ER
Gene Expression in Human Primary Osteoblasts: Evidence for the Expression of Two Receptor Proteins
Stefanie Denger,
George Reid,
Martin Ko
,
Gilles Flouriot,
Dominik Parsch,
Heike Brand,
Kenneth S. Korach,
Vera Sonntag-Buck and
Frank Gannon
European Molecular Biology Laboratory (S.D., G.R., M.K., H.B.,
V.S.-B., F.G.), 69117 Heidelberg, Germany; Endocrinologie
Moléculaire de la Reproduction (G.F.), UPRES-A CNRS 6026, Campus
de Beaulieu, 35042 Rennes cedex, France; Department of Orthopedics
(D.P.), University of Heidelberg, 69118 Heidelberg, Germany; and
National Institute of Environmental Health Sciences (K.S.K.), Research
Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Stefanie Denger, EMBL, Meyerhofstrasse 1, 69012 Heidelberg, Germany. E-mail:
denger{at}embl-heidelberg.de
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ABSTRACT
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The beneficial influence of E2 in the maintenance of healthy bone
is well recognized. However, the way in which the actions of this
hormone are mediated is less clearly understood. Western blot analysis
of ER
in osteoblasts clearly demonstrated that the well
characterized 66-kDa ER
was only one of the ER
isoforms present.
Here we describe a 46-kDa isoform of ER
, expressed at a level
similar to the 66-kDa isoform, that is also present in human primary
osteoblasts. This shorter isoform is generated by alternative splicing
of an ER
gene product, which results in exon 1 being skipped with a
start codon in exon 2 used to initiate translation of the protein.
Consequently, the transactivation domain AF-1 of this ER
isoform is
absent. Functional analysis revealed that human (h)ER
46 is able to
heterodimerize with the full-length ER
and also with ERß. Further,
a DNA-binding complex that corresponds to hER
46 is detectable in
human osteoblasts. We have shown that hER
46 is a strong inhibitor of
hER
66 when they are coexpressed in the human osteosarcoma cell line
SaOs. As a functional consequence, proliferation of the transfected
cells is inhibited when increasing amounts of hER
46 are
cotransfected with hER
66. In addition to human bone, the expression
of the alternatively spliced ER
mRNA variant is also detectable in
bone of ER
knockout mice.
These data suggest that, in osteoblasts, E2 can act in part through an
ER
isoform that is markedly different from the 66-kDa receptor. The
expression of two ER
protein isoforms may account, in part, for the
differential action that estrogens and estrogen analogs have in
different tissues. In particular, the current models of the action of
estrogens should be reevaluated to take account of the presence of at
least two ER
protein isoforms in bone and perhaps in other
tissues.
 |
INTRODUCTION
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OSTEOPOROSIS AFFECTS ONE third of the
postmenopausal female population and arises as a consequence of
accelerated bone loss that follows menopause or ovariectomy. The
development of osteoporosis can be prevented by early hormone
replacement therapy (1, 2). However, the detailed cellular
and molecular mechanisms underlying the positive effect that estrogens
have in maintaining bone density are poorly defined. Estrogens act to
decrease bone resorption and by doing so, contribute to the prevention
of osteoporosis. E2 binds to specific intracellular receptors, the ERs.
To date, two ERs (ER
and ERß), encoded by different genes, have
been characterized (3, 4, 5). Both belong to the nuclear
receptor superfamily of ligand-inducible transcription factors that
include the steroid, thyroid hormone, and retinoic acid receptors.
Nuclear receptors regulate gene expression by interacting directly with
cognate DNA sequences [responsive elements (6, 7)] or
through protein/protein interactions with other transcriptional factors
(8, 9). ERs are modular proteins and can be subdivided
into six distinct regions (AF) (10) based on homology
with other members of the nuclear hormone superfamily. Regions C and E
are responsible for DNA and hormone binding, respectively. The A/B
region contains a ligand-independent transactivation domain
[activating function 1 (AF-1)] whereas a hormone-inducible
transcription activating function (AF-2) is present in the
hormone-binding domain.
Antiestrogens, such as tamoxifen and raloxifene [also known as
selective ER modulators (SERMs)] are clinically used to block the
actions of estrogen in ER-positive breast tumors while conferring
agonist-like effects in bone tissue (11, 12).
It was recently shown that the ER
gene in human (h), chicken (c),
and mouse (m) is a complex genomic unit showing differential promoter
usage and alternative splicing (13, 14, 15). This results in a
cell-specific variation in the expression level of the ER
gene in
different tissues. Generally, the consequence of alternative splicing
has been the expression of the classical and well studied 66-kDa ER
(hER
66). In addition, we have recently described a 61-kDa ER
(cER
61) that is present in chicken liver and which is transcribed
from a specific promoter (16). However, in contrast to the
isoforms described in this study, the responsible promoter is located
in a region downstream from the translation start site in exon 1 of the
66-kDa chicken ER
. The resulting cER
61 protein is characterized
by an increased basal level of ligand-independent activity
(16). However, the internal ATG that is responsible for
the expression of cER
61 is exclusively conserved in oviparous
species and is not found in mammals.
In this study we evaluated the expression profile of ER
present in
human osteoblasts and show that an alternative hER
isoform, 46 kDa
in size (hER
46), is expressed in osteoblasts at a significant level
and constitutes a significant proportion of the total ER
. Analogous
studies showed that MCF-7 cells also have the potential to express
hER
46 (17), albeit to a lower level than in
osteoblasts. The hER
46 arises from an alternative splicing event
that excludes the first exon of hER
. The differential expression of
this isoform in primary osteoblasts suggests alternative explanations
for the differential action of SERMs in a variety of tissues.
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RESULTS
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Selective Amplification and Quantitative Analysis of ER
cDNA
Demonstrates the Expression of Alternatively Spliced Receptor
Variants
To determine the promoter(s) used in the expression of hER
in
osteoblasts and in bone tissue, RT-PCR analysis was performed using
specific upstream primers from the different 5'-exons of ER
(AF)
(13). The only mRNA isoforms detected using RNA directly
isolated from bone tissue or from cultivated primary osteoblast cells
were derived from the F-hER
promoter (Fig. 1B
). It should be noted that exon 1F
splices to exon 1E before splicing to a common splice site within exon
1 at position +163. Therefore, all F-hER
variants include a partial
sequence of exon 1E. All exon-specific 5'-untranslated regions (UTRs)
(AF) were detected in the MCF-7 mRNA. Primary smooth muscle cells,
isolated from the medial layer of human aorta and used as an
alternative primary culture, showed a PCR band with A- and C-specific
primers, whereas no signal was obtained with the other variants tested
(Fig. 1B
). In the osteoblast-like cell line SaOs, both E-hER
and
F-hER
variants were detectable. S1 nuclease protection results
confirmed that the F-hER
mRNA variant was the only ER
mRNA
transcript observed in osteoblasts (Fig. 2
) and indicates that the F-hER
mRNA
plays the major role in the generation of ER
in bone.
As is clear from RT-PCR analysis, shorter products of F-hER
cDNAs
were amplified from primary osteoblasts, SaOs and bone tissue. To
characterize these products, an S1 nuclease protection analysis,
designed to detect potential alternative internal splicing where the
F-transcript spliced directly to exon 2, was performed. It was found
that this alternative mRNA splice variant was present in osteoblast
cells and also in MCF-7 cells (Fig. 3
).
The resulting F-hER
mRNA transcripts from exon 2 to exon 8 are
therefore identical to the previously described full-length
transcript. In contrast to MCF-7, where splicing of exon 1F to
exon 2 represents only a minority of the transcripts (as shown by the
ratio of the specific splice fragment to that of all other forms of
ER
), this splice form corresponds to approximately 50% of total
transcripts in osteoblasts. These data were also confirmed by a
quantitative real-time RT-PCR approach (Fig. 3B
), where it is
demonstrated that hER
66 mRNA levels in MCF-7 cells are 200-fold more
abundant as compared with primary osteoblasts. The alternative splicing
event of F splicing to exon 2 results in the formation of an mRNA
transcript that encodes hER
46 and represents a minor component in
MCF-7 cells, whereas in primary osteoblasts this splicing event
generates 50% of total transcripts coding for ER
. In light of this
observation, it was then investigated whether this alternative splicing
event resulted in the expression of an alternative ER
protein at
significant levels.
Alternative Splicing Events Result in the Expression of ER
Isoforms in Human Primary Osteoblasts
Two internal ATG codons, in frame with the remainder of the hER
open reading frame, occur in exon 2 at positions +752 and +758.
Examination of the sequence surrounding these ATGs showed that both
have a favorable Kozak sequence for translation initiation
(18). Use of either of these ATGs would result in a hER
protein with a predicted size of approximately 46 kDa lacking the first
173 amino acids of the hER
66. Human ER
cDNA starting from exon 2
was cloned into an eukaryotic expression vector (pSG5) to test whether
this protein could be expressed. As a positive control, the hER
cDNA
expression vector (HEO), which generates a hER
protein of 66 kDa,
was used. Analysis of the in vitro translation products that
resulted from HEO (IVT66) showed a protein of 66 kDa (hER
66) and a
shorter hER
protein of 46 kDa (hER
46) at a significantly lower
level. This shorter hER
isoform was the only product expressed by
pSGhER
46, confirming that translation could begin from
the downstream initiation codon. To investigate whether the high levels
of F/2 transcript correspond to the expression of a hER
46 protein
isoform in vivo, immunoprecipitation of radiolabeled total
protein from osteoblasts using the ER
-specific H222 monoclonal
antibody [which is directed against the hormone-binding domain
of the hER
protein] was performed. As shown in Fig. 4
, the alternative isoform hER
46 is
expressed to a very significant level in human osteoblasts.
Densitometric evaluation of the hER
66 and hER
46 protein levels
showed similar amounts of each in osteoblasts. Surprisingly, another
protein of approximately 39 kDa was also immunoprecipitated. The origin
of this band is currently under investigation. In summary, the hER
F
promoter is the predominant promoter used by osteoblasts, and this
results in the expression of either the 66-kDa hER
protein or a
46-kDa hER
isoform that lacks the A/B domain of the full-length
receptor.
Heterodimerization of hER
46 with hER
66 and ERß
ER acts as a dimer when it activates transcription via DNA
binding. To ascertain whether hER
46 is a functionally active ER
protein isoform, the ability of hER
46 to form homodimers or
heterodimers with hER
66 and with hERß that are able, in turn, to
bind to a responsive element [estrogen response element (ERE)] was
evaluated in vitro by EMSAs. Heterodimers were synthesized
in vitro by rabbit reticulocyte lysates using extracts
containing equimolar concentrations of constructs encoding
hER
66/hER
46, hER
66/hERß, or hERß/hER
46. Homodimers were
generated by including the appropriate single constructs. As shown in
Fig. 5
, these in vitro
translated extracts were able to form stable DNA/protein complexes with
a radiolabeled consensus ERE. The specificity of these complexes was
confirmed by supershift experiments using antibodies directed against
the N terminus (ER21) and C terminus (H222) of ER
. The potential to
form hER
66/hERß heterodimers (19) was confirmed.
Interestingly, hER
46 was able to form heterodimers with both
hER
66 and hERß in addition to forming homodimers. A shift of the
heterodimer complexes hER
46/hER
66 and hER
46/hERß was
observed using the H222 monoclonal antibody. As H222 does not recognize
ERß, the supershift of hER
46/hERß complexes is due to H222
binding to the hER
46 component of the heterodimer. A complex of
intermediate mobility was generated when hER
46 and hER
66 were
cotranslated, resulting from the formation of hER
46/hER
66
heterodimers. When the complexes were incubated with the polyclonal
antibody ER21, which is directed against the N terminus of ER-
, a
supershift was detectable only in complexes where hER
66 was present
(hER
66 homodimers and hER
66/46 heterodimers, respectively). Due
to the lack of the A/B domain, hER
46 homodimers were not
supershifted in the presence of ER21. Although the hERß protein is
not expressed at detectable levels in osteoblasts (Denger, S.,
manuscript in preparation), the formation of hER
46/hERß complexes
that are detectable in vitro could be involved in other
cell- and tissue-specific ER signaling processes in which hER
and
hERß are synthesized concomitantly (21).

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Figure 5. In Vitro Heterodimerization of
hER 46 with hER 66 and hERß
Plasmids (0.75 µg) containing either vector alone (pSG5),
pSGhER 46, pSGhER 66, or hERß were in vitro
transcribed and translated using the reticulocyte lysate system. Four
microliters of each product were incubated with 60,000 cpm of labeled
ERE. Specificity was determined by supershift using monoclonal H222
antibody, directed against the C terminus of ER (A) and the
polyclonal antibody ER21, which is directed against the N terminus (B).
Both antibodies show no cross-reactivity to ERß. The unspecific
band corresponds to the vector (pSG5) alone. The positions of the
specific hER 46, hER 66, and ERß homodimers complexed with
ERE, the heterodimers consisting of hER 46/66, hER 66/ß,
or hER 46/ß bound to ERE as well as the supershifts are indicated
by arrows.
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hER
46 Is Expressed in Primary Human Osteoblasts ex
Vivo and Binds to an ERE
We evaluated, using a gel shift assay, which ER
protein
isoforms that are expressed in human osteoblasts ex vivo
bind to an ERE. In vitro translated constructs coding
for hER
66 and hER
46 served as controls. Interestingly, a
homodimer complex in the same region of hER
46 was detectable after
incubation of whole cell extracts of osteoblasts with radiolabeled ERE
in addition to higher and lower complexes (Fig. 6
), demonstrating that the shorter ER
isoform is functionally active and can bind to an ERE. In contrast, no
hER
66/ERE complex was detectable in extracts prepared from
osteoblasts. This is in agreement with other results, which show that
the hER
46 has a higher affinity for DNA compared with the
full-length receptor and, consequently, hER
46 homodimers are
preferentially formed as shown in Fig. 5
(Denger, S., and G.
Flouriot, unpublished results). The formation of the additional smaller
complex could be due to homodimerization of the 39-kDa isoform that was
seen in immunoprecipitation of osteoblast ER
protein, although this
complex needs to be further characterized (Fig. 6
).
Inhibition of hER
66 Transactivation by hER
46 in SaOs
Both hER
66 and hER
46 receptor isoforms were evaluated in
transient transfection assays using a luciferase reporter gene
construct in which two EREs are placed upstream of the thymidine kinase
minimal promoter (ERE-tk-Luc). The osteosarcoma cell line SaOs was used
in these experiments as primary osteoblasts proved refractory to
transfection. As hER
46 is devoid of the A/B domain, it lacks one of
the major transactivation functions (AF-1) of hER
66. The results
show that hER
46 can transactivate ERE-mediated reporter gene
expression in an estrogen-dependent manner in this cell context,
although to a lower extent than to hER
66 (Fig. 7
). To assess the ability of hER
46 to
influence hER
66 transactivation of reporter genes, both hER
receptors were cotransfected into SaOs cells in various ratios. The
results obtained demonstrated that hER
46 acts as a potent inhibitor
of hER
66 transactivation with a dose- dependent mode of action.
In addition, the effects of the SERM 4-hydroxytamoxifen (4-OHT) on the
transactivation capability of both isoforms were evaluated. While 4-OHT
had no effect on reporter gene activation of hER
46, a moderate
transactivation of hER
66 was detectable in the presence of 4-OHT. In
contrast to the result in the presence of E2, increasing concentrations
of hER
46 had only a minor inhibitory effect on luciferase expression
modulated by hER
66 in the presence of 4-OHT, which indicates a
distinct, differential action of SERMs on these two ER
isoforms. In
SaOs, hER
46 was unable to transactivate luciferase reporter gene
expression from an ERE in response to 4-OHT, in keeping with the
described agonistic effects of 4-OHT that are mediated through AF-1
(22). In contrast, hER
46 was able to transactivate
reporter gene expression in response to E2 through AF-2. These results
show that transactivation of reporter genes in SaOs is mediated by both
transactivation domains, AF-1 and AF-2.
Cotransfection of hER
66 and hER
46 Results in Decreased
Proliferation of SaOs Cells in a Dose-Dependent Manner
We next addressed whether the coexpression of both hER
isoforms
would have a functional impact on SaOs cells. Proliferation of the
cells was evaluated after immunostaining using an antibody directed
against the proliferation marker Ki67 (MIB). As shown in Fig. 8
, proliferation of SaOs cells was
slightly increased when low concentrations of hER
46 were
cotransfected with a constant amount of hER
66. When higher
concentrations of hER
46 were cotransfected, proliferation decreased
in a dose-dependent manner. This suggests that hER
46 acts as a
regulatory element that can control the proliferative activity of
hER
66.
To rule out that the inhibitory action of hER
46 was due to a
squelching effect, we performed transfection experiments with a total
of 300 ng of DNA, a concentration that had been previously reported to
be an optimal concentration for maximal transactivation without an
inhibitory effect in SaOs cells (23). Similar to the
previous results shown in Figs. 7
and 8
, we observed increased cellular
proliferation at low ratios of hER
46 to hER
66, followed by a
decrease when the ratios transfected increased such that hER
46 was
the predominant isoform present (data not shown). Therefore, we
conclude that a squelching effect does not occur under these
experimental conditions and that the observed effects are due to a
synergistic effect of hER
46 when present at low concentrations and
an inhibitory activity when this isoform is the predominant ER
present.
hER
46 Is Expressed in ERKO Bone
The generation of a mouse knockout model for ER
(
ERKO) has
been a useful tool to study the impact of ER on different tissues
including bone. It was assumed that the
ERKO mouse would be
completely devoid of ER
expression due to the insertion of a
neomycin cassette in exon 1 (24). As it has been
demonstrated that alternative splicing events in human bone result in
the formation of hER
46 (Fig. 4
), the question of whether a
corresponding mER
variant could be expressed in
ERKO mice was
addressed. Therefore, an RT-PCR analysis of primary
ERKO mouse bone
cells using primers from the 5'-untranslated exon 1F and 3' primers
from exon 2 of the mER
coding region was performed using sequences
obtained for the mER
(15). PCR amplification resulted
in a fragment of 250 bp. Using Southern blotting, the amplification
products were screened with oligonucleotides corresponding to exon 1
and to exon 2 of mER
, respectively. As shown in Fig. 9
, Southern blotting resulted in a
positive signal when the PCR products were probed with an
oligonucleotide primer from exon 2. Alternatively, a primer sequence
from exon 1 was used as a probe in keeping with the construct used for
the generation of the
ERKO mouse. No hybridization of the smaller
product was detectable when a primer from exon 1 was used as a probe in
ERKO tissue, whereas the wild-type control resulted in a positive
signal. Thus an alternatively spliced mRNA, which potentially encodes a
protein equivalent to the hER
46, also exists in wild-type and in
ERKO mice.
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DISCUSSION
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The importance of ER
in human bone metabolism is supported by
the report of a man unresponsive to estrogen as a consequence of a
homozygous mutation in both ER
alleles that results phenotypically
in a decrease in bone mineral density and in an increase in bone
turnover (25). Sequence analysis revealed an identical
mutation of a single base pair within the second exon of both copies of
his hER
gene, causing a premature stop codon upstream of the start
codon for the hER
46. Despite the importance of ER in a wide range of
tissues, this mutation did not result in lethality to the individual.
We show evidence that the mild effects observed may be explained by the
potential expression of hER
46 in this individual due to the
alternative splicing event to exon 2. It is possible that this
surprising phenotype is due to a compensating role played by hER
46,
which can be generated within this individual.
A detailed analysis of the ER status in bone has not been reported to
date but is required particularly as there is a growing trend to use
hormone replacement therapy or SERMs for the treatment and prevention
of osteoporosis. Such studies are hampered by the fact that bone tissue
consists of a mixture of cell types and by the low expression of ER. In
this study, we have focused predominantly on osteoblasts obtained from
primary culture of human bone. Using methods of greater sensitivity,
the relative roles of different ER
isoforms have been clarified.
Previous work described the existence of a new 46-kDa isoform of ER
(17). This hER
46 protein isoform is identical to the
well characterized hER
66 (3) except that it lacks the
A/B domain and therefore is devoid of AF-1 activity. However, as the
DNA-binding and ligand-binding domains are not affected by deletion of
this region, hER
46 may act as a ligand-inducible transcription
factor (17). S1 nuclease protection assays and
immunoprecipitation indicate that hER
46 mRNA and protein are
expressed at a level similar to hER
66 in osteoblasts. This contrasts
with the previous study in MCF-7 in which hER
46 was a minor
component and shows that hER
46 has the potential to be a major
element in some tissues (perhaps not only in osteoblasts) as the
mediator of SERM or hormone action. These findings provide, at least in
part, a potential explanation for the selective action of E2 and SERMs
in a variety of physiological and pathological processes. In the light
of these results, the role of ER in bone must be re-evaluated to take
into account the presence of all ER
protein isoforms that can
influence bone metabolism.
In this paper we show that both hER
isoforms are functionally
active. In addition to having the capacity to transactivate a
luciferase reporter through an ERE, hER
46 was also able to influence
the proliferation of SaOs cells in a dose-dependent manner (Fig. 8
).
Dimerization of full-length ER
and ERß has been previously
demonstrated (19). Our results further show that in
vitro heterodimerization of hER
46 and hER
66 complexes can
occur. The results shown in Fig. 6
demonstrate that the hER
46/46
homodimer and hER
46/66 heterodimer preferentially bind to the ERE.
This is also reflected in the extract prepared from osteoblasts, in
which shorter isoforms of ER
appear to preferentially bind to the
ERE.
The demonstration of the significant level of expression of hER
46 in
osteoblasts may shed a light on open questions regarding how estrogens
act in a cell-specific manner in bone and in other tissues. Obviously,
the 66-kDa ER
receptor is only one component of the pool of ER
proteins in osteoblasts. Recently, we have demonstrated that hER
46
expression is not restricted to osteoblasts but is also present in
other cells. For example, hER
46 is expressed in MCF-7 cells,
although a different expression profile of both hER
isoforms occurs
compared with bone, as a consequence of other promoters being used
preferentially in these cells (17). Internal ribosome
entry in exon 2 of the full-length mRNA that encodes hER
66 can also
result in the generation of hER
46. Such a mechanism has recently
been described (26) and is likely to be responsible for
the presence of low amounts of hER
46 that are seen when plasmid DNA
coding for hER
66 is translated in vitro (Fig. 5
). This
indicates that alternative splicing of exon 1F to exon 2 is the major
mechanism by which hER
46 is generated in osteoblasts, although both
mechanisms result in the formation of hER
46. In osteoblasts,
however, hER
66 and hER
46 are present at similar levels, as are
the respective mRNA species that directly encode these proteins. Other
tissues need to be reexamined to determine the levels of hER
46 as
this can have functional consequences.
Recent studies indicate that ERß does not make a significant
contribution to the actions of estrogens in bone (20). The
current data suggest that at least two isoforms of ER
must be
considered in the response of osteoblasts to estrogens. However,
hER
46 can also form heterodimers with ERß, suggesting that a broad
functional activity of this shorter ER
isoform within ER-mediated
signaling in a tissue-specific manner is possible. Therefore, although
the ER
/ERß function plays a minor role in osteoblasts, heterodimer
formation of ER
and ERß may reflect the in vivo
situation in other tissues such as testis (20) or during
embryogenesis (27), where both receptors are
expressed.
The N-terminal domain of hER
is important for ligand-independent
activation through the MAPK pathway (28). The hER
46
isoform lacks the N-terminal 173 amino acids. However, ligand-inducible
transactivation occurs despite the deletion of the A/B domain in
SaOs cells. Therefore, hER
46 can act as a transcription factor that
can bypass this cytokine- induced signaling while maintaining
intact the ligand-dependent activation property of hER
(Fig. 7
).
Other experiments show that, in some cellular contexts, such as HepG2,
where transactivation domain 1 (AF-1) is predominantly used in ER
signaling, hER
46 does not have a transactivation effect
(17). In contrast, analysis of hER
46-mediated
transactivation demonstrated that a cell context such as HeLa, which is
mainly sensitive to AF-2, showed that hER
46 induced transcriptional
activity in a ligand-dependent manner to a level similar to hER
66
(17). The relative contributions of both AF-1 and AF-2 on
transcriptional control is known to vary in a cell- and
promoter-specific manner (29, 30). One consequence of
these data and of the present study is that the presence of an AF-2
domain in hER
appears to be sufficient to activate transcription
after binding of ligand. The presence of AF-2 can therefore provide a
sufficient response to E2 to allow, albeit limited, function and
development. In transient transfection experiments performed in this
study, coexpression of hER
46 and hER
66 revealed that hER
46 can
act as a powerful competitor that efficiently suppresses the
transactivation capacity of hER
66 in SaOs cells in a dose-dependent
manner. These results show that, in SaOs cells, transfected ER
isoforms mediate their action via both AF-1 and AF-2 and consequently
hER
46 is able to transactivate ERE-mediated reporter gene expression
after binding of estrogen. This indicates that the influence of the
cell context on the spectrum of action of ERs is more complex than
previously anticipated with three scenarios now described: HeLa as an
AF-2 context, HepG2 as an AF-1 context, and SaOs acting in a mixed
AF-1/AF-2 context. As a functional consequence of hER
isoform
expression, the proliferation of SaOs cells is altered in a
dose-dependent manner.
The generation of transgenic mice devoid of ER
was an important
experiment by which to define the role of ER
(24). One
possible outcome could have been that the
ERKO would be lethal given
the broad range of tissues influenced by E2 action through ER. However,
both sexes of the homozygous
ERKO mice show no grossly altered
phenotype and develop normally, with the exception of being infertile.
After the identification of ERß, this result and that of the human
individual with a disrupted ER
gene have been ascribed to the rescue
of the lack of functional ER
by ERß (31, 32). From
our data and reports of other groups (33, 34), it is now
evident that ERß plays only a minor role in bone. In the construction
used for the knockout mouse, the disruption of the ER
gene was
performed within mouse exon 2, which corresponds to hER
exon 1. Our
data indicate that the modest effects on bone in the
ERKO mouse may
be due, in part, to the occurrence of shorter isoforms whose
translation is initiated downstream from the insertional disruption. In
this regard, uterine tissue from
ERKO mice was able to specifically
bind 3H-E2 (up to 10% of levels detected in the
wild-type mice) that could be immunoprecipitated by H222 antibody
(35). Recently, we performed a detailed analysis of the
5'-upstream region of the mER
gene, which showed that it is a
complex genomic unit exhibiting multiple alternative splicing and
promoter usage (15). The results obtained from primary
ERKO bone cells demonstrate that the mER
gene also generates
alternatively spliced transcripts that are characterized by a deletion
of the A/B domain and therefore correspond to the shorter hER
isoform in bone that is described in this study. Other transcripts have
also been described and could yield open reading frames that contribute
to an overall minor impact of the
ERKO (35, 36). Recent
data demonstrate that another ER
isoform is expressed in the uterus
of ERKO mice (Ko
, M., unpublished data); therefore, it can be
speculated that other mER
isoforms can mediate the effects of
estrogens in other tissues such as bone.
Further work addressing ER isoform expression in osteoblasts and in
osteoclasts is required to fully elucidate the physiological role of
these two receptor proteins in the cell lineages that regulate the
balance of bone metabolism. The benefit of studying ER in tissues, as
opposed to the standard cell lines in culture, is also highlighted. We
present evidence that 1) hER
46 is expressed at a significant level
and 2) is able to form heterodimers with hER
66 and hERß and 3) has
the capacity to modulate ER
- and ERß-mediated transactivation and
increases the complexity of ER signaling. The results of this
study contribute to the knowledge and understanding of how E2 and E2
analogs mediate their pleiotropic effects in a variety of physiological
and pathological processes.
 |
MATERIALS AND METHODS
|
---|
Cells and Tissues
All cell lines were maintained in DMEM (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% FCS
(Life Technologies, Inc.), penicillin (100 U/ml,
Life Technologies, Inc.), and streptomycin (100 µg/ml,
Life Technologies, Inc.) at 37 C and 5%
CO2. Human primary osteoblasts were isolated from
trabecular bone samples from patients who underwent hip or knee
operations under sterile conditions. Outgrowing osteoblasts were
cultivated in DMEM + 20% FCS and characterized by staining for
alkaline phosphatase activity (Sigma, Deisenhofen,
Germany). Primary osteoblasts were trypsinized and propagated when
local confluency was obtained and were used in experiments until
passage 3.
ER
Expression Vector Preparation
The expression vector plasmid HEO (kindly provided by P.
Chambon) was used to obtain the full-length hER
receptor protein
with a molecular mass of 66 kDa. To create the expression vector coding
for hER
of 46 kDa (hER
46), the hER
coding region from position
+519 up to +1,788 was amplified by PCR, using HEO as template DNA.
Primers were designed to introduce BamHI restriction sites
at the ends of the resulting PCR product. The nested primers contained
EcoRI and BamHI restriction sites, which were
used to subclone the amplified fragment into the polylinker of pSG5
expression plasmid, downstream of SV40 promoter to obtain the
expression vectors pSGhER
66 and pSGhER
46. All cloned PCR products
were validated by dideoxy sequencing.
S1 Nuclease Assay
A modified S1 nuclease mapping procedure was followed as
described by Flouriot et al. (37). This method
allows the generation of highly labeled single-stranded DNA probes by
extension from a specific primer by T7 DNA polymerase in the presence
of [
32-P]deoxy-CTP (3,000 Ci/mmol). The
probes are then hybridized with the appropriate RNA sample and
subjected to S1 nuclease digestion. To prepare templates for different
probes, RT-PCR reactions were performed. The PCR products were
subcloned into the TA cloning vector pCR 2.1 (Invitrogen,
San Diego, CA). A PCR reaction was then performed using a biotinylated
forward primer together with a reverse primer either from vector (M13)
or hER
coding region. To characterize the abundance of the different
5'-variants, the probes were designed to contain specific 5'-sequences
of exon 1A-1F up to exon 2. All probes were constructed by PCR with a
5'-biotinylated primer located within the vector sequence to allow
discrimination of free probe from protected fragments.
RNA Isolation and RT-PCR of hER
Isoforms
Expression of different hER
isoforms was determined using
reverse transcription of RNA followed by PCR and Southern blotting.
Reverse transcription of total RNA was performed using 1 µg of total
RNA, an oligonucleotide primer (PRT) from exon 8,
located within the 3'-UTR of hER
(5'-TTGGCTAAAGTGGTGCATGATGAGG) with
50 U of Expand reverse transcriptase (Roche Diagnostics,
Mannheim, Germany) following the protocol of the supplier. Two
microliters of this reaction were then used for two rounds of 35 cycles
of PCR amplification. The 5'-primers and nested primers used for the
amplification of hER
isoforms A, B, C, E, and F were A1
(5'-CTCGCGTGTCGGCGGGACAT and A2 (5'-GCTGCG TCGCCTCTAACCTC), B1
(5'-CTGGCCGTGAAACTCA GCCT) and B2 (5'-ATCCAGCAGCGACGACAAGT), C1
(5'-TCTCTCGGCCCTTGACTTCT and C2 (5'-CAAGCCCATGGAACATTTCTG), E1
(5'-AGCCTCAAATATCTCCAAAATCT) and E2 (5'-AATTATATTCTGTAGCTACCAAAGAAG)
and F1 (5'-TTCTAT- AGCAT AAG AAGACAG) and F2 (5'-GAGTGATAATCTTC),
respectively. The 3'-primer P1 (5'-ATTATCTGAACCGTGTGGGAG) was
chosen within exon 8 of the hER
gene. A nested primer P2
(5'-CGTGAAGTACGACATGTCTAC) was selected upstream of primer P1. The
Expand Long Template PCR system (Roche Diagnostics) was
used for amplification as recommended by the manufacturer. Five
microliters of each reaction were analyzed on a 1% agarose gel.
Southern Blotting
After separation on agarose gels, PCR products were transferred
to nylon membranes (Hybond N+, Amersham Pharmacia Biotech,
Arlington Heights, IL) with 6x saline sodium citrate (SSC) as transfer
solution. The membranes were incubated in a prehybridization buffer
containing 6x SSC, 5x Denhardts solution, 0.05% sodium
pyrophosphate, 100 µg/ml salmon sperm DNA, and 0.5% SDS at 37 C for
1 h. Then, the membranes were hybridized in 6x SSC, 1x
Denhardts solution, 0.05% sodium pyrophosphate, 100 µg/ml yeast
tRNA with an oligonucleotide probe selected in exon 1, which had been
end-labeled using T4 polynucleotide kinase and
[
-32P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech). The most stringent wash was carried out for
1 min at 55 C in 6x SSC, 0.05% sodium pyrophosphate. Specific PCR
products were visualized by exposing the membranes to x-ray films.
For Southern blotting of
ERKO bone, primers from mER
exon 1 and
exon 2 were used to probe the PCR products that were amplified from
mouse 5'-upstream exon 1F to exon 2.
Quantitative RT-PCR
To quantitate hER
mRNA levels, we used a quantitative RT-PCR
approach, based on the incorporation of a labeled fluorogenic probe.
After RNA isolation, reverse transcription was performed with Expand
reverse transcriptase (Roche Diagnostics) using a primer
located within the 3'-UTR of ER
as described above. For the
amplification of hER
F-mRNA variants, where exon 1F splices either
to the common splice site at position +163 in exon 1 or directly to
position +685 in exon 2, PCR was performed in a SMARTCycler
(Eurogentec, Seraing, Belgium) using the downstream primers
5'-GCAGGGCAGAAGGCTCAGAA-3' (exon 1) and 5'-CCACCCTGGCGTCGATTATCT-3'
(exon 2) and an upstream primer from exon 1F
5'-TGCAGGCTCCATGCTCAGAA-3'. The sequence of the probe
(5'-TET-CCATGCTCCTTTCTCCTGCCCATTC-DABSYL-3') was chosen as a nested
primer to the exon 1F-primer. The amplification of
glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal
standard for normalization.
In Vitro Transcription and Translation
In vitro transcription and translation were
accomplished with the TNT coupled Reticulocyte Lysate system from
Promega Corp. (Madison, WI) following the manufacturers
protocol. The expression vectors pSGhER66 and pSGhER46 were used as
templates for transcription with T7 RNA polymerase followed by
translation to generate human ER
proteins (hER
66 and
hER
46).
Immunoprecipitation
Human primary osteoblasts were cultivated in 15-cm TC-plates in
DMEM+ 10% FCS until subconfluency. After washing twice with PBS, the
cells were incubated in methionine-free DMEM + 10% methionine-free FCS
for 10 h. Then, 1,000 µCi radiolabeled Pro-mix
(35S-methionine/
35S-cysteine-mix, Amersham Pharmacia Biotech) were added to each plate, and the cells were incubated
overnight at 37 C and 5% CO2. After washing with
cold PBS, the cells were harvested and lysed in 1 ml RIPA buffer (50
mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS) for 30 min at 4 C. The in
vitro transcibed and translated hER
46 and hER
66, which were
translated in the presence of 35S-labeled
methionine and served as positive controls, as well as the osteoblast
whole cell proteins, were incubated with an unspecific antibody for
1 h (anti-
-Actin, 1 µg/ml, Roche Diagnostics),
followed by binding to 5% protein-A-sepharose (Amersham Pharmacia Biotech) and centrifuged for 5 min at 10,000 rpm. The
supernatants were incubated with hER
-specific antibody (H222, 1
µg/ml), followed by binding to protein A-sepharose for 1 h at 4
C. After extensive washes, Laemmli buffer was added to the
precipitates, and the samples were boiled for 5 min. After
centrifugation, the supernatants were separated on a 10% SDS-PAGE gel.
The gel was then dried and subjected to autoradiography.
Transient Transfections
For transfection experiments, SaOs cells were plated into 6-cm
dishes (Nunc, Wiesbaden, Germany) at a density of 1 x
105 cells per plate and grown in DMEM media
supplemented with 10% FCS. After 3 d, the cells were washed with
1x PBS, and the medium was replaced by phenol-red free DMEM + 2.5%
charcoal stripped, E2-free FCS. After an additional 24 h,
transient transfections were carried out using the calcium phosphate
precipitate method (38). Transfections were performed
using 5 µg of luciferase reporter plasmid containing two EREs
(EREtkLuc) together with 0.25 µg of a CAT plasmid to correct for
transfection efficiency along with 0.5 µg of pSG5 expression plasmid
encoding hER
66 and hER
46. After overnight incubation, the
transfection media was removed, the cells were washed twice with PBS,
and 3 ml of phenol-red free DMEM supplemented with 2.5% charcoal
stripped FCS and 10-8 M E2 were
added. After 48 h cells were harvested and luciferase assays and
CAT ELISAs were performed using commercial kits (Roche
Diagnostics). Quantification of cellular proliferation was performed by
counting immunostained SaOs using the KI67 proliferation antigen that
is localized in the nucleus. The number of Ki67-positive cells per 150
cells per visual field was evaluated using an Axiophot fluorescence
microscope (Carl Zeiss, Jena, Germany).
EMSA
In vitro translated products or extracts of human
primary osteoblasts were preincubated in GSA buffer [10
mM Tris-HCl (pH 8), 1 mM
EDTA, 3 mM MgCl2, 12%
glycerol, 100 µg/ml BSA, 1 mM DTT,
107 M E2, 100
mM KCl] with 1 µg of poly (dI/dC) for 5
min at room temperature. The samples were then incubated for 20 min at
room temperature with 1 ng of radioactive oligonucleotide probe (6
x 104 cpm) end labeled with
[
-32P]ATP (3,000
Ci/mM) using T4 polynucleotide kinase
(Roche). For supershift experiments, ER
-specific
antibodies (1 mg/ml) were added 10 min after initial start of
incubation. Protein-DNA complexes were then separated from free probe
by nondenaturing electrophoresis on 4% polyacrylamide gels in 1x TBE.
The gels were prerun at 4 C for 30 min followed by electrophoresis for
2 h at 200 V, dried subsequently, and exposed to
Kodak Biomax film (Eastman Kodak Co.,
Rochester, NY). The sequence of the consensus ERE 30-bp oligonucleotide
was derived from the 5'-flanking region of chicken apo-VLDL II gene
(-186 to -156) (39). The nucleotide sequence was
5'-ctgtgctcaGGTCAgacTGACCttccatta-3' with the wild-type consensus ERE
palindrome shown in capital letters.
 |
ACKNOWLEDGMENTS
|
---|
The helpful discussions of Dr. Gaetano Clavenna, Dr. Francesco
Colotta and Dr. Gianfranco Caselli contributed to the development of
the work presented in this report. We also thank Dr. Geoffrey Greene
for the generous gift of hER
antibodies.
 |
FOOTNOTES
|
---|
This work, which was supported by the European Molecular Biology
Organization, forms part of the GENOSPORA network program funded by the
EC 5th framework program. S.D. received a fellowship from
Dompé SPA (LAquila, Italy).
Abbreviations: AF-1, AF-2, Activating function-1 and -2; CAT,
chloramphenicol acetyltransferase;
ERKO, mouse knockout model for
ER
; 4-OHT, 4-hydroxytamoxifen; SERM, selective ER modulator; SSC,
saline sodium citrate; UTR, untranslated region.
Received for publication February 23, 2001.
Accepted for publication August 22, 2001.
 |
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