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Department of Orthopedic Surgery (M.-B.M., G.G.) and Division of Oral and Maxillofacial Radiology (A.G.), University of Connecticut Health Center, Farmington, Connecticut 06032
Address all correspondence and requests for reprints to: Gloria Gronowicz, Ph.D., Department of Orthopedic Surgery, MC 1110, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: gronowicz{at}nso1.uchc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol. In 7-day-old mice,
administration of varying concentrations of dexamethasone for 72 h
resulted in a dose-dependent increase in the number of apoptotic
osteoblasts as demonstrated by in situ terminal
deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling
staining of calvaria. A maximum of 22 ± 1% apoptotic osteoblasts
on the bone surface was found with 1 mg/kg BW dexamethasone compared
with 2 ± 1% in vehicle-treated mice. Injection of varying
concentrations of 17ß-estradiol (0.5–5 mg/kg BW), but not
17-estradiol, with 1 mg/kg dexamethasone produced a dose-dependent
decrease in the number of apoptotic osteoblasts to 5 ± 1% with 5
mg/kg 17ß-estradiol. Thus, glucocorticoid-induced apoptosis of
osteoblasts may be prevented at least in part by 17ß-estradiol. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogen deficiency also has marked effects on bone metabolism and results in osteoporosis (1, 7, 8, 9). Estrogen withdrawal primarily causes enhanced bone resorption (10, 11). Estrogen depletion after menopause is strongly associated with a reduction in bone mass and is often accompanied by a remodeling imbalance (12). Osteoclastic activity is enhanced with an impaired ability of osteoblasts to refill osteoclastic resorption spaces (9). However, estrogen replacement therapy can prevent bone loss in postmenopausal women (13).
Many of the in vivo effects of glucocorticoids and estrogen
on bone formation have been confirmed by in vitro studies.
In bones cultured for 24 h, glucocorticoids stimulate collagen
synthesis, but at later time points glucocorticoids inhibit collagen,
fibronectin and DNA synthesis (14, 15, 16). In cultured rat osteoblastic
cells, glucocorticoids inhibit cell replication and DNA content (17).
In contrast, estrogen increases DNA content and alkaline phosphatase
activity in osteoblastic cells (18). Estrogens also stimulate messenger
RNA levels for 1(I) procollagen and synthesis of type I collagen
(19), which is associated with an increase in insulin-like growth
factor I and II secretion (20). In vivo data in mice and
rats demonstrate differences in the response of bone to
glucocorticoids. In rats, numerous investigators have found decreased
bone formation parameters and bone mass (21, 22, 23). However, a few
studies have shown decreased bone formation without a decrease in bone
mass mainly due to effects of glucocorticoids on cartilage and bone
resorption (24, 25). In mice, a decrease in bone mass appears to result
from decreased bone formation (26, 27).
Another mechanism by which glucocorticoids and estrogen may affect osteoblast function is through the control of programmed cell death. Glucocorticoids have been shown to decrease the number of osteoblasts and osteocytes by apoptosis (28, 29, 30). Glucocorticoids also induce apoptosis in mouse thymocytes and lymphocytes by a receptor-mediated process (31). Apoptosis is a form of cell death in which the cell actively triggers an intrinsic suicide mechanism that results in cell shrinkage, blebbing of the plasma membrane followed by chromatin condensation, DNA fragmentation, and formation of apoptotic bodies. The apoptotic bodies are rapidly phagocytosed by neighboring cells and macrophages without the release of intracellular contents, and thus, there is no inflammation. In contrast to glucocorticoids, estrogen prevents apoptosis in ovarian granulosa cells (32) and decreases apoptosis of peripheral blood mononuclear cells (33). In bone, estrogen has been shown to induce apoptosis of osteoclasts (34, 35), but estrogen’s effects on osteoblast apoptosis has not been studied.
As glucocorticoids and estrogen appear to have opposite effects on cell survival, and osteoblasts have glucocorticoid and estrogen receptors that can directly modulate osteoblast gene expression (36, 37, 38), we hypothesized that estrogen may be able to prevent glucocorticoid-induced apoptosis in bone. To determine which factors and genes are involved in osteoblast apoptotic pathways, osteoblast cultures were examined for Bcl-2 and Bax, which are related proteins associated with apoptosis (39). Expression of Bcl-2 protein can prevent programmed cell death induced by a variety of stimuli, including growth factor depletion, stress and many chemotherapeutic agents. Expression of Bcl-2 in murine T cell lines and lymphoma cell lines protects these cells from glucocorticoid-induced apoptosis (40, 41). Bax promotes apoptosis, and Bcl-2 protects cells from programmed cell death. Bcl-2 can form homodimers or heterodimers with Bax (Bcl-2-associated X protein). When the level of Bax increases, Bax homodimers predominate, and cells undergo apoptosis (42). When Bcl-2 is in excess, Bcl-2 homodimers predominate, and cells survive. This study demonstrates that glucocorticoids and estrogen alter Bcl-2 and Bax levels in osteoblasts. Estrogen is shown to prevent glucocorticoid-induced apoptosis, both in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(I) procollagen and alkaline
phosphatase messenger RNA and increased collagen synthesis compared
with F1 cells (our unpublished data). The phenotype of these
fractions had been previously characterized (44). Thus, F3 appears to
be a population of osteoblast progenitors and young osteoblasts,
whereas F1 are mainly fibroblasts. At confluence, the cells were
trypsinized and plated at a density of 14,000 cells/cm2 in
phenol red-free F-12 medium with 1% serum and 100 µg/ml kanamycin.
After 24 h of preculture, cells were treated with varying doses of
corticosterone (1–1000 nM), the endogenous glucocorticoid
of the rat; estrogen (0.01–1.0 nM 17ß-estradiol and 0.1
nM 17-estradiol); or 10 µg/ml lipopolysaccharide
(Sigma Chemical Co., St. Louis, MO) in the presence of
0.2% serum. The low concentration of serum is required to maintain
cell attachment to the culture plate. In addition, mouse osteoblasts, F3, were obtained from 6-day-old mouse calvaria by a similar method of sequential digestion with 0.2% collagenase and 0.1% hyaluronidase in F-12 medium and cultured as described above for rat osteoblasts.
Acridine orange/ethidium bromide staining
Osteoblasts were cultured in four-well Nunc plates (Nunclon,
Copenhagen, Denmark) at a density of 40,000 cells/well. The cells were
labeled using the nucleic acid-binding dye mix of 100 µg/ml acridine
orange and 100 µg/ml ethidium bromide (Sigma Chemical Co.) in PBS. The cells were examined by fluorescence light
microscopy. Viable cells had green fluorescent nuclei with organized
structure. The early apoptotic cells had yellow chromatin in nuclei
that were highly condensed or fragmented. Apoptotic cells also
exhibited membrane blebbing. The late apoptotic cells had orange
chromatin with nuclei that were highly condensed and fragmented. The
necrotic cells had bright orange chromatin in round nuclei. Only cells
with yellow, condensed, or fragmented nuclei were counted as apoptotic
cells in a blinded, nonbiased manner. For each sample, at least 500
cells/well and 4 wells/condition were counted, and the percentage of
apoptotic cells was determined: % of apoptotic cells = (total
number of apoptotic cells/total number of cells counted) x
100.
In vivo treatment of mice
Seven-day-old neonatal CD-1 mice (Charles River Laboratories, Inc., Boston, MA) were used for this study. Stock
solutions of 1 mg/ml dexamethasone, 5 mg/ml 17ß-estradiol, and 5
mg/ml 17-estradiol were prepared in ethanol. Dosing solutions were
prepared by diluting the stock solution with normal saline. After
measuring their weight, mice were given daily sc injections of
dexamethasone (0.1, 0.3, or 1.0 mg/kg BW) and/or 17ß-estradiol and/or
17-estradiol (0.5, 3, and 5 mg/kg BW). At 72 h, mice were
weighed and killed. The entire calvarium was removed for histological,
and terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick
end labeling (TUNEL) analysis. Six mice were used in each group, and
the experiment was repeated at least three times.
TUNEL staining of apoptotic cells
To detect apoptosis in osteoblasts, immunochemical staining of
DNA fragments by TUNEL was performed with the Oncor Apoptag Plus Kit
(Oncor, Gaithersburg, MD). Briefly, calvaria were fixed with 10%
neutral-buffered formalin for 24 h and then embedded in paraffin.
The sections were subsequently cleared in xylene and digested with 5
µg/ml proteinase K in 10 mM Tris-0.1 mM EDTA
buffer for 15 min at room temperature. The residues of
digoxigenin-nucleotide were added to the fragmented DNA by terminal
deoxynuceotidyl transferase at 37 C for 1 h. The fragmented DNA
was subsequently labeled with antidigoxigenin antibody conjugated to a
fluorophore in a humidified chamber for 30 min at room temperature. The
tissue sections were counterstained with propidium iodide/Antifade
(Oncor). Apoptotic cells were visualized in a Nikon
Optiphot microscope (Melville, NY).
After staining, static histomorphometric measurements were performed using the Bioquant program (Bioquant-True Color Windows, R & M Biometrics, Inc., Nashville, TN) in a blinded nonbiased manner. One section per animal was selected for the absence of tears or folding. The number of apoptotic cells, the total number of osteoblasts along the bone surface, and the perimeter of the bone (0.7–0.8 mm/section) were determined at x20 magnification. Then, the percentage of apoptotic cells was determined as a percentage of apoptotic cells per total number of osteoblasts. For each treatment group, 3.2 mm bone surface were analyzed. For in vitro studies, cells were fixed in 2% paraformaldehyde for 30 min and then digested in 5 µg/ml proteinase K for 10 min. The cells were stained similarly as the tissue samples. Each in vivo experiment had six animals per group and was repeated three times.
Western blot analysis
Protein was extracted from cell cultures with 10 mM
Tris, 0.1% SDS, and protease inhibitors (1 µg/ml leupeptin, 0.5
µg/ml pepstatin, and 0.7 µg/ml aprotonin) and measured with the
bicinchoninic acid protein assay kit (Pierce Chemical Co.,
Rockford, IL). Seventy micrograms of protein per lane was loaded onto a
10–20% SDS-polyacrylamide gel. Protein was electrophoretically
transferred to Immobilon-P membranes (Millipore Corp.,
Bedford, MA) in 25 mM Tris base, 192 mM
glycine, and 15% methanol (TBS; vol/vol). Membranes were blocked with
1% TBS containing 0.1% Tween (T-TBS) and 5% skim milk overnight at 4
C. After washing in T-TBS, blots were incubated for 1 h with
either a 1:150 dilution of Bcl-2 or Bax rabbit polyclonal antibodies
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in
blocking buffer, followed by 1 h with 1:20,000 dilution of goat
antirabbit IgG conjugated to horseradish peroxidase in blocking buffer
(Pierce Chemical Co.). Positive bands were identified
using a Pierce Chemical Co. chemiluminescent kit and
Fuji Photo Film Co. Ltd. film (Tokyo, Japan). After
detection, the membranes were stripped by incubation in 100
mM 2-mercaptoethanol, 2% SDS, and 62.5 mM
Tris, pH 6.8, for 45 min at 50 C, blocked, and then washed in T-TBS and
reprobed. Western blots were performed three times.
Statistical analysis
Statistical analysis was performed by an one-way ANOVA, followed
by Student-Newman-Keuls test to determine significance between groups.
In the text, significant differences refer to P <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Treatment with 100
nM corticosterone for 72 h produced apoptotic cells
that had yellow chromatin in condensed nuclei and often had membrane
blebbing (Fig. 1B). Necrotic cells had orange nuclei, and there was no
significant increase in the number of necrotic cells with
glucocorticoid treatment. This was also confirmed by trypan blue
exclusion. Simultaneous administration of 100 nM
corticosterone and 0.01 nM 17ß-estradiol diminished the
number of apoptotic osteoblasts (Fig. 1C). Neither the administration
of 17ß-estradiol nor 17-estradiol alone affected the number of
apoptotic cells (data not shown).
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. Apoptotic osteoblasts were rarely seen in control
cultures (Fig. 1D). Treatment with 0.01 nM 17ß-estradiol
prevented apoptosis in osteoblast cultures treated with 100
nM corticosterone (Fig. 1F). No effect on the number of
apoptotic cells was seen in glucocorticoid-treated cultures with 0.01
nM 17-estradiol or 17ß-estradiol (data not shown).
To quantitate the number of apoptotic cells at various time points, 100
nM corticosterone was administered for 24, 48, 72, and
96 h (Fig. 2A). A maximal effect was
found at 72 h. Treatment with varying concentrations of
corticosterone at 72 h demonstrated a dose-dependent effect with
100 nM corticosterone producing 31 ± 2% apoptotic
cells compared with 3.4 ± 0.5% in control cultures (Fig. 2B).
Higher concentrations of corticosterone or longer incubations with
corticosterone caused cell lifting of late apoptotic cells, which made
it difficult to accurately quantitate apoptotic cells. Therefore, the
number of apoptotic cells appeared to decrease on the culture dish at
96 h or with 1000 nM corticosterone (Fig. 2, A and
B). To determine the cell specificity of the glucocorticoid effect,
fibroblastic F1 and osteoblastic F3 cells were treated with 100
nM corticosterone or vehicle for 72 h. In contrast to
F3 cells, the F1 cells did not undergo glucocorticoid-induced apoptosis
(Fig. 2C). We also examined glucocorticoid’s effect on primary mouse
osteoblasts. Mouse osteoblasts, F3, demonstrated a similar
dose-response curve as corticosterone; however, the maximal effect was
26 ± 3% of apoptotic cells with 100 nM
corticosterone at 72 h (Fig. 2D).
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). Treatment with
1 nM 17ß-estradiol and 100 nM corticosterone
inhibited glucocorticoid-induced apoptosis by approximately 57%. No
significant change was seen with 17-estradiol. Estrogen also
decreased the number of mouse apoptotic cells induced by
glucocorticoids to the same extent as rat osteoblasts (data not
shown).
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). Treatment of F3
murine osteoblasts with 10 µg/ml lipopolysaccharide for 72 h
caused apoptosis in 42.4 ± 3.3% of the osteoblasts (Fig. 4).
Simultaneous treatment of cells with the same concentration of
lipopolysaccharide and 0.1 nM 17ß-estradiol reduced
apoptosis to 10.4 ± 1.3%.
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, A–D, with the densitometric scans of
three blots in the middle and the Bcl-2/Bax ratio of the
three blots at the bottom of each graph. Cells were treated
with varying concentrations of corticosterone for 72 h (Fig. 5A).
Corticosterone caused a dose-dependent decrease in the levels of Bcl-2
protein. Corticosterone at 100 and 1000 nM demonstrated
significant reductions of 60% and 82% in Bcl-2 protein levels,
respectively, compared with vehicle-treated cells. Corticosterone at
100 nM caused a significant 16% increase in Bax protein.
Because the Bcl-2/Bax ratio determines whether a cell will undergo
apoptosis (42), densitometric scans of the bands were performed to
determine the mean ratio of Bcl-2 to Bax levels in cells treated with
corticosterone or vehicle in three experiments. The Bcl-2/Bax ratio in
control cells was arbitrarily set at 1. Corticosterone decreased the
Bcl-2/Bax ratio, with a maximal decrease to 0.18 with 1000
nM.
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). At 6 h,
no significant difference in Bcl-2 was found in control vs.
corticosterone-treated cells. However, Bax levels were increased
significantly by 20.0 ± 3.1%. By 12 h, a significant
decrease of 54.4 ± 2.0% was seen for Bcl-2. The graph of the
densitometric scans of the Bcl-2 and Bax levels in Fig. 5 are the means
of three separate experiments and showed that the average decrease in
Bcl-2 was 51% at 24 h. For the three experiments the Bcl-2/Bax
ratio in control cells at 6 h was arbitrarily set at 1.
Corticosterone significantly decreased the Bcl-2/Bax ratio to 0.83 at
6 h and to 0.47 by 12 h, where it remained for 72 h. A
time-dependent decrease in Bcl-2 and Bax levels in control cells was
also seen in Western blots, perhaps due to the presence of only 0.2%
serum in these primary osteoblast cultures.
The effect of estrogen on Bcl-2 and Bax levels were examined in F3
cells at 72 h (Fig. 5C). Corticosterone (100 nM)
significantly decreased Bcl-2 levels by 58%, whereas 17ß-estradiol
alone and 17-estradiol alone had no effect on Bcl-2 levels.
Treatment of osteoblastic cells with 0.1 nM 17ß-estradiol
and 100 nM corticosterone blocked the
corticosterone-induced decrease in Bcl-2 protein levels. The increase
in Bax protein after glucocorticoid treatment was also prevented by
simultaneous treatment with estrogen. The Bcl-2/Bax ratio, which was
reduced to 0.37 in glucocorticoid-treated cells, increased to 1.19 with
administration of 17ß-estradiol. In contrast, 17-estradiol had no
effect on glucocorticoid-induced apoptosis. Thus, 17ß-estradiol
prevented the glucocorticoid-induced decrease in the Bcl-2/Bax
ratio.
Analysis of Western blots for Bcl-2 and Bax levels in F1 cell cultures
also demonstrated that F1 cells did not undergo apoptosis in response
to glucocorticoids or estrogen (Fig. 5D). These results also confirmed
data obtained by histology, as illustrated in Fig. 2C.
As it has been shown that 1 mg/kg BW dexamethasone suppresses bone
collagen synthesis at 24 h in neonatal mice (45), 7-day-old mice
were injected with dexamethasone to study the effect of glucocorticoids
on osteoblast apoptosis. Mice were injected sc with dexamethasone (0.1,
0.3, and 1 mg/kg BW) or vehicle, 17ß-estradiol, and/or
17-estradiol for 72 h. To determine the population of cells in
mouse calvaria that undergoes apoptosis after dexamethasone treatment,
staining of fragmented DNA was performed by the TUNEL technique (Fig. 6). In situ staining of
calvaria demonstrated apoptosis in the mature osteoblasts lining the
mineralized matrix in mice treated with 0.3 mg/kg BW (Fig. 6B) and 1
mg/kg BW (Fig. 6C) dexamethasone for 72 h compared with control
(Fig. 6A). Usually apoptotic osteoblasts were seen as a contiguous
layer along the bone, as shown in Fig. 6C. Apoptotic cells were rarely
seen in the periosteum. In sutures of the neonatal mouse calvaria where
the periosteum is thicker by several cell layers, no increase in
TUNEL-labeled periosteal cells was seen with dexamethasone (data not
shown). In addition, no apoptotic cells were seen among the osteocytes.
With 5 mg/kg BW 17ß-estradiol and 1 mg/kg BW dexamethasone, a
decrease in apoptotic osteoblasts was apparent, and few apoptotic
osteoblasts were seen (Fig. 6D). Neither 17ß-estradiol alone (Fig. 6E) nor 17-estradiol alone (not shown) had any visible effect on the
number of apoptotic cells.
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), quantitation of TUNEL-labeled
osteoblasts demonstrated an approximately 8-fold increase in the number
of apoptotic osteoblasts (21.6 ± 1.5%) along the bone surface of
the calvaria compared with that in vehicle-treated mice (2.2 ±
0.7%). With 0.3 mg/kg BW dexamethasone, a 4- to 5-fold increase in
apoptotic osteoblasts was seen, whereas 0.1 mg/kg BW had a small, but
insignificant, effect at 72 h (data not shown). The average length
of the osteoblast on the bone surface was 8.4 µm, which did not vary
significantly with treatment. Seven-day-old mice were injected with 1
mg/kg BW dexamethasone and varying concentrations (0.5, 3, and 5 mg/kg
BW) of 17ß-estradiol or 17-estradiol (Fig. 7). A dose-dependent
decrease in the number of apoptotic osteoblasts induced by
glucocorticoids was found with 3 mg/kg BW 17ß-estradiol producing a
significant decrease and 5 mg/kg BW 17ß-estradiol reducing the number
(4.8 ± 0.9%) almost to vehicle-treated levels at 72 h. In
contrast, 17-estradiol alone or with dexamethasone had no
significant effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol had no effect. Glucocorticoids reduced Bcl-2 levels and
increased Bax protein levels by 12 h of treatment in murine
osteoblast cultures, resulting in a dramatic decrease in the Bcl-2 to
Bax ratio, which is involved in regulating apoptosis. The reduction in
Bcl-2/Bax could be prevented by treating osteoblasts simultaneously
with varying concentrations of 17ß-estradiol (0.1–1.0
nM). In humans, the concentration of cortisol is 220–600 nmol/liter, and this level is greater with high dose glucocorticoid therapy, although glucocorticoid-binding proteins in the serum lower the effective concentration (46). In 21-day-old rats, the serum concentration of its natural glucocorticoid, corticosterone, is 0.3 µM (14). Therefore, 1–100 nM corticosterone is supraphysiological, but may be pertinent to the in vivo state of patients receiving high dose glucocorticoid therapy. The levels of estradiol in humans is 7–220 pmol/liter, with an ovulatory surge resulting in concentrations of greater than 740 pmol/liter (46). In our studies, the most effective dose of 17ß-estradiol in preventing apoptosis was 0.1–0.01 nM; therefore, this dose is physiologically relevant.
Glucocorticoid-induced apoptosis appears to be specific for osteoblasts
from both mouse and rat cell cultures. Glucocorticoids failed to induce
apoptosis in fibroblastic F1 cells derived from fetal rat calvaria, as
demonstrated by acridine orange/ethidium bromide staining and Western
blot analysis of Bcl-2 and Bax levels. In contrast, F3 cells, which are
known to be osteoblast-like and respond to PTH, underwent apoptosis
(43). In addition, corticosterone with or without 17ß-estradiol had
no significant effect on Bcl-2 or Bax levels in F1 cells. McCarthy
et al. (44) extensively characterized these cell populations
and showed that F3 cells express more type I collagen and alkaline
phosphatase than F1 cells. The targeting of only osteoblasts for
glucocorticoid-induced apoptosis was confirmed in vivo,
where only osteoblasts along the bone demonstrated an increase in the
number of apoptotic cells in response to glucocorticoids.
Glucocorticoids appeared to have no significant effect on periosteal
cells in vivo. The finding of a glucocorticoid-specific
effect on osteoblasts and not fibroblasts is supported by previous work
in our laboratory demonstrating the corticosterone decreases cell
attachment and fibronectin integrin levels in F3, but not F1 cells (47, 48). As the fibronectin integrin, 5ß1, has
been shown by others to be important in cell survival, and its
down-regulation induces apoptosis (49), these results suggest that
integrins may be involved in the apoptotic pathway induced by
glucocorticoids. In contrast to Weinstein et al. (30), we
did not see any apoptotic osteocytes. This may be explained by
differences in the glucocorticoid used (prednisolone vs.
corticosterone) and/or the dose and duration of treatment (27 days
vs. 3 days in our system). In addition, osteocyte apoptosis
was restricted to a small group of cells in the center of the
metaphysis of the femur, according to Weinstein et al. (30).
They were absent from vertebral cortical bone, even though apoptotic
osteoblasts were found. Therefore, our combined data suggest that a
primary target for glucocorticoid-induced apoptosis in the bone cell
lineage is the osteoblast.
Glucocorticoid treatment also failed to induce apoptosis in transformed or immortalized osteoblast-like lines such as Saos-2 cells, ROS 17/2.8 cells, and MC3T3 cells (our unpublished data), which suggests that the glucocorticoid effect is specific for primary, untransformed osteoblasts. Thus, it appears that normal cell function and signaling are necessary for glucocorticoid-induced apoptosis in osteoblasts. Perhaps a factor is altered by transformation and thereby prevents transformed cells from undergoing apoptosis.
Estrogen protects osteoblasts from apoptosis, as it not only prevented
apoptosis induced by glucocorticoids but also that caused by
lipopolysaccharide treatment. Pretreatment of osteoblasts with
17ß-estradiol for 1 h further reduced the number of
glucocorticoid-induced apoptotic cells compared with that in cells
treated with glucocorticoids and estrogen simultaneously in murine
osteoblasts (our unpublished data). Thus, future experiments
will involve pretreatment of osteoblasts with estrogen for varying
times to prevent completely glucocorticoid-induced apoptosis. The
effect of estrogen also appears to be mediated through the classical
steroid receptor for estrogen, as the inactive isomer of estrogen,
17-estradiol, did not affect the number of apoptotic cells produced
by glucocorticoids in vitro or in vivo. As
estrogen also has the ability to promote osteoclast apoptosis (34, 35),
regulation of the life span of bone cells may be one of the mechanisms
by which estrogen affects bone remodeling and bone mass.
Apoptosis was commonly found in neighboring osteoblasts. In bone,
specific groups of contiguous osteoblasts lining the mineralized matrix
exhibited apoptosis with glucocorticoid treatment, whereas most of the
osteoblasts appeared viable. The focal localization of apoptotic cells
has also been found in neighboring cells in the tip of villus
epithelium of normal intestine (50). As osteoblasts are known to
communicate with each other and osteocytes through gap junctions,
factors involved in apoptosis in one osteoblast may be able to
stimulate apoptosis in neighboring osteoblasts and osteocytes. In cell
cultures stained with acridine orange/ethidium bromide or TUNEL,
apoptosis was most apparent in semiconfluent cultures where small
aggregates of cells exhibited DNA fragmentation, as shown in Fig. 1.
However, individual apoptotic cells were also apparent amid viable
cells. These aggregates of apoptotic cells would often detach from
their substrate, especially with the TUNEL technique, which requires
numerous washing steps. Cell adhesion receptors, such as integrins,
have been shown to be involved in fibroblast apoptosis, and loss of
integrin-mediated attachment of the fibronectin integrin can induce
apoptosis (49), which may also be occurring in osteoblast cultures.
In this study, estrogen reversed the glucocorticoid-induced decrease in the Bcl-2/Bax ratio. As Bcl-2 levels remain elevated with estrogen treatment, estrogen may prevent apoptosis induced by other factors besides lipopolysaccharides, such as aging, growth factor depletion, and UV radiation. Interestingly, a time-dependent decrease in Bcl-2 and Bax levels in control cells was seen with Western blots, probably due to the presence of only 0.2% serum in these primary osteoblast cultures, which are serum dependent for survival. Or, Bcl-2 and Bax levels may be induced when cells are trypsinized and replated in culture, and once they attach, spread, and proliferate, their levels decrease. With a low level of serum, F3 cells are viable for at least 2 weeks and then start to lift from the dish. In B cells and other tissues, the Bcl-2 protein appears to protect cells from undergoing apoptosis in response to many stimuli (51). Bcl-2 protects cells from apoptosis by binding to the proapoptotic proteins, such as Bax, Bcl-xs, and Bad; thus, it is the Bcl-2/Bax ratio that plays an important role in determining a cell’s fate (42, 53). When the level of Bcl-2 is high, Bcl-2 homodimers and Bcl-2/Bax heterodimers predominate, and cells survive. When the level of Bcl-2 is low and/or the level of Bax is high, Bax homodimers predominate, and cells undergo apoptosis. Programmed cell death has been shown to be prevented by increasing Bcl-2 levels in numerous cell types by transfection or other molecular techniques (53, 54). Acridine orange/ethidium bromide staining of glucocorticoid-treated osteoblast cultures demonstrated that the decrease in the Bcl-2/Bax ratio preceded DNA fragmentation visualized by 48 h. The Bcl-2 levels were decreased by 54% at 12 h, and Bax levels were up-regulated as early as 6 h, suggesting that the Bcl-2/Bax ratio may play an important role in osteoblast survival. In rats, a dose of 5 mg/kg dexamethasone injection can induce DNA fragmentation in thymocytes as early as 2 h after steroid treatment (56).
Estrogen has been shown to be effective in preventing osteoporosis (9). Long term estrogen treatment has been shown to reduce the incidence of fractures of vertebrae, distal forearm, and hip by 50% (57, 58). Estrogen has been shown to promote apoptosis of murine osteoclasts in vitro and in vivo (35), thus inhibiting bone resorption and reducing bone loss. The differential effect of estrogen on osteoblasts vs. osteoclasts has precedence in other cell types and with other hormones, such as the effect of glucocorticoids on apoptosis of lymphoid cells, which is not only cell type specific but also cell stage specific (56). Our data suggest that another beneficial effect of estrogen may be to prolong the life of the osteoblast on the bone surface.
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Footnotes |
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Received April 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in SLE but not in normal cultures. Clin Immunol Immunopathol 82:258–262[CrossRef][Medline]
5ß1 integrin supports survival of cells on
fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 92:6161–6165This article has been cited by other articles:
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A. Grey, Q. Chen, K. Callon, X. Xu, I. R. Reid, and J. Cornish The Phospholipids Sphingosine-1-Phosphate and Lysophosphatidic Acid Prevent Apoptosis in Osteoblastic Cells via a Signaling Pathway Involving Gi Proteins and Phosphatidylinositol-3 Kinase Endocrinology, December 1, 2002; 143(12): 4755 - 4763. [Abstract] [Full Text] [PDF]
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 B. L. Riggs, S. Khosla, and L. J. Melton III Sex Steroids and the Construction and Conservation of the Adult Skeleton Endocr. Rev., June 1, 2002; 23(3): 279 - 302. [Abstract] [Full Text] [PDF]
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 M. W. Knoferl, D. Jarrar, M. K. Angele, A. Ayala, M. G. Schwacha, K. I. Bland, and I. H. Chaudry 17beta -Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1131 - C1138. [Abstract] [Full Text] [PDF]
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 J. Lemonnier, E. Hay, P. Delannoy, O. Fromigue, A. Lomri, D. Modrowski, and P. J. Marie Increased Osteoblast Apoptosis in Apert Craniosynostosis : Role of Protein Kinase C and Interleukin-1 Am. J. Pathol., May 1, 2001; 158(5): 1833 - 1842. [Abstract] [Full Text] [PDF]
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H. J. Chae, S. W. Chae, J. S. Kang, B. G. Bang, S. B. Cho, R. K. Park, H. S. So, Y. K. Kim, H. M. Kim, and H. R. Kim Dexamethasone Suppresses Tumor Necrosis Factor-{alpha}-Induced Apoptosis in Osteoblasts: Possible Role for Ceramide Endocrinology, August 1, 2000; 141(8): 2904 - 2913. [Abstract] [Full Text] [PDF]
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