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1
First Department of Internal Medicine, University of Tokushima School of Medicine, Tokushima, Japan
Address all correspondence and requests for reprints to: Daisuke Inoue, M.D., First Department of Internal Medicine, University of Tokushima School of Medicine, 3–18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail: inoued{at}clin.med.tokushima-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) subtype because stable overexpression of
ER, but not of ERß, enhanced the OPG induction by
17ß-E2. Moreover, estrogen withdrawal after 5-day
pretreatment, mimicking the event occurring in vivo at
menopause, dramatically diminished the expression of OPG. These
findings suggest that down-regulation of OPG after estrogen withdrawal
contributes to the enhanced osteoclastic bone resorption and bone loss
after menopause by enhancing RANK ligand-RANK system that lies
downstream of a large number of bone-resorbing cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(11, 12), and monocyte/macrophage colony
stimulating factor (M-CSF) (12, 13) that are known to
stimulate osteoclastogenesis and to be down-regulated by estrogen. A
decrease in an unknown inhibitory factor of osteoclastic bone
resorption has also been suggested as a possible mechanism of enhanced
resorption upon estrogen withdrawal (14). These factors
are likely to act not solely but in a combinatorial way with others to
develop postmenopausal bone loss. However, relative importance of these
factors is yet to be determined. Recently, two groups have independently identified a novel member of the TNF receptor superfamily, termed osteoprotegerin (OPG) or osteoclastogenesis-inhibitory factor (15, 16), that regulates bone mass through an inhibitory action on osteoclast differentiation and function. OPG is a secreted, soluble receptor-type of protein that lacks a transmembrane domain as predicted by its primary amino acid sequence (16, 17). Subsequently, OPG has been found to bind to RANK ligand (18, 19), a long-sought regulator of osteoclastogenesis expressed on the surface of osteoblastic/stromal cells in response to various bone resorptive cytokines and hormones. OPG acts as a decoy receptor for RANK ligand and blocks its interaction with its functional receptor RANK (20, 21) expressed on the cell surface of osteoclast progenitors, and thereby inhibits osteoclast differentiation. Because a majority of signals from bone resorptive factors converge on the RANK ligand-RANK system (18, 19, 22, 23), it is plausible to assume that OPG plays a significant role in physiological regulation and pathological changes of bone metabolism as a key modulator of the function of RANK ligand. In fact, it has been shown that OPG increases bone mass in vivo either by administration of recombinant protein to normal rats (15, 16) or by overexpression in transgenic mice (16). Moreover, deletion of mouse OPG gene by homologous recombination resulted in severe osteoporosis due to increased osteoclast activities (24, 25). Therefore, OPG is a physiological negative regulator of bone resorption that participates in the maintenance of bone homeostasis. However, its pathogenetic roles in the development of metabolic bone diseases including postmenopausal osteoporosis are as yet unclear. Recently, Hofbauer et al. (26) has reported a stimulatory effect of estrogen on OPG expression by human osteoblastic cell lines. These cells constitutively expressed functional estrogen receptors and showed an approximately 3-fold induction of OPG expression in response to 17ß-E2. This study has raised a possibility that deregulated expression of OPG may contribute to the pathogenesis of osteoporosis due to estrogen deficiency. However, the mechanism by which estrogen stimulates OPG messenger RNA (mRNA) expression remains totally unknown.
In the present study, we examined effects of estrogen on OPG
expression by a mouse stromal cell line in vitro to
determine its potential role in the alterations of bone metabolism
caused by estrogen deficiency. We found that estrogen up-regulated OPG
expression at both the mRNA and protein levels. The effect of estrogen
was steroid-specific and was likely to involve its genomic action
through estrogen receptor (ER)-. Furthermore, we also demonstrated
its down-regulation by deprivation of estrogen after 5-day
pretreatment, which mimicked estrogen withdrawal upon menopause
in vivo. Taken together, these results suggest that
decreased expression of OPG by stromal or osteoblastic cells contribute
to the enhanced osteoclastic bone resorption due to estrogen
deficiency.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-E2, and testosterone were purchased from
Wako Pure Chemical Co. (Osaka, Japan); and geneticin,
cycloheximide (CHX) and 5,
6-dichloro-1-ß-D-ribofuranosylbenzimidazol (DRB) from
Sigma (St. Louis, MO). CHX was dissolved in 99.5%
ethanol, and DRB in dimethyl sulfoxide, and stored at -20 C until use.
ICI 182,780, a pure estrogen antagonist, was obtained from AstraZeneca
(Cheshire, UK). All the DNA-modifying enzymes used in this study were
from New England Biolabs, Inc. (Beverly, MA) unless
otherwise specified. A pan-specific neutralizing antibody against
transforming growth factor (TGF)-ß was obtained from R&D Systems
(Minneapolis, MN).
Cell culture
ST-2, a mouse bone marrow stromal cell line, was obtained
from Riken cell bank (Ibaragi, Japan) and was maintained in -MEM
(Life Technologies, Inc., Rockville, MD) containing 10%
FBS (Sanko Junyaku Co., Tokyo, Japan) and penicillin/streptomycin
(Life Technologies, Inc.). For experiments, 3–5 x
105 cells of ST-2 (passage 7–12) were plated in
10-cm culture dish and grown for 24 h. Then, the culture medium
was changed to phenol red-free -MEM containing 1% charcoal-treated
FBS. Twenty four hours later, cells were treated with reagents
including various concentrations of 17ß-E2 for
indicated periods and used for expression analysis.
Stable transfection
Human estrogen receptor (hER)-expression vectors
containing neomycin-resistant gene were generous gifts from Dr.
Shigeaki Kato (University of Tokyo, Tokyo, Japan). ST-2 cells were
transfected with an hER- or hERß-expression vector using the
liposome method according to the instruction provided by the
manufacturer. In brief, ST-2 cells were cultured for 18–24 h and grown
to a 50–80% confluency, and then treated with a mixed solution of DNA
and Lipofectamine Reagent (Life Technologies, Inc.) under
a serum-free condition. After 5 h of transfection period, an
isovolume of medium containing 20% FBS was added to make a final serum
concentration of 10%, and further cultured for 24 h. Seventy-two
hours after initial transfection, the transfected cells were
subcultured onto new plates in complete growth medium supplemented with
1 mg/ml geneticin for about 10 days until individual clones became
visible. Several colonies of neomycin-resistant cells were isolated
with a cloning ring and expanded. To reselect clones with high levels
of expression, cells were grown in the presence of 3 mg/ml of geneticin
for another round of selection. Thus established cell lines were
maintained in the presence of 0.1–0.3 mg/ml geneticin to prevent loss
of the integrated gene.
RNA analysis
Total RNA was extracted from ST-2 cells using TRIZOL
reagent (Life Technologies, Inc.), and mRNA expression of
OPG and other genes was examined using RT-PCR and ribonuclease (RNase)
protection assay. For RT-PCR, 2 µg of total RNA was
reverse-transcribed using Superscript RT (Life Technologies, Inc.) and random primers (Promega Corp., Madison,
WI). Two microliters out of a 20 µl RT reaction were used for PCR
analysis. Primer sets used for amplification were as follows: mouse OPG
sense: 5'-CTGAGGTTTCTCGAGGACCACAATG-3' (nucleotide 69–93) and
antisense: 5'-CCAGTGACGGATCCTAGTTATAAGCAGC-3' (nucleotide 1313–1286);
mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense:
5'-TGTCTTCACCACCATGGAGAAGG-3' and antisense:
5'-GTGGATGCAGGGATGATGTTCTG-3'; ER sense: 5'-GTGCCCTACTACCTGGAGAAC-3'
and antisense: 5'-GTAGCGAGTCTCCTTGGCAG-3'. The primers for ER were
derived from completely conserved sequences between human and
mouse genes so that both gene transcripts can be amplified. To detect
expression of the transfected hERß gene, we used human-specific
nested primers as follows: ERß sense1:
5'-GGTCCATCGCCAGTTATCACATCTG-3' (nucleotide 688–712); ERß
antisense1: 5'-CATCCTGGGTCGCTGTGACCAGAG-3' (nucleotide 1676–1653);
ERß sense2: 5'-GCTCTTGGATGGAGGTGTTA-3' (nucleotide 1416–1436); and
ERß antisense2: 5'-CTTGAAGTAGTTGCCAGGAG-3' (nucleotide 1575–1556).
Amplified products were separated on a 1.5–2% agarose gel and stained
with ethidium bromide for visualization.
The cycle number of PCR was determined empirically so that quantitative
information is not lost: 26 cycles for OPG, 30 cycles for ER and 22
cycles for GAPDH. For hERß, 35 cycles of first PCR was performed with
primers sense1 and antisense1 and 2 out of 50 µl was reamplified with
primers sense2 and antisense2. For RNase protection assay, fragments of
mouse OPG complementary DNA (cDNA (nucleotide: 79–436) and RANK ligand
cDNA (nucleotide: 480-1030) were subcloned into pBluescript SKII(+)
(Stratagene, La Jolla, CA). The resultant plasmids were
linearized and used for generation of a complementary RNA probe using
MAXIscript in vitro Transcription Kit (Ambion, Inc., Austin, TX). The control probe templates for GAPDH and
ß-actin were purchased from Ambion, Inc. RNase
protection assays were performed using RPA II kit (Ambion, Inc.) according to the manufacturer’s instructions. Briefly,
RNA samples were incubated with
[-32P]-labeled complementary RNA probes at
42 C for 18 h and digested with an RNase A/T1 mixture. RNA/RNA
hybrids were precipitated, separated on a 6% polyacrylamide gel
containing 8 M urea, dried and autoradiographed.
In some experiments, detected bands were quantified by
densitometry.
Protein analysis
For the collection of conditioned media, ST-2 cells cultured in
phenol red-free -MEM containing 1% charcoal-treated FBS for 24
h were treated with 17ß-E2 or vehicle alone for
48 h. The conditioned media from ST-2 cell cultures were
electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a
polyvinylidene difluoride membrane (ATTO Corp., Tokyo, Japan). The
membrane was sequentially incubated with a rabbit anti-OPG polyclonal
antibody (Imgenex, San Diego, CA) for 2 h and then with a goat
antirabbit IgG secondary antibody for 1 h, and OPG protein was
visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ) following the suggested
protocol.
Dual luciferase assay
Estrogen-induced transcriptional activities were monitored by a
reporter gene assay using an ERE-luc vector with a PGL3 (Promega Corp.) backbone (27) kindly provided by Dr.
Shigeaki Kato (University of Tokyo, Tokyo, Japan), which contains three
tandem estrogen response elements derived from human vitellogenin gene
upstream of a firefly luciferase cDNA cassette. Transient transfection
was performed using GenePORTER transfection reagent (Gene Therapy
System, Inc., San Diego, CA) according to the instruction provided by
the manufacturer. Briefly, cells were first cultured in phenol red-free
-MEM containing 10% charcoal-treated FBS for 48 h in 6-well
plates, and then incubated with a mixture of 15 µl of GenePORTER, 3
µg ERE-luc plasmid, and 0.1 mg Renilla luciferase expression vector
as an internal control for 5 h in the presence of 1%
charcoal-treated FBS. After 48 h, cells were harvested, lysed in
1xpassive lysis buffer, subject to three rounds of freeze-and-thaw and
analyzed for luciferase activities using Dual Luciferase Reporter Assay
System (Promega Corp.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the time course study
using RNase protection assay revealed that 1 nM
17ß-E2 enhanced OPG mRNA expression as early as
8 h after treatment. The effects of 17ß-E2
reached the maximum of approximately 2-fold induction at 24 h, and
persisted for up to 48 h. Similar results were obtained with
semiquantitative RT-PCR analysis (data not shown), showing its
quantitative reliability under these experimental conditions, compared
with the quantitative RNase protection assay. Thus, dose-response of
the effects of 17ß-E2 on OPG mRNA expression
was examined using semiquantitative RT-PCR analysis. As shown in Fig. 2, treatment of ST-2 cells with various
concentrations of 17ß-E2 for 24 h resulted
in a dose-dependent increase in the steady-state levels of OPG mRNA,
expressed as a ratio to GAPDH mRNA as an internal control. The effect
of 17ß-E2 was readily detectable at 0.01
nM, reached a peak of nearly 2-fold induction at 1–10
nM, but higher concentrations of
17ß-E2 was less potent. These results
demonstrate that 17ß-E2 induces OPG mRNA
expression by ST-2 cells in a time- and dose-dependent manner.
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and the recently identified receptor ERß (28).
Given the fact that estrogen up-regulates OPG expression by ST-2 cells,
we next asked which receptor subtype, ER or ERß, mediated the
stimulatory effect of 17ß-E2 on OPG expression.
For this purpose, we established ST-2-derived cell lines stably
overexpressing hER or hERß under the control of CMV promoter, and
tested estrogen effects on these cells. To ensure overexpression of
ER, we first examined ER mRNA expression in stable transfectants
as well as parental cells by RT-PCR. Primers for PCR were derived from
cDNA sequences completely conserved between human and mouse ER genes
so that we could compare the endogenous and exogenous ER gene
expression. As shown in Fig. 3A, in
parental ST-2 cells, the expression of mouse ER was minimally
detectable under the experimental condition used here, and an
hER-transfected cell line expressed an apparently higher level of
expression as expected. This increase in ER expression was also
confirmed at the protein level by Western blot analysis shown in Fig. 3B. Similar results were obtained with two other cell lines stably
overexpressing hER (data not shown). As to ERß, we confirmed
expression of hERß mRNA by RT-PCR using nested primers as shown in
Fig. 3C. Overexpression of functional ER and ERß in the stable
transfectants was verified by enhanced induction of ERE-dependent
transcription by 17ß-E2 (Fig. 3D). As a result
of overexpression of hER, the stimulatory effect of
17ß-E2 on OPG mRNA expression was enhanced
(Fig. 4, A and B). In contrast,
overexpression of ERß did not enhance but even reversed the effect of
17ß-E2. These results are consistent with the
notion that up-regulation of OPG mRNA expression by
17ß-E2 is dependent on its binding to ER,
and imply distinct negative regulation by
E2-ERß complex on OPG expression.
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or hERß were analyzed for OPG
protein content by Western blot analysis. As shown in Fig. 4C
, under
reducing conditions, the mouse OPG protein was well detectable at a
little above 50 kDa as a monomer in straight conditioned medium. As
expected, an hER-overexpressing cell line showed a substantial
induction (approximately 1.6 fold) by treatment with
17ß-E2, whereas no positive effects on OPG
protein secretion were observed with an hERß-stable clone, consistent
with the results of the mRNA expression studies shown in Fig. 4A.
Therefore, induction of OPG expression by
17ß-E2 occurs at both the mRNA and protein
levels via ER. Based on these results, we used an
ER-overexpressing cell line with good responses to estrogen
(ER-1C) in the following experiments.
Steroid specificity of the estrogen effect
To examine if the stimulatory effect on OPG mRNA expression is
specific to 17ß-E2, the hER-transfected ST-2
cells were treated with 1 nM
17ß-E2, 17-E2, an
inactive stereoisomer of 17ß-E2, or
testosterone for 24 h. As shown in Fig. 5A, whereas
17ß-E2 apparently induced OPG mRNA expression,
17-E2 and testosterone had little effect on
the expression of OPG mRNA. Similar results were obtained by
semiquantitative RT-PCR analysis (data not shown). The estrogen
specificity was further confirmed by a pure ER-antagonist, ICI 182,780.
As shown in Fig. 5B, the stimulatory effect of
17ß-E2 on OPG expression by
hER-overexpressing ST-2 cells was completely blocked by ICI 182,780.
In contrast, neither 17ß-E2 nor ICI 182,780 had
any effects on OPG expression by hERß-cells. Therefore,
17ß-E2 stimulates OPG expression through
binding to ER in a steroid-specific manner.
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, when cells were pretreated with
CHX, the effect of 17ß-E2 was completely lost.
Notably, the control level of expression also dramatically declined,
suggesting that the basal transcription of the OPG gene is dependent on
a relatively unstable protein(s). Moreover, inhibition of RNA
polymerase II-dependent transcription by DRB also resulted in loss of
the effect of 17ß-E2. Therefore,
17ß-E2 action on OPG mRNA requires ongoing
protein synthesis and involves a transcriptional event. These results
suggested that the stimulatory effects of estrogen on OPG expression
might be mediated by another factor produced by ST-2 cells in response
to estrogen. One good candidate for such factors is TGF-ß because it
has been shown to be inducible by estrogen and to up-regulate OPG
expression in osteoblastic cells (29). We therefore tested
the effect of a pan-specific neutralizing antibody against TGF-ß on
the estrogen effects. As shown in Fig. 7, addition of the TGF-ß-blocking antibody caused no appreciable effects
on the OPG induction by estrogen. These results indicated that TGF-ß
is not a major mediator of the estrogen effect under these experimental
conditions.
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-overexpressing ST-2 cells were first grown for 5 days in the
presence of 1 nM of 17ß-E2, and
then cultured for another 48 h in the absence or presence of
17ß-E2. As shown in Fig. 8, cells under estrogen removal showed a
clear-cut reduction in the level of OPG mRNA expression within 48
h compared with cells continuously exposed to estrogen. From these
observations, it is plausible to conclude that the regulation of OPG
expression by 17ß-E2 in cultured stromal cells
is relevant to the event occurring in pre- and postmenopausal
women.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteoclasts differentiate from hematopoietic stem cells through multiple steps, and the whole differentiational process is subject to complex regulation by various factors including local cytokines and systemic hormones (30, 31). Recent studies have identified two critical factors directly regulating osteoclastogenesis: M-CSF (32, 33), and RANK ligand (18). Both factors act directly on osteoclast progenitors through their cognate receptors and stimulate osteoclast differentiation and survival (34, 35). Moreover, substantial evidence indicates that signals from a majority of bone resorptive agents converge on RANK ligand expression on the surface of osteoblastic/stromal cells (18, 19, 22, 23, 35). Natural inactivating point mutation in the mouse M-CSF gene (32) and deletion of the RANK ligand gene by homologous recombination in knockout mice (36) both result in osteopetrosis due to a defect in osteoclastogenesis. Conversely, osteoclast precursor cell lines as well as primary spleen cells are able to differentiate into mature osteoclast-like cells when treated with M-CSF and RANK ligand in the absence of osteoblastic/stromal cells (18). Therefore, these factors are not only dispensable but, when combined together, sufficient for osteoclast differentiation. Of the two direct regulators of osteoclast differentiation M-CSF has already been shown to be a cellular target of estrogen action in stromal/osteoblastic cells and thus could be a mediator of osteoporosis caused by estrogen deficiency (12). The present study demonstrates that estrogen also up-regulates OPG production by marrow stromal cells. Although ST-2 cells used in this study are known to express RANK ligand in response to several factors (18, 29, 37), we did not see any significant effects of estrogen on the expression of RANK ligand expression in these cells (data not shown). Because the activity of RANK ligand-RANK system is determined by a balance between the expression of RANK ligand itself and its decoy receptor OPG (38), our findings indicate that estrogen withdrawal enhances RANK ligand-RANK signal by down-regulating OPG expression. Because most of bone-resorbing cytokines that are shown to be up-regulated by estrogen deficiency enhance bone resorption by stimulating RANK ligand expression, the present observations unveil an important stimulatory mechanism of bone resorption that can enforce the actions of these factors after estrogen withdrawal. Although the amplitude of OPG induction by 17ß-E2 was relatively small in ST-2 cells, it has been suggested that modest decrease in the level of OPG expression is sufficient to enhance bone resorption in vivo because heterozygous OPG gene mutant mice exhibited significant bone loss (24).
Bone has long been recognized as a direct target organ of estrogen
since the demonstration of the presence of classical estrogen receptor
now called ER in osteoblasts (39, 40). Recent discovery
of a novel estrogen receptor subtype ERß has added a higher level of
complexity to the regulation of bone metabolism by estrogen. It has
already been reported that ERß is abundantly expressed in bone, and
that in osteoblastic cells its expression is subject to a distinct mode
of regulation from that of ER (41, 42). However,
selective action of each subtype of estrogen receptor on bone is
currently unclear. According to our present results, enhanced induction
of OPG expression by 17ß-estradiol was only observed in
ER-overexpressing ST-2 cells but not in ERß-transfected cells.
Therefore, ER appears to participate in the inductive effects of
estrogen on OPG expression by osteoblastic/stromal cells. However, in
view of the fact that ER gene-deleted female mice did not show
reduced bone mass (43, 44), clarification of the spectrum
of targets for each receptor subtype in bone requires further
investigation.
The stimulatory effect of estrogen on OPG expression was completely lost in the presence of a protein synthesis inhibitor, cycloheximide, raising a possibility that the effect is not a direct result of genomic action of estrogen on the OPG promoter but is mediated by an unknown factor. Among good candidates for such factors is TGF-ß because its expression is up-regulated by estrogen (45) and it potently enhances OPG expression by osteoblastic/stromal cells (29). However, mediation by TGF-ß appears unlikely because of the following reasons: firstly, TGF-ß is secreted from osteoblasts as an inactive form and mostly remains inactivated in vitro due to a lack of an efficient activator (46). Secondly, our results indicated that neutralizing pan-specific antibodies against TGF-ß had no detectable effects on the stimulation of OPG expression by estrogen in ST-2 cells. Thus, estrogen may increase an unknown stimulator of OPG expression other than TGF-ß. Alternatively, OPG expression by ST-2 cells may be dependent on an unstable intracellular protein with a relatively short half life. Molecular basis of the estrogen effect on OPG expression is yet to be elucidated.
In conclusion, we have identified OPG as a novel target of estrogen as an inhibitor of bone resorption. Although estrogen withdrawal enhances the secretion of bone-resorbing cytokines as well, its effect on OPG expression appears to be of great importance because it directly modifies RANK ligand-RANK system which lies downstream of a large number of bone-resorbing signals. Further studies will be necessary to determine relative contribution of OPG in the pathogenesis of osteoporosis caused by estrogen withdrawal.
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Acknowledgments |
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Footnotes |
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Received January 5, 2001.
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References |
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