| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
in Human Bone
Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrookes Hospital (E.O.A., A.H., J.E.C.), Cambridge; Department of Clinical Chemistry, Royal Liverpool University Teaching Hospital (E.O.A.), Liverpool L7 8XP; and MRC Bone Research Group, Nuffield Orthopaedic Centre, University of Oxford, Redcliffe Hospital (V.K., J.T.T.), Oxford, United Kingdom
Address correspondence and requests for reprints to: Dr. Emmanuel Abu, Department of Clinical Chemistry, Royal Liverpool University Teaching Hospital, Duncan Building, Prescot Street, Liverpool L7 8XP, United Kingdom. E-mail: eoa{at}liverpool.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsDiscussionReferences |
|---|
In this study, we have used specific affinity purified polyclonal
antibodies to the functional glucocorticoid receptor 
(GR) to
investigate its expression in both developing and adult human bone
using sections of neonatal rib, calvarial, and vertebral bones, tibial
growth plates from adolescents, and iliac crest biopsies from adults
who were to undergo liver transplantation.
In the tibial growth plates, GR was predominantly expressed in the
hypertrophic chondrocytes within the cartilage. In the primary
spongiosa, the receptor was highly expressed by osteoblasts at sites of
bone modeling. Within the bone marrow, receptors were also detected in
mononuclear cells and in endothelial cells of blood vessels. In the
neonatal rib and vertebrae, GR was widely distributed at sites of
endochondral bone formation in resting, proliferating, mature, and
hypertrophic chondrocytes. They were also highly expressed in
osteoblasts at sites of bone modeling. At sites of intramembranous
ossification in neonatal calvarial bone and rib periosteum, GR was
widely expressed in cells within the fibrous tissue and in osteoblasts
at both the bone-forming surface and at modeling sites. In the iliac
crests from adults, GR was predominantly expressed in osteocytes.
The receptors were not detected in osteoclasts.
Our results show for the first time the presence of the functional
GR in human bone in situ and suggest that the actions
of glucocorticoids on bone may be mediated, in part, directly via the
GR at different stages of life. The absence of receptor expression in
osteoclasts also suggests that the effects of glucocorticoids on bone
resorption may be mediated indirectly.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsDiscussionReferences |
|---|
Glucocorticoids exert profound effects on many skeletal growth factors
and cytokines. They decrease insulin-like growth factor (IGF)-I and
IGF-II receptor transcription; IGF binding protein (IGFBP)-3, -4, and
-5 expression; and increase IGFBP-6 transcription. They also activate
transforming growth factor ß, but shift the binding of transforming
growth factor ß to the nonsignal transducing betaglycan (8, 9, 10, 11, 12, 13).
Although these in vitro effects are well-documented, the
molecular mechanisms mediating these actions are still poorly
understood. The actions of glucocorticoids are mediated, at least in
part, via specific glucocorticoid receptors (GRs). GRs are ligand
dependent DNA-binding nuclear proteins and belong to the superfamily of
steroid/thyroid/retinoic acid receptors (14). The human GR was first
cloned and sequenced by Hollenberg et al. (15). They
demonstrated two isoforms of the receptor, GR and GRß, that
diverge at amino acid 727 and contain additional distinct open reading
frames of 50 and 15 amino acids, respectively, at their carboxy
termini. In contrast to the ß isoform, the isoform is
predominantly expressed in most tissues. It also binds ligand and,
therefore, is functional, whereas the GRß does not bind ligand. The
presence of GRs have been demonstrated in a mouse osteoblastic cell
line (MC3T3-E1) and in primary human osteoblastic cells in
vitro using ligand binding studies (16, 17). In human osteoblastic
cells, the binding sites for the glucocorticoid dexamethasone was
several times greater than for dihydrotestosterone, estradiol, and
1,25-(OH)2 vitamin D3.
However, the presence of GRs has not been demonstrated in human bone
in situ. In this study, we have used specific affinity
purified polyclonal antibodies to the functional GR to investigate
its expression in both developing and adult human bone.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsDiscussionReferences |
|---|
Ethical approval was obtained from the local ethics committee, and informed written consent was also obtained. The neonatal bone samples included calvaria, vertebrae, and ribs and were obtained within 48 h postmortem (n = 4). They were snap frozen and stored at -70 C. The adolescent bone samples consist of tibial growth plates and were obtained from three males (11, 15, and 15 yr of age) and two females (9 and 12 yr of age) during surgery for corrective osteotomy. The adult bone samples were iliac crest biopsies obtained from patients prior to liver transplantation (n = 5, three males and two females). The tibial growth plates and the iliac crest biopsies were processed and embedded in paraffin.
Antibody
The antibody used was raised in rabbits against specific peptide
sequences (750–769) of the carboxy terminus of the functional GR
and does not cross-react with the GRß. It was purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA; catalogue
no. SC-1002) with the control peptide (catalogue no. SC-1002P)
used for immunoneutralization.
Immunocytochemistry
Immunolocalization was performed as described previously (18). Briefly, the frozen neonatal bone sections were fixed in 4% paraformaldehyde, whereas the paraffin-embedded sections were dewaxed in inhibisol. Blocking serum was applied for 30 min and after washing, the sections were incubated in 2.5 µg/mL primary antibody overnight at 4 C. The sections were then washed and incubated with biotinylated goat antirabbit antibody (Vector Laboratories, Inc., Burlingame, CA) at a final concentration of 5 µg/mL in phosphate-buffered saline for 1 h. After washing, the sections were incubated with avidin-biotin complex (Vector Laboratories, Inc.) for 30 min, and the signal was detected using a 3,3'-diaminobenzidine substrate (Vector Laboratories, Inc.). The sections were then washed, air-dried, cleared with inhibisol, and mounted using Depex (BDH, Inc., Toronto, Ontario, Canada). Photographs were taken using ektachrome 64 films.
Negative control experiments in which the primary antibody was neutralized with the control peptides used as the immunogen at 10 times the concentration of the primary antibody (25 µg/mL) were also performed. Control experiments were also performed in which the primary antibody was omitted from the staining procedure.
Cytochemistry
Osteoclasts and osteoblasts in the neonatal frozen sections were identified by tartrate-resistant acid phosphatase and alkaline phosphatase (ALP), respectively, using serial unfixed and undecalcified sections. For the tartrate-resistant acid phosphatase reaction, the sections were incubated in 0.1 M citrate buffer (pH 4.5) containing 1 mM naphthol AS-BI phosphate (Sigma, Poole, UK) and 10 mM sodium tartrate (19). The sections were washed in cold distilled water containing 50 mM sodium fluoride, post-coupled in 0.1 mM acetate buffer (pH 6.2) containing 2.2 mM Fast Garnet GBC (Sigma) at 22 C for 30 sec, and washed in distilled water before mounting in aqueous mountant.
For the ALP reaction, sections were incubated for 2 min at 22 C in a
2% solution (w/v) of sodium barbitone containing magnesium chloride
(0.2 mM), -naphthyl acid phosphate (0.16 M;
Sigma), and Fast Red TR (4.0 mM;
Sigma) at a final pH of 9.0 (20). The sections were washed
in distilled water, counter-stained with 0.01% methyl green, and
mounted in aqueous mountant.
Histological staining
Cells in the sections of growth plates and iliac crest biopsies were identified morphologically using 1% toluidine blue (pH 4.5).
Results
Morphologically, the sections of neonatal rib and vertebrae were
similar, consisting of areas of endochondral ossification with
undifferentiated, proliferating, mature, and hypertrophic chondrocytes
and numerous osteoblasts at the primary spongiosa (Fig. 1
, a and b). In the rib periosteum (Fig. 1c) and calvarial bone (Fig. 1d), areas of intramembranous bone
formation were observed with cells in the fibrous tissue and numerous
osteoblasts at the bone-forming surfaces and at modeling sites. The
tibial growth plates consist of areas of endochondral bone formation
and were similar to the sections of neonatal vertebrae and rib (Fig. 1, e and f). The iliac crest biopsy sections consisted of two cortices and
intervening cancellous bone, with many osteocytes and only a few
osteoblasts. Multinucleated osteoclasts were not observed. An area of
the cortex with osteocytes is shown in Fig. 1g.
|
In the tibial growth plate, GR was highly
expressed in the hypertrophic chondrocytes and in a few mature
chondrocytes. The receptors were not observed in the proliferating or
undifferentiated chondrocytes (Fig. 2a).
GR expression in the hypertrophic chondrocytes was abolished when
the primary antibody was neutralized with the specific peptide
immunogen (Fig. 2b). In the primary spongiosa, GR was highly
expressed in osteoblasts at sites of bone modeling (Fig. 2c) and also
in osteocytes and endothelial cells of blood vessels. The receptors
were predominantly localized to the nuclei of osteoblasts (Fig. 2, d
and e). They were not observed in osteoclasts (Fig. 2, f and g), but
were expressed in mononuclear cells within the bone marrow (Fig. 2h).
|
In contrast to the pattern of expression in the tibial growth
plate, GR was highly and widely distributed throughout the cartilage
at sites of endochondral ossification in the neonatal rib and vertebrae
in undifferentiated, proliferating, mature, and hypertrophic
chondrocytes (Fig. 3, a and b). The
receptors were similarly expressed in osteoblasts at sites of bone
modeling in both bone samples (Fig. 3, c and d). They were also
localized in lining cells at sites of bone modeling in the ribs (Fig. 3e). At sites of intramembranous ossification in both the calvarial
bone and rib periosteum, GR was widely distributed in osteoblasts at
the bone-forming surface and at sites of bone modeling and in cells
within the fibrous tissue (Fig. 3, f and h). Serial sections of the rib
periosteum and calvarial bone showing osteoblasts expressing ALP are
shown in Fig. 3, i and j.
|
In the iliac crest biopsies, GR was predominantly expressed in
osteocytes (Fig. 4) both within the
cortex and in cancellous bone.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsDiscussionReferences |
|---|
receptors in human bone in
situ at different stages of life.
At sites of endochondral ossification in the tibial growth plate, GR
receptors were expressed predominantly in the hypertrophic
chondrocytes, whereas at the same sites in the neonatal rib and
vertebrae they were observed throughout the cartilage in the resting,
proliferating, mature, and hypertrophic chondrocytes. The reason for
this difference in distribution is unlikely to be related to the method
of fixation and masking of the expression of the GR in some
chondrocytes of the tibial growth plates, since in the same sections
the receptors were observed in hypertrophic chondrocytes and were
highly expressed in osteoblasts at sites of new bone formation. The
results suggest that glucocorticoids may be involved in chondrocyte
proliferation, maturation, and differentiation earlier in life, whereas
at puberty they may be involved primarily in chondrocyte
differentiation and hypertrophy. Pharmocological doses of
glucocorticoids cause stunted growth in children (21). In
vitro, glucocorticoids inhibit the basal expression of tissue
inhibitor of metalloproteinase-3 (22), which may result in cartilage
degradation. In contrast, glucocorticoids also suppress the synthesis
of inducible nitric oxide in equine chondrocytes (23). Nitric oxide is
a mediator of articular damage and inhibits cartilage formation (24).
The effects of glucocorticoids on cartilage may also depend on the
stage of chondrocyte differentiation. Thus, dexamethasone inhibits the
production of fibronectin in the prehypertrophic chondrocyte, but not
in the hypertrophic and mineralizing chondrocytes (25). The mechanism
of action of glucocorticoids on cartilage, therefore, requires further
investigation.
At sites of intramembranous ossification in the neonatal calvaria and
rib periosteum, GR was detected in osteoblasts at the bone-forming
surface, as well as at modeling sites. These results suggest that
glucocorticoids may influence bone formation and remodeling at sites of
intramembranous ossification. The effects of glucocorticoids on
appositional bone growth in contrast to longitudinal bone growth are
less well understood. However, several studies have demonstrated
effects of glucocorticoids on rat calvarial osteoblasts. Cortisol
down-regulates osteoblast -1 procollagen, messenger RNA, IGF-I, and
ß1 integrins, but increases interstitial collagenase expression in
rat calvarial osteoblastic cultures (26). More recently, dexamethasone
has been shown to suppress in vivo levels of bone collagen
synthesis in mice (27) and to induce fetal rat calvarial osteoblast
differentiation by bone morphogenetic protein-6 (28). Whether these
in vitro actions reflect the actions of glucocorticoids
in vivo is not certain.
In the adult iliac crest samples, GR was predominantly expressed in
osteocytes both in cortical and cancellous bone. The effect of
glucocorticoids on osteocyte function is poorly understood. In organ
cultures of rat parietal bones, glucocorticoids inhibit ß1 integrin
production, disrupt osteoblast organization, and decrease the number of
osteocytes (29). More recently, Weinstein et al. (30) showed
that glucocorticoids inhibit osteoblastogenesis and promote apoptosis
in osteoblasts and osteocytes in mice. Osteocytes transduce mechanical
stimuli in bone, and glucocorticoids have been shown to reduce bone
loading-bearing capacity in rats (31). Additional studies are required
to characterize the effects and mechanisms of glucocorticoids on
osteocyte function. However, the expression of GRs in osteocytes in
adult human bone suggests that the deleterious effects of
pharmacological doses of glucocorticoids on bone may be mediated by
osteocytes, both in cortical and cancellous bone.
In this study, GRs were not detected in osteoclasts. The mechanism of induction of bone resorption by glucocorticoids is controversial. Early studies using fetal limb bones in culture (32) and studies using isolated osteoclasts (33, 34) suggested that glucocorticoids decrease osteoclast function and inhibit bone resorption. In contrast, in cocultures of mouse bone marrow-derived cells and spleen cells (35) and in bone marrow cultures (36) dexamethasone enhances 1,25 (OH)2D3-induced osteoclast-like cell differentiation and increases the capacity of the cells to promote pit formation on dentine slices. Stimulation of bone resorption has also been observed in organ culture studies following glucocorticoid administration to fetal rat parietal bones and neonatal mouse calvariae (37, 38, 39, 40). However, it is not certain whether this results from a direct effect on osteoclasts or is mediated indirectly by actions on osteoblasts. Recently, Dempster et al. (41) showed that glucocorticoids inhibit bone resorption by isolated rat osteoclasts by enhancing their apoptosis. They, however, noted that the effects of glucocorticoids on rat bones may not necessarily correspond to their actions on the human skeleton. In addition, dexamethasone has been shown to stimulate osteoclast-like cell formation in spleen (suggesting a direct effect) as well as in mouse bone marrow cultures, with further enhancement of this effect by PTH and prostaglandin (42). However, in this study, dexamethasone did not affect the bone-resorbing activity of mature osteoclasts.
Clinical studies suggest that secondary hyperparathyroidism may play a
role in mediating glucocorticoid-induced osteoporosis (4). In our
study, the absence of GRs in multinucleated osteoclasts and their high
expression in osteoblasts at sites of bone modeling and remodeling
would be consistent with an indirect effect of glucocorticoids on
mature osteoclast activity via osteoblasts. The localization of GR
in mononuclear cells in the bone marrow may support a role for
glucocorticoids in osteoclast differentiation (36, 42).
The presence of GR in neonatal vertebrae and in cancellous and
cortical bone of the adult iliac crest supports an action of
glucocorticoids on both cancellous and cortical bone and is consistent
with patterns of bone loss in subjects receiving glucocorticoid therapy
(43, 44). Our results indicate that glucocorticoids have direct effects
on bone during its development and growth and also on the mature
skeleton. Additional studies are required to investigate the mechanism
of action and, in particular, the effects of glucocorticoids on
osteocyte and osteoclast function.
Received September 16, 1999.
Revised September 30, 1999.
Accepted November 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsDiscussionReferences |
|---|
,
25-dihydroxycholecalciferol receptors. J Androl. 15:194–199.This article has been cited by other articles:
 |
|
 |
|
  M. Sbaihi, K. Rousseau, S. Baloche, F. Meunier, M. Fouchereau-Peron, and S. Dufour Cortisol mobilizes mineral stores from vertebral skeleton in the European eel: an ancestral origin for glucocorticoid-induced osteoporosis? J. Endocrinol., May 1, 2009; 201(2): 241 - 252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P Tynjala, P Lahdenne, P Vahasalo, H Kautiainen, and V Honkanen Impact of anti-TNF treatment on growth in severe juvenile idiopathic arthritis Ann Rheum Dis, August 1, 2006; 65(8): 1044 - 1049. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C. J. van der Eerden, M. Karperien, and J. M. Wit Systemic and Local Regulation of the Growth Plate Endocr. Rev., December 1, 2003; 24(6): 782 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Walsh, G. R. Jordan, C. Jefferiss, K. Stewart, and J. N. Beresford High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid-induced osteoporosis Rheumatology, January 1, 2001; 40(1): 74 - 83. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
