Download Steroid hormone receptor expression and action

Clinical Science (2000) 98, 217–240 (Printed in Great Britain)
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Steroid hormone receptor expression and
action in bone
Rosemary BLAND
Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
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The skeleton is a complex tissue, and hormonal control of bone remodelling is elaborate. The
important role that steroid hormones play in bone cell development and in the maintenance of
normal bone architecture is well established, but it is only relatively recently that it has become
possible to describe their precise mechanism of action. This review focuses not only on the
steroid hormones (oestrogens, corticosteroids, androgens and progesterone), but also on
related hormones (vitamin D, thyroid hormone and the retinoids), all of which act via
structurally homologous nuclear receptors that form part of the steroid/thyroid receptor
superfamily. By examining the actions of all of these hormones in vivo and in vitro, this review
gives a general overview of the current understanding of steroid hormone action in bone. In
addition, a comprehensive review of steroid hormone receptor expression in bone cells is
included. Finally, the role that future developments, such as steroid hormone receptor knockout
mice, will play in our understanding of steroid hormone action in bone is considered.
INTRODUCTION
Bone is a highly metabolically active tissue in which the
processes of osteoblastic bone formation and osteoclastic
resorption are continuous throughout life. Coupling of
osteoblast and osteoclast action ensures that normal bone
structure is maintained. A loss of bone homoeostasis may
result in a decrease in bone mass and deterioration of the
microarchitecture of the skeleton (osteoporosis), or a
defect in the mineralization of bone, such as that seen in
osteomalacia. Osteoporosis has huge health implications
affecting both men and women. It has been estimated
that, in the U.S.A., at least 90 % of all hip and spine
fractures among elderly white women and more than
70 % of those among elderly white men may be attributed
to osteoporosis [1]. Although there is a gradual loss of
bone during life, which is accelerated in postmenopausal
women, two factors that affect the degree of osteoporosis
are the peak bone mass achieved and the rate of bone loss.
Many factors influence the peak bone mass and the rate of
bone loss, including gender, environmental factors such
as diet, calcium intake and the level of exercise, and
genetic factors ; these have been reviewed in detail
elsewhere [2,3].
The control of bone formation and resorption is
influenced locally by factors such as cytokines (both
paracrine and autocrine), growth factors, mechanical
loading, nitric oxide, and cell–cell communications [4–8].
Systemic control is exerted through the action of cytokines, growth factors and hormones [9,10]. Endocrine
regulation occurs either by hormones acting at the cell
membrane, for example the classical bone hormones
parathyroid hormone and calcitonin [11,12], or by other
hormones, such as growth hormone and insulin [9].
Key words : bone, osteoblast, receptors, retinoids, steroids, thyroid hormone, vitamin D.
Abbreviations : AP, alkaline phosphatase ; AR, androgen receptor ; BMD, bone mineral density ; E , 17β-oestradiol ; ER, oestrogen
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receptor ; GR, glucocorticoid receptor ; IGF, insulin-like growth factor ; IGFBP, IGF binding protein ; IL-6, interleukin-6 ; MR,
mineralocorticoid receptor ; 1,25(OH) D , 1α,25-dihydroxyvitamin D ; PPAR, peroxisome-proliferator-activated receptor ; PR,
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progesterone receptor ; RT-PCR, reverse transcriptase–PCR ; RA, retinoic acid ; RAR, RA receptor ; RXR, retinoid X receptor ; T ,
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3,3h,5-tri-iodothyronine ; TGF, transforming growth factor ; T R, thyroid hormone receptor ; VDR, vitamin D receptor.
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Correspondence : Dr Rosemary Bland, Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH,
U.K. (e-mail R.Bland!bham.ac.uk).
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Additionally, evidence from a number of in vitro, in vivo
and clinical studies clearly indicates a role for steroid
hormones in the regulation of normal bone development
and the maintenance of intact bone.
SKELETAL ACTIONS OF STEROID
HORMONES
Bone remodelling is a complex process involving a
number of stages. Quiescent bone is covered by flat
bone-lining cells. In response to a bone-resorbing stimulus, osteoclastic migration and bone resorption are
activated. Osteoclasts then begin to remove both organic
matrix and mineral content of bone to produce a pit
(Howslip’s lacuna). During the reversal phase, macrophage-like mononuclear cells smooth the surface and
begin to deposit a cement-like substance to aid binding of
old bone to new bone. In the formation phase, osteoblasts
deposit osteoid in the pit, which is then mineralized
under osteoblastic control. Quiescence is restored at
completion of the cycle. Steroid hormones can influence
bone remodelling in a number of ways at any stage
throughout the remodelling cycle. They can act directly
on osteoblasts and osteoclasts to alter either bone
resorption or bone formation. However, it is important
to remember that, in vivo, normal bone structure is
maintained by complex interactions between osteoblasts
and osteoclasts, and it is difficult to consider effects on a
single cell type. Steroid hormones may also alter cellular
differentiation and\or proliferation and regulate the
expression of osteoblast target genes, such as those
encoding alkaline phosphatase (AP), osteopontin and
osteocalcin. Effects on matrix production or calcification,
and cell migration, have also been described.
The actions of steroid hormones on bone in vitro have
been studied in a variety of cell systems. A number of
osteoblastic cell lines exist ; the most commonly used
ones are mouse MC3T3-E1 cells [13], ROS 17\2.8 and
UMR 106 rat osteosarcoma [14,15], and the human cell
lines TE85 (HOS-TE85), MG-63 and SaOS-2 [16–18].
Primary cultures are normally prepared from calvarial or
trabecular bone. Studies into osteoclast activity have
proved to be more difficult. Osteoclasts are difficult to
isolate from bone, and until recently no true osteoclastic
cell lines existed. Consequently bone marrow cultures,
haematopoietic cell lines and cells derived from giant-cell
tumours have been used as model systems in which to
study osteoclast differentiation and activity. Transgenic
mouse technology has now allowed the generation of
two osteoclastic cell lines (reviewed in [19]). So far the
number of studies using these systems is very limited
compared with those on osteoblastic cells.
The actions of the steroid hormones, retinoids, vitamin
D and thyroid hormones are mediated by hormone
binding to structurally homologous nuclear receptors,
which act as ligand-dependent transcription factors to
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either activate or repress target gene expression [20,21].
Members of the steroid\thyroid receptor superfamily
regulate gene transcription by binding to similar hormone response element DNA sequences, as either homodimers or heterodimers [22,23], with the retinoid X
receptor (RXR) functioning as a common heterodimer
[24]. The expression of nuclear receptors in bone cells has
been studied to varying degrees, with the majority of data
focusing on the expression of steroid receptors in
osteoblasts. Far fewer studies have examined receptor
expression in other bone cells (Table 1). The importance
of steroid receptor expression in bone is highlighted in
receptor knockout animals. This is an emerging area of
research that should provide important insight into the
physiological requirement for steroid receptor expression
for normal skeletal development.
CORTICOSTEROID HORMONES
Glucocorticoids
In humans, glucocorticoids enhance bone resorption and
decrease bone formation, and consequently lead to a
decrease in bone mass [25,26]. Excess glucocorticoids in
vivo, as a result of either prolonged steroid therapy or
Cushing’s syndrome, are associated with a decrease in
bone mineral density (BMD) and in osteopenia, leading
to the development of osteoporosis in 30–50 % of cases
[27–30]. The degree of bone loss, which is greater in
trabecular than in cortical bone, appears to be related to
the duration and dose of treatment [25].
Robust animal models in which to study osteoporosis
are limited [31], and this is particularly the case for
studies of the effects of glucocorticoids on bone. In
common with other situations, the rat has been the most
popular animal system for studies both in vivo and in
vitro. However, results from in vivo studies in rats have
been inconsistent, with glucocorticoids inducing both
positive and negative effects on rat bone [32–35]. In
addition, the effects of glucocorticoids on bone in vitro
are clearly species-dependent. In rats, glucocorticoids
increase the differentiation of osteoblasts and osteoblast
progenitors [32,36,37], resulting in an induction of the
ability of the cells to produce mineralized matrix and
bone nodules [37–40]. Boden et al. [36] demonstrated
that some of these differentiation effects are probably
mediated via bone morphogenic protein-6. In addition,
insulin-like growth factors (IGFs) and their binding
proteins (IGFBPs) are important in bone development,
and may be involved in the glucocorticoid regulation of
osteoblast function [41–44]. This increase in osteoblast
differentiation is associated with an induction of osteoblast marker genes. AP mRNA and activity are
stimulated by glucocorticoids [39,45], as are osteopontin,
osteocalcin and bone sialoprotein [39,40,46,47]. Glucocorticoids also have a significant influence on collagen
Steroid receptors in bone
synthesis. They either increase [39,48] or decrease [48,49]
collagen production, and also cause an osteoblast-specific
decrease in the adhesion of bone cells to collagen type I
and fibronectin [50]. In addition to stimulating osteoblast
activity, glucocorticoids have also been shown to decrease
bone resorption in rats, which was paralleled by a
decrease in osteoclast numbers, most probably due to
osteoclast apoptosis [51].
In mice, however, the principal effect of glucocorticoids is to stimulate bone resorption and osteoclast
formation [52,53], an effect which is mediated directly via
the glucocorticoid receptor (GR) [52]. Glucocorticoids
are also able to modulate osteoclast responsiveness to
calcitonin, and this occurs possibly via regulation of
calcitonin receptor expression [54]. Additionally,
Weinstein et al. [55] demonstrated that glucocorticoid
treatment resulted in an inhibition of osteoblastogenesis
and the promotion of osteoblast apoptosis, and Bellows
et al. [32] demonstrated a glucocorticoid-mediated inhibition of the proliferation and differentiation of osteoprogenitors. These results were confirmed by a reduction
in osteoblast activity, as shown by decreases in AP
activity, osteocalcin mRNA and secretion, collagen
mRNA levels and synthesis and calcium accumulation
[56,57]. Glucocorticoid-mediated responses operate during a very specific time period, and the effects of
dexamethasone on differentiation, in both rats and mice,
are most potent when acting on proliferating cells [40,57].
Results generated from in vitro studies on human
bone cells conflict with in vivo observations and indicate that, contrary to being detrimental, glucocorticoids
are actually required for normal osteoblast differentiation and may prevent osteoblast apoptosis under
certain conditions [58]. In human osteoblast cultures,
dexamethasone increased AP activity [59–63] and was
required for differentiation into a mineralizing, boneproducing osteoblast [61,63]. The effect of dexamethasone on osteocalcin levels was more variable
[59,60,62], and in primary cultures of osteoblasts the
responses may be dependent upon the age of the donor
[62]. One possible explanation for this conflicting evidence is that glucocorticoids, and steroids in general,
exert differential effects on the developing compared
with the adult skeleton. Additionally, in vitro studies
generally assume that bone cells respond uniformly to
hormones, whereas in vivo the response to systemic
stimuli is probably highly localized.
GR expression
In humans, alternative splicing of the GR mRNA
produces two highly similar isoforms, GRα and GRβ, in
which amino acids 1–727 are identical [64]. However,
GRβ does not bind ligand and, although it may act as a
dominant-negative regulator of GRα activity [65], it has
not been studied in any detail ; thus the data discussed
here refer to the GRα isoform. Cytosolic binding studies
have shown specific glucocorticoid binding sites in rat
and mouse osteoblastic cells [66–71]. Specific binding has
also been demonstrated in human osteoblasts and human
osteosarcoma cells [60,72–74]. GR mRNA has been
detected in human osteoblasts and in the human cells
lines SaOS-2, MG-63 and TE85 [60,74–77]. The only
evidence for GR protein has been provided by Abu et al.
[78], who detected GRs in a number of human bone cells,
including osteoblasts, osteocytes and chondrocytes, but
they were unable to detect GR expression in osteoclasts.
Mineralocorticoids
There have been very few studies of mineralocorticoid
action in bone. Agarwal et al. [79] found that aldosterone
significantly inhibited AP activity and enhanced the
proliferation of rat calvaria osteoblastic cells, and this was
inhibited by two specific mineralocorticoid receptor
(MR) antagonists. Some other evidence for the potential
role of mineralocorticoids in bone comes from patients
with apparent mineralocorticoid excess, an inherited
form of hypertension. Apparent mineralocorticoid excess
is caused by a deficiency of 11β-hydroxysteroid dehydrogenase, the enzyme responsible for the interconversion of hormonally active cortisol into inactive
cortisone [80]. Bone disease was reported in two affected
cases, possibly as a result of increased mineralocorticoid
activity [81,82].
MR expression
In osteoblastic cells derived from newborn-rat calvaria,
MR mRNA has been detected by Northern analysis and
reverse transcriptase–PCR (RT-PCR). The MR protein
was demonstrated by immunoprecipitation and Western
blotting, and was localized to the cytosol by immunocytochemistry [79]. RT-PCR and Northern analysis have
also demonstrated the presence of mRNA encoding the
MR in primary cultures of human osteoblasts and human
osteosarcoma cells [74], although specific MR binding
was not detected in any of the osteosarcoma cell lines
[74]. Recently we have demonstrated mRNAs for both
the GR and the MR in the differentiating human foetal
osteoblast cell line SV-HFO (R. Bland and M. Hewison,
unpublished work). In a study examining receptor
expression in human foetal tissue, both GR and MR
mRNAs were localized to osteoblasts at bone-forming
sites. The MR was also present in chondrocytes of
developing ribs and long bones. However, unlike the GR,
which is present throughout gestation, MR mRNA was
undetectable after 16 weeks [77].
It is interesting to note that glucocorticoids act
primarily through the GR ; however, in vitro they are also
able to bind to the MR with a similar affinity as
mineralocorticoids [83]. In classical MR target tissues
(kidney, colon), the MR is aldosterone-selective, because
glucocorticoids are inactivated by 11β-hydroxysteroid
dehydrogenase. Consequently, glucocorticoids can act
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Table 1
Expression of members of the steroid/thyroid hormone receptor superfamily in bone cells
Numbers denote references, and letters in square brackets indicate the detection method used : [a] RT-PCR, [b] Northern blot analysis/RNase protection assays, [c] in
situ hybridization, [d] in situ PCR, [e] Western blot analysis, [f] immunohistochemistry/immunocytochemistry, [g] binding assays. Abbreviations : OB, osteoblasts ; OC,
osteoclasts.
(1) Steroid hormones
Cell type
Human OB
Human OC
Human osteocytes
Human chondrocytes
Human growth plate
Human foetal bone
Human foetal cell line (SV-HFO)
Human foetal OB
Human foetal OC
Human foetal chondrocytes
HOS-8603
U2-OS
GR
MR
60 [b,g], 72 [g], 74 74 [a]
[a], 76 [a], 78 [f]
78 [f]
78 [f]
77 [c], 78 [f]
ERα
ERβ
AR
PR
72 [g], 116 [b,g],
118 [g], 127 [c],
136 [d], 184 [b,g]
143 [f], 144 [f]
136 [d]
127 [c]
137 [f]
129 [a]
132 [a]
394 [b,e,f]
72 [g], 184 [b,g],
188 [g], 190 [f]
116 [g], 211 [a,f],
215 [f]
77 [c]
77 [c]
74 [a,b,g]
74 [a,b]
117 [g], 119 [g],
122 [b,e,f]
TE85 (HOS-TE85)
74 [a,b,g]
74 [a,b]
MG-63
Human pre-OC cell lines
Human giant stromal cells
Human giant-cell tumour
Rat calvarial osteoblastic cells
74 [a,b,g], 75 [b]
74 [a,b]
66 [g], 67 [g], 68
[g], 69 [g]
79 [e,f]
112 [b,g], 117 [g],
120 [b,e,f]
75 [b], 212 [a]
149 [a,e], 150 [c,f]
147 [a]
142 [b,g], 145 [g]
124 [a], 125 [a],
126 [a]
112 [b,g], 113 [b,g],
114 [b,g], 121 [a],
125 [a]
114 [b,e,g], 130 [a]
123 [a]
148 [c]
138 [f]
71 [b,g]
70 [g], 115 [g], 128
[a], 129 [a], 130
[a]
ROS 17/2.8
Mouse OC-like cells
Mouse growth plate
Mouse bone marrow stromal cells
Rabbit OC
Rabbit growth plate
Chicken OC
Pig and guinea-pig osteocytes
144 [f]
190 [f]
190 [f]
73 [g]
SaOS-2
UMR 106
Rat calvarial periosteum
Rat pre-OC
Rat growth plate
Mouse osteosarcoma
MC3T3-E1
394 [f]
394 [f]
139 [f]
137 [c,f]
131 [a]
132 [a]
133 [c]
133 [c]
133 [c]
71 [g]
70 [g]
394 [b,e]
394 [b,e]
183 [b,e,g], 185 [g], 185 [g]
187 [b,e,g]
183 [b,e,g], 185 [g], 117 [g], 119 [g],
185 [g], 221 [a,f]
186 [e,g], 187
[b,e,g], 212 [a]
177 [b,g], 187
117 [g], 210 [b,g],
[b,e,g]
211 [a,f], 213 [a]
211 [a,f], 212 [a]
149 [e]
145 [g]
125 [a]
125 [a]
130 [a], 394 [e]
185 [g]
128 [a], 129 [a],
130 [a]
70 [g], 130 [a], 189
[b,e,f,g]
191 [f]
115 [g], 130 [a,f]
146 [b]
127 [c], 138 [f]
140 [b,e,g], 141
[a,e]
135 [f]
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137 [c,f]
130 [a,f]
130 [a,f]
179 [g]
Steroid receptors in bone
Table 1
(cont.)
(2) Related non-steroid hormones
Cell type
VDR
Human OB
Human OC
Human chondrocytes/ osteocytes
Human foetal chondrocytes
U2-OS
SaOS-2
MG-63
Rat OB
Rat OC
Rat osteocytes
Rat tibia
Rat chondrocytes
72 [g], 76 [a], 235 [d]
235 [d]
236 [e]
18 [g], 231 [g], 236 [e]
75 [b], 236 [e], 366 [b,g]
234 [c], 238 [f], 239 [f], 240
[f]
240 [f]
234 [c]
T3R
RAR
RXR
282 [f] (α1, α2, β1)
281 [b,f] (α1, α2, β1),
282 [f] (α2, β1)
282 [f] (α2, β1)
283 [g]
330 [a] (α, β, γ)
330 [a] (α)
330 [a] (α, β, γ)
75 [b]
236 [a] (α)
236 [a] (α), 330 [a] (α)
236 [a] (α)
328 [b] (α, β, γ)
293 [b] (α, γ)
328 [b] (α, β)
293 [b] (α, β)
237 [e]
327 [b] (α)
224 [b] (α, β, γ), 237 [e],
327 [b] (α, γ)
232 [b] (α, β, γ), 237 [e],
303 [b] (α, β, γ)
237 [e] (α)
232 [b] (α, β), 237 [e]
(α)
232 [b] (α, β), 237 [e]
(α)
71 [b] (α, γ)
302 [b], 329 [b] (α, γ)
329 [b] (α, β)
281 [b,f] (α1, α2, β1)
285 [a,e] (α1, β1) [b]
(α2)
284 [f] (α1, β1)
237 [e] (α1, α2, β1)
Rat growth plate (various cells)
Rat calvaria
388 [g], 389 [g], 390 [g]
Rat pre-OB
ROS series of rat osteosarcomas 230 [g], 232 [b], 237 [e], 241 232 [b,e] (α1, α2, β1),
[g], 364 [g]
256 [g], 257 [g]
UMR series of rat osteosarcomas 130 [a], 232 [b], 233 [b,g],
130 [a] (α, β), 232 [b,e]
237 [e], 391 [g]
(α1, α2, β1), 261 [g],
281 [b] (α1, α2, β1),
[f] (α)
Mouse calvarial cells
392 [g]
280 [g]
Mouse osteosarcoma cells
71 [g]
MC3T3-E1
115 [g], 130 [a], 233 [b,g],
130 [a] (α, β), 258 [g]
393 [g]
Mouse limb buds, teeth,
cartilage
Mouse bone marrow stromal
115 [g], 130 [a,f]
130 [a,f] (α, β)
cells
Rabbit OC
Chicken calvaria
388 [g]
through the GR or, depending on the activity of the
locally expressed 11β-hydroxysteroid dehydrogenase,
the MR.
SEX STEROIDS
This group of steroids includes oestrogen, the hormone
that is most closely associated with effects on bone.
However, androgens and progesterone also play an
important role in the maintenance of bone structure.
Oestrogens
Oestrogens are important for the development, maturation and maintenance of the skeleton (reviewed in
[84,85]). Oestrogen deficiency results in osteoporosis.
Postmenopausal bone changes can be prevented or
326 [c] (γ)
298 [b] (α)
298 [b] (β)
reversed following treatment with oestrogen, and the use
of oral contraceptives may exert a beneficial influence on
bone mass [84,86]. Oestrogens have important effects on
bone in both males and females. The pubertal growth
spurt of both sexes is driven primarily by oestrogens [87].
The importance of oestrogens for the male skeleton is
confirmed by the single case report of complete oestrogen
resistance in a male [88]. The man, who had a homozygous truncation of the oestrogen receptor (ER) due to
a point mutation which produced a premature stop
codon in the ER gene, had increased bone turnover,
osteoporosis, unfused epiphyses, delayed bone age and
continued linear growth into adulthood [88]. This phenotype is very similar to that seen in two aromatasedeficient men (discussed later).
In species other than the human, oestrogens produce
effects on bone similar to those seen in humans, and the
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ovariectomized rat is an useful model for postmenopausal
bone loss [84,89]. Interestingly, Gouveia et al. [90] found
that, in rats, physiological doses of thyroxine prevented
the bone loss associated with ovariectomy. However, it is
not yet known if the cellular and molecular mechanisms
of oestrogen action in rodents are the same as in humans.
Oestrogens appear to have effects on both osteoblasts
and osteoclasts. 17β-Oestradiol (E ) has been reported to
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both stimulate [91,92] and inhibit [93,94] osteoblast
proliferation. This divergent effect may be dependent
upon cell density [94]. E stimulates the mRNA ex#
pression and\or activity of a number of osteoblast marker
genes, including collagen type I, osteocalcin, osteopontin,
osteonectin and AP [92–96]. This change in osteoblast
markers correlates with an increase in osteoblast differentiation and increased deposition and mineralization
of matrix [92]. However, oestrogens primarily regulate
bone remodelling by modulating the production of
cytokines and growth factors from bone marrow and
bone cells (reviewed in [10,84,97]). In osteoblasts derived
from various sources, E has been shown to increase
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IGF-I levels [91], decrease interleukin-6 (IL-6) mRNA
expression and inhibit the induction of IL-6 by tumour
necrosis factor-α [98]. The decrease in BMD generated by
ovariectomy in rats is associated with a decrease in
transforming growth factor (TGF)-β3 mRNA expression, which is restored by E therapy [99]. Similar
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responses have also been seen in a series of experiments
using a range of human foetal osteoblastic cell lines transfected with ERs. E has been shown to increase IGF-I
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mRNA and bone morphogenic protein-6 mRNA and
protein, but did not alter the expression of IGF-II, bone
morphogenic protein-1–5 and 7 or TGF-β1 and -β2 [100,
101]. Conversely, E inhibited IL-6 production [102].
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Oestrogen also exerts a significant influence on osteoclast function. A loss of oestrogen stimulates osteoclastogenesis, and this is thought to be mediated primarily
by an induction of IL-6 [103], although there is evidence
to suggest that the oestrogenic induction of osteoclast
differentiation is mediated via osteoblasts and depends on
the presence of IL-6 receptors on osteoblastic cells
[97,104]. It is currently thought that the protective effect
of oestrogens in bone may be mediated via regulation of
bone cell apoptosis. E induces apoptosis of isolated
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osteoclasts [105], and it has been suggested that this is
mediated via TGF-β1 [106]. Similarly, oestrogen loss in
ovariectomized rats and in humans undergoing oestrogen
withdrawal therapy leads to an increase in the number of
apoptotic osteocytes [107,108]. Conversely, in foetal
human osteoblasts E increased the expression of TIEG
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(TGF-β-inducible early gene), which has a role in
regulating apoptosis [109].
ER expression
Originally it had been thought that there was only a
single ER. However, it is now apparent that, in addition
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to the original ER (ERα), there is a second receptor
subtype, ERβ, which is able to bind oestrogenic compounds [110,111]. ERα and ERβ are not isoforms of each
other, but two distinct proteins encoded by separate
genes located on different chromosomes. Therefore,
unless ERβ is specified, all the previous studies concentrated on ERα. Despite the obvious importance of
oestrogens in bone, ERs have proved difficult to detect.
Binding assays have revealed the presence of ERs in rat
osteosarcoma cells [112–114], mouse osteoblastic cells
[70,71,115] and human osteoblasts and osteoblastic-like
cell lines [71,112,116–119]. Kd values for oestradiol
binding range from 0.1 nM [119] to 1 nM [112], and the
number of binding sites per cell is generally low (65–825)
[71,112], although greater numbers of receptors per cell
have been reported in some cell systems [70,118].
Several studies have confirmed the presence of mRNA
encoding the ER in rat, mouse and human osteosarcoma
cell lines [71,75,112–114,120–122], rat calvarial cells
[123–126] and rabbit and human osteoblasts and chondrocytes [127]. Western blotting has demonstrated ER
protein in human osteosarcoma cells [120,122].
Few studies have so far examined the expression of
ERβ in bone. ERβ mRNA has been detected in rat
calvarial cells, in rat and mouse osteoblastic cells
[125,128–130], in the human differentiating osteoblastic
cell line SV-HFO and in human foetal tissue [131–133].
The expression of the ER isoforms appears to be
differently regulated during osteoblast differentiation. In
rat calvarial cells, increasing ERα expression is correlated
with increasing osteoblast differentiation [124–126], and
in MC3T3-E1 cells decreasing ERβ mRNA expression
was associated with osteoblast maturation [129]. However, in the human SV-HFO cells it is increased expression of ERβ that is associated with increasing
differentiation, while levels of ERα mRNA remain
constant [132].
Although receptor binding assays and Northern and
Western blotting have demonstrated the presence of ERs
in osteoblasts, attempts to identify ERs in bone by
immunocytochemistry have proved difficult [134].
Ikegami et al. [120] and Sutherland et al. [122] found
some nuclear staining in human osteosarcoma cells.
However, Braidman and co-workers [135] were unable
to detect ER protein in human osteoblasts, although very
low levels of ER mRNA in human osteoblasts and
osteocytes were detected using in situ RT-PCR [136].
ERs have also been detected in pig and guinea-pig
osteocytes [135], and species-specific expression of
mRNA and protein for ERα and ERβ has been shown in
rat, rabbit, mouse and human growth plates [137,138].
However, in contrast with ERα expression, which is seen
in resting, proliferative and hypertrophic chondrocytes
[127], ERβ is expressed exclusively in hypertrophic
chondrocytes [139]. In mice, ERβ mRNA and protein
were detected in early hypertrophic chondrocytes, but
Steroid receptors in bone
ERα was not seen. However, in human bone both ERα
and ERβ were present in resting and proliferating
chondrocytes and in the early hypertrophic zone
[133,137].
ER expression in osteoclasts is controversial. Several
reports have demonstrated ERs in osteoclasts. Both
Oursler et al. [140] and Pederson et al. [141] have
demonstrated ER mRNA, protein and binding in avian
osteoclasts. Oursler et al. [142] also detected ER protein
in human osteoclasts isolated from giant-cell tumours, as
did Gunhan et al. [143]. Pensler et al. [144] showed
immunostaining of ERs in normal human osteoclasts.
Other groups have shown either very low or undetectable
levels of ERs in rat, rabbit and human osteoclasts
[133,145–148]. However, ERs have been demonstrated in
pre-osteoclastic cells : osteoclast precursor cells [148],
bone marrow- or giant-cell-tumour-derived stromal cells
[115,145,147,130] or the pre-osteoclastic cell lines FLG
29.1 and TCG 51 [149,150]. Connor et al. [133] also
demonstrated ERβ mRNA in human foetal osteoclasts.
Androgens
The role of androgens in the maintenance of a normal
bone structure has been reviewed previously [151–153].
Serum androgen levels in females are positively correlated
with bone density both pre- and post-menopause
[154–156]. Patients with defective androgen receptors
(ARs) often develop osteopenia [157], and BMD is
decreased in females with androgen-insensitivity syndrome, despite appropriate treatment with oestrogen and
progesterone [158,159]. In contrast, the male Tfm (testicular feminization) rat, which is androgen-resistant,
develops a female bone phenotype, but is not osteoporotic. It has been suggested that the high oestrogen
concentrations produced by aromatization (discussed
later) of the non-functional androgens may limit bone
defects. This is endorsed by the observation that, if Tfm
rats are orchidectomized (and therefore lose the testicular
source of oestrogens), they do not achieve a normal
trabecular bone volume [160]. Hypogonadism in men is
associated with increased bone turnover, which may
result in osteoporosis [161,162]. Testosterone supplementation has been found to increase BMD in eugonadal
osteoporotic men [163–165]. It has even been suggested
that glucocorticoid-induced bone loss may be mediated
via a decrease in the levels of free testosterone in males
[166]. In both asthmatic men and female rats (with
cortisol-induced bone loss), testosterone was able to
reverse the effects of glucocorticoids [167,168].
The most commonly used animal system is the
orchidectomized male rat [169]. An imbalance between
bone formation and resorption results in the loss of
cancellous and cortical bone [170–173]. The bone loss
due to orchidectomy can be prevented by treatment with
testosterone and oestrogens [169–171]. Interestingly, it
has also been shown that androgens play a significant role
in maintaining bone volume in oestrogen-deficient rats
[174].
Most in vitro studies have demonstrated that
androgens stimulate proliferation and differentiation of
osteoblasts [70,175–177] and osteoblast precursors [37],
and increase AP activity [176]. These responses may be
due to alterations in TGF-β mRNA expression and
activity [176–178]. Androgens also mediate osteoclast
function. Treatment of osteoclasts with either testosterone or dihydrotestosterone produces a decrease in
their bone resorption ability and an increase in TGF-β
secretion [179]. Additionally, androgen withdrawal (orchidectomy) results in an increase in osteoclast numbers
[180]. This increase in osteoclastogenesis is also seen in
bone-marrow-derived stromal cells and is probably due
to an increase in IL-6 production [180].
AR expression
Evidence from the oophorectomized rat and studies
using AR antagonists, either in vivo in mice or with
human osteoblastic cells in vitro, suggest that the effects
of androgens on bone are caused by a direct action on the
AR [176,181], possibly via an increase in AR numbers or
mRNA expression [182,183]. In keeping with this, ARs
have been detected in osteoblasts and osteoclast-like
cells.
In 1989 Colvard et al. [184] detected nuclear AR
binding in human osteoblast cells. Binding has since been
demonstrated in human osteoblasts and osteosarcoma
cells [72,177,185–188]. AR binding has also been demonstrated in the rat osteosarcoma cell line UMR 106.01
[185] and in the mouse osteoblastic cell line MC3T3-E1
[70,189]. The number of binding sites per cell appears to
vary greatly and is species-specific, with as few as 70
binding sites in UMR 106.01 cells [185] to more than
14 000 in MC3T3-E1 cells [70]. AR mRNA and protein
have also been detected in human osteosarcoma cells
[183,187] and MC3T3-E1 cells [130,189]. Immunohistochemistry of normal, developing human bone localized
ARs to chondrocytes and osteoblasts at sites of bone
formation, and to a lesser extent to osteocytes and
mononuclear cells within the bone marrow [190]. In a
further study, Mizuno et al. [191] detected AR protein in
osteoclast-like multinucleated cells obtained from cocultures of mouse osteoblast and bone marrow cells, and
AR binding has been demonstrated in avian osteoclasts
[179].
It is important to remember that, as well as activating
the AR, androgens can be converted into oestrogens by
the enzyme aromatase [192], and therefore can exert their
effects via the AR or via the ER. Females with aromatase
deficiency exhibit delayed bone age and have no growth
spurt, despite elevated androgen levels [193]. Inhibition
of aromatase activity in male rats impairs skeletal
modelling and bone maintenance in both growing and
aged rats and mimics the effects of orchidectomy
# 2000 The Biochemical Society and the Medical Research Society
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224
R. Bland
[194,195], and the aromatase knockout mouse develops
osteopenia [196]. Two men with mutations in the
aromatase P450 gene demonstrated tall stature, continued
growth, delayed skeletal maturation and osteopenia,
despite high levels of circulating androgens and the
ability to respond to oestradiol [197,198]. These
observations are similar to those seen in the man with a
mutation in the ER, re-inforcing the importance of
oestrogens for the development of the male skeleton. The
relative contribution of androgens\oestrogens in male
skeletal homoeostasis has been reviewed recently [199].
Progesterone
Clinical studies using progesterone treatment have
suggested a protective effect on bone mass [200,201].
Additionally, in glucocorticoid-induced osteoporosis,
progesterone treatment caused an increase in trabecular
bone density [202], suggesting that progesterone antagonizes the glucocorticoid-mediated effects, thereby
preventing glucocorticoid-induced bone loss [203]. This
has also been demonstrated in vitro, where progesterone
inhibited the glucocorticoid-induced release of %&Ca from
mouse calvarial bone [52]. In postmenopausal women,
levels of progesterone as well as oestrogen decrease, and
postmenopausal osteoporosis may be due in part to the
loss of progesterone. In other situations of low circulating
oestrogens, for example in ovariectomized rats (supplemented with progesterone) or pseudopregnant rats, it
appears that the bone mass is protected by the high levels
of progesterone present [204,205].
In cultured rat calvaria, progesterone increases cell
proliferation and [$H]proline incorporation into collagen
proteins [206]. It also increases osteoblast differentiation,
as measured by the increase in AP activity, osteocalcin
secretion and the number and area of bone nodules [207].
In bone cell cultures derived from adult rat vertebrae,
progesterone stimulates the proliferation and differentiation of osteoprogenitors, increasing the number of
bone nodules and AP-positive colonies [208]. Interestingly, this differentiation appears to be sex-dependent,
with the effects of progesterone being seen only in
osteoprogenitor populations derived from adult female
rats, and not in cell populations derived from adult male
rats [37]. In contrast with the regulatory effects of the
other hormones, IL-6 mRNA and protein were not
affected by progesterone in human osteoblasts or human
bone marrow stromal cells [209].
Progesterone receptor (PR) expression
The presence of PRs in human bone has been demonstrated in both osteoblasts and osteoclasts. Progesterone
binding was first detected in primary cultures of human
osteoblast-like cells [116], and has since been detected in
human osteosarcoma cell lines [131,133,220]. However,
Orwoll et al. [185] and Wei et al. [210] were unable to
detect binding in either UMR 106.01 cells or SaOS-2
# 2000 The Biochemical Society and the Medical Research Society
cells. PR mRNA and protein have also been detected in
human osteoblastic cells [185,210–212], and MacNamara
and Loughrey [213] have recently shown that transcripts
encoding the A and B forms of the PR (two isoforms
transcribed from distinct promoters within the same gene
[214]) are present. Immunohistochemical analysis of
human bone localized PR protein to both osteoblasts
[215] and osteoclasts [144]. Fiorelli et al. [149] have
demonstrated specific progesterone binding in the preosteoclastic cell line FLG 29.1.
OTHER RELATED (NON-STEROID)
HORMONES
This group of hormones includes vitamin D, thyroid
hormone and the retinoids. Although these hormones are
not steroids, they function via nuclear receptors which
form part of the steroid\thyroid receptor superfamily.
Vitamin D
Vitamin D is required for normal bone development,
and vitamin D deficiency is associated with rickets in
children and osteomalacia in adults (reviewed in [216]).
Disorders of resistance to vitamin D also clearly indicate the requirement for 1α,25-dihydroxyvitamin D
$
[1,25(OH) D ] for normal bone development [217].
# $
Early studies assumed that the predominant effects of
1,25(OH) D on bone were mediated via the osteoblast.
# $
The evidence for this came partly from the presumed lack
of vitamin D receptor (VDR) expression in osteoclasts
(discussed below), coupled with the fact that, in response
to 1,25(OH) D , osteoblasts secrete factors which stimu# $
late osteoclastic bone resorption and osteoclast differentiation [218,219]. Consequently studies have focused
on the action of 1,25(OH) D in osteoblastic cells.
# $
1,25(OH) D modulates the expression of a number of
# $
osteoblastic marker genes, including osteocalcin
[57,220–222], osteopontin [223] and AP [224]. Additionally, 1,25(OH) D inhibits collagen synthesis [225–227].
# $
However, 1,25(OH) D has a profound effect on the
# $
differentiation and proliferation of both osteoblasts and
osteoclasts [220,228]. In common with many other
steroids, IL-6 appears to be important, possibly mediating the induction of osteoclastogenesis by 1,25(OH) D
# $
[229].
VDR expression
Clear evidence for the importance of VDR expression for
normal bone development is seen in the condition of
hereditary vitamin D-resistant rickets [217]. Numerous
studies have demonstrated the presence of VDRs in rat,
mouse and human bone cells using 1,25(OH) D binding
# $
assays (detailed in Table 1). Generally the number of
binding sites per cell is quite high, reaching 18 000 in ROS
17\2.8s cells [230] and 37 000 in mouse osteosarcoma cells
[71], although it has been suggested that the number of
Steroid receptors in bone
binding sites per cell depends on the cell density [231].
VDR mRNA has also been demonstrated in human, rat
and mouse osteoblastic cells [75,76,130,232,233] and
localized to rat and human osteoblasts by in situ
hybridization [234] and in situ RT-PCR [235]. VDR
protein has also been detected in rat and human osteosarcoma cells [236,237] and has been localized to the
osteoblasts in rat bone by immunocytochemistry
[238–240]. Johnson et al. [240] specifically studied the
expression of the VDR in rats throughout skeletal
development. Immunostaining was demonstrated in the
mesenchyme condensing to form the vertebral column
and limbs on day 13 of gestation, and in the chondrocytes
and osteoblasts of limb buds on day 17 of gestation.
There is evidence to suggest that the osteoblastic
response to 1,25(OH) D varies in relation to the stage of
# $
osteoblast development [40,222]. It also appears that the
expression of VDRs may be dependent upon the stage of
osteoblast development. In less well differentiated rat
osteoblast-like cells, 1,25(OH) D binding was variable.
# $
No binding was detected in ROS 24\1 cells [230], and in
ROS 2\3 cells binding has been reported to be both
present [230] and absent [241]. Additionally, Bland et al.
[237] also demonstrated that the level of VDR protein
expressed varied depending on the stage of osteoblast
differentiation, with VDR expression highest in the least
well differentiated cells.
It has generally been accepted that osteoclasts do not
have VDRs [238]. However, Johnson et al. [240] detected
VDR protein in cells which resembled osteoclasts, and
VDR mRNA has been detected in bone marrow cells and
osteoclasts using the highly sensitive technique of in situ
RT-PCR [235]. More recently, Gruber et al. [130]
detected VDR mRNA in mouse bone marrow cells.
Thyroid hormones
The response to thyroid hormones varies depending on
the skeletal site [242]. However, thyroid hormones
clearly play a role in normal bone development. Increased
osteoblast and osteoclast activity in hyperthyroidism
leads to increased bone turnover, which may result in
osteoporosis [243–245]. Infantile hyperthyroidism is a
disorder sometimes accompanied by secondary craniostenosis, and it appears that the increased bone formation,
which leads to premature closure of the sagittal suture, is
caused by the thyroid hormone excess [246]. Juvenile
hypothyroidism results in delayed skeletal maturation
and epiphyseal dysgenesis with long bone growth retardation. In adults, hypothyroidism is associated with
reduced bone turnover and osteosclerosis [243,244],
which is often corrected following thyroid hormone
therapy [247]. Although thyroid replacement therapy has
been associated with bone loss, this occurs primarily in
patients who were previously thyrotoxic and have been
rendered hypothyroid by treatment [248]. Perhaps the
best evidence for a requirement for 3,3h,5-tri-iodo-
thyronine (T ) is seen in syndromes of thyroid hormone
$
resistance, where a mutation in the gene encoding the β
form of the thyroid hormone receptor (T R) often results
$
in abnormal bone development, which may be characterized by short stature, delayed skeletal maturation and
abnormal epiphyseal fusion with stippling [249].
Rats in which hypothyroidism has been induced show
an increase in bone volume [250] and changes to the
epiphyseal growth plate [251]. Conversely, hyperthyroid
rats show a small increase in the number of eroded
surfaces [250] and also demonstrate a decrease in BMD,
which varies according to the skeletal site [90,252].
Although thyroid hormones stimulate bone resorption
directly (as measured by the release of previously
incorporated %&Ca) in foetal rat bones [253], it is unlikely
that osteoclasts are the primary T target cell. Isolated
$
osteoclasts are unable to respond to T and require co$
culture with other bone cells [244,254,255].
Thyroid hormones have also been shown to regulate a
number of osteoblast genes. In rat and mouse osteoblasts,
T induced AP activity as well as osteocalcin and collagen
$
production [256–260]. Interestingly, AP activity was
unaffected in UMR 106 cells [261]. Thyroid hormones
also regulate certain growth factors and cytokines. T
$
induces IGF-I and IGFBP-2 mRNA expression in rat
primary cultures and osteoblastic cell lines [262,263],
producing a corresponding rise in protein levels [263].
This increase appears to be dose-dependent, with high
concentrations abolishing the effect [264]. IGFBP-4
transcription was increased in MC3T3-E1 cells [265], and
it is possible that this contributes to the anti-proliferative
effects of T . T increased IL-6 and IL-8 mRNA and
$ $
protein levels in human bone marrow stromal cells and
MG-63 cells, although no response was seen in SaOS-2
cells or primary human osteoblasts, and no regulation of
IL-1β, IL-3, IL-2 or IL-4 was observed [266]. In contrast,
T reduced the induction of IL-6 synthesis produced by
$
activators of both the protein kinase C and the protein
kinase A pathways in MC3T3-E1 cells [267].
The regulation of these genes by thyroid hormones
appears to be site-specific. Following treatment of rats
with thyroxine, in situ hybridization detected a rise in
osteocalcin and AP mRNAs in femoral, but not vertebral,
osteoblasts [252]. In bone marrow cultures of committed
osteoblasts obtained from femoral bone, T increased
$
osteocalcin and collagen type I mRNA levels, with only
a slight increase in IGF-I mRNA. However, in similar
cultures obtained from vertebral bones, T had no effect
$
on osteocalcin or collagen type I, but IGF-I mRNA was
markedly increased [268]. AP activity was also found to
vary between sites [268]. It is probable that this anatomically localized response is not limited to thyroid
hormones, but is likely to be common to most steroids
(localized bone changes in the oestrogen knockout mouse
are discussed later). This would provide an important
mechanism for the specificity of hormone action in bone.
# 2000 The Biochemical Society and the Medical Research Society
225
226
R. Bland
At high concentrations, T normally decreases cell
$
replication in rat and mouse osteoblastic cell lines
[257,258,261,269] and primary cultures of rat osteoblasts
[259,260]. Varga et al. [270] found that T did not inhibit
$
DNA synthesis, but increased the number of apoptotic
cells. Thyroid hormones appear to have a profound effect
on bone cell development. Responses to thyroid
hormones vary depending on the stage of osteoblast
development [232], and T has been shown to prevent the
$
acquisition of an osteoblast phenotype by MC3T3-E1
cells [269]. It also suppresses the differentiation of
osteoprogenitor cells to osteoblasts [271]. Both T and to
$
a lesser extent thyroxine induce chondrocyte hypertrophy and matrix formation [272,273] ; the effects of
thyroid hormones on chondrocytes have been reviewed
recently [274].
T3R expression
T Rs are encoded by two c-erbA genes (α and β) located
$
on different chromosomes [275]. Alternative splicing of
the T Rα gene leads to the generation of two mature
$
mRNAs that are translated into two proteins : T Rα1 and
$
c-erbAα2 [276,277]. Alternative splicing of the β gene
produces T Rβ1 and T Rβ2 [278,279].
$
$
Nuclear T binding has been shown in rat osteo$
sarcoma cell lines [256,257,261] and mouse osteoblastic
cells [258,280], with Kd values ranging from 0.1 nM
[257,258] to 5 nM [256] and with approx. 2000–2500
binding sites per cell [257,258]. Functional T R mRNA
$
and protein (T Rα1, T Rβ1, c-erbAα2) was first demon$
$
strated by Northern and Western analysis in rat osteosarcoma cell lines [232]. T Rα1, T Rβ1 and c-erbAα2
$
$
mRNAs have since been found in human osteosarcoma
cells [281]. Immunocytochemical analysis has detected
T Rα, T Rβ and c-erbAα2 protein in the rat and human
$
$
osteosarcoma cells and sections of human bone [281,282].
T R proteins (T Rα1, T Rβ1 and c-erbAα2) have also
$
$
$
been demonstrated by Western blotting in primary
cultures of neonatal rat calvaria [237].
Very little work has been done on the expression of
T Rs in osteoclasts. However, using immunohisto$
chemistry Allain et al. [281] demonstrated staining
corresponding to T Rα and T Rβ proteins in osteoclasts
$
$
of normal human bone, and Abu et al. [282] confirmed
low expression of T Rβ1 and c-erbAα2 in osteoclasts and
$
some osteocytes. Chondrocytes exhibit specific T nu$
clear binding [283] and immunohistochemistry ; Northern and Western analyses have demonstrated T Rβ1 and
$
T Rα1 expression in human chondrocytes and the rat
$
growth plate [130,282,284,285].
Retinoids
The vertebrate limb initially consists of a mesenchymal
core covered by a layer of ectoderm. During the
elongation of the limbs the apparently homogeneous
population of the mesenchymal cells gives rise to different
# 2000 The Biochemical Society and the Medical Research Society
cell types (cartilage, bone and muscle), which develop in
appropriate spatial relationships to constitute the developed limb [286]. Retinoic acid (RA) may be the
natural morphogen involved in vertebrate limb generation (reviewed in [287] ; the role of retinoids in skeletal
morphogenesis has been reviewed recently [288]). Administration of RA to pregnant mice causes limb dysmorphogenesis in their offspring [289]. In rats, chronic
vitamin A toxicosis causes various bone defects, including
decreased shaft length and width, metaphyseal flaring,
cartilaginous degeneration, osteopenia and fractures
[290]. Rats on a high-retinol diet suffered increased bone
resorption, osteoporosis, cartilage degeneration and increased metaphyseal vascular penetration [290]. Addition
of RA (via the diet) to an in vivo rat model of
endochondral bone formation reduced mesenchymal cell
proliferation and diminished endochondral bone growth
and development [291].
Chondrocytes, osteoblasts and osteoclasts are all
targets for RA action, where it regulates proliferation,
differentiation and gene expression. Treatment of chondrocyte cultures with RA and 9-cis-RA rapidly decreases
the proliferation and energy status of these cells [292,293].
RA decreased proliferation of and AP activity in MC3T3E1 cells [294], and in UMR 106 and UMR 106-06 cells
RA decreased proliferation and induced morphological
changes consistent with a less metabolically active cell
type [295,296]. In contrast, RA increased the proliferation
of cells obtained from giant-cell tumours, but decreased
bone resorption [297]. However, in unfractionated rabbit
bone cell cultures RA increased the bone-resorbing
activity [298]. Additionally, in similar mouse bone cell
cultures, RA stimulated the formation of osteoclast-like
cells, and also stimulated osteopontin mRNA expression
in isolated osteoclasts [299].
RA treatment of immature chondrocytes rapidly induced the expression of genes (osteopontin, osteonectin
and AP) characteristic of fully mature hypertrophic
chondrocytes [300]. In rat and mouse osteoblastic cells,
AP mRNA and activity was induced by RA [301–303].
However, in organ cultures of mouse calvaria and
cultures of rat calvaria, AP activity and mRNA levels
decreased following treatment with RA, and varied
according to the stage of development [304,305]. RA
increased the expression of matrix γ-carboxyglutamic
acid-containing protein in human osteoblasts, chondrocytes and fibroblasts [306]. RA stimulated the secretion of osteocalcin in MG-63 cells [307], but had no
effect on osteocalcin secretion in ROS 17\2 cells [308].
However, RA in combination with 9-cis-RA decreased
osteocalcin and collagen type I(α1) mRNA levels in ROS
17\2.8 cells [222]. In comparison, RA either alone or in
combination with 9-cis-RA increased osteopontin
mRNA expression in ROS 25\1 cells and rat calvarial
cells [222,305].
One important effect of RA is on collagen homoeo-
Steroid receptors in bone
stasis, either by a direct effect on collagen synthesis or via
the regulation of collagenase. In human skin fibroblasts
and osteoblast-rich rat calvarial cultures, the production
of procollagen is decreased by RA [309,310]. Collagen
breakdown (measured by the release of [$H]hydroxyproline) in osteoblast-rich calvarial cultures is stimulated
by RA [311]. Collagenase activity and mRNA expression
are increased by RA in UMR 106-01 cells [312] and
osteoblast-rich calvarial cultures [311]. In contrast, in
chondrocytes RA treatment is associated with an increase
in the expression of type X [313] and type III collagen
mRNAs [314], but with a decrease in type II collagen
transcription and mRNA levels [314].
The effects of RA on cellular differentiation may be
mediated by changes in the expression of growth factors,
in particular modulation of the IGF\IGFBP axis. In rat
foetal calvarial cultures RA induced IGFBP-6 mRNA
levels [315], but decreased the levels of IGF-I and IGF-II
mRNAs, although it induced IGF-II polypeptide levels
[316]. In various human osteoblast cell systems RA
consistently induced IGFBP-6 mRNA, but had variable
effects on IGFBP-3, -4 and -5 mRNAs [317].
Retinoid receptor expression
There are two groups of retinoid receptors : the RA
receptors (RARs), whose ligand is all-trans-RA, and the
RXRs, whose ligand is 9-cis-RA. 9-cis-RA is not a unique
ligand for RXRs. While it binds and activates RXRs, it
also binds the RARs with higher affinity [318]. There are
at least three RAR genes : RARα [319], RARβ [320] and
RARγ [321]. There are also three RXR genes : RXRα,
RXRβ and RXRγ [322]. Each RAR gene generates
multiple isoforms [323], and preliminary evidence indicates that multiple isoforms of the RXRβ and RXRγ
genes also exist [324,325].
The first description of RAR expression in bone cells
was in 1990 by Nakayama et al. [302], who demonstrated
the presence of mRNA encoding RARs in MC3T3-E1
cells, although the authors were not able to conclusively
state which isoform was expressed. Also in 1990, Ruberte
et al. [326] mapped the expression of RARγ during mouse
embryogenesis to limb buds, all cartilages and teeth.
Since then a number of reports have demonstrated
variable expression of mRNAs encoding the various
RAR isoforms in rat and mouse osteoblastic cells (see
Table 1) [71,232,303,327–329]. Human osteoblastic cells
(both primary cultures and SaOS-2 cells) were shown to
contain mRNAs for RARα, RARβ and RARγ [330].
Mahonen and Maenpaa [75] demonstrated RAR mRNA
expression in MG-63 cells, but did not specify the
isoform. The important role of retinoids on chondrocytes
is illustrated by the expression of RARα, RARγ, RXRα
and RXRβ mRNAs in cultured chondrocytes [293].
The first demonstration of an mRNA encoding a RXR
was by Kindmark et al. [330], who demonstrated RXRα
mRNA expression in primary cultures of human osteo-
blastic cells and the human cell line SaOS-2. These results
were later confirmed by Jaaskelainen et al. [236]. mRNAs
encoding RXRα and RXRβ have been demonstrated in
rat osteosarcoma cells [232], rat bone [328] and MC3T3E1 cells [329]. Western blotting demonstrated the expression of RARα and RXR proteins in rat osteoblasts
and osteosarcoma cells [237]. Using isolated osteoclasts,
Saneshige et al. [298] have demonstrated expression of
mRNAs for RARα and RXRβ.
Peroxisome-proliferator-activated receptors
(PPARs)
The PPAR family consists of three receptors : PPARα,
PPARβ (also called δ or NUC-1) and PPARγ [331,332].
The PPARs appear to play an important role in regulating
the differentiation of osteoblastic cells into adipocytelike cells [333,334]. In situ studies in the rat have indicated
that PPARα is expressed exclusively in osteoblasts,
especially of young animals. PPARβ can be found in
osteoblasts and hypertrophic chondrocytes, and scattered within the haemopoietic marrow. PPARγ is also
present in osteoblasts and hypertrophic chondrocytes
and in bone marrow stromal cells [335–338]. Human
osteoblast-like cells (SaOS-2, MG-63 and primary
cultures) express PPARγ [333,334], MC3T3-E1 cells
express PPARγ1 but not PPARγ2, whereas ROS17\2.8
express PPARδ [333,339].
STEROID RECEPTOR KNOCKOUT ANIMALS
The true importance of steroid receptor action in bone
will become apparent with the advent of receptor
knockout animals, which may suffer from dysfunction of
bone development. This is an area of research that is
currently being developed. The bone phenotype of GR,
MR and AR knockout animals has yet to be examined,
and initial reports have indicated that the PR knockout
mouse has no apparent bone defects [340].
Female ERα knockout mice exhibit decreases in femur
length and diameter, but only slight, localized decreases
in femur BMD and bone mineral content. In contrast,
males show a significant change in BMD, but a lesser
change in femur length [341,342]. It has been suggested
that the decrease in BMD is possibly a consequence of
increased resorption, as ERα knockout mice have been
shown to have a 20 % increase in the numbers of
osteoclasts [343]. However, as the decrease in mineralized
area and BMD is only approx. 10 % [344] and as no gross
abnormalities are detected, the implication is that the
ERα is not required, or can be compensated for, during
bone development [345]. In addition, the bone loss seen
in ERα knockout animals varies depending on the region
of the femur examined and increases only selectively with
oestrogen therapy, again indicating the potential importance of ERβ [346]. Interestingly, the decrease in
# 2000 The Biochemical Society and the Medical Research Society
227
228
R. Bland
femur length seen in ERα knockout mice contrasts with
the phenotypes of the ERα-negative man and the two
aromatase-deficient men, who exhibited tall stature and
unfused epiphyses [88,197,198]. It had been hoped that
the development of an ERβ knockout mouse would
clarify the situation. However, initial studies of the bone
phenotype of the ERβ knockout mouse have shown that
prepubertal animals and adult males are similar to wildtype mice. In contrast, adult females exhibit changes in
their cortical, but not trabecular, bone structure, indicating that ERβ is required for pubertal feminization of
the skeleton in mice, but not for the protective effects of
oestrogen on trabecular bone [347]. Perhaps the demonstration of ERα and ERβ expression in growth plate
chondrocytes [127,139] may go some way towards
explaining these results. Oestradiol decreases the recruitment, proliferation and synthetic activity of chondrocytes, leading to maturation of the growth plate and
inhibition of long bone growth [84]. Therefore the
inability to respond to oestradiol in the ERα-negative
man results in unfused epiphyses and a tall stature.
Kennedy et al. [138] have recently shown that, in rabbits
(a species in which, like humans, epiphyseal closure
occurs), ERα is present in the epiphyseal growth plates of
both mature males and females. However, in the rat (a
species in which epiphyseal closure does not occur), the
ERα is only present in immature animals. Therefore
developmental loss of ERα expression is associated with
loss of the ability to undergo epiphyseal closure. As mice
are rodents and therefore would not normally undergo
epiphyseal closure, the loss of ERα expression in the
knockout animals would perhaps not be expected to
induce long bone growth, as it appears to do in humans.
VDR knockout mice display a phenotype that is very
similar to that seen in patients with the syndrome of
hereditary 1,25(OH)D -resistant rickets, although, un$
like the VDR knockout mice, this disease is not lethal
[217,348,349]. However, what is particularly interesting
is that the foetuses of VDR knockout mice appear normal,
and the knockout phenotype only becomes apparent
after weaning (at approx. 3 weeks), becoming more
pronounced with age [349,350]. Post-weaning VDR-null
mutant mice show marked growth retardation such that,
at 6 weeks of age, the homozygote animals have a body
weight of only 50 % of that of the wild-type animals.
They have low serum calcium and phosphate, and
increased AP activity. They exhibit a low bone density
(decreased by 40 % compared with the wild type). The
tibia and fibula show features typical of advanced rickets,
such as inadequate mineralization, a loss of the orderly
arrangement of chondrocytes and widening of the layers
of chondrocytes in the epiphyseal growth plates
[348–350]. Supplementation with calcium significantly
improves the impaired mineralization, but not the disordered proliferation\differentiation of chondrocytes
[348]. The time of onset of a knockout phenotype may be
# 2000 The Biochemical Society and the Medical Research Society
related to the mechanism of calcium absorption. In rats
calcium is absorbed by a 1,25(OH) D -independent
# $
mechanism for the first 18 days of life, which is then
gradually replaced by a 1,25(OH) D -dependent com# $
ponent [349]. Although 1,25(OH) D plays a role in the
# $
formation of osteoclasts, the number of osteoclasts
appeared to be normal in the VDR knockout mice [348].
Recent evidence has suggested that, although
1,25(OH) D -driven osteoclastogenesis is mediated by
# $
VDR-expressing osteoblasts, osteoclasts can also be
formed under the influence of parathyroid hormone or
IL-1α in the absence of VDR-mediated effects [351].
Deletion of T Rβ has demonstrated the importance of
$
the T Rβ gene for auditory function, but T Rβ1 knockout
$
$
animals exhibit no major bone phenotype [352]. However, animals in which the T Rα gene has been disrupted,
$
thereby preventing production of both T Rα1 and c$
erbAα2, exhibit impaired mineralization, delayed ossification of epiphysis and diaphysis, and early lethality
[353]. In particular, the growth cartilage was thinner and
contained more chondrocytes, but there was a decrease in
the number of hypertrophic chondrocytes, and the
chondrocyte column was disrupted and randomly
arranged. The calcium content of the skeletons was also
lower [353]. These effects could be rescued by thyroxine
treatment, presumably acting via the T Rβ. A more
$
recent development is mice lacking both T Rα1 and T Rβ
$
$
[354]. These mice suffer from decreased weight and
shorter femurs and tibias. Both bone mineral content and
bone area of the femur, tibia and vertebrae were significantly decreased, although there was no difference in
BMD. Additionally, the growth plates were disorganized
with no columnar structure and cartilage in the epiphysis,
which leads to delayed ossification [354].
The number of receptor isoforms and the possible
combination of receptor knockouts complicates the
situation in retinoid receptor knockout animals. Initial
studies looked at single-receptor deletions. RARα1,
RARβ2 and RARγ2 knockout mice all appear normal
[355–358] and demonstrate no skeletal abnormalities.
Mice deficient in all isoforms of RARα displayed webbed
digits on the forelimbs and hindlimbs, but the defects
were restricted to soft tissues [356]. Interestingly, mice in
which RARα1 was overexpressed developed limb defects
similar to those seen in animals administered high doses
of RA [359]. It was found that overexpression of RARα1
interfered with chondrogenesis in the hindlimb by
delaying chondroprogenitor differentiation, and the
authors suggested that continued expression of RARα in
cells destined to become chondroblasts contributes to a
different cellular fate [359]. In contrast, animals deficient
in all isoforms of RARγ were growth-deficient and
exhibited a number of skeletal malformations [355].
Defects included ossified fusion between the basioccipital
and anterior arch of the atlas, fusion of the first and
second ribs, fused vertebrae and homoeotic trans-
Steroid receptors in bone
formations along the antero-posterior axis, indicating
that RA is required for proper specification of some
cervical and thoracic vertebrae. However, as in the RARα
knockout mice, no limb malformations were detected.
Additionally, the defects in the RARγ mice demonstrated
incomplete penetrance and expressivity, suggesting that
some functional redundancy exists between the receptors.
This was confirmed by the generation of double knockouts. RARα\γ mutants consistently exhibited skeletal
malformations [360]. Lohnes et al. [360] generated
various RAR double mutants, all of which exhibited bone
defects. Abnormalities of the axial skeleton occurred in
all mutants, and included a number of homoeotic
transformations and various malformations such as rib
fusion, basioccipital–exoccipital fusion, defects in cervical
neural arches and ectopic bone production. Craniofacial
abnormalities were detected in all mutants except RARα\
β2, RARα1\β2 and RARβ2\γ. Interestingly, the limbs of
all double mutants were essentially normal, with the
exception of RARα\γ knockout mice, which demonstrated a number of malformations [360]. The various
defects exhibited by RAR knockout animals have been
reviewed by Lohnes et al. [361].
RXRα knockout mice have normal limb development
(although other tissues are affected) and are resistant to
RA-induced limb deformities, thereby implicating RXRα
as a component in the teratogenic process in limbs [362].
Triple RXRα\β\γ knockout mice were shown to be
growth-deficient, but exhibited no gross abnormalities
[363].
FUTURE CONSIDERATIONS
Previously, work has concentrated on the investigation
of osteoblast function. However, there is now an increasing awareness of the important role that other bone
cells, such as chondrocytes and osteoblast and osteoclast
precursor cells, play in steroid-mediated responses. It is
also important to remember that, in vivo, the response to
steroids will occur as a result of a combination of factors,
acting on a mixture of bone cells. For example, despite the
facts that androgens clearly regulate both osteoblast and
osteoclast function and that ARs are present in both cell
types, it appears that cells of the osteoblast lineage are
required to mediate the effects of androgen withdrawal.
Orchidectomy of SAMP6 mice (a model of defective
osteoblast development) failed to produce the expected
bone loss [364]. Furthermore, in addition to modulating
target gene transcription, steroids also regulate the
numbers, binding capacity and expression of their own
and other receptors within the receptor superfamily. For
example, 1,25(OH) D increases ER numbers [115],
# $
while oestradiol increases VDR binding and augments
1,25(OH) D -mediated responses [365]. Glucocorticoids
# $
also increase VDR numbers and stimulate cell respon-
siveness to 1,25(OH) D in rat osteoblasts [366], but
# $
decrease VDR expression in human osteosarcoma cells
[367].
It is also important to determine the cellular expression
of all members of the steroid\thyroid receptor family.
Although it is the RXR heterodimers that are thought to
be the predominantly functionally active complexes
[368,369], the steroid receptors are also capable of
promiscuous interactions with other members of the
steroid receptor superfamily [370–372]. Therefore the
co-expression of more than one steroid receptor per
cell could provide an array of possible heterodimeric
complexes, which would form the basis for complex
interaction among their signalling pathways, and
competition between steroid receptors for heterodimer
partners will regulate steroid hormone responsiveness.
Another component that may prove to be important
for the development of bone disease is the genetic
variability in steroid receptors. It had been suggested that
polymorphisms in the VDR could be used to predict
bone turnover [373]. However, the relationship between
VDR polymorphisms and bone density remains controversial ; much of the data has been reviewed by Cooper
and Umbach [374], who concluded that VDR polymorphisms represent just one genetic determinant of
BMD. Kobayashi et al. [375] suggested a similar relationship between polymorphisms of the ER gene and
BMD, although in similar studies by Mizunuma et al.
[376] and Gennari et al. [377] only a slight, nonsignificant relationship was observed. It appears likely
that individual polymorphisms are not so important, but
can lead jointly to increased susceptibility [378].
The field of steroid hormone action has become
increasingly more complex. Although the steroid receptors have a very important role to fulfil, they are just
one of a number of factors that must be considered. Until
recently our established understanding of receptor action
was that liganded steroid receptors bind to specific DNA
sequences, as either homodimers or heterodimers, to
regulate gene transcription [22]. However, it is now
apparent that this initial concept is very simplistic and
that a range of accessory proteins (including co-repressors and co-activators) also participate in the process
(reviewed in [379]). A number of these proteins have now
been identified, some of which maybe receptor-specific
[380]. It is also possible that structural differences
between receptor isoforms, such as those seen in ERα and
ERβ [111], will influence the ability of receptors to
interact with co-repressors and co-activators, and therefore modulate steroid responsiveness. In addition to the
genomic actions of steroid hormones, there is increasing
evidence suggesting that steroids are also able to elicit
rapid, non-genomic actions (reviewed in [381]). These
effects have been demonstrated for virtually all steroids
and probably occur via specific membrane receptors
[382]. Membrane binding sites for oestrogen have been
# 2000 The Biochemical Society and the Medical Research Society
229
230
R. Bland
identified in a human pre-osteoclastic cell line [383], and
recent studies have identified membrane receptors for
1,25(OH) D and 24,25(OH) D in rat chondrocytes and
# $
# $
chick tibia [384–386]. The physiological importance of
these non-genomic actions has yet to be determined.
As our understanding of the mechanisms of steroid
action in bone grows, hopefully we will be able to design
novel therapeutic agents. Non-steroidal selective
modulators of the ER (SERMs) are now beginning to be
used therapeutically to prevent postmenopausal bone
loss [387]. However, there may better receptor targets,
and the discovery of further receptor isoforms (the
expression of which may be site- and developmentspecific) and novel ligands may provide a range of
effective modulators of bone homoeostasis.
17
18
19
20
21
22
23
REFERENCES
24
1 Melton, L. J., Thamer, M., Ray, N. F. et al. (1997)
Fractures attributable to osteoporosis : report from the
national osteoporosis foundation. J. Bone Miner. Res. 12,
16–23
2 Kelly, P. J., Eisman, J. A. and Sambrook, P. N. (1990)
Interaction of genetic and environmental influences on
peak bone mass. Osteoporosis Int. 1, 56–60
3 NIH (1994) Optimal calcium intake : NIH consensus
development panel on optimal calcium intake.
J. Am. Med. Assoc. 272, 1942–1948
4 Manolagas, S. C. and Jilka, R. L. (1995) Bone marrow,
cytokines and bone remodeling. N. Engl. J. Med. 332,
305–311
5 Mundy, G. R., Boyce, B., Hughes, D. et al. (1995) The
effects of cytokines and growth factors on osteoblastic
cells. Bone 17, 71S–75S
6 Etherington, J., Harris, P. A., Nandra, D. et al. (1996) The
effect of weight-bearing exercise on bone mineral density :
a study of female ex-elite athletes and the general
population. J. Bone Miner. Res. 11, 1333–1338
7 Evans, D. M. and Ralston, S. H. (1996) Nitric oxide and
bone. J. Bone Miner. Res. 11, 300–305
8 Civitelli, R. (1995) Cell–cell communication in bone.
Calcif. Tissue Int. 56, S29–S31
9 Canalis, E. (1996) Regulation of bone remodeling. In
Primer on the Metabolic Bone Diseases and Disorders of
Mineral Metabolism, 3rd edn (Favus, M. J., ed.), pp. 23–26,
Lippincott-Raven, Philadelphia
10 Pacifici, R. (1998) Cytokines, estrogen, and
postmenopausal osteoporosis – the second decade.
Endocrinology 139, 2659–2661
11 Kronenberg, H. M. (1996) Parathyroid hormone :
mechanism of action. In Primer on the Metabolic Bone
Diseases and Disorders of Mineral Metabolism, 3rd edn
(Favus, M. J., ed.), pp. 68–70, Lippincott-Raven,
Philadelphia
12 Deftos, L. J. (1996) Calcitonin. In Primer on the Metabolic
Bone Diseases and Disorders of Mineral Metabolism, 3rd
edn (Favus, M. J., ed.), pp. 82–87, Lippincott-Raven,
Philadelphia
13 Sudo, H., Kodama, H. A., Amagai, Y., Yamamoto, S. and
Kasai, S. (1983) In vitro differentiation and calcification in
a new clonal osteogenic cell line derived from newborn
mouse calvaria. J. Cell Biol. 96, 191–198
14 Majeska, R. J., Rodan, S. B. and Rodan, G. A. (1980)
Parathyroid hormone-responsive clonal cell lines from rat
osteosarcoma. Endocrinology 107, 1494–1503
15 Partridge, N. C., Alcorn, D., Michelangeli, V. P., Ryan, G.
and Martin, T. J. (1983) Morphological and biochemical
characterization of four clonal osteogenic sarcoma cell
lines of rat origin. Cancer Res. 43, 4308–4314
16 McAllister, R. M., Gardner, M. B., Greene, A. E., Bradt,
C., Nichols, W. W. and Landing, B. H. (1971) Cultivation
# 2000 The Biochemical Society and the Medical Research Society
25
26
27
28
29
30
31
32
33
34
35
36
37
in vitro of cells derived from a human osteosarcoma.
Cancer Res. 27, 397–402
Heremans, H., Billiau, A., Cassiman, J. J., Mulier, J. C. and
de Somer, P. (1978) In vitro cultivation of human tumor
tissues II. Morphological and virological characterization
of three cell lines. Oncology 35, 246–252
Murray, E., Provvedini, D., Curran, D., Catherwood, B.,
Sussman, H. and Manolagas, S. (1987) Characterization of
a human osteoblastic osteosarcoma cell line (SAOS-2) with
high bone alkaline phosphatase activity. J. Bone Miner.
Res. 2, 231–238
Roodman, G. D. (1999) Cell biology of the osteoclast.
Exp. Hematol. 27, 1229–1241
Evans, R. M. (1988) The steroid and thyroid hormone
receptor superfamily. Science 240, 889–895
Mangelsdorf, D. J., Thummel, C., Beato, M. et al. (1995)
The nuclear receptor superfamily : the second decade. Cell
83, 835–839
Carson-Jurica, M. A., Schrader, W. T. and O’Malley, B. W.
(1990) Steroid receptor family : structure and functions.
Endocr. Rev. 11, 201–220
Williams, G. R. and Brent, G. A. (1992) Specificity of
nuclear hormone receptor action : who conducts the
orchestra ? J. Endocrinol. 135, 191–194
Kliewer, S. S. A., Umesono, K., Mangeldorf, D. J. and
Evans, R. M. (1992) Retinoid X receptor interacts with
nuclear receptors in retinoic acid, thyroid hormone and
vitamin D signalling. Nature (London) 355, 446–449
Canalis, E.$ (1996) Mechanisms of glucocorticoid action in
bone : implications to glucocorticoid-induced osteoporosis.
J. Clin. Endocrinol. Metab. 81, 3441–3447
Ishida, Y. and Heersche, J. N. M. (1998) Glucocorticoidinduced osteoporosis : both in vivo and in vitro
concentrations of glucocorticoids higher than physiological
levels attenuate osteoblast differentiation. J. Bone Miner.
Res. 13, 1822–1826
Lukert, B. P. and Raisz, L. G. (1990) Glucocorticoidinduced osteoporosis : pathogenesis and management. Ann.
Intern. Med. 112, 352–364
Henderson, N. K. and Sambrook, P. N. (1996)
Relationship between osteoporosis and arthritis and effect
of corticosteroids and other drugs on bone. Curr. Opin.
Rheumatol. 8, 365–369
Osella, G., Terzolo, M., Reimondo, G. et al. (1997) Serum
markers of bone and collagen turnover in patients with
Cushing’s syndrome and in subjects with adrenal
incidentalomas. J. Clin. Endocrinol. Metab. 82, 3303–3307
Pearce, G., Tabensky, D. A., Delmas, P. D., Baker,
H. W. G. and Seeman, E. (1998) Corticosteroid-induced
bone loss in men. J. Clin. Endocrinol. Metab. 83, 801–806
Newman, E., Turner, A. S. and Wark, J. D. (1995) The
potential of sheep for the study of osteopenia : current
status and comparison with other animal models. Bone 16,
277S–284S
Bellows, C. G., Ciaccia, A. and Heersche, J. N. M. (1998)
Osteoprogenitor cells in cell populations derived from
mouse and rat calvaria differ in the response to
corticosterone, cortisol, and cortisone. Bone 23, 119–125
Ferretti, J. L., Vazquez, S. O., Delgado, C. J., Capozza, R.
and Coitry, G. (1992) Biphasic dose–response curves of
cortisol effects on rat diaphyseal bone biomechanics.
Calcif. Tissue Int. 50, 49–54
Li, M., Shen, Y., Halloran, B. P., Baumann, B. D., Miller,
K. and Wronski, T. J. (1996) Skeletal response to
corticosteroid deficiency and excess in growing male rats.
Bone 19, 81–88
Shen, V., Birchman, R., Liang, X. G., Wu, D. D., Lindsay,
R. and Dempster, D. W. (1997) Prednisolone alone, or in
combination with estrogen or dietary calcium deficiency or
immobilization, inhibits bone formation but does not
induce bone loss in mature rats. Bone 21, 345–351
Boden, S. D., Hair, G., Titus, L. et al. (1997)
Glucocorticoid-induced differentiation of fetal rat calvarial
osteoblasts is mediated by bone morphogenic protein-6.
Endocrinology 138, 2820–2828
Ishida, Y. and Heersche, J. N. (1997) Progesterone
stimulates proliferation and differentiation of
osteoprogenitor cells in bone cell populations derived from
adult female but not from adult male rats. Bone 20, 17–25
Steroid receptors in bone
38 Bellows, C. G., Heersche, J. N. M. and Aubin, J. E. (1990)
Determination of the capacity for proliferation and
differentiation of osteoprogenitor cells in the presence and
absence of dexamethasone. Dev. Biol. 140, 132–138
39 Kasugai, S., Todescan, R., Nagata, T., Yao, K. L., Butler,
W. T. and Sodek, J. (1991) Expression of bone-matrix
proteins associated with mineralized tissue formation by
adult-rat bone-marrow cells in vitro – inductive effects of
dexamethasone on the osteoblast phenotype. J. Cell.
Physiol. 147, 111–120
40 Pockwinse, S. M., Stein, J. L., Lian, J. B. and Stein, G. S.
(1995) Developmental stage-specific cellular responses to
vitamin D and glucocorticoids during differentiation of the
osteoblast phenotype : interrelationship of morphology and
gene expression by in situ hybridization. Exp. Cell Res.
216, 244–260
41 Delany, A. M. and Canalis, E. (1995) Transcriptional
repression of insulin-like growth factor I by
glucocorticoids in rat bone cells. Endocrinology 136,
4776–4781
42 Chevalley, T., Strong, D. D., Mohan, S., Baylink, D. and
Linkhart, T. A. (1996) Evidence for a role for insulin-like
growth factor binding proteins in glucocorticoid inhibition
of normal human osteoblast-like cell proliferation. Eur. J.
Endocrinol. 134, 591–601
43 Conover, C. A., Lee, P. D., Riggs, B. L. and Powell, D. R.
(1996) Insulin-like growth factor-binding protein-1
expression in cultured human bone cells : regulation by
insulin and glucocorticoid. Endocrinology 137, 3295–3301
44 Kream, B. E., Tetradis, S., Lafrancis, D., Fall, P. M., Feyen,
J. H. M. and Raisz, L. G. (1997) Modulation of the effects
of glucocorticoids on collagen synthesis in fetal rat
calvariae by insulin-like growth factor binding protein-2.
J. Bone Miner. Res. 12, 889–895
45 Green, E., Todd, B. and Heath, D. (1990) Mechanism of
glucocorticoid regulation of alkaline phosphatase gene
expression in osteoblast-like cells. Eur. J. Biochem. 188,
147–153
46 Ogata, Y., Yamauchi, M., Kim, R. H., Li, J. J., Freedman,
L. P. and Sodek, J. (1995) Glucocorticoid regulation of
bone sialoprotein (BSP) gene-expression – identification of
a glucocorticoid response element in the bone sialoprotein
gene promoter. Eur. J. Biochem. 230, 183–192
47 Shalhoub, V., Aslam, F., Breen, E. et al. (1998) Multiple
levels of steroid hormone-dependent control of osteocalcin
during osteoblast differentiation : glucocorticoid regulation
of basal and vitamin D stimulated gene expression. J. Cell.
Biochem. 69, 154–168
48 Dietrich, J. W., Canalis, E. M., Maina, M. and Raisz, L. G.
(1979) Effects of glucocorticoids on fetal rat bone collagen
synthesis in vitro. Endocrinology 104, 715–721
49 Delany, A. M., Gabbitas, B. Y. and Canalis, E. (1995)
Cortisol down-regulates osteoblast α1(I) procollagen
mRNA by transcriptional and posttranscriptional
mechanisms. J. Cell. Biochem. 57, 488–494
50 Gronowicz, G. A. and McCarthy, M. B. (1995)
Glucocorticoids inhibit the attachment of osteoblasts to
bone extracellular matrix proteins and decrease beta 1integrin levels. Endocrinology 136, 598–608
51 Dempster, D. W., Moonga, B. S., Stein, L. S., Horbert,
W. R. and Antakly, T. (1997) Glucocorticoids inhibit bone
resorption by isolated rat osteoclasts by enhancing
apoptosis. J. Endocrinol. 154, 397–406
52 Conaway, H. H., Grigorie, D. and Lerner, U. H. (1996)
Stimulation of neonatal mouse calvarial bone resorption by
the glucocorticoids hydrocortisone and dexamethasone.
J. Bone Miner. Res. 11, 1419–1429
53 Kaji, H., Sugimoto, T., Kanatani, M., Nishiyama, K. and
Chihara, K. (1997) Dexamethasone stimulates osteoclastlike cell formation by directly acting on hemopoietic blast
cells and enhances osteoclast-like cell formation stimulated
by parathyroid hormone and prostaglandin E . J. Bone
#
Miner. Res. 12, 734–741
54 Wada, S., Udagawa, N., Akatsu, T., Nagata, N., Martin,
T. J. and Findley, D. M. (1997) Regulation by calcitonin
and glucocorticoids of calcitonin receptor gene expression
in mouse osteoclasts. Endocrinology 138, 521–529
55 Weinstein, R. S., Jilka, R. L., Parfitt, M. and Manolagas,
S. C. (1998) Inhibition of osteoblastogenesis and promotion
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
of apoptosis of osteoblasts and osteocytes by
glucocorticoids. J. Clin. Invest. 102, 272–282
Advani, S., Lafrancis, D., Bogdanovic, E., Taxel, P., Raisz,
L. G. and Kream, B. E. (1997) Dexamethasone suppresses
in vivo levels of bone collagen synthesis in neonatal mice.
Bone Miner. 20, 41–46
Lian, J. B., Shalhoub, V., Aslam, F. et al. (1997) Speciesspecific glucocorticoid and 1,25-dihydroxyvitamin D
responsiveness in mouse MC3T3-E1 osteoblasts :
dexamethasone inhibits osteoblast differentiation and
vitamin D down-regulates osteocalcin gene expression.
Endocrinology 138, 2117–2127
Nakashima, T., Sasaki, H., Tsuboi, M. et al. (1998)
Inhibitory effect of glucocorticoid for osteoblast apoptosis
induced by activated peripheral blood mononuclear cells.
Endocrinology 139, 2032–2040
Wong, M. M., Rao, L. G., Ly, H. et al. (1990) Long-term
effects of physiologic concentrations of dexamethasone in
human bone-derived cells. J. Bone Miner. Res. 5, 803–813
Subramaniam, M., Colvard, D., Keeting, P. E., Rasmussen,
K., Riggs, B. L. and Spelsberg, T. C. (1992) Glucocorticoid
regulation of alkaline phosphatase, osteocalcin, and protooncogenes in normal human osteoblast-like cells. J. Cell.
Biochem. 50, 411–424
Iba, K., Chiba, H., Sawada, N., Hirota, S., Ishii, S. and
Mori, M. (1995) Glucocorticoids induce mineralization
coupled with bone protein expression without influence on
growth of a human osteoblastic cell line. Cell Struct.
Funct. 20, 319–330
Sutherland, M. S., Rao, L. G., Muzaffar, S. A. et al. (1995)
Age-dependent expression of osteoblastic phenotypic
markers in normal human osteoblasts cultured long-term
in the presence of dexamethasone. Osteoporosis Int. 5,
335–343
Janssen, J. M. M. F., Bland, R., Hewison, M. et al. (1999)
Estradiol formation by human osteoblasts via multiple
pathways : relation to osteoblast function. J. Cell. Biochem.
75, 528–537
Hollenberg, S. M., Weinberger, C., Ong, E. S. et al. (1985)
Primary structure and expression of a functional human
glucocorticoid receptor cDNA. Nature (London) 318,
635–641
Bamerger, C. M., Schulte, H. M. and Chrousos, G. P.
(1996) Molecular determinants of glucocorticoid receptor
function and tissue sensitivity to glucocorticoids. Endocr.
Rev. 17, 245–261
Chen, T. L., Aronow, L. and Feldman, D. (1977)
Glucocorticoid receptors and inhibition of bone cell
growth in primary culture. Endocrinology 100, 619–628
Manolagas, S. C. and Anderson, D. C. (1978) Detection of
high-affinity glucocorticoid binding in rat bone.
J. Endocrinol. 76, 379–380
Chen, T. L. and Feldman, D. (1979) Glucocorticoid
receptors and actions in subpopulations of cultured rat
bone cells. J. Clin. Invest. 63, 750–757
Haussler, M. R., Manolagas, S. C. and Deftos, L. J. (1980)
Glucocorticoid receptor in clonal osteosarcoma cell lines : a
novel system for investigating bone active hormones.
Biochem. Biophys. Res. Commun. 94, 373–380
Masuyama, A., Ouchi, Y., Sato, F., Hosoi, T., Nakamura,
T. and Orimo, H. (1992) Characteristics of steroid
hormone receptors in cultured MC3T3-E1 osteoblastic
cells and effect of steroid hormones on cell proliferation.
Calcif. Tissue Int. 51, 376–381
Suzuki, S., Koga, M., Takaoka, K., Ono, K. and Sato, B.
(1993) Effects of retinoic acid on steroid and vitamin D
$
receptors in cultured mouse osteosarcoma cells. Bone 14,
7–12
Liesegang, P., Romalo, G., Sudmann, M., Wolf, L. and
Schweikert, H. U. (1994) Human osteoblast-like cells
contain specific, saturable, high-affinity glucocorticoid,
androgen, estrogen, and 1α,25-dihydroxycholecalciferol
receptors. J. Androl. 15, 194–199
Song, L. N. (1994) Effects of retinoic acid and
dexamethasone on proliferation, differentiation, and
glucocorticoid receptor expression in cultured human
osteosarcoma cells. Oncol. Res. 6, 111–118
Bland, R., Worker, C. A., Noble, B. S. et al. (1999)
# 2000 The Biochemical Society and the Medical Research Society
231
232
R. Bland
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
Characterisation of 11β-hydroxysteroid dehydrogenase
activity and corticosteroid receptor expression in human
osteosarcoma cell lines. J. Endocrinol. 161, 455–464
Mahonen, A. and Maenpaa, P. H. (1994) Steroid hormone
modulation of vitamin D receptor levels in human MG-63
osteosarcoma cells. Biochem. Biophys. Res. Commun. 205,
1179–1186
Tanaka, S., Haji, M., Takayanagi, R., Tanaka, S., Sugioka,
Y. and Nawata, H. (1996) 1,25-Dihydroxyvitamin D
enhances the enzymatic activity and expression of the$
messenger ribonucleic acid for aromatase cytochrome P450
synergistically with dexamethasone depending on the
vitamin D receptor level in cultured human osteoblasts.
Endocrinology 137, 1860–1869
Condon, J., Gosden, C., Gardener, D. et al. (1998)
Expression of type 2 11β-hydroxysteroid dehydrogenase
and corticosteroid hormone receptors in early human fetal
life. J. Clin. Endocrinol. Metab. 83, 4490–4497
Abu, E. O., Horner, A., Kusec, V., Triffitt, J. T. and
Compston, J. E. (1997) The localisation of glucocorticoid
receptors in human bone. J. Bone Miner. Res. 12, P16
Agarwal, M. K., Mirshahi, F., Mirshahi, M. et al. (1996)
Evidence for receptor-mediated mineralocorticoid action
in rat osteoblastic cells. Am. J. Physiol. 270, C1088–C1095
Stewart, P. M. and Krozowski, Z. S. (1999) 11βHydroxysteroid dehydrogenase. Vitam. Horm. Adv. Res.
Appl. 57, 249–324
Batista, M. C., Mendonca, B. B., Kater, C. E. et al. (1986)
Spironolactone-reversible rickets associated with 11βhydroxysteroid dehydrogenase deficiency syndrome.
J. Pediatr. 109, 989–993
Milford, D. V., Shackleton, C. H. and Stewart, P. M.
(1995) Mineralocorticoid hypertension and congenital
deficiency of 11β-hydroxysteroid dehydrogenase in a
family with the syndrome of ‘ apparent ’ mineralocorticoid
excess. Clin. Endocrinol. 43, 241–246
Arriza, J. L., Weinberger, C., Cerelli, G. et al. (1987)
Cloning of human mineralocorticoid receptor
complementary DNA : structural and functional kinship
with the glucocorticoid receptor. Science 237, 268–275
Turner, R. T., Riggs, B. L. and Spelsberg, T. C. (1994)
Skeletal effects of estrogen. Endocr. Rev. 15, 275–300
Prince, R. L. (1994) Estrogen effects on calcitropic
hormones and calcium homeostasis. Endocr. Rev. 15,
301–309
Garnero, P., Sornay-Rendu, E. and Delmas, P. D. (1995)
Decreased bone turnover in oral contraceptive users. Bone
16, 499–503
Cutler, G. B. (1997) The role of estrogen in bone growth
and maturation during childhood and adolescence.
J. Steroid Biochem. Mol. Biol. 61, 141–144
Smith, E. P., Boyd, J., Frank, G. R. et al. (1994) Estrogen
resistance caused by a mutation in the estrogen-receptor
gene in a man. N. Engl. J. Med. 331, 1056–1061
Dick, I. M., St. John, A., Heal, S. and Prince, R. L. (1996)
The effect of estrogen deficiency on bone mineral density,
renal calcium and phosphorus handling and calcitropic
hormones in the rat. Calcif. Tissue Int. 59, 174–178
Gouveia, C. H., Jorgetti, V. and Bianco, A. C. (1997)
Effects of thyroid hormone administration and estrogen
deficiency on bone mass of female rats. J. Bone Miner.
Res. 12, 2098–2107
Ernst, M., Heath, J. K. and Rodan, G. A. (1989) Estradiol
effects on proliferation, messenger ribonucleic acid for
collagen and insulin-like growth factor-I, and parathyroid
hormone-stimulated adenylate cyclase activity in
osteoblastic cells from calvariae and long bones.
Endocrinology 125, 825–833
Qu, Q., Perala-Heape, M., Kapanen, A. et al. (1998)
Estrogen enhances differentiation of osteoblasts in mouse
bone marrow culture. Bone 22, 201–209
Gray, T. K., Flynn, T. C., Gray, K. M. and Nabell, L. M.
(1987) 17β-Estradiol acts directly on the clonal osteoblastic
cell line UMR106. Proc. Natl. Acad. Sci. U.S.A. 84,
6267–6271
Robinson, J. A., Harris, S. A., Riggs, B. L. and Spelsberg,
T. C. (1997) Estrogen regulation of human osteoblastic cell
proliferation and differentiation. Endocrinology 138,
2919–2927
# 2000 The Biochemical Society and the Medical Research Society
95 Ernst, M., Schmid, C. H. and Froesch, E. R. (1988)
Enhanced osteoblast proliferation and collagen gene
expression by estradiol. Proc. Natl. Acad. Sci. U.S.A. 85,
2307–2310
96 Turner, R. T., Backup, P., Sherman, P. J., Hills, E., Evans,
G. L. and Spelsberg, T. C. (1992) Mechanism of action of
estrogen on intramembranous bone formation : regulation
of osteoblast differentiation and activity. Endocrinology
131, 883–889
97 Jilka, R. L. (1998) Cytokines, bone remodeling, and
estrogen deficiency : a 1998 update. Bone 23, 75–81
98 Girasole, G., Jilka, R. L., Passeri, G. et al. (1992) 17βEstradiol inhibits interleukin-6 production by bone
marrow-derived stromal cells and osteoblasts in vitro : a
potential mechanism for the anti-osteoporotic effect of
estrogens. J. Clin. Invest. 89, 883–891
99 Yang, N. N., Bryant, H. U., Hardikar, S. et al. (1996)
Estrogen and raloxifene stimulate transforming growth
factor-β3 gene expression in rat bone : a potential
mechanism for estrogen- or raloxifene-mediated bone
maintenance. Endocrinology 137, 2075–2084
100 Kassem, M., Okazaki, R., Harris, S. A., Spelsberg, T. C.,
Conover, C. A. and Riggs, B. L. (1998) Estrogen effects
on insulin-like growth factor gene expression in a human
osteoblastic cell line with high levels of estrogen receptor.
Calcif. Tissue Int. 62, 60–66
101 Rickard, D. J., Hofbauer, L. C., Bonde, S. K., Gori, F.,
Spelsberg, T. C. and Riggs, B. L. (1998) Bone
morphogenic protein-6 production in human osteoblastic
cell lines. Selective regulation by estrogen. J. Clin. Invest.
101, 413–422
102 Kassem, M., Harris, S. A. and Spelsberg, T. C. (1996)
Estrogen inhibits interleukin-6 production and gene
expression in a human osteoblastic cell line with high
levels of estrogen receptors. J. Bone Miner. Res. 11,
193–199
103 Jilka, R. L., Hangoc, G., Girasole, G. et al. (1992)
Increased osteoclast development after estrogen loss :
mediation by interleukin-6. Science 257, 88–91
104 Udagawa, N., Takahashi, N., Katagiri, T. et al. (1995)
Interleukin (IL)-6 induction of osteoclast differentiation
depends on IL-6 receptors expressed on osteoblastic cells
but not on osteoclast progenitors. J. Exp. Med. 182,
1461–1468
105 Kameda, T., Mano, H., Yuasa, T. et al. (1997) Estrogen
inhibits bone resorption by directly inducing apoptosis of
the bone-resorbing osteoclasts. J. Exp. Med. 186, 489–495
106 Hughes, D. E., Dai, A., Tiffee, J. C., Li, H. H., Mundy,
G. R. and Boyce, B. F. (1996) Estrogen promotes
apoptosis of murine osteoclasts mediated by TGF-β. Nat.
Med. (N. Y.) 2, 1132–1136
107 Tomkinson, A., Reeve, J., Shaw, R. W. and Noble, B. S.
(1997) The death of osteocytes via apoptosis accompanies
estrogen withdrawal in human bone. J. Clin. Endocrinol.
Metab. 82, 3128–3135
108 Tomkinson, A., Gevers, E. F., Wit, J. M., Reeve, J. and
Noble, B. S. (1998) The role of estrogen in the control of
rat osteocyte apoptosis. J. Bone Miner. Res. 13,
1243–1250
109 Tau, K. R., Hefferan, T. E., Waters, K. M. et al. (1998)
Estrogen regulation of a transforming growth factor-β
inducible early gene that inhibits deoxyribonucleic acid
synthesis in human osteoblasts. Endocrinology 139,
1346–1353
110 Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M.,
Nilsson, S. and Gustafsson, J. A. (1996) Cloning of a
novel estrogen receptor expressed in rat prostate and
ovary. Proc. Natl. Acad. Sci. U.S.A. 93, 5925–5930
111 Mosselman, S., Polman, J. and Dijkema, R. (1996) ERβ :
identification and characterization of a novel human
estrogen receptor. FEBS Lett. 392, 49–53
112 Komm, B. S., Terpening, C. M., Benz, D. J. et al. (1988)
Estrogen binding, receptor mRNA, and biologic response
in osteoblast-like osteosarcoma cells. Science 241, 81–84
113 Migliaccio, S., Davis, V. L., Gibson, M. K., Gray, T. K.
and Korach, K. S. (1992) Estrogens modulate the
responsiveness of osteoblast-like cells (ROS 17\2.8)
stably transfected with estrogen receptor. Endocrinology
130, 2617–2624
Steroid receptors in bone
114 Davis, V., Couse, J. F., Gray, T. K. and Korach, K. S.
(1994) Correlation between low levels of estrogen
receptors and estrogen responsiveness in two rat
osteoblast-like cell lines. J. Bone Miner. Res. 9, 983–991
115 Bellido, T., Girasole, G., Passeri, G. et al. (1993)
Demonstration of estrogen and vitamin D receptors in
bone marrow-derived stromal cells : up-regulation of the
estrogen receptor by 1,25-dihydroxyvitamin-D .
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Endocrinology 133, 553–562
116 Eriksen, E. F., Colvard, D. S., Berg, N. J. et al. (1988)
Evidence of estrogen receptors in normal human
osteoblast-like cells. Science 241, 84–86
117 Etienne, M. C., Fischel, J. L., Milano, G. et al. (1990)
Steroid receptors in human osteoblast-like cells. Eur. J.
Cancer 26, 807–810
118 Benz, D. J., Haussler, M. R. and Komm, B. S. (1991)
Estrogen binding and estrogenic responses in normal
human osteoblast-like cells. J. Bone Miner. Res. 6,
531–541
119 Slootweg, M. C., Ederveen, A. G. H., Schot, L. P. C.,
Schoonen, W. G. E. J. and Kloosterboer, H. J. (1992)
Oestrogen and progesterone synergistically stimulate
human and rat osteoblast proliferation. J. Endocrinol.
133, R5–R8
120 Ikegami, A., Inoue, S., Hosoi, T. et al. (1994) Cell cycledependent expression of estrogen receptor and effect of
estrogen on proliferation of synchronized human
osteoblast-like osteosarcoma cells. Endocrinology 135,
782–789
121 Hoshino, S., Inoue, S., Hosoi, T. et al. (1995)
Demonstration of isoforms of the estrogen receptor in
the bone tissues and in osteoblastic cells. Calcif. Tissue
Int. 57, 466–468
122 Sutherland, M. K., Hui, D. U., Rao, L. G., Wylie, J. N.
and Murray, T. M. (1996) Immunohistochemical
localization of the estrogen receptor in human
osteoblastic SaOS-2 cells : association of receptor levels
with alkaline phosphatase activity. Bone 18, 361–369
123 Westerlind, K. C., Sarkar, G., Bolander, M. E. and
Turner, R. T. (1995) Estrogen receptor mRNA is
expressed in vivo in rat calvarial periosteum. Steroids 60,
484–487
124 Komm, B., Henderson, R., Bodine, P., Green, J., Lian, J.
and Stein, G. (1996) The estrogen receptor is
developmentally up-regulated during osteoblast
differentiation. J. Bone Miner. Res. 11, M308
125 Onoe, Y., Miyaura, C., Ohta, H., Nozawa, S. and Suda,
T. (1997) Expression of estrogen receptor β in rat bone.
Endocrinology 138, 4509–4512
126 Bodine, P. V. N., Henderson, R. A., Green, J. et al. (1998)
Estrogen receptor-α is developmentally regulated during
osteoblast differentiation and contributes to selective
responsiveness of gene expression. Endocrinology 139,
2048–2057
127 Kusec, V., Virdi, A. S., Prince, R. and Triffitt, J. T. (1998)
Localization of estrogen receptor-alpha in human and
rabbit skeletal tissues. J. Clin. Endocrinol. Metab. 83,
2421–2428
128 Chen, X. W., Garner, S. C. and Anderson, J. J. (1998)
Messenger RNAs for α and β estrogen receptors
expressed in MC3T3 osteoblast-like cells in culture. Bone
23, F044
129 Ishibashi, O., Saitoh, Y., Hara, K. and Kawashima, H.
(1998) Estrogen receptors α and β are differentially
expressed during osteoblast maturation. Bone 23, F043
130 Gruber, R., Czerwenka, F., Wolf, F., Ho, G. M.,
Willheim, M. and Perterlik, M. (1999) Expression of the
vitamin D receptor, of estrogen and thyroid hormone
receptor alpha- and beta-isoforms, and of the androgen
receptor in cultures of native mouse bone marrow and of
stromal\osteoblastic cells. Bone 24, 465–473
131 Brandenberger, A. W., Tee, M. K., Lee, J. Y., Chao, V.
and Jaffe, R. B. (1997) Tissue distribution of estrogen
receptors alpha (ER-α) and beta (ER-β) mRNA in the
midgestational human fetus. J. Clin. Endocrinol. Metab.
82, 3509–3512
132 Arts, J., Kuiper, G. G. J. M., Janssen, J. M. M. F. et al.
(1997) Differential expression of estrogen receptors α and
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
β mRNA during differentiation of human osteoblast SVHFO cells. Endocrinology 138, 5067–5070
Connor, J. R., Dodds, R. A., Nuttall, M. E., Prichett,
W. P., Yhu, Y. and Gowen, M. (1998) Osteoblasts in human
bone express higher levels of estrogen receptor β mRNA
than estrogen receptor α mRNA. Bone 23, F049
Colston, K. W., King, R. J. B., Hayward, J. et al. (1989)
Estrogen receptors and human bone cells :
immunocytochemical studies. J. Bone Miner. Res. 4,
625–631
Braidman, I. P., Davenport, L. K., Carter, D. H., Selby,
P. L., Mawer, E. B. and Freemont, A. J. (1995)
Preliminary in situ identification of estrogen target cells
in bone. J. Bone Miner. Res. 10, 74–80
Hoyland, J. A., Mee, A. P., Baird, P., Braidman, I. P.,
Mawer, E. B. and Freemont, A. J. (1997) Demonstration
of estrogen receptor mRNA in bone using in situ reversetranscriptase polymerase chain reaction. Bone 20, 87–92
Nilsson, O., Grigelioniene, G., Arai, N. et al. (1998)
Expression of estrogen receptors in mouse and human
growth plates. Bone 23, SA191
Kennedy, J., Baris, C., Hoyland, J. A., Selby, P. L.,
Freemont, A. J. and Braidman, I. P. (1999)
Immunofluorescent localization of estrogen receptoralpha in growth plates of rabbits, but not in rats, at sexual
maturity. Bone 24, 9–16
Nilsson, L. O., Boman, A., Savendahl, L. et al. (1999)
Demonstration of estrogen receptor-beta
immunoreactivity in human growth plate cartilage. J.
Clin. Endocrinol. Metab. 84, 370–373
Oursler, M. J., Osdoby, P., Pyfferoen, J., Riggs, B. L. and
Spelsberg, T. C. (1991) Avian osteoclasts as estrogen
target cells. Proc. Natl. Acad. Sci. U.S.A. 88, 6613–6617
Pederson, L., Kremer, M., Foged, N. T. et al. (1997)
Evidence of a correlation of estrogen receptor level and
avian osteoclast estrogen responsiveness. J. Bone Miner.
Res. 12, 742–752
Oursler, M. J., Pederson, L., Fitzpatrick, L., Riggs, B. L.
and Spelsberg, T. (1994) Human giant-cell tumours of the
bone (osteoclastomas) are estrogen target-cells. Proc.
Natl. Acad. Sci. U.S.A. 91, 5227–5231
Gunhan, M., Gunhan, O., Celasun, B., Mutlu, M. and
Bostanci, H. (1998) Estrogen and progesterone receptors
in the giant cell granulomas of the oral cavity. J. Oral Sci.
40, 57–60
Pensler, J. M., Radosevich, J. A., Higbee, R. and
Langman, C. B. (1990) Osteoclasts isolated from
membranous bone in children exhibit nuclear estrogen
and progesterone receptors. J. Bone Miner. Res. 5,
797–802
Malawer, M., Bray, M. and Kass, M. (1984) Fluorescent
histochemical demonstration of estrogen and
progesterone binding in giant cell tumors of bone :
preliminary observations. J. Surg. Oncol. 25, 148–152
Mano, H., Yuasa, T., Kameda, T. et al. (1996) Mammalian
mature osteoclasts as estrogen target cells. Biochem.
Biophys. Res. Commun. 223, 637–642
Collier, F. M., Huang, W. H., Holloway, W. R. et al.
(1998) Osteoclasts from human giant cell tumours of
bone lack estrogen receptors. Endocrinology 139,
1258–1267
Huang, W. H., Lau, A. T., Daniels, L. L. et al. (1998)
Detection of estrogen receptor α, carbonic anhydrase II
and tartrate-resistant acid phosphatase mRNAs in
putative mononuclear osteoclast precursor cells of
neonatal rats by fluorescence in situ hybridization. J. Mol.
Endocrinol. 20, 211–219
Fiorelli, G., Gori, F., Petilli, M. et al. (1995) Functional
estrogen receptors in a human preosteoclastic cell line.
Proc. Natl. Acad. Sci. U.S.A. 92, 2672–2676
Oreffo, R. O., Kusec, V., Virdi, A. S. et al. (1999)
Expression of estrogen receptor-alpha in cells of the
osteoclastic lineage. Histochem. Cell Biol. 111, 125–133
Vanderschueren, D. and Bouillon, R. (1995) Androgens
and bone. Calcif. Tissue Int. 56, 341–346
Orwoll, E. S. (1996) Androgens as anabolic agents for
bone. Trends Endocrinol. Metab. 7, 77–84
Hofbauer, L. C. and Khosla, S. (1999) Androgen effects
# 2000 The Biochemical Society and the Medical Research Society
233
234
R. Bland
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
on bone metabolism : recent progress and controversies.
Eur. J. Endocrinol. 140, 271–286
Buchanan, J. R., Hospodar, P., Myers, C., Leuenberger,
P. and Demers, L. M. (1988) Effect of excess endogenous
androgens on bone density in young women. J. Clin.
Endocrinol. Metab. 67, 937–943
Steingberg, K. K., Freni-Titulaer, L. W., Depuey, E. G. et
al. (1989) Sex steroids and bone density in premenopausal
and perimenopausal women. J. Clin. Endocrinol. Metab.
69, 533–539
Davidson, B. J., Ross, R. K., Paganini-Hill, A.,
Hammond, G. D., Siiteri, P. K. and Judd, H. L. (1982)
Total and free estrogens and androgens in
postmenopausal women with hip fractures. J. Clin.
Endocrinol. Metab. 54, 115–120
Soule, S. G., Conway, G., Prelevic, G. M., Prentice, M.,
Ginsburg, J. and Jacobs, H. S. (1995) Osteopenia as a
feature of the androgen insensitivity syndrome. Clin.
Endocrinol. 43, 671–675
Munoz-Torres, M., Jodar, E., Quesada, M. and EscobarJimenez, F. (1995) Bone mass in androgen-insensitivity
syndrome : response to hormonal replacement therapy.
Calcif. Tissue Int. 57, 94–96
MacLean, H. E., Chu, S., Joske, F., Warne, G. L. and
Zajac, J. D. (1995) Androgen receptor binding studies on
heterozygotes in a family with androgen insensitivity
syndrome. Biochem. Mol. Med. 55, 31–37
Vanderschueren, D., van Herck, E., Suiker, A. M. et al.
(1993) Bone and mineral metabolism in the androgenresistant (testicular feminized) male rat. J. Bone Miner.
Res. 8, 801–809
Seeman, E., Melton, L. J., O’Fallon, W. M. and Riggs,
B. L. (1983) Risk factors for spinal osteoporosis in men.
Am. J. Med. 75, 977–983
Stanley, H. L., Schmitt, B. P., Poses, R. M. and Deiss,
W. P. (1991) Does hypogonadism contribute to the
occurrence of a minimal trauma hip fracture in elderly
men ? J. Am. Geriatr. Soc. 39, 766–771
Anderson, F. H., Francis, R. M. and Faulkner, K. (1996)
Androgen supplementation in eugonadal men with
osteoporosis – effects of 6 months of treatment on bone
mineral density and cardiovascular risk factors. Bone 18,
171–177
Behre, H. M., Kliesch, S., Leifke, E., Link, T. M. and
Nieschlag, E. (1997) Long-term effect of testosterone
therapy on bone mineral density in hypogonadal men.
J. Clin. Endocrinol. Metab. 82, 2386–2390
Francis, R. M. (1999) The effects of testosterone on
osteoporosis in men. Clin. Endocrinol. 50, 411–414
Fitzgerald, R. C., Skingle, S. J. and Crisp, A. J. (1997)
Testosterone concentrations in men on chronic
glucocorticosteroid therapy. J.R. Coll. Physicians London
31, 168–170
Reid, I. R., Wattie, D. J., Evans, M. C. and Stapleton, J. P.
(1996) Testosterone therapy in glucocorticoid-treated
men. Arch. Intern. Med. 156, 1133–1134
Wimalawansa, S. J. and Simmons, D. J. (1998) Prevention
of corticosteroid-induced bone loss with alendronate.
Proc. Soc. Exp. Biol. Med. 217, 162–167
Vanderschueren, D., van Herck, E., Schot, P. et al. (1993)
The aged male rat as a model for human osteoporosis :
evaluation by nondestructive measurements and
biomedical testing. Calcif. Tissue Int. 53, 342–347
Wakley, G. K., Schutte, Jr., H. D., Hannon, K. S. and
Turner, R. T. (1991) Androgen treatment prevents loss of
cancellous bone in the orchidectomized rat. J. Bone
Miner. Res. 6, 325–330
Vanderschueren, D., van Herck, E., Suiker, A. M. H.,
Visser, W. J., Schot, L. P. C. and Bouillon, R. (1992) Bone
and mineral metabolism in aged male rats : short and long
term effects of androgen deficiency. Endocrinology 130,
2906–2916
Danielsen, C. C., Mosekilde, L. and Andreassen, T. T.
(1992) Long-term effect of orchidectomy on cortical bone
from rat femur : bone mass and mechanical properties.
Calcif. Tissue Int. 50, 169–174
Gunness, M. and Orwoll, E. (1995) Early induction of
alterations in cancellous and cortical bone histology after
# 2000 The Biochemical Society and the Medical Research Society
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
orchidectomy in mature rats. J. Bone Miner. Res. 10,
1735–1744
Lea, C. K. and Flanagan, A. M. (1999) Ovarian androgens
protect against bone loss in rats made oestrogen deficient
by treatment with ICI 182,780. J. Endocrinol. 169,
111–117
Kasperk, C. H., Wergedal, J. E., Farley, J. R., Linkhart,
T. A., Turner, R. T. and Baylink, D. J. (1989) Androgens
directly stimulate proliferation of bone cells in vitro.
Endocrinology 124, 1576–1578
Kasperk, C. H., Wakley, G. K., Hierl, T. and Ziegler, R.
(1997) Gonadal and adrenal androgens are potent
regulators of human bone cell metabolism in vitro.
J. Bone Miner. Res. 12, 464–471
Benz, D. J., Haussler, M. R., Thomas, M. A., Speelman,
B. and Komm, B. S. (1991) High-affinity androgen
binding and androgenic regulation of α1(I)-procollagen
and transforming growth factor-β steady state messenger
ribonucleic acid levels in human osteoblast-like
osteosarcoma cells. Endocrinology 128, 2723–2730
Bodine, P. V., Riggs, B. L. and Spelsberg, T. C. (1995)
Regulation of c-fos expression and TGF-β production by
gonadal and adrenal androgens in normal human
osteoblastic cells. J. Steroid Biochem. Mol. Biol. 52,
149–158
Pederson, L., Kremer, M., Judd, J. et al. (1999)
Androgens regulate bone resorption activity of isolated
osteoclasts in vitro. Proc. Natl. Acad. Sci. U.S.A. 96,
505–510
Bellido, T., Jilka, R. L., Boyce, B. F. et al. (1995)
Regulation of interleukin-6, osteoclastogenesis, and bone
mass by androgens. The role of the androgen receptor.
J. Clin. Invest. 95, 2886–2895
Mason, R. A. and Morris, H. A. (1997) Effects of
dihydrotestosterone on bone biochemical markers in
sham and oophorectomized rats. J. Bone Miner. Res. 12,
1431–1437
Takeuchi, M., Kakushi, H. and Tohkin, M. (1994)
Androgens directly stimulate mineralization and increase
androgen receptors in osteoblast-like osteosarcoma cells.
Biochem. Biophys. Res. Commun. 204, 905–911
Wiren, K., Keenan, E., Zhang, X., Ramsey, B. and
Orwoll, E. (1999) Homologous androgen receptor upregulation in osteoblastic cells may be associated with
enhanced functional androgen responsiveness.
Endocrinology 140, 3114–3124
Colvard, D., Eriksen, E., Keeting, P. et al. (1989)
Identification of androgen receptors in normal human
osteoblast-like cells. Proc. Natl. Acad. Sci. U.S.A. 86,
854–857
Orwoll, E. S., Stribrska, L., Ramsey, E. E. and Keenan,
E. J. (1991) Androgen receptors in osteoblast-like cell lines.
Calcif. Tissue Int. 49, 183–187
Keenan, E. J., Ramsey, E., Wiren, K. and Orwoll, E.
(1996) Androgen receptor promoter regulation in
osteoblasts : characterization of androgenic progestins.
J. Bone Miner. Res. 11, T385
Czerwiec, F. S., Liaw, J. J., Liu, S. B. et al. (1997) Absence
of androgen-mediated transcriptional effects in
osteoblastic cells despite presence of androgen receptors.
Bone 21, 49–56
Kasperk, C., Helmboldt, A., Borcsok, I. et al. (1997)
Skeletal site-dependent expression of the androgen
receptor in human osteoblastic cell populations. Calcif.
Tissue Int. 61, 464–473
Nakano, Y., Morimoto, I., Ishida, O. et al. (1994) The
receptor, metabolism and effects of androgen in
osteoblastic MC3T3-E1 cells. Bone Miner. 26, 245–259
Abu, E. O., Horner, A., Kusec, V., Triffitt, J. T. and
Compston, J. E. (1997) The localisation of androgen
receptors in human bone. J. Clin. Endocrinol. Metab. 82,
3493–3497
Mizuno, Y., Hosoi, T., Inoue, S. et al. (1994)
Immunocytochemical identification of androgen receptor
in mouse osteoclast-like multinucleated cells. Calcif.
Tissue Int. 54, 325–326
Simpson, E. R., Mahendroo, M. S., Means, G. D. et al.
(1994) Aromatase cytochrome P450, the enzyme
Steroid receptors in bone
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
responsible for estrogen biosynthesis. Endocr. Rev. 15,
342–355
Conte, F. A., Grumbach, M. M., Ito, Y., Fisher, C. R. and
Simpson, E. R. (1994) A syndrome of female
pseudohermaphrodism, hypogonadism, and multicystic
ovaries associated with missense mutations in the gene
encoding aromatase (P450arom). J. Clin. Endocrinol.
Metab. 78, 1287–1292
Vanderschueren, D., Van Herck, E., De Coster, R. and
Bouillon, R. (1996) Aromatization of androgens is
important for skeletal maintenance of aged male rats.
Calcif. Tissue Int. 59, 179–183
Vanderschueren, D., Van Herck, E., Nijs, J., Ederveen,
A. G. H., de Coster, R. and Bouillon, R. (1997) Aromatase
inhibition impairs skeletal modeling and decreases bone
mineral density in growing male rats. Endocrinology 138,
2301–2307
Orhan, K. O., Fisher, C., Graves, K., Nanu, L., Zerwekh,
J. and Simpson, E. R. (1998) Alterations in bone
histomorphometry and growth in aromatase deficient
(ArKO) mice. Proc. 80th Am. Endocr. Soc. OR7-2
Morishima, A., Grumbach, M. M., Simpson, E. R., Fisher,
C. and Qin, K. (1995) Aromatase deficiency in male and
female siblings caused by a novel mutation and the
physiological role of estrogens. J. Clin. Endocrinol.
Metab. 80, 3689–3698
Carani, C., Qin, K., Simoni, M. et al. (1997) Effect of
testosterone and estradiol in a man with aromatase
deficiency. N. Engl. J. Med. 337, 91–95
Vanderschueren, D., Boonen, S. and Bouillon, R. (1998)
Action of androgens versus estrogens in male skeletal
homeostasis. Bone 23, 391–394
Christiansen, C., Nilas, L., Riis, B. J., Rodbro, P. and
Deftos, L. (1985) Uncoupling of bone formation and
resorption by combined oestrogen and progestagen
therapy in postmenopausal osteoporosis. Lancet ii,
800–801
Tremollieres, F., Pouilles, J. M. and Ribot, C. (1993)
Effect of long-term administration of progesterone on
post-menopausal bone loss : result of a two-year,
controlled randomized study. Clin. Endocrinol. 38,
627–631
Grecu, E. O., Weinshelbaum, A. and Simmons, R. (1990)
Effective therapy of glucocorticoid-induced osteoporosis
with medroxyprogesterone acetate. Calcif. Tissue Int. 46,
294–299
Prior, J. C. (1990) Progesterone as a bone-tropic
hormone. Endocr. Rev. 11, 386–398
Barengolts, E. I., Kouznetsova, T., Segalene, A. et al.
(1996) Effects of progesterone on serum levels of IGF-1
and on femur IGF-1 mRNA in ovariectomized rats.
J. Bone Miner. Res. 11, 1406–1412
Bowman, B. M. and Miller, S. C. (1996) Elevated
progesterone during pseudopregnancy may prevent bone
loss associated with low estrogen. J. Bone Miner. Res. 11,
15–21
Manzi, D. L., Pilbeam, C. C. and Raisz, L. G. (1994) The
anabolic effects of progesterone on fetal rat calvaria in
tissue culture. J. Soc. Gynecol. Invest. 1, 302–309
Chen, L. and Foged, N. T. (1996) Differentiation of
osteoblast in vitro is regulated by progesterone. J. Tongji
Med. Univ. 16, 83–86
Ishida, Y., Tertinegg, I. and Heersche, J. N. M. (1996)
Progesterone and dexamethasone stimulate proliferation
and differentiation of osteoprogenitors and progenitors
for adipocytes and macrophages in cell populations
derived from adult rat vertebrae. J. Bone Miner. Res. 11,
921–930
Rifas, L., Kenney, J. S., Marcelli, M. et al. (1995)
Production of interleukin-6 in human osteoblasts and
human bone marrow cells : evidence that induction by
interleukin-1 and tumor necrosis factor-α is not regulated
by ovarian steroids. Endocrinology 136, 4056–4067
Wei, L. L., Leach, M. W., Miner, R. S. and Demers, L. M.
(1993) Evidence for progesterone receptors in human
osteoblast-like cells. Biochem. Biophys. Res. Commun.
195, 525–532
MacNamara, P., O’Shaughnessy, C., Manduca, P. P. and
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
Loughrey, H. C. (1995) Progesterone receptors are
expressed in human osteoblast-like cell lines and in
primary human osteoblast cultures. Calcif. Tissue Int. 57,
436–441
Saito, H. and Yanaihara, T. (1998) Steroid formation in
osteoblast-like cells. J. Int. Med. Res. 26, 1–12
MacNamara, P. and Loughrey, H. C. (1998) Progesterone
receptor A and B isoform expression in human
osteoblasts. Calcif. Tissue Int. 63, 39–46
Kastner, P., Krust, A., Turcotte, B. et al. (1990) Two
distinct estrogen-regulated promoters generate transcripts
encoding the two functionally different human
progesterone receptor forms A and B. EMBO J. 9,
1603–1614
Boivin, G., Anthoine-Terrier, C. and Morel, G. (1994)
Ultrastructural localization of endogenous hormones and
receptors in bone tissue : an immunocytological approach
in frozen sections. Micron 25, 15–27
St-Arnaud, R. and Glorieux, F. H. (1997) Vitamin D and
bone development. In Vitamin D (Feldman, D., Glorieux,
F. H. and Pike, J. W., eds.), pp. 293–303, Academic Press,
San Diego
Malloy, P. J., Pike, J. W. and Feldman, D. (1999) The
vitamin D receptor and the syndrome of hereditary 1,25dihydroxyvitamin D-resistant rickets. Endocr. Rev. 20,
156–188
McSheehy, P. M. J. and Chambers, T. J. (1987) 1,25Dihydroxyvitamin D stimulates rat osteoblastic cells to
$ that increases osteoclastic bone
release a soluble factor
resorption. J. Clin. Invest. 80, 425–429
Suda, T., Udagawa, N., Nakamura, I., Miyaura, C. and
Takahashi, N. (1995) Modulation of osteoclast
differentiation by local factors. Bone 17, 87S–91S
Skjodt, H., Gallagher, J. A., Beresford, J. N., Couch, M.,
Poser, J. W. and Russell, R. G. G. (1985) Vitamin D
metabolites regulate osteocalcin synthesis and
proliferation of human bone cells in vitro. J. Endocrinol.
105, 391–396
Demay, M. B., Gerardi, J. M., DeLuca, H. F. and
Kronenberg, H. M. (1990) DNA sequences in the rat
osteocalcin gene that bond the 1,25-dihydroxyvitamin D
$
receptor and confer responsiveness to 1,25dihydroxyvitamin D . Proc. Natl. Acad. Sci. U.S.A. 87,
$
369–373
Williams, G. R., Bland, R. and Sheppard, M. C. (1995)
Retinoids modify regulation of endogenous gene
expression by vitamin D and thyroid hormone in three
$
osteosarcoma cell lines. Endocrinology
136, 4304–4314
Noda, M., Vogel, R. L., Craig, A. M., Prahl, J., DeLuca,
H. F. and Denhardt, D. T. (1990) Identification of a
DNA sequence responsible for binding of the 1,25dihydroxyvitamin D receptor and 1,25dihydroxyvitamin D$ enhancement of mouse secreted
$
phosphoprotein 1 (Spp-1
or osteopontin) gene expression.
Proc. Natl. Acad. Sci. U.S.A. 87, 9995–9999
Pols, H. A. P., Schilte, H. P., Herrmann-Erlee, N. M. P.,
Visser, T. J. and Birkenhager, J. C. (1986) The effect of
1,25-dihydroxyvitamin D on growth, alkaline
phosphatase and adenylate$ cyclase of rat osteoblast-like
cells. Bone Miner. 1, 397–405
Rowe, D. W. and Kream, B. E. (1982) Regulation of
collagen synthesis in fetal rat calvaria by 1,25dihydroxyvitamin D . J. Biol. Chem. 257, 8009–8015
Kream, B. E., Rowe,$D., Smith, M. D., Maher, V. and
Majeska, R. (1986) Hormonal regulation of collagen
synthesis in a clonal rat osteosarcoma cell line.
Endocrinology 119, 1922–1928
Harrison, J. R., Petersen, D. N., Lichtler, A. C., Mador,
A. T., Rowe, D. W. and Kream, B. E. (1989) 1,25Dihydroxyvitamin D inhibits transcription of type I
collagen genes in the $rat osteosarcoma cell line ROS
17\2.8. Endocrinology 125, 327–333
Owen, T. A., Aronow, M. S., Barone, L. M., Bettencourt,
B., Stein, G. S. and Lian, J. B. (1991) Pleiotropic effects of
vitamin D on osteoblast gene expression are related to the
proliferative and differentiated state of the bone cell
phenotype : dependency upon basal levels of gene
expression, duration of exposure, and bone matrix
# 2000 The Biochemical Society and the Medical Research Society
235
236
R. Bland
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
competency in normal rat osteoblast cultures.
Endocrinology 128, 1496–1504
Schiller, C., Gruber, R., Ho, G. M. et al. (1998)
Interaction of triiodothyronine with 1 alpha,25dihydroxyvitamin D3 on interleukin-6-dependent
osteoclast-like cell formation in mouse bone marrow cell
cultures. Bone 22, 341–346
Dokoh, S., Donaldson, C. A. and Haussler, M. R. (1984)
Influence of 1,25-dihydroxyvitamin D on cultured
$ the 1,25osteogenic sarcoma cells : correlation with
dihydroxyvitamin D receptor. Cancer Res. 44,
$
2103–2109
Rodan, S. B., Imai, Y., Thiede, M. A. et al. (1987)
Characterization of a human osteosarcoma cell line
(Saos-2) with osteoblastic properties. Cancer Res. 47,
4961–4966
Williams, G. R., Bland, R. and Sheppard, M. C. (1994)
Characterization of thyroid hormone (T ) receptors in
$
three osteosarcoma cell lines of distinct osteoblast
phenotype : interactions among T , vitamin D , and
$
retinoid signaling. Endocrinology$135, 2375–2385
Krishnan, A. V., Cramer, S. D., Bringhurst, F. R. and
Feldman, D. (1995) Regulation of 1,25-dihydroxyvitamin
D receptors by parathyroid hormone in osteoblastic
$ : role of second messenger pathways. Endocrinology
cells
136, 705–712
Davideau, J. L., Papagerakis, P., Hotton, D., Lezot, F.
and Berdal, A. (1996) In situ investigation of vitamin D
receptor, alkaline phosphatase, and osteocalcin expression
in oro-facial mineralized tissues. Endocrinology 137,
3577–3585
Mee, A. P., Hoyland, J. A., Braidman, I. P., Freemont,
A. J., Davies, M. and Mawer, E. B. (1996) Demonstration
of vitamin D receptor transcripts in actively resorbing
osteoclasts in bone sections. Bone 18, 295–299
Jaaskelainen, T., Pirskanen, A., Ryhanen, S., Palvimo,
J. J., DeLuca, H. F. and Maenpaa, P. H. (1994) Functional
interference between AP-1 and the vitamin D receptor on
osteocalcin gene expression in human osteosarcoma cells.
Eur. J. Biochem. 224, 11–20
Bland, R., Sammons, R. L., Sheppard, M. C. and
Williams, G. R. (1997) Thyroid hormone, vitamin D and
retinoid receptor expression and signalling in primary
cultures of rat osteoblastic and immortalised
osteosarcoma cells. J. Endocrinol. 154, 63–74
Narbaitz, R., Stumpf, W. E., Sar, M., Huang, S. and
DeLuca, H. F. (1983) Autoradiographic localization of
target cells for 1α,25-dihydroxyvitamin D in bones from
fetal rats. Calcif. Tissue Int. 35, 177–182 $
Clemens, T. L., Garrett, K. P., Zhou, X. Y., Pike, J. W.,
Haussler, M. R. and Dempster, D. W. (1988)
Immunocytochemical localization of the 1,25dihydroxyvitamin D receptor in target cells.
$
Endocrinology 122, 1224–1230
Johnson, L. A., Grande, J. P., Roche, P. C. and Kumar, R.
(1996) Ontogeny of the 1,25-dihydroxyvitamin D
receptor in fetal rat bone. J. Bone Miner. Res. 11, $56–61
Manolagas, S. C., Haussler, M. R. and Deftos, L. J. (1980)
1,25-Dihydroxyvitamin D receptor-like macromolecule
$ lines. J. Biol. Chem. 255,
in rat osteogenic sarcoma cell
4414–4417
Suwanwalaikorn, S., Ongphiphadhanakul, B., Braverman,
L. and Baran, D. T. (1996) Differential responses of
femoral and vertebral bones to long-term excessive lthyroxine administration in adult rats. Eur. J. Endocrinol.
134, 655–659
Mosekilde, L., Eriksen, E. F. and Charles, P. (1990)
Effects of thyroid hormone on bone and mineral
metabolism. Endocrinol. Metab. Clin. North Am. 19,
35–62
Allain, T. J. and McGregor, A. M. (1993) Thyroid
hormones and bone. J. Endocrinol. 139, 9–18
Stern, P. H. (1996) Thyroid hormone and bone. In
Principles of Bone Biology (Bilezikian, J. P., Raisz, L. G.
and Rodan, G. A., eds.), pp. 521–531, Academic Press,
San Diego
Akita, S., Nakamura, T., Hirano, A., Fujii, T. and
Yamashita, S. (1994) Thyroid hormone action on rat
calvarial studies. Thyroid 4, 99–106
# 2000 The Biochemical Society and the Medical Research Society
247 Ross, D. S. (1994) Hyperthyroidism, thyroid hormone
therapy, and bone. Thyroid 4, 319–326
248 Franklyn, J., Betteridge, J., Holder, R., Daykin, J., Lilley,
J. and Sheppard, M. (1994) Bone mineral density in
thyroxine treated females with or without a previous
history of thyrotoxicosis. Clin. Endocrinol. 41, 425–432
249 Weiss, R. E. and Refetoff, S. (1996) Effect of thyroid
hormone on growth. Endocrinol. Metab. Clin. North
Am. 25, 719–730
250 Allain, T. J., Thomas, M. R., McGregor, A. M. and
Salisbury, J. R. (1995) A histomorphometric study of
bone changes in thyroid dysfunction in rats. Bone 16,
505–509
251 Ren, S. G., Huang, Z., Sweet, D. E., Malozowski, S. and
Cassorla, F. (1990) Biphasic response of rat tibial growth
to thyroxine administration. Acta. Endocrinol. 122,
336–340
252 Suwanwalaikorn, S., van Auken, M., Kang, M. I., Alex, S.,
Braverman, L. E. and Baran, D. T. (1997) Site selectivity
of osteoblast gene expression response to thyroid
hormone localized by in situ hybridization. Am. J.
Physiol. 272, E212–E217
253 Mundy, G. R., Shapiro, J. L., Bandelin, J. G., Canalis,
E. M. and Raisz, L. G. (1976) Direct stimulation of bone
resorption by thyroid hormones. J. Clin. Invest. 58,
529–534
254 Allain, T. J., Chambers, T. J., Flanagan, A. M. and
McGregor, A. M. (1992) Tri-iodothyronine stimulates rat
osteoclastic bone resorption by an indirect effect.
J. Endocrinol. 133, 327–331
255 Britto, J. M., Fenton, A. J., Holloway, W. R. and
Nicholson, G. C. (1994) Osteoblasts mediate thyroid
hormone stimulation of osteoclastic bone resorption.
Endocrinology 134, 169–176
256 Rizzoli, R., Poser, J. and Burgi, U. (1986) Nuclear
thyroid hormone receptors in cultured bone cells. Metab.
Clin. Exp. 35, 71–74
257 Sato, K., Han, D. C., Fujii, Y., Tsushima, T. and Shizume,
K. (1987) Thyroid hormone stimulates alkaline
phosphatase activity in cultured rat osteoblastic cells
(ROS 17\2.8) through 3,5,3h-triiodo-L-thyronine nuclear
receptors. Endocrinology 120, 1873–1881
258 Kasono, K., Sato, K., Han, D. C., Fujii, Y., Tsushima, T.
and Shizume, K. (1988) Stimulation of alkaline
phosphatase activity by thyroid hormone in mouse
osteoblast-like cells (MC3T3-E1) : a possible mechanism
of hyper-alkaline phosphatasia in hyperthyroidism. Bone
Miner. 4, 355–363
259 Egrise, D., Martin, D., Neve, P., Verhas, M. and
Schoutens, A. (1990) Effects and interactions of 17βestradiol, T and 1,25(OH) D on cultured osteoblasts
$ rats. Bone Miner.
# $ 11, 273–283
from mature
260 Oishi, K., Ishida, H., Nishikawa, S., Hamasaki, A. and
Wakano, Y. (1990) The effect of triiodothyronine on cell
growth and alkaline phosphatase activity of isolated rat
calvarial cells. J. Bone Miner. Res. 5, S229
261 LeBron, B. A., Pekary, E., Mirell, C., Hahn, T. J. and
Hershman, J. M. (1989) Thyroid hormone 5h-deiodinase
activity, nuclear binding, and effects on mitogenesis in
UMR-106 osteoblastic osteosarcoma cells. J. Bone Miner.
Res. 4, 173–178
262 Varga, F., Rumpler, M. and Klaushofer, K. (1994)
Thyroid hormones increase insulin-like growth factor
mRNA levels in the clonal osteoblastic cell line MC3T3E1. FEBS Lett. 345, 67–70
263 Schmid, C., Schlapfer, I., Futo, E. et al. (1992)
Triiodothyronine (T ) stimulates insulin-like growth
$ binding protein (IGFBP)-2
factor (IGF)-1 and IGF
production by rat osteoblasts in vitro. Acta Endocrinol.
126, 467–473
264 Lakatos, P., Caplice, M. D., Khanna, V. and Stern, P. H.
(1993) Thyroid hormones increase insulin-like growth
factor I content in the medium of rat bone tissue. J. Bone
Miner. Res. 8, 1475–1481
265 Glantschnig, H., Varga, F. and Klaushofer, K. (1996)
Thyroid hormone and retinoic acid induce the synthesis
of insulin-like growth factor-binding protein-4 in mouse
osteoblastic cells. Endocrinology 137, 281–286
266 Siddiqi, A., Burrin, J. M., Wood, D. F. and Monson, J. P.
Steroid receptors in bone
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
(1998) Tri-iodothyronine regulates the production of
interleukin-6 and interleukin-8 in human bone marrow
stromal and osteoblast-like cells. J. Endocrinol. 157,
453–461
Tokuda, H., Kozawa, O., Harada, A., Isobe, K. I. and
Uematsu, T. (1998) Triiodothyronine modulates
interleukin-6 synthesis in osteoblasts : inhibitions in
protein kinase A and C pathways. Endocrinology 139,
1300–1305
Milne, M., Kang, M. I., Quail, J. M. and Baran, D. T.
(1998) Thyroid hormone excess increases insulin-like
growth factor I transcripts in bone marrow cell cultures :
divergent effects on vertebral and femoral cell cultures.
Endocrinology 139, 2527–2534
Fratzl-Zelman, N., Horandner, H., Luegmayr, E. et al.
(1997) Effects of triiodothyronine on the morphology of
cells and matrix, the localization of alkaline phosphatase,
and the frequency of apoptosis in long-term cultures of
MC3T3-E1 cells. Bone 20, 225–236
Varga, F., Luegmayr, E., Fratzl-Zelman, N. et al. (1999)
Tri-iodothyronine inhibits multilayer formation of the
osteoblastic cell line, MC3T3-E1, by promoting
apoptosis. J. Endocrinol. 160, 57–65
Ohishi, K., Ishida, H., Nagata, T. et al. (1994) Thyroid
hormone suppresses the differentiation of
osteoprogenitor cells to osteoblasts, but enhances
functional activities of mature osteoblasts in cultured rat
calvaria cells. J. Cell. Physiol. 161, 544–552
Alini, M., Kofsky, Y., Wu, W., Pidoux, I. and Poole,
A. R. (1996) In serum-free culture thyroid hormones can
induce full expression of chondrocyte hypertrophy
leading to matrix calcification. J. Bone Miner. Res. 11,
105–113
Ishikawa, Y., Genge, B. R., Wuthier, R. E. and Wu,
L. N. Y. (1998) Thyroid hormone inhibits growth and
stimulates terminal differentiation of epiphyseal growth
plate chondrocytes. J. Bone Miner. Res. 13, 1398–1411
Williams, G. R., Robson, H. and Shalet, S. M. (1998)
Thyroid hormone actions on cartilage and bone :
interactions with other hormones at the epiphyseal plate
and effects on linear growth. J. Endocrinol. 157, 391–403
Sap, J., Munoz, A., Damm, K. et al. (1986) The c-erb-A
protein is a high-affinity receptor for thyroid hormone.
Nature (London) 324, 635–640
Izumo, S. and Mahdavi, V. (1988) Thyroid hormone
receptor alpha isoforms generated by alternative splicing
differentially activate myosin HC gene transcription.
Nature (London) 334, 539–542
Yen, P. M. and Chin, W. W. (1994) New advances in
understanding the molecular mechanisms of thyroid
hormone action. Trends Endocrinol. Metab. 5, 65–72
Koenig, R. J., Warne, R. L., Brent, G. A., Harney, J. W.,
Larsen, P. R. and Moore, D. D. (1988) Isolation of a
cDNA clone encoding a biologically active thyroid
hormone receptor. Proc. Natl. Acad. Sci. U.S.A. 85,
5031–5035
Hodin, R. A., Lazar, M. A., Wintman, B. I. et al. (1989)
Identification of a thyroid hormone receptor that is
pituitary specific. Science 244, 76–79
Krieger, N. S., Stappenbeck, T. S. and Stern, P. H. (1988)
Characterization of specific thyroid hormone receptors in
bone. J. Bone Miner. Res. 3, 473–478
Allain, T. J., Yen, P. M., Flanagan, A. M. and McGregor,
A. M. (1996) The isoform-specific expression of the triiodothyronine receptor in osteoblasts and osteoclasts.
Eur. J. Clin. Invest. 26, 418–425
Abu, E. O., Bord, S., Horner, A., Chatterjee, V. K. K.
and Compston, J. E. (1997) The expression of thyroid
hormone receptors in human bone. Bone 21, 137–142
Carrascosa, A., Ferrandez, M. A., Audi, L. and Ballabriga,
A. (1992) Effects of triiodothyronine (T ) and
$ sites in
identification of specific nuclear T -binding
$
cultured human fetal epiphyseal chondrocytes.
J. Clin.
Endocrinol. Metab. 75, 140–144
Robson, H., Hasserjian, R. P., Anderson, E., Shalet, S. M.
and Williams, G. R. (1997) Localisation of thyroid
hormone receptor (T R) isoforms α1 and β1 in rat tibial
growth plate cartilage$ and adjacent bone. J. Endocrinol.
155, O26
285 Ballock, R. T., Mita, B. C., Zhou, X., Chen, D. H. C. and
Mink, L. M. (1999) Expression of thyroid hormone
receptor isoforms in rat growth plate cartilage in vivo.
J. Bone Miner. Res. 14, 1550–1556
286 Ruberte, E., Nakshatri, H., Kastner, P. and Chambon, P.
(1992) Retinoic acid receptors and binding proteins in
mouse limb development. In Retinoids in Normal
Development and Teratogenesis (Morriss-Kay, G., ed.),
pp. 99–111, Oxford University Press, New York
287 Tabin, C. J. (1991) Retinoids, homoboxes, and growth
factors : toward molecular models for limb development.
Cell 66, 199–217
288 Underhill, T. M. and Weston, A. D. (1998) Retinoids and
their receptors in skeletal development. Microsc. Res.
Tech. 43, 137–155
289 Satre, M. A. and Kochhar, D. M. (1989) Elevations in the
endogenous levels of the putative morphogen retinoic
acid in embryonic mouse limb-buds associated with limb
dysmorphogenesis. Dev. Biol. 133, 529–536
290 Hough, S., Avioli, L. V., Muir, H. et al. (1988) Effects of
hypervitaminosis A on the bone and mineral metabolism
of the rat. Endocrinology 122, 2933–2939
291 DeSimone, D. P. and Reddi, A. H. (1983) Influence of
vitamin A on matrix-induced endochondral bone
formation. Calcif. Tissue Int. 35, 732–739
292 Shapiro, I. M., Debolt, K., Hatori, M., Iwamoto, M. and
Pacifici, M. (1994) Retinoic acid induces a shift in the
energetic state of hypertrophic chondrocytes. J. Bone
Miner. Res. 9, 1229–1237
293 Hagiwara, H., Inoue, A., Nakajo, S., Nakaya, K., Kojima,
S. and Hirose, S. (1996) Inhibition of proliferation of
chondrocytes by specific receptors in response to
retinoids. Biochem. Biophys. Res. Commun. 222,
220–224
294 Kaji, H., Sugimoto, T., Kanatani, M., Kano, J., Fukase,
M. and Chihara, K. (1995) Effect of retinoic acid on the
proliferation and alkaline phosphatase activity of
osteoblastic MC3T3-E1 cells by modulating the release of
local regulators from monocytes. Exp. Clin. Endocrinol.
Diabetes 103, 297–302
295 Oreffo, R. O. C., Francis, M. J. A. and Triffitt, J. T.
(1985) Vitamin A effects on UMR 106 osteosarcoma cells
are not mediated by specific cytosolic receptors. Biochem.
J. 232, 599–603
296 Ng, K. W., Livesey, S. A., Collier, F., Gummer, P. R. and
Martin, T. J. (1985) Effect of retinoids on the growth,
ultrastructure, and cytoskeletal structure of malignant rat
osteoblasts. Cancer Res. 45, 5106–5113
297 Colucci, S., Grano, M., Mori, G. et al. (1996) Retinoic
acid induces cell proliferation and modulates gelatinases
activity in human osteoclast-like cells. Biochem. Biophys.
Res. Commun. 227, 47–52
298 Saneshige, S., Mano, H., Tezuka, K. I. et al. (1995)
Retinoic acid directly stimulates osteoclastic bone
resorption and gene expression of cathepsin K\OC-2.
Biochem. J. 309, 721–724
299 Kaji, H., Sugimoto, T., Kanatani, M., Fukase, M.,
Kumegawa, M. and Chihara, K. (1995) Retinoic acid
induces osteoclast-like cell formation by directly acting
on hemopoietic blast cells and stimulates osteopontin
mRNA expression in isolated osteoclasts. Life Sci. 56,
1903–1913
300 Iwamoto, M., Shapiro, I. M., Yamami, K. et al. (1993)
Retinoic acid induces rapid mineralization and expression
of mineralization-related genes in chondrocytes. Exp.
Cell Res. 207, 413–420
301 Zhou, H., Manji, S. S., Findlay, D. M., Martin, T. J.,
Heath, J. K. and Ng, K. W. (1994) Novel action of
retinoic acid. Stabilization of newly synthesized alkaline
phosphatase transcripts. J. Biol. Chem. 269, 22433–22439
302 Nakayama, Y., Takahashi, K., Noji, S., Muto, K.,
Nishijima, K. and Taniguchi, S. (1990) Functional modes
of retinoic acid in mouse osteoblastic clone MC3T3-E1,
proved as a target cell for retinoic acid. FEBS Lett. 261,
93–96
303 Zhou, H., Hammonds, R. G., Findlay, D. M., Fuller,
P. J., Martin, T. J. and Ng, K. W. (1991) Retinoic acid
modulation of mRNA levels in malignant,
# 2000 The Biochemical Society and the Medical Research Society
237
238
R. Bland
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
nontransformed, and immortalized osteoblasts. J. Bone
Miner. Res. 6, 767–777
Togari, A., Kondo, M., Arai, M. and Matsumoto, S.
(1991) Effects of retinoic acid on bone formation and
resorption in cultured mouse calvaria. Gen. Pharmacol.
22, 287–292
Ohishi, K., Nishikawa, S., Nagata, T. et al. (1995)
Physiological concentrations of retinoic acid suppress the
osteoblastic differentiation of fetal rat calvaria cells in
vitro. Eur. J. Endocrinol. 133, 335–341
Cancela, M. L. and Price, P. A. (1992) Retinoic acid
induces matrix Gla protein gene expression in human
cells. Endocrinology 130, 102–108
Szulc, P. and Delmas, P. D. (1996) Influence of vitamin D
and retinoids on the gammacarboxylation of osteocalcin
in human osteosarcoma MG63 cells. Bone 19, 615–620
Nishimoto, S. K., Salka, C. and Nimni, M. E. (1987)
Retinoic acid and glucocorticoids enhance the effect of
1,25-dihydroxyvitamin D on bone gamma$ synthesis by rat
carboxyglutamic acid protein
osteosarcoma cells. J. Bone Miner. Res. 2, 571–577
Oikarinen, H., Oikarinen, A. I., Tan, E. M. L. et al.
(1985) Modulation of procollagen gene expression by
retinoids. J. Clin. Invest. 75, 1545–1553
Kim, H. T. and Chen, T. L. (1989) 1,25Dihydroxyvitamin D interaction with dexamethasone
$ on procollagen messenger
and retinoic acid : effects
ribonucleic acid levels in rat osteoblast-like cells. Mol.
Endocrinol. 3, 97–104
Varghese, S., Rydiziel, S., Jeffrey, J. J. and Canalis, E.
(1994) Regulation of interstitial collagenase expression
and collagen degradation by retinoic acid in bone cells.
Endocrinology 134, 2438–2444
Connolly, T. J., Clohisy, J. C., Shilt, J. S., Bergman,
K. D., Partridge, N. C. and Quinn, C. O. (1994) Retinoic
acid stimulates interstitial collagenase messenger
ribonucleic acid in osteosarcoma cells. Endocrinology
135, 2542–2548
Pacifici, M., Golden, E. B., Iwamoto, M. and Adams,
S. L. (1991) Retinoic acid treatment induces type X collagen
gene expression in cultured chick chondrocytes. Exp. Cell
Res. 195, 38–46
Horton, W. E., Yamada, Y. and Hassell, J. R. (1987)
Retinoic acid rapidly reduces cartilage matrix synthesis
by altering gene transcription in chondrocytes. Dev. Biol.
123, 508–516
Gabbitas, B. and Canalis, E. (1996) Retinoic acid
stimulates the transcription of insulin-like growth factor
binding protein-6 in skeletal cells. J. Cell. Physiol. 169,
15–22
Gabbitas, B. and Canalis, E. (1997) Retinoic acid
regulates the expression of insulin-like growth factors I
and II in osteoblasts. J. Cell. Physiol. 172, 253–264
Zhou, Y. H., Mohan, S., Linkhart, T. A., Baylink, D. J.
and Strong, D. D. (1996) Retinoic acid regulates insulinlike growth factor-binding protein expression in human
osteoblast cells. Endocrinology 137, 975–983
Allenby, G., Bocquel, M. T., Saunders, M. et al. (1993)
Retinoic acid receptors and retinoid X receptors :
interactions with endogenous retinoic acids. Proc. Natl.
Acad. Sci. U.S.A. 90, 30–34
Petkovich, M., Brand, N. J., Krust, A. and Chambon, P.
(1987) A human retinoic acid receptor which belongs to
the family of nuclear receptors. Nature (London) 330,
444–450
Brand, N., Petkovich, M., Krust, A. et al. (1988)
Identification of a second human retinoic acid receptor.
Nature (London) 332, 850–853
Zelent, A., Krust, A., Petkovich, M., Kastner, P. and
Chambon, P. (1989) Cloning of murine alpha and beta
retinoic acid receptors and a novel receptor gamma
predominantly expressed in skin. Nature (London) 339,
714–717
Mangelsdorf, D. J., Borgmeyer, R. A., Heyman, R. A. et
al. (1992) Characterization of three RXR genes that
mediate the action of 9-cis retinoic acid. Genes Dev. 6,
329–344
# 2000 The Biochemical Society and the Medical Research Society
323 Giguere, V. (1994) Retinoic acid receptors and cellular
retinoid binding proteins : complex interplay in retinoid
signaling. Endocr. Rev. 15, 61–79
324 Leid, M., Kastner, P., Lyons, R. et al. (1992) Purification,
cloning, and RXR identity of the HeLa cell factor with
which RAR or TR heterodimerizes to bind target
sequences efficiently. Cell 68, 377–395
325 Liu, Q. and Linney, E. (1993) The mouse retinoid-X
receptor-gamma gene : genomic organization and evidence
for functional isoforms. Mol. Endocrinol. 7, 651–658
326 Ruberte, E., Dolle, P., Krust, A., Zelent, A., Morriss-Kay,
G. and Chambon, P. (1990) Specific spatial and temporal
distribution of retinoic acid receptor gamma transcripts
during mouse embryogenesis. Development 108, 213–222
327 Suva, L. J., Ernst, M. and Rodan, G. A. (1991) Retinoic
acid increases zif268 early gene expression in rat
preosteoblastic cells. Mol. Cell. Biol. 11, 2503–2510
328 Harada, H., Miki, R., Masushige, S. and Kato, S. (1995)
Gene expression of retinoic acid receptors, retinoid-X
receptors, and cellular retinol-binding protein I in bone
and its regulation by vitamin A. Endocrinology 136,
5329–5335
329 Chen, Y., Takeshita, A., Ozaki, K., Kitano, S. and
Hanazawa, S. (1996) Transcriptional regulation by
transforming growth factor β of the expression of retinoic
acid and retinoid X receptor genes in osteoblastic cells is
mediated through AP-1. J. Biol. Chem. 271, 31602–31606
330 Kindmark, A., Torma, H., Johansson, A., Ljunghall, S.
and Melhus, H. (1993) Reverse transcription–polymerase
chain reaction assay demonstrates that the 9-cis retinoic
acid receptor α is expressed in human osteoblasts.
Biochem. Biophys. Res. Commun. 192, 1367–1372
331 Dreyer, C., Keller, H., Mahfoudi, A., Laudet, V., Krey,
G. and Wahli, W. (1993) Positive regulation of the
peroxisomal beta oxidation pathway by fatty acids
through activation of peroxisome proliferator-activated
receptors (PPAR). Biol. Cell 77, 67–76
332 Braissant, O., Foufelle, F., Scotto, C., Dauca, M. and
Wahli, W. (1996) Differential expression of peroxisome
proliferator-activated receptors (PPARs) : tissue
distribution of PPAR-α, -β, and -γ in the adult rat.
Endocrinology 137, 354–366
333 Diascro, D. D., Vogel, R. L., Johnson, T. E. et al. (1998)
High fatty acid content in rabbit serum is responsible for
the differentiation of osteoblasts into adipocyte-like cells.
J. Bone Miner. Res. 13, 96–106
334 Nuttall, M. E., Patton, A. J., Olivera, D. L., Nadeau,
D. P. and Gowen, M. (1998) Human trabecular bone
cells are able to express both osteoblastic and adipocytic
phenotype : implications for osteopenic disorders. J. Bone
Miner. Res. 13, 371–382
335 Cecchini, M. G., Portenier, J., Wetterwald, A., Braissant,
O. and Wahli, W. (1997) Expression of peroxisome
proliferator-activated receptors in rat bone. J. Bone
Miner. Res. 12, S420
336 Greene, M. E., Blumberg, B., McBride, O. W. et al.
(1995) Isolation of the human peroxisome proliferator
activated receptor gamma cDNA : expression in
hematopoietic cells and chromosomal mapping. Gene
Expression 4, 281–289
337 Elbrecht, A., Chen, Y., Cullinan, C. A. et al. (1996)
Molecular cloning, expression and characterization of
human peroxisome proliferator activated receptors γ1 and
γ2. Biochem. Biophys. Res. Commun. 224, 431–437
338 Gimble, J. M., Robinson, C. E., Wu, X. et al. (1996)
Peroxisome proliferator-activated receptor-γ activation by
thiazolidinediones induces adipogenesis in bone marrow
stromal cells. Mol. Pharmacol. 50, 1087–1094
339 Okazaki, R., Nakatsu, M., Arai, M. et al. (1997)
Activation of peroxisome proliferator activated receptor γ
inhibits osteoblastic differentiation. J. Bone Miner. Res.
12, S421
340 Bain, S. D., Lydon, J. P., Tibbetts, T., Strachan, M. J.,
Puerner, D. A. and O’Malley, B. W. (1997) Mice lacking
functional progesterone receptors have no apparent
alterations in peak bone mass or bone histomorphometry.
J. Bone Miner. Res. 12, S432
Steroid receptors in bone
341 Korach, K. S., Couse, J. F., Curtis, S. W. et al. (1996)
Estrogen receptor gene disruption : molecular
characterization and experimental and clinical
phenotypes. Recent Prog. Horm. Res. 51, 159–188
342 Couse, J. F. and Korach, K. S. (1999) Estrogen receptor
null mice : what have we learned and where will they lead
us ? Endocr. Rev. 20, 358–417
343 Pan, L. C., Ke, H. Z., Simmons, H. A. et al. (1997)
Estrogen receptor-α knockout (ERKO) mice lose
trabecular and cortical bone following ovariectomy.
J. Bone Miner. Res. 12, 126
344 Taki, M., Kimbro, K. S., Washburn, T. F. et al. (1997)
Effects of estrogen receptor destruction on femurs from
male mice. J. Bone Miner. Res. 12, S430
345 Kimbro, K. S., Taki, M., Pan, L., Ke, H. Z., Thompson,
D. and Korach, K. S. (1997) The effects of estrogen
receptor gene disruption on bone. Proc. 79th Am.
Endocr. Soc., OR27-3
346 Kimbro, K. S., Taki, M., Beamer, G. and Korach, K. S.
(1998) Ovariectomized female estrogen receptor-α
knockout and wild type mice respond differently to
estrogen and dihydrotestosterone treatment. Proc. 80th
Am. Endocr. Soc., OR7-4
347 Vidal, O., Windahl, S., Andersson, G., Gustavsson, J. A.
and Phlsson, C. (1999) Increased cortical bone mineral
content but unchanged trabecular bone mineral density in
adult female mice lacking estrogen receptor-beta. J. Bone
Miner. Res. 14, S141, absst. 103
348 Kato, S., Takeyama, K.-I., Kitanaka, S., Murayama, A.,
Sekine, K. and Yoshizawa, T. (1999) In vivo function of
VDR in gene expression-VDR knock-out mice. J. Steroid
Biochem. Mol. Biol. 69, 247–251
349 Li, Y. C., Pirro, A. E., Amling, M. et al. (1997) Targeted
ablation of the vitamin D receptor : an animal model of
vitamin D-dependent rickets type II with alopecia. Proc.
Natl. Acad. Sci. U.S.A. 94, 9831–9835
350 Yoshizawa, T., Handa, Y., Uematsu, Y. et al. (1997) Mice
lacking the vitamin D receptor exhibit impaired bone
formation, uterine hypoplasia and growth retardation
after weaning. Nat. Genet. 16, 391–396
351 Takeda, S., Yoshizawa, T., Fukumoto, S. et al. (1997)
Bone metabolism and in vitro osteoclast formation in
VDR knock-out mice. J. Bone Miner. Res. 12, S110,
absst. 32
352 Forrest, D., Golarai, G., Connor, J. and Curran, T. (1996)
Genetic analysis of thyroid hormone receptors in
development and disease. Recent Prog. Horm. Res. 51,
1–22
353 Fraichard, A., Chassande, O., Plateroti, M. et al. (1997)
The T3Ralpha gene encoding a thyroid hormone receptor
is essential for post-natal development and thyroid
hormone production. EMBO J. 16, 4412–4420
354 Gothe, S., Wang, Z., Ng, L. et al. (1999) Mice devoid of
all known thyroid hormone receptors are viable but
exhibit disorders of the pituitary–thyroid axis, growth,
and bone maturation. Genes Dev. 13, 1329–1341
355 Lohnes, D., Kastner, P., Dierich, A., Mark, M., LeMeur,
M. and Chambon, P. (1993) Function of retinoic acid
receptor gamma in the mouse. Cell 73, 643–658
356 Lufkin, T., Lohnes, D., Mark, M. et al. (1993) High
postnatal lethality and testis degeneration in retinoic acid
receptor α mutant mice. Proc. Natl. Acad. Sci. U.S.A. 90,
7225–7229
357 Li, E., Sucov, H. M., Lee, K. F., Evans, R. M. and
Jaenisch, R. (1993) Normal development and growth of
mice carrying a targeted disruption of the alpha 1 retinoic
acid receptor gene. Proc. Natl. Acad. Sci. U.S.A. 90,
1590–1594
358 Mendelsohn, C., Mark, M., Dolle, P. et al. (1994)
Retinoic acid receptor β2 (RAR β2) null mutant mice
appear normal. Dev. Biol. 166, 246–258
359 Cash, D. E., Bock, C. B., Schughart, K., Linney, E. and
Underhill, T. M. (1997) Retinoic acid receptor alpha
function in vertebrate limb skeletogenesis : a modulator of
chondrogenesis. J. Cell Biol. 136, 445–457
360 Lohnes, D., Mark, M., Mendelsohn, C. et al. (1994)
Function of the retinoic acid receptors (RARS) during
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
development. 1. Craniofacial and skeletal abnormalities in
RAR double mutants. Development 120, 2723–2748
Lohnes, D., Mark, M., Mendelsohn, C. et al. (1995)
Developmental roles of the retinoic acid receptors.
J. Steroid Biochem. Mol. Biol. 53, 475–486
Sucov, H. M., Izpisua-Belmonte, J. C., Ganan, Y. and
Evans, R. M. (1995) Mouse embryos lacking RXR alpha
are resistant to retinoic-acid-induced limb defects.
Development 121, 3997–4003
Krezel, W., Dupe, V., Mark, M., Dierich, A., Kastner, P.
and Chambon, P. (1996) RXRγ null mice are apparently
normal and compound RXRα+/−\RXRβ−/−\RXRγ−/−
mutant mice are viable. Proc. Natl. Acad. Sci. U.S.A. 93,
9010–9014
Weinstein, R. S., Jilka, R. L., Parfitt, A. M. and
Manolagas, S. C. (1997) The effects of androgen
deficiency on murine bone remodeling and bone mineral
density are mediated via cells of the osteoblastic lineage.
Endocrinology 138, 4013–4021
Liel, Y., Kraus, S., Levy, J. and Shany, S. (1992) Evidence
that estrogens modulate activity and increase the number
of 1,25-dihydroxyvitamin D receptors in osteoblast-like
cells (ROS 17\2.8). Endocrinology 130, 2597–2601
Chen, T. L., Hauschka, P. V. and Feldman, D. (1986)
Dexamethasone increases 1,25-dihydroxyvitamin D
receptor levels and augments bioresponses in rat $
osteoblast-like cells. Endocrinology 118, 1119–1126
Godschalk, M., Levey, J. R. and Downs, Jr., R. W. (1992)
Glucocorticoids decrease vitamin D receptor number and
gene expression in human osteosarcoma cells. J. Bone
Miner. Res. 7, 21–27
Hsu, J. H., Zavacki, A. M., Harney, J. W. and Brent,
G. A. (1995) Retinoid-X receptor (RXR) differentially
augments thyroid hormone response in cell lines as a
function of the response element and endogenous RXR
content. Endocrinology 136, 421–430
Lemon, B. D. and Freedman, L. P. (1996) Selective effects
of ligands on vitamin D receptor- and retinoid X
receptor-mediated gene $activation in vivo. Mol. Cell.
Biol. 16, 1006–1016
Green, S. (1993) Promiscuous liaisons. Nature (London)
361, 590–591
Schrader, M., Muller, K. M., Nayeri, S., Kahlen, J. P. and
Carlberg, C. (1994) Vitamin D –thyroid hormone
$
receptor heterodimer polarity directs
ligand sensitivity of
transactivation. Nature (London) 370, 382–386
Yen, P. M., Liu, Y., Palvimo, J. J. et al. (1997) Mutant and
wild-type androgen receptors exhibit cross-talk on
androgen-, glucocorticoid-, and progesterone-mediated
transcription. Mol. Endocrinol. 11, 162–171
Morrison, N. A., Qi, J. C., Tokita, A. et al. (1994)
Prediction of bone density from vitamin D receptor
alleles. Nature (London) 367, 284–287
Cooper, G. S. and Umbach, D. M. (1996) Are vitamin D
receptor polymorphisms associated with bone mineral
density ? A meta-analysis. J. Bone Miner. Res. 11,
1841–1849
Kobayashi, S., Inoue, S., Hosoi, T., Ouchi, Y., Shiraki, M.
and Orimo, H. (1996) Association of bone mineral
density with polymorphism of the estrogen receptor
gene. J. Bone Miner. Res. 11, 306–311
Mizunuma, H., Hosoi, T., Okano, H. et al. (1997)
Estrogen receptor gene polymorphism and bone mineral
density at the lumbar spine of pre- and postmenopausal
women. Bone 21, 379–383
Gennari, L., Becherini, L., Masi, L. et al. (1998) Vitamin
D and estrogen receptor allelic variants in Italian
postmenopausal women : evidence of multiple gene
contribution to bone mineral density. J. Clin. Endocrinol.
Metab. 83, 939–944
Willing, M., Sowers, M., Aron, D. et al. (1998) Bone
mineral density and its change in white women : estrogen
and vitamin D receptor genotypes and their interaction.
J. Bone Miner. Res. 13, 695–705
McKenna, N. J., Rainer, B. L. and O’Malley, B. W. (1999)
Nuclear receptor coregulators : cellular and molecular
biology. Endocr. Rev. 20, 321–344
# 2000 The Biochemical Society and the Medical Research Society
239
240
R. Bland
380 Montano, M. M., Chang, W. and Katzenellenbogen, B. S.
(1998) An estrogen receptor-selective corepressor :
cloning and characterization. Proc. 80th Am. Endocr.
Soc., OR34-2
381 Christ, M., Haseroth, K., Falkenstein, E. and Wehling, M.
(1999) Nongenomic steroid actions : fact or fantasy ?
Vitam. Horm. 57, 325–373
382 Picard, D. (1998) Steroids tickle cells inside and out.
Nature (London) 392, 437–438
383 Fiorelli, G., Gori, F., Frediani, U. et al. (1996) Membrane
binding sites and non-genomic effects of estrogen in
cultured human pre-osteoclastic cells. J. Steroid Biochem.
Mol. Biol. 59, 233–240
384 Nemere, I., Schwartz, Z., Pedrozo, H., Sylvia, V. L.,
Dean, D. D. and Boyan, B. D. (1998) Identification of a
membrane receptor for 1,25-dihydroxyvitamin D which
$
mediates rapid activation of protein kinase C. J. Bone
Miner. Res. 13, 1353–1359
385 Kato, A., Bishop, J. E. and Norman, A. W. (1998)
Evidence for a 1alpha,25-dihydroxyvitamin D
$ isolated
receptor\binding protein in a membrane fraction
from a chick tibial fracture-healing callus. Biochem.
Biophys. Res. Commun. 244, 724–727
386 Pedrozo, H. A., Schwartz, Z., Rimes, S. et al. (1999)
Physiological importance of the 1,25(OH) D membrane
# $ specific
receptor and evidence for a membrane receptor
for 24,25(OH) D . J. Bone Miner. Res. 14, 856–867
# Lindsay,
$
387 Cosman, F. and
R. (1999) Selective estrogen
# 2000 The Biochemical Society and the Medical Research Society
388
389
390
391
392
393
394
receptor modulators : clinical spectrum. Endocr. Rev. 20,
418–434
Kream, B. E., Jose, M., Yamada, S. and DeLuca, H. F.
(1977) A specific high-affinity binding macromolecule for
1,25-dihydroxyvitamin D in fetal bone. Science 197,
$
1086–1088
Manolagas, S. C. and Anderson, D. C. (1979)
Glucocorticoids regulate the concentration of 1,25dihydroxycholecalciferol receptors in bone. Nature
(London) 277, 314–315
Chen, T. L., Cone, C. M., Morey-Holton, E. and
Feldman, D. (1983) 1α,25-Dihydroxyvitamin D
receptors in cultured rat osteoblast-like cells. J. $Biol.
Chem. 258, 4350–4355
Partridge, N. C., Frampton, R. J., Eisman, J. A. et al.
(1980) Receptors for 1,25(OH) -vitamin D enriched in
#
$ cells. FEBS
cloned osteoblast-like rat osteogenic
sarcoma
Lett. 115, 139–142
Chen, T. L. and Feldman, D. (1981) Regulation of 1,25dihydroxyvitamin D receptors in cultured mouse bone
$ 5561–5566
cells. J. Biol. Chem. 256,
Chen, T. L., Hirst, M. A. and Feldman, D. (1979) A
receptor-like binding macromolecule for 1α,25dihydroxycholecalciferol in cultured mouse bone cells.
J. Biol. Chem. 254, 7491–7494
Vidal, O., Kindblom, L. G. and Ohlsson, C. (1999)
Expression and localization of estrogen receptor-beta in
murine and human bone. J. Bone Miner. Res. 14, 923–929