By
From the * Department of Orthodontics, Nippon Dental University School of Dentistry at Niigata,
Niigata 951, Japan; Department of Oral Anatomy, Meikai University School of Dentistry, Sakado,
Saitama 350-02, Japan; and § Endocrine Research, Eli Lilly & Co., Indianapolis, Indiana 46285
Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy.
Using a recently developed technique for isolation of highly purified mammalian osteoclasts,
we showed that 17 -estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At
concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast
apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition
of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that
the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by
the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor-
mediated mechanism.
Estrogen deficiency, caused by either menopause or
ovariectomy, results in pathological bone loss, which
can be prevented by estrogen replacement therapy (1, 2).
Although it is believed that estrogen's main action in preventing bone loss is through inhibition of osteoclastic bone
resorption, the precise mechanism of such effects is not
clear, largely due to technical difficulties in obtaining purified functional osteoclasts (3, 4). Osteoclasts are terminally
differentiated multinucleate cells the main function of which
is to dissolve bone matrix and minerals in the resorption phase of bone remodeling (5). Recruitment, differentiation, and activity of osteoclasts are tightly controlled by systemic and local factors. For instance, vitamin D3, prostaglandins,
TGF- Estrogen effects on osteoclasts are thought to be mediated indirectly through nonosteoclastic cells. For instance,
loss of estrogen at menopause or by ovariectomy is associated with increased secretion of IL-1, IL-6, and TNF- Osteoclast Preparation.
Purified rabbit osteoclasts were prepared by the method of Kakudo et al. (25) from unfractionated
bone cells obtained according to the procedure described by Takada et al. (26). Briefly, cell suspensions obtained from minced
long bones of 10-d-old rabbits (Japan White; Saitama Experimental Animals Supply Co., Saitama, Japan) were agitated by vortexing and plated in 10-cm tissue culture dishes (Becton Dickinson
Labware, Lincoln Park, NJ) coated with 0.24% collagen gel
(Nitta Gelatin Co., Tokyo, Japan). After a 3-h incubation, adherent nonosteoclast cells were removed from the collagen gel by sequential treatment with 0.001% pronase E and 0.01% collagenase
(Wako Pure Chemical Industries, Osaka, Japan). The remaining
osteoclasts were then collected by 0.1% collagenase solution treatment and replated. When these cell suspensions were seeded onto
tissue culture dishes, osteoclasts attached and spread out on the
dishes. By staining these cells for tartrate-resistant acid phosphatase (TRAcP, a marker of osteoclasts) activity using a leukocyte acid phosphatase kit (Sigma Chemical Co., St. Louis, MO)
after a 2-h incubation, we estimated that the purity of the TRAcP-positive multinucleate cells (>3 nuclei) was >99% (Fig. 1). When the cells harvested from collagen gels were cultured on
dentine slices, they formed resorption pits as judged by scanning electron microscopic observation (25). We used these pure osteoclast cell suspensions for all experiments.
Assay for Osteoclastic Bone-resorbing Activity.
In an assay to evaluate
osteoclastic bone-resorbing activity, resorption pits formed by osteoclasts on dentine slices were measured for area, and their number was counted. Briefly, purified osteoclasts were replated onto
dentine slices (circular, 6 mm in diameter) in each well of 96-well
plates (Becton Dickinson) at 150 cells/well and cultured for 24 h
in phenol red-free Osteoclast Apoptosis.
The method used to detect osteoclast
apoptosis by fluorescence microscopy was described previously
(27). After treatment with E2, recombinant human TGF- Northern Blot Analysis.
For assessment of the mRNA expression of osteoclast-specific genes (cathepsin K [Cat K] and carbonic anhydrase II [ECA II]), total RNA was extracted by the
AGPC method (30) from osteoclasts cultured on dentine slices in
phenol red-free TUNEL Assay.
To detect in situ DNA fragmentation of
apoptotic osteoclasts, we employed the TUNEL (TdT-mediated
dUTP-biotin nick end-labeling) assay by using a TACS Blue LabelTM kit (Trevigen, Inc., Gaithersburg, MD) according to the
procedure recommended by the manufacturer.
Direct reduction of osteoclastic bone resorption by E2 started within 6 h
of treatment (data not shown), and great reduction was observed by 24 h (Fig. 2, A-C). The ratio of pit area per pit
number was reduced by the treatment with E2, indicating
that E2 inhibition of bone resorption occurred at the level
of individual osteoclast activity (Fig. 2 C, inset). The level of
inhibition of osteoclastic bone resorption by E2 was comparable to that obtained for calcitonin (Fig. 2 C).
To verify the molecular mechanisms of estrogen-inhibited osteoclastic bone-resorbing activity, we examined
whether E2 would affect the mRNA of Cat K and CA II,
enzymes involved in the bone-resorbing activity of osteoclasts. E2 reduced mRNA levels of both Cat K and CA II
in a time-dependent manner (Fig. 3, A and B). Such reduction was correlated with E2 inhibition of formation of resorption pits by the osteoclasts (data not shown). These
data corresponded to the results that E2 inhibition of bone
resorption occurred at the level of individual osteoclast activity (Fig. 2 C, inset).
To understand the mechanism of estrogen-induced osteoclast inactivation, we evaluated the morphological changes in osteoclasts upon estrogen treatment at the subcellular level.
Apoptosis is quite different from necrosis, which is accompanied by cell disintegration, and provides an organized
means of cell death to minimize damage to the surrounding
cells (32). To examine direct effects of estrogen on osteoclast apoptosis, we observed pure authentic osteoclasts attached to dentine slices in this study, as >90% of the osteoclasts plated remained attached to the dentine slices after 24 h
of cultures. Moreover, we confirmed by video monitoring
that osteoclasts did not detach easily even during the morphological change to the apoptotic appearance, especially
up to 24 h (data not shown). After 24 h of E2 treatment,
osteoclasts displayed characteristics of apoptosis, including
chromatin condensation and grossly changed cellular and
nuclear morphology as detected by fluorescence microscopy (Fig. 4 A). These observations were further confirmed
by transmission electron microscopy (Fig. 4 C). E2 treatment did not change the morphology of intracellular organelles of osteoclasts, and cell membranes remained intact
(Fig. 4 C). Such features are characteristic of apoptosis, and
clearly indicated that the cause of cell death was not necrosis, which is accompanied by cellular disintegration (14,
15). Furthermore, in situ DNA fragmentation in these cells
was detected by the TUNEL method (Fig. 4 B). The number of DNA fragment-bearing osteoclasts (TUNEL-positive cells) was correlated with the number of morphological
apoptotic osteoclasts detected by Hoechst 33258 staining. In
unfractionated bone cells, E2 also induced osteoclast apoptosis without causing changes in nonosteoclastic cells (data
not shown).
Quantification of E2-induced osteoclast apoptosis showed
a dose-dependent increase (Fig. 5 A), which correlated
with the dose range of E2 for inhibition of bone resorption
(Fig. 2 B). In contrast to estrogen, calcitonin, which inhibits osteoclast activity directly through its receptors on osteoclasts, did not cause osteoclasts to undergo apoptosis,
suggesting that inhibition of osteoclastic bone resorption by
E2 is mediated by a distinctively different mechanism than
that used by calcitonin. As for detached cells, significant
cellular disintegration prevented us from detecting osteoclast apoptosis in them in 24-h and older cultures. E2-induced osteoclast apoptosis was also time dependent (Fig. 5 B). E2
(0.1 nM) inhibition of formation of resorption pits by osteoclasts was correlated with a reduction in the expression
of mRNAs for Ca K and CA II and with an increased induction of osteoclast apoptosis in a time course study.
These findings suggest that E2 directly acts on osteoclasts
and inhibits osteoclastic bone resorption by causing osteoclast inactivation partially due to apoptosis. A recent report
indicated that estrogen promoted TGF-
Using a pure or a partial antagonist which can block estrogen effects by binding to the ER, we examined whether
these estrogen effects on osteoclasts were ER mediated or
not. ICI, a pure antiestrogen, had no effect on pit formation when expressed in terms of pit area, whereas TAM, a
partial estrogen antagonist, partially inhibited the resorption
activity of purified osteoclasts (Fig. 6 A). When combined
with E2, both ICI and TAM reversed the inhibitory effect of E2, indicating that E2 inhibited osteoclastic bone-resorbing activity through ER. In contrast, the inhibitory effect
of calcitonin was not affected by either ICI or TAM (Fig. 6
A). Consistent with their lack of, or partial, ability to inhibit pit formation, ICI (0.001-100 nM) did not induce
apoptosis of osteoclasts, whereas TAM induced osteoclast
apoptosis partially at 1 nM , but not in a statistically significant manner, at 0.001-0.1 nM or at 10-100 nM (Fig. 6 B,
data not shown). These contradictory effects of antiestrogens might be due to variety of ERs and coregulators (33,
34). When combined with E2, ICI and TAM blocked E2-induced osteoclast apoptosis as a pure or partial antagonist,
respectively (Fig. 6 B, actual number of attached osteoclasts, plated at 150 cells, onto dentine slice after 24 h of
culture: control, 143.5 ± 5.2; 1 nM ICI, 139.5 ± 7.3; 1 nM TAM, 141.0 ± 7.8; 0.1 nM E2, 137.0 ± 10.9; 0.1 nM
E2 + 1 nM ICI, 141.0 ± 7.2; 0.1 nM E2 + 1 nM TAM,
136.9 ± 7.8). These results suggest that estrogen effects on
osteoclasts might be mediated by the ER and not be a consequence of toxicity. Only a high dose of TAM was reported to be able to induce osteoclast apoptosis in rat osteoclasts as well as E2 treatment (35). In this culture system,
however, the optimal concentration of TAM for both inducing osteoclast apoptosis and rescuing osteoclasts from
undergoing apoptosis by the treatment with E2 was 1 nM,
which might be due to partial mimicking of estrogen effects. TAM may act on osteoclasts as a partial agonist rather
than as a partial antagonist.
Several isoforms of ER In conclusion, the data presented in this study demonstrate that in addition to the indirect effects of estrogen on
bone resorption mediated by soluble factors secreted from
nonosteoclastic cells and cell-to-cell contact with nonosteoclastic cells, estrogen can function directly on osteoclasts to
inhibit their bone-resorbing activity. The ability of estrogen to induce apoptosis of osteoclasts at correlative concentrations effective for inhibition of bone resorption may be
the mechanism underlying such effects. The effects of the
antiestrogens also suggest that osteoclast apoptosis induced by estrogen is mediated by the ER. These findings, therefore, may shed new light on our understanding of the cellular mechanism by which estrogen provides protective effects on the skeleton, and support the validity of using
estrogen replacement therapy for treating postmenopausal
osteoporosis.
, IL-1, IL-6, and TNF-
stimulate osteoclast differentiation and activity via direct or indirect mechanisms,
whereas calcitonin directly inhibits osteoclast activity (6).
The fate of osteoclasts after bone resorption is largely unknown. Certain factors, such as calcitonin, inactivate osteoclasts without induction of cell death, whereas other factors, such as bisphosphonates and vitamin K2, are suggested
to induce apoptosis of osteoclasts (11). Apoptosis, or
programmed cell death, is characterized by nucleosomal
DNA fragmentation and grossly changed morphology of
the nuclei without a change in the morphology of the intracellular organelles (14, 15).
from the peripheral blood monocytes, bone marrow stromal cells, or osteoblasts, and decreased expression of TGF-
in
bone (16). Elevated levels of these factors result in increased osteoclastogenesis (20). Moreover, Hughes et al. (21) recently reported that estrogen promoted apoptosis of
murine osteoclast-like cells mediated by TGF-
in a mixed
cell population in culture. Using a recently developed
technique for isolation of highly purified authentic osteoclasts, we showed in this study the direct effects of estrogen
on osteoclastic bone-resorbing activity and osteoclast apoptosis. These estrogen effects on osteoclasts were blocked
by treatment with antiestrogens. Moreover, Northern blot
analysis demonstrated abundant estrogen receptor
(ER
)1
mRNA expression in isolated osteoclasts, suggesting that
estrogen may also have a direct impact on osteoclasts (22-
24). The data presented here indicate that estrogen inhibits
osteoclastic bone resorption activity in part by targeting osteoclasts directly to undergo apoptosis through ER-mediated mechanisms.
Fig. 1.
Purified osteoclasts harvested
from collagen gels. 2,000 osteoclasts were
plated on a dentine slice and stained for
TRAcP activity after a 2-h incubation.
(×100).
[View Larger Version of this Image (100K GIF file)]
-MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 0.1% BSA (Sigma Chemical Co.).
Dentine slices were then brushed with a rubber policeman to remove cells after observation under a fluorescence microscope,
and stained with acid hematoxylin (Sigma Chemical Co.) for 5 min. Total number of pits (reflecting active osteoclast number)
was counted under a light microscope, and total area of resorption pits (reflecting bone-resorbing activity) was quantified by
densitometric analysis of images of the whole area of dentine
slices put into a computer. 17
-Estradiol (E2) and ICI164,384 (ICI)
were provided by Shionogi Pharmaceuticals Inc. (Osaka, Japan)
and Dr. A.E. Wakeling (ZENECA Pharmaceuticals, Cheshire,
UK), respectively. Tamoxifen (TAM) was obtained from Sigma
Chemical Co. At the point of replating, agents were added to the
cultures at the desired concentrations. At the end of the culture
period, osteoclasts were fixed with formalin-acetone solution (37% formalin: acetone: 18 mM citric acid/19 mM Na Citrate/
12 mM NaCl; 7:65:25) and stained for TRAcP activity to determine the osteoclast number on the dentine slices.
1
(R&D Systems, Minneapolis, MN) or salmon calcitonin (CT;
28), cells were fixed with 2% glutaraldehyde solution (Wako Pure
Chemical Co.) for 10 min, and stained with 0.2 mM Hoechst
33258 for visualization of chromatin condensation under a fluorescence microscope (VANOX AHB-LB, Olympus Co., Tokyo, Japan). Transmission electron microscopy was performed as follows. After fixation of osteoclasts on dentine slices with 2%
glutaraldehyde solution for 1 h, the cells were immersed in 0.16 M
EDTA (pH 7.2) and 0.2 M sucrose at 4°C for 2 wk to decalcify
the dentine slices (29), and postfixed in 1% osmium tetraoxide solution (Electron Microscopy Science, Fort Washington, PA) for
1 h. After dehydration in graded ethanol solutions, the cells were
embedded in epoxy resin. Sections were cut, stained with 4%
uranyl acetate, and examined under a transmission electron microscope (JEM-100CX, JEOL).
-MEM supplemented with 0.1% BSA with or
without 0.1 nM E2 for 6 or 24 h, and for examination of the
mRNA expression of ERs, osteoclasts isolated by the treatment
with 0.001% pronase E solution (31) were cultured on tissue culture dishes for 3 h in phenol red-free
-MEM supplemented with 0.1% BSA, and then total RNA was extracted by the same
method. The RNA was blotted onto a nitrocellulose membrane
after formaldehyde agarose gel electrophoresis, and Northern blotting was carried out. 32P-labeled radioactive probes were prepared
by the random primer labeling procedure, and the blot was hybridized in 50% formamide/5× SSPE/5× Denhart's reagent/0.2
mg/ml salmon sperm DNA/labeled probe. After hybridization,
the membrane was washed under stringent washing conditions
(0.1× SSPE/0.1% NaPPi/1% SDS) at 65°C before autoradiography. The hybridization probes were rabbit Cat K cDNA (30),
rabbit CA II cDNA, and human ER
cDNA, all of which were
obtained from American Type Culture Collection (Rockville,
MD). A rat ER
cDNA was cloned by RT-PCR method from
rat testes. A human
-actin cDNA probe was used as a reference.
Estrogen Directly Impacts Osteoclast Function.
Fig. 2.
Estrogen inhibition of bone resorption by purified rabbit osteoclasts. Purified osteoclasts were incubated with medium (A) lacking or (B)
containing 0.1 nM E2 for 24 h on dentine slices (×40). After removal of osteoclasts, resorption pits formed by osteoclasts were stained with acid hematoxylin and observed under a light microscope. (C) Inhibitory effects of E2 on osteoclast-mediated bone resorption. Purified osteoclasts were cultured on
dentine slices (150 cells/slice) in medium (Con) or in medium containing 0.001-1 nM E2 (E2) or 1 nM salmon calcitonin (CT). Osteoclastic bone resorption activity was measured in terms of pit area formed by purified osteoclasts after 24 h of incubation. Pit area per pit number is also indicated (inset). Values are means ± SD, n = 4. *P <0.05, **P <0.01, ***P <0.005 compared with control groups. Data are representatives of those obtained in three additional independent experiments.
[View Larger Versions of these Images (122 + 25K GIF file)]
Fig. 3.
Negative regulation of mRNA levels for Cat K and CA II by
estrogen. (A) Northern blot analysis. Total RNAs (3 µg) from ~25,000 purified osteoclasts cultured on dentine slices (150 cells/dentine slices)
with (E2) or without 0.1 nM E2 (Con) for 6 or 24 h were used as samples.
(B) Relative abundance of Cat K and CA II mRNAs. The relative abundance of Cat K and CA II mRNAs on Northern blotting was evaluated
from the values of densitometric scanning. The values shown were normalized with respect to the abundance of -actin (ACT), and expressed as
means ± SD of samples from three other independent experiments.
[View Larger Version of this Image (35K GIF file)]
Fig. 4.
Estrogen-induced osteoclast apoptosis. Osteoclasts were cultured on dentine slices with 0.1 nM E2 for 24 h. (A) A fluorescence micrograph
shows normal and apoptotic osteoclasts (arrowhead) attached to a dentine slice in a 24-h culture. ×250. (B) The TUNEL assay indicates DNA fragmentation in an apoptotic osteoclast. ×250. (C) This micrograph, obtained by electron transmission microscopy, demonstrates the gross morphological changes
in an apoptotic osteoclast. Nuclear fragments are indicated by the arrowheads. ×2,000.
[View Larger Versions of these Images (153 + 157K GIF file)]
-mediated apoptosis of in vitro-developed murine osteoclast-like cells in mixed cell cultures (21). TGF-
1 (10 ng/ml) induces apoptosis of our pure authentic osteoclasts. However, the incidence was lower than that by E2 treatment in our mature
cells or that of TGF-
-induced apoptosis in murine mixed
cell cultures (Fig. 5 A, reference 21). The causes of this discrepancy might be partially due to their different species;
however, these results suggest that estrogen-enhanced osteoclast apoptosis mediated by TGF-
might occur in an
indirect manner. The direct induction of osteoclast apoptosis by estrogen besides indirect induction might exist.
Fig. 5.
Dose- and time-dependent manner of estrogen-induced osteoclast apoptosis. (A) Purified osteoclasts were cultured on dentine slices
(150 cells/slice) for 24 h in medium (Con) or in medium containing
0.001-1 nM E2, 1-20 ng/ml TGF-1, or 1 nM CT. Apoptotic osteoclasts
were quantified under a fluorescence microscope. (B) Time-dependent
effects of E2 on osteoclast apoptosis and osteoclast number. Under the
same culture conditions, purified osteoclasts were incubated in medium
without 0.1 nM E2 (apoptosis:
; cell number:
) or with 0.1 nM E2
(apoptosis:
, cell number:
) for 6, 12, or 24 h. Apoptotic osteoclasts
are expressed as a percentage of total number of adherent osteoclasts. Values are means ± SD, n = 4. *P <0.05, **P <0.005 compared with time = 0 groups. Data are representative of those of three additional independent
experiments.
[View Larger Version of this Image (20K GIF file)]
Fig. 6.
Estrogen affects osteoclasts through ER-mediated mechanisms. (A) Antiestrogens recovered E2-inhibited osteoclastic bone resorption. ICI at
1 nM or 1 nM TAM was added to osteoclast cultures in the presence or absence of 0.1 nM E2 or 1 nM salmon CT. 24 h later, osteoclastic bone resorption activity was measured in terms of pit area. (B) Antiestrogens blocked E2-induced osteoclast apoptosis. Under the same culture conditions, purified osteoclasts were incubated in medium with or without 1 nM ICI or 1 nM TAM in the presence or absence of 0.1 nM E2, and osteoclast apoptosis was measured after a 24-h treatment. Apoptotic osteoclasts are expressed as a percentage of total number of adherent osteoclasts. (C) Northern blot analysis of
ER and ER
mRNA expression in osteoclasts. Total RNA (50 µg) from ~5 × 105 purified osteoclasts cultured on tissue culture dishes with 0.1 nM
E2 for 6 h were used as samples. Values are means ± SD, n = 4. *P <0.05, **P <0.01, ***P <0.005 compared with control or estrogen-treated groups
for A and B. Data are representative of those of three additional independent experiments.
[View Larger Versions of these Images (26 + 16K GIF file)]
and one subtype of ER, called
ER
, have been reported (36). In rabbit osteoclasts, we
previously demonstrated the expression of mRNAs (~1.5,
2, and 6 kb with the 6-kb mRNA considered to be putative ER
mRNA) which hybridized with ER
cDNA in
Northern blots (22). In this study, Northern blot analysis
revealed that osteoclasts expressed ER
mRNA, but ER
mRNA was undetectable (Fig. 6 C). ER cDNA-hybridizing mRNAs besides putative ER
in pure osteoclasts might
be isoforms of ER
mRNA or some other mRNA but not
ER
mRNA. These data suggest that rabbit osteoclasts
might be controlled by estrogen through ER
s.
Address correspondence to Dr. Masayoshi Kumegawa, Department of Oral Anatomy, Meikai University School of Dentistry, Keyakidai 1-1, Sakado, Saitama 350-02, Japan. Phone: 81-492-85-5511; FAX: 81-492-71-3523; E-mail: o-anat-1{at}dent.meikai.ac.jp
Received for publication 9 September 1996 and in revised form 11 June 1997.
Dr. T. Kameda and Dr. H. Mano contributed equally to this work.We thank Ms. Yoko Katagiri and Ms. Sachiko Ishii for technical assistance and preparation of the manuscript, respectively. We are also grateful to Ms. Yumiko Kanda for her assistance with the transmission electron microscopy.
1. | Lindsay, R., D.M. Hart, J.M. Aitken, E.B. MacDonald, J.B. Anderson, and A.C. Clarke. 1976. Long-term prevention of postmenopausal osteoporosis by oestrogen. Evidence for an increased bone mass after delayed onset of oestrogen treatment. Lancet. 1: 1038-1041 [Medline]. |
2. | Ettinger, B., H.K. Genant, and C.E. Cann. 1985. Long-term estrogen replacement therapy prevents bone loss and fractures. Ann. Intern. Med. 102: 319-324 [Medline]. |
3. | Heaney, R.P., R.R. Recker, and P.D. Saville. 1978. Menopausal changes in bone remodeling. J. Lab. Clin. Med. 92: 964-970 [Medline]. |
4. | Selby, P.L., M. Peacock, S.A. Barkworth, W.B. Brown, and G.A. Taylor. 1985. Early effects of ethinyloestradiol and norethisterone treatment in post-menopausal women on bone resorption and calcium regulating hormones. Clin. Sci. (Lond.). 69: 265-271 [Medline]. |
5. | Vaes, G.. 1988. Cellular biology and biochemical mechanism of bone resorption. A review of recent developments on the formation, activation, and mode of action of osteoclasts. Clin. Orthop. Relat. Res. 231: 239-271 [Medline]. |
6. |
Dewhirst, F.E.,
P.P. Stashenko,
J.E. Mole, and
T. Tsurumachi.
1985.
Purification and partial sequence of human osteoclast-activating factor: identity with interleukin 1 beta.
J. Immunol.
135:
2562-2568
|
7. | Pfeilschifter, J., C. Chenu, A. Bird, G.R. Mundy, and G.D. Roodman. 1989. Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclast-like cells in vitro. J. Bone Miner. Res. 4: 113-118 [Medline]. |
8. | Bertolini, D.R., G.E. Nedwin, T.S. Bringman, D.D. Smith, and G.R. Mundy. 1986. Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors. Nature (Lond.). 319: 516-518 [Medline]. |
9. | Lowik, C.W., G. van der Pluijm, H. Bloys, K. Hoekman, O.L. Bijvoet, L.A. Aarden, and S.E. Papapoulos. 1989. Parathyroid hormone (PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in osteoclastogenesis. Biochem. Biophys. Res. Commun. 162: 1546-1552 [Medline]. |
10. | Chambers, T.J., N.A. Athanasou, and K. Fuller. 1984. Effect of parathyroid hormone and calcitonin on the cytoplasmic spreading of isolated osteoclasts. J. Endocrinol. 102: 281-286 [Abstract]. |
11. | Zaidi, M., H.K. Datta, B.S. Moonga, and I. MacIntyre. 1990. Evidence that the action of calcitonin on rat osteoclasts is mediated by two G proteins acting via separate post-receptor pathways. J. Endocrinol. 126: 473-481 [Abstract]. |
12. | Kameda, T., K. Miyazawa, Y. Mori, T. Yuasa, M. Shiokawa, Y. Nakamaru, H. Mano, Y. Hakeda, A. Kameda, and M. Kumegawa. 1996. Vitamin K2 inhibits osteoclastic bone resorption by inducing osteoclast apoptosis. Biochem. Biophys. Res. Commun. 220: 515-519 [Medline]. |
13. | Hughes, D.E., K.R. Wright, H.L. Uy, A. Sasaki, T. Yoneda, G.D. Roodman, G.R. Mundy, and B.F. Boyce. 1995. Bisphosphonates promote apoptosis in murine osteoclasts in vivo and in vitro. J. Bone Miner. Res. 10: 1478-1487 [Medline]. |
14. | Wyllie, A.H., J.F. Kerr, and A.R. Currie. 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 6: 251-306 . |
15. | Wyllie, A.H., R.G. Morris, A.L. Smith, and D. Dunlop. 1984. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142: 67-77 [Medline]. |
16. | Pacifici, R., L. Rifas, S. Teitelbaum, E. Slatopolsky, R. McCracken, M. Bergfeld, W. Lee, L.V. Avioli, and W.A. Peck. 1987. Spontaneous release of interleukin 1 from human blood monocytes reflects bone formation in idiopathic osteoporosis. Proc. Natl. Acad. Sci. USA. 84: 4616-4620 [Abstract]. |
17. | Cohen-Solal, M.E., A.M. Graulet, M.A. Denne, J. Gueris, D. Baylink, and M.C. de Vernejoul. 1993. Peripheral monocyte culture supernatants of menopausal women can induce bone resorption: involvement of cytokines. J. Clin. Endocrinol. Metab. 77: 1648-1653 [Abstract]. |
18. | Jilka, R.L., G. Hangoc, G. Girasole, G. Passeri, D.C. Williams, J.S. Abrams, B. Boyce, H. Broxmeyer, and S.C. Manolagas. 1992. Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science (Wash. DC). 257: 88-91 [Medline]. |
19. | Ikeda, T., C. Shigeno, R. Kasai, H. Kohno, S. Ohta, H. Okumura, J. Konishi, and T. Yamamuro. 1993. Ovariectomy decreases the mRNA levels of transforming growth factor-beta 1 and increases the mRNA levels of osteocalcin in rat bone in vivo. Biochem. Biophys. Res. Commun. 194: 1228-1233 [Medline]. |
20. | Horowitz, M.C.. 1993. Cytokines and estrogen in bone: anti-osteoporotic effects. Science (Wash. DC). 260: 626-627 [Medline]. |
21. |
Hughes, D.E.,
A. Dai,
J.C. Tiffee,
H.H. Li,
G.R. Mundy, and
B.F. Boyce.
1996.
Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-![]() |
22. | Mano, H., T. Yuasa, T. Kameda, K. Miyazawa, Y. Nakamaru, M. Shiokawa, Y. Mori, T. Yamada, K. Miyata, H. Shindo, et al . 1996. Mammalian mature osteoclasts as estrogen target cells. Biochem. Biophys. Res. Commun. 223: 637-642 [Medline]. |
23. | Oursler, M.J., L. Pederson, L. Fitzpatrick, B.L. Riggs, and T. Spelsberg. 1994. Human giant cell tumors of the bone (osteoclastomas) are estrogen target cells. Proc. Natl. Acad. Sci. USA. 91: 5227-5231 [Abstract]. |
24. | Oursler, M.J., P. Osdoby, J. Pyfferoen, B.L. Riggs, and T.C. Spelsberg. 1991. Avian osteoclasts as estrogen target cells. Proc. Natl. Acad. Sci. USA. 88: 6613-6617 [Abstract]. |
25. | Kakudo, S., K. Miyazawa, T. Kameda, H. Mano, Y. Mori, T. Yuasa, I. Nakamaru, M. Shiokawa, K. Nagahira, S. Tokunaga, et al . 1996. Isolation of highly enriched rabbit osteoclasts from collagen gels: a new assay system for bone-resorbing activity of mature osteoclasts. J. Bone Miner. Metab. 14: 129-136 . |
26. | Takada, Y., M. Kusuda, K. Hiura, T. Sato, H. Mochizuki, Y. Nagao, M. Tomura, M. Yahiro, Y. Hakeda, H. Kawashima, and M. Kumegawa. 1992. A simple method to assess osteoclast-mediated bone resorption using unfractionated bone cells. Bone Miner. 17: 347-359 [Medline]. |
27. | Kameda, T., H. Ishikawa, and T. Tsutsui. 1995. Detection and characterization of apoptosis in osteoclasts in vitro. Biochem. Biophys. Res. Commun. 207: 753-760 [Medline]. |
28. | Iida, S., S. Kakudo, Y. Mori, M. Matsui, K. Magota, Y. Kitajima, N. Nakamura, H. Mano, Y. Hakeda, H. Azuma, et al . 1996. Human calcitonin has the same inhibitory effect on osteoclastic bone resorption by human giant cell tumor cells as salmon calcitonin. Calcif. Tissue Int. 59: 100-104 [Medline]. |
29. | Warshawsky, H., and G. Moore. 1967. A technique for the fixation and decalcification of rat incisors for electron microscopy. J. Histochem. Cytochem. 15: 542-549 [Medline]. |
30. |
Tezuka, K.,
Y. Tezuka,
A. Maejima,
T. Sato,
K. Nemoto,
H. Kamioka,
Y. Hakeda, and
M. Kumegawa.
1994.
Molecular
cloning of a possible cysteine proteinase predominantly expressed in osteoclasts.
J. Biol. Chem.
269:
1106-1109
|
31. | Tezuka, K., T. Sato, H. Kamioka, P.J. Nijweide, K. Tanaka, T. Matsuo, M. Ohta, N. Kurihara, Y. Hakeda, and M. Kumegawa. 1992. Identification of osteopontin in isolated rabbit osteoclasts. Biochem. Biophys. Res. Commun. 186: 911-917 [Medline]. |
32. | Walker, N.I., B.V. Harmon, G.C. Gobe, and J.F. Kerr. 1988. Patterns of cell death. Methods Achiev. Exp. Pathol. 13: 18-54 [Medline]. |
33. | Halachmi, S., E. Marden, G. Martin, H. Mackay, C. Abbondanza, and M. Brown. 1994. Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. Science (Wash. DC). 264: 1455-1458 [Medline]. |
34. | Kamei, Y., L. Xu, T. Heinzel, J. Torchia, R. Kurokawa, B. Gloss, S.-C. Lin, R.A. Heyman, D.W. Rose, C.K. Glass, and M.G. Rosenfeld. 1996. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell. 85: 403-414 [Medline]. |
35. | Arnett, T.R., R. Lindsay, J.M. Kilb, B.S. Moonga, M. Spowage, and D.W. Dempster. 1996. Selective toxic effects of tamoxifen on osteoclasts: comparison with the effects of oestrogen. J. Endocrinol. 149: 503-508 [Abstract]. |
36. | Friend, K.E., L.W. Ang, and M.A. Shupnik. 1995. Estrogen regulates the expression of several different estrogen receptor mRNA isoforms in rat pituitary. Proc. Natl. Acad. Sci. USA. 92: 4367-4371 [Abstract]. |
37. | Inoue, S., S. Hoshino, H. Miyoshi, M. Akishita, T. Hosoi, H. Orimo, and Y. Ouchi. 1996. Identification of a novel isoform of estrogen receptor, a potential inhibitor of estrogen action, in vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 219: 766-772 [Medline]. |
38. |
Kuiper, G.G.J.M.,
E. Enmark,
M. Pelto-Huikko,
S. Nilsson, and
J-Å. Gustafsson.
1996.
Cloning of a novel estrogen receptor expressed in rat prostate and ovary.
Proc. Natl. Acad.
Sci. USA.
93:
5925-5930
|