bones of the transgenic and the littermate rats.
Representative x-ray of femurs of transgenic rats (line 3) and those of
control littermates of 7-month-old are shown. C,
histological evaluation of cancellous bone morphology in 7-month-old
wild-type and transgenic rats. Longitudinal sections were made through
the proximal tibia and fluorescent micrographs of the double-labeled
mineralization fronts were shown. Only calcein label is visible
(green), and the demeclocyclin label (yellow) is
too faint to detect at this magnification.
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Histomorphometrical Evaluation of the Bone Phenotypes--
To
investigate the effects of the inhibition of both ER and ER
signaling pathways in the bone in detail, the histomorphometrical analysis was performed by double labeling with calcein and
tetracycline, markers of the amount of newly formed bone (33). When
sham-operated, the cancellous bone volume (BV/TV) of the proximal tibia
displayed no significant difference between transgenic rats and their
littermates (Fig. 5A).
Ovariectomy significantly reduced BV/TV of transgenic rats and their
littermates (Fig. 5A). The BV/TV was maintained in control
littermates by E2 supplement; however, it remained decreased in both lines of the transgenic rats even if E2
was administrated after ovariectomy (Fig. 5A). This finding
is in accordance with the BMD changes of the distal femur. The
cancellous bone formation rate (BFR/BS) as well as the osteoclast
surface as percentages of total bone surface (OcS/BS) were increased
after ovariectomy both in transgenic rats and the littermates,
indicating a high bone turnover state (Fig. 5, B and
C). Interestingly, only transgenic rats in both lines
sustained rather high values of BFR/BS and OcS/BS in response to
E2 administration (Fig. 5, B and C),
keeping a high turnover state.
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Fig. 5.
Bone remodeling rate and trabecular density
changes in the transgenic rats. Bars represent
means ± standard deviation. Significance of difference between
groups was evaluated with Student's t test. For 7 months,
n = 5-6; *, p < 0.05, significantly
different from the sham-operated rats within the each strain; **,
p < 0.05, significantly different from the
ovariectomized rats within each strain. A, quantitative
representation of the trabecular bone density in the metaphyseal region
of the tibia expressed as percentages of the total tissue area
(BV%TV). B, measurement of bone-formation rate
in the transgenic and the littermate rats. C, osteoclast
surface as percentages of bone surface (OcS%BS) in the
tibial metaphysis.
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Increased Serum E2 Concentration in Sham-operated
Transgenic Rats--
The average body and uterine weight revealed no
significant difference in any treated groups between transgenic and
littermate rats (Table I). The serum
osteocalcin values in the ovariectomized littermate rats were
significantly higher than the values in the sham-operated group.
However, the significant increase of the serum osteocalcin level did
not decline when treated with E2 in both transgenic lines.
In contrast to the sham-operated littermates, the serum level of
E2 was significantly higher (about 1.4-fold) in
sham-operated transgenic rats, suggesting impaired negative feedback
due to E2 insensitivity. Ovariectomy decreased and
E2 administration increased the serum E2 level
without any difference in all groups. The serum calcium and phosphorus
levels were not significantly changed among any treated groups.
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Table I
Body and uterine weights, blood chemistry, and bone metabolic markers
in wild-type and transgenic rats
OVX, ovariectomized. Values are means ± S.E. (n = 5) and were obtained in intact control and transgenic rats.
*, p < 0.05 between sham and OVX rats within
each group;  , p < 0.05 between OVX and
OVX + E2 rats within each group;  ,
p < 0.05 between sham-operated wild-type and
transgenic rats. All these differences are evaluated by two-sided
Student's t test.
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DISCUSSION |
In this study, we have established the rats antagonizing both
ER and ER pathways in vivo and demonstrated for the
first time that the ovariectomy-induced bone loss was prevented by the ER-mediated signaling pathways.
Southern blot analysis of the DNA from each of the transgenic rat line
indicated different sites of insertion of the transgene (data not
shown). This allows us to conclude that the bone phenotypes observed
were consequences of expression of transgene rather than insertional
mutagenesis. The rER-(1-535) transcript was expressed in various
kinds of tissues including bone. Assuming that the expression of the
transgene remains rather constant throughout the target tissues, the
relative ratio of transgene product to endogenous product should become
greater in the tissues where the expression of endogenous ERs is lower.
For example, in transgenic primary osteoblast-like cells, the
rER-(1-535) protein detected by Western blotting is abundant enough
to cause the complete inhibition of the
E2-dependent transactivation by endogenous ERs.
We could not detect the endogenous ER proteins in osteoblast-like cells using anti-ER and anti-ER antibodies by Western blotting (data not shown). Thus, it is suggested that the expression of the endogenous ER proteins confirmed by functional analysis here are under threshold levels of Western blot analysis. 1.4-fold higher serum E2
level in transgenic rats, although it may not seem so remarkable
compared with ERKO mice, is statistically significant and reflects
the reduced estrogen responsiveness. In the case of human, only a man
with a point mutation of ER was reported (14) and his serum E2 level (119 pg/ml) was remained approximately twice that
the normal male serum concentration (50 pg/ml). The absence of
reproductive abnormalities including uterine weight in transgenic rat
might be partly because the differences in the dose-response curves for
ER agonism and antagonism by the dominant negative transgene (34).
Further studies are required for clarifying the ER-mediated transcriptional inhibition in other cells and tissues.
The bone mineral measurements clearly revealed that the
ovariectomy-induced bone loss was prevented through ER-mediated
signaling pathway, and the data presented here are consistent with the
hypothesis that ER plays a major role in the bone physiology (16).
Histomorphometrical analysis supports the mechanism that the prevention
from the high bone turnover due to ovariectomy was mediated by the ER
cascades. The serum osteocalcin levels of the transgenic rats remained
increased after ovariectomy followed by E2 administration,
reflecting the high turnover state where both bone formation and its
resorption are accelerated (25, 35, 36). The reasons why the difference of BMD was not observed between sham-operated transgenic rats and their
littermates still remain to be resolved. Taken together with that the
bone density in the femurs is not significantly decreased in the
ERKO (16, 37) as well as ERKO (38, 39) female mice, the
compensation of BMD might be achieved by ER-independent mechanisms such
as the bioactivity of estrogen-regulated cytokines or by a novel
estrogen-responsive pathway distinct from ER and ER. This
possibility is also supported from our results; the intact transgenic
rats and littermates exhibited no difference in the distal femur BMD in
the presence of pharmacological (100 nM) E2
concentration. Alternatively, it may be possible that the dominant
negative activity of rER-(1-535) is not enough to inhibit ER-mediated pathways completely in this condition.
Several studies have focused on the possible involvement of bone
resorbing cytokines such as interleukin-1 (IL-1) (40), interleukin-6
(IL-6) (41), and tumor necrosis factor (42) in the stimulation of bone
resorption due to estrogen deficiency (43-45). Estrogen is supposed to
suppress the production of these cytokines by osteoblasts and bone
marrow stromal cells (44, 45). Although substantial lines of evidence
regarding the protective effects against the bone loss caused by
ovariectomy have been accumulated from the several transgenic animal
models such as IL-6 (33), IL-1 receptor type I (IL-1R1) (46) knockout
mice, and soluble tumor necrosis factor- receptor type I transgenic mice (35), specific contribution of these cytokines to the bone loss
due to estrogen deficiency still remains controversial. Moreover, whether ERs are directly responsible for the regulation of these estrogen-regulated cytokines is completely unknown.
Our results demonstrated that the inhibition of ER and ER still
induced bone loss after ovariectomy to the same degree as that of
littermates. If we assume that the dominant negative activity is
sufficient to inhibit ER pathways as shown in the case of
E2 treatment after ovariectomy, the regulation of the
bone-resorbing cytokines by estrogen may be involved in
ovariectomy-induced bone loss rather than the classical
ER/ERE-dependent regulation. Alternatively, ovariectomy may
cause some changes other than the decline of E2 concentration in vivo, influencing the crucial effects in
the bone metabolism. These possibilities are also supported by the results of the ERKO (37, 47) and ERKO (39) mice showing the bone
loss due to ovariectomy.
In the present study, we have created a transgenic rat line with
impaired estrogen sensitivity by expressing a dominant negative mutant
against both ER and ER in bone and other tissues. Taking advantage of the analytical usefulness of the rat as species, we thus
opened a way to examine the physiological roles of estrogen through
ER-dependent and ER-independent signaling pathways.
Moreover, this study provides a mechanism for diseases caused by the
dominant negative phenotypes of the mutations of nuclear receptors
including ER. In practice, some patients with thyroid hormone
resistance are caused by the heterozygous mutation of thyroid hormone
receptor to its dominant negative mutant (48, 49). The forthcoming analyses of the bone phenotypes of the ER/ double-knockout mice and the comparison with our animal model described here will give us
further insights into the physiological and pathophysiological actions
of estrogen in the bone metabolism.
We thank Dr. J. Å. Gustafsson for the gift
of rat ER plasmid. We are grateful to M. Suzuki and K. Horikiri
(Saitama medical School) for breeding rats.
The abbreviations used are:
ER, estrogen
receptor;
ERE, estrogen response element;
RT-PCR, reverse transcription
PCR;
BMD, bone mineral density;
BFR, bone formation rate;
BV, bone
volume;
TV, trabecular bone volume;
BS, trabecular bone surface;
dLS, double-labeled perimeter;
sLS, single-labeled perimeter;
MS, mineralizing surface;
MAR, mineral apposition rate;
OcS, trabecular
osteoclast surface;
bp, base pair(s);
IL, interleukin;
E2, 17-estradiol.
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ABSTRACT
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