1 Department of Oral Cell Biology, Academic Center for Dentistry Amsterdam Vrije Universiteit; and Departments of 2 Endocrinology and 3 Orthopedics, Academic Hospital Vrije Universiteit, 1081 BT Amsterdam, The Netherlands
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ABSTRACT |
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Several studies indicate that
estrogen may enhance the effects of mechanical loading on bone mineral
density in elderly women. This stimulating effect of estrogen could be
due to increased sensitivity of bone cells to mechanical stress in the
presence of estrogen. The present study was performed to determine
whether 17-estradiol (E2) enhances mechanical
stress-induced prostaglandin production and cyclooxygenase (COX)-2 mRNA
expression. We subjected bone cells from seven nonosteoporotic women
between 56 and 75 yr of age for 1 h to pulsating fluid flow (PFF)
in the presence or absence of 10
11 M E2 and
measured prostaglandin production and COX-1 and COX-2 mRNA expression.
One hour of PFF stimulated prostaglandin (PG)E2 production
threefold, PGI2 production twofold, and COX-2, but not
COX-1, mRNA expression 2.9-fold. Addition of E2 further
enhanced PFF-stimulated PGE2 production by 1.9-fold but did
not significantly affect PGI2 production or COX-2 or COX-1
mRNA expression. E2 by itself did not affect any of the
parameters measured. These results suggest that estrogen modulates bone
cell mechanosensitivity via the prostaglandin synthetic pathway
independently of COX mRNA expression.
cyclooxygenase; 17-estradiol; fluid shear stress; human bone
cells
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INTRODUCTION |
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MECHANICAL LOADING OF BONE is an important regulator of bone structure and bone mass. Several animal studies have demonstrated the anabolic effect of additional loading (2, 3) and the bone resorbing effect of unloading of bone (20, 30).
Although the anabolic effect of additional loading is well known, the nature of the mechanical signal is still not completely characterized. Several studies suggest that osteocytes are the professional stress-sensing cells of bone and that flow of interstitial fluid in the lacunocanalicular network, caused by loading-derived bone strain, provides the mechanical signal for osteocyte mechanosensing (15, 16, 29). We showed earlier that bone cells in vitro respond to fluid flow of ~1 Pa, calculated to be of physiological magnitude (31), with the rapid production of prostaglandins and nitric oxide (13, 15, 28).
Several studies have shown that prostaglandins are involved in the
signaling pathway leading to adaptive bone formation (4, 9). Prostaglandins are produced by osteoblastic cells and are abundant in bone (8, 21). Moreover, bone cells respond to mechanical stress with an increase in prostaglandin production, and
osteocytes have been shown to be even more sensitive than osteoblasts
(1, 15, 32). Prostaglandins are generated by phospholipase
A2-mediated release of arachidonic acid from phospholipids in the cell membrane, followed by conversion of arachidonic acid into
prostaglandin (PG)G2 and subsequently into
PGH2. PGH2 is then isomerized to the
biologically active prostanoids, including PGE2,
PGI2, and PGF2. The enzyme prostaglandin G/H
synthase, or cyclooxygenase (COX), is involved in the rate-limiting
conversion of PGG2 into PGH2 (26).
COX mRNA levels, enzyme synthesis, and enzyme activity are upregulated
in various cell types by several factors, including growth factors,
inflammatory mediators, and hormones (7). COX is present
in two isoforms: a constitutive form (COX-1) and an inducible form
(COX-2). Although COX-1 is constitutively expressed in several cell
types, including endothelial and murine calvarial cells, it can be
modulated by serum factors (11, 18). COX-2 is not
constitutively present in most tissues, but COX-2 mRNA expression can
be induced rapidly and transiently among other factors by serum factors
(22, 23). We showed earlier that primary human
bone cells respond to pulsating fluid flow (PFF) with an increase in
COX-2, but not COX-1, mRNA expression (13). Interestingly,
an animal study by Forwood (9) had demonstrated the
crucial role of COX-2 in loading-adaptive osteogenic responses of rat
bone in vivo. Therefore, the in vitro induction of COX-2 by fluid flow
seems to mimic an early event in loading-adaptive bone formation.
Although mechanical stress acts locally, estrogen, as a systemic hormone, may generally enhance the effect of local loading (3, 12, 17) and abolish the effect of unloading (33). It is still unclear at what level estrogen interacts with the signaling pathway leading to functional adaptation. It has been suggested that estrogen influences comparative late processes in the mechanoadaptive response of bone tissue (12). On the other hand, it cannot be excluded that estrogen also modulates the strain-sensing bone cells themselves or their mechanically induced responses (24).
In this study, we investigated whether estrogen modulation of adaptive
bone formation occurs also at the level of cell mechanosensing. Therefore, we studied the effect of 17-estradiol on fluid
flow-stimulated prostaglandin production and COX-1 and COX-2 mRNA
expression by human bone cells in vitro.
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MATERIALS AND METHODS |
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Donors. Transiliac bone biopsies were obtained from seven postmenopausal women (age 56-75 yr) who were not osteoporotic, were without other metabolic bone disease, and had entered the hospital for orthopedic surgery (elective joint replacement). The Ethics Board of the Academic Hospital Vrije Universiteit approved the protocol, and all subjects gave informed consent.
Culture of primary human bone cells. Cell cultures were established as described earlier (28). Briefly, the biopsies were placed in sterile PBS at 4°C and were dissected within 1 h after removal. They were chopped into small fragments, washed extensively and repeatedly with PBS, and incubated with 2 mg/ml of collagenase IA (Sigma, St. Louis, MO) for 2 h at 37°C in a shaking waterbath to remove all soft tissue from the bone chip's surface. After collagenase treatment, the denuded bone fragments were washed once with medium containing 10% fetal bovine serum (FBS; GIBCO, Paisley, UK) to inhibit further collagenase activity and then transferred to 25-cm2 flasks (Nunc, Roskilde, Denmark). They were cultured in DMEM (GIBCO) supplemented with 100 U/ml of penicillin (Sigma), 50 µg/ml of streptomycin sulfate (Sigma), 50 µg/ml of gentamycin (GIBCO), 1.25 µg/ml of fungizone (GIBCO), 100 µg/ml of ascorbate (Merck, Darmstadt, Germany), and 10% FBS. Culture medium was replaced 3 times/wk. When the cell monolayer growing from the bone fragments reached confluency, cells were trypsinized, plated at 25 × 103 cells/well in 6-well culture dishes (Costar, Cambridge, MA), and cultured in medium as described above.
Cell characterization: 1,25-dihydroxyvitamin D3
challenge, expression of von Willebrand factor.
To test their osteoblastic phenotype, bone-derived cell cultures were
incubated for 2 days in the presence or absence of 108 M
1,25-dihydroxyvitamin D3 in medium supplemented with 0.2%
BSA. Osteocalcin was measured in the conditioned medium by
radioimmunoassay (Incstar, Stillwater, MN) by use of an antibody raised
against bovine osteocalcin. The detection limit was 0.2 ng/ml. All
osteocalcin values were corrected for the amount of osteocalcin in
medium with 0.2% BSA (Sigma) and for the total amount of protein
present in the cell layer. Alkaline phosphatase activity was determined in the cell lysate by use of p-nitrophenyl phosphate (Merck)
as a substrate at pH 10.3, according to the method described by Lowry (19). The assay was performed in 96-well microtiter
plates, and the absorbance was read at 410 nm by a Dynatech MR7000
microplate reader (Dynatech, Billinghurst, UK). To test the presence of
endothelial cells in the bone cell cultures, cells were stained for
expression of the endothelial cell-specific factor VIII, von Willebrand
factor (vWF), by immunofluorescence with the use of a monoclonal vWF antibody. Cultures of primary human umbilical cord-derived endothelial cells served as positive control for the human bone cell cultures.
PFF.
Two days before PFF treatment, the culture medium was replaced by DMEM
without phenol red (GIBCO) containing 1011 M
17
-estradiol (E2) or vehicle and supplemented with 0.2%
BSA to avoid serum stimulation of COX mRNA expression
(14). Antibiotics and ascorbate were added as usual.
Similar medium, with or without E2, was used during PFF
experiments. PFF (5 Hz) was generated by pumping 12 ml of culture
medium by means of a roller pump through a parallel-plate flow chamber
containing the bone cells as described previously (15).
The fluid shear stress was 0.6 ± 0.3 Pa, and the estimated peak
stress rate was 8.4 Pa/s. Control cultures were kept under stationary
conditions in a petri dish, containing the same amount of medium as in
the PFF apparatus, at 37°C in a humidified atmosphere of 5%
CO2 in air. Stationary and PFF-treated cultures, either in
the presence or absence of E2, were studied simultaneously.
After 1 h of fluid flow treatment, the conditioned medium was
collected and assayed for PGE2, PGI2, and
PGF2
production. The cells were harvested to isolate
total DNA and RNA, as described in Total RNA, DNA, and
protein.
PGE2. PGE2 release in the conditioned medium was measured by an enzyme immunoassay (EIA) system (Amersham, Buckinghamshire, UK) with the use of an antibody raised against mouse PGE2. The detection limit was 16 pg/ml. Absorbance at 450 nm was determined with the Dynatech MR7000 microplate reader.
PGI2.
Because PGI2 rapidly and spontaneously hydrolyzes to its
stable metabolite 6-keto-PGF1, this metabolite was
measured by an EIA system with the use of an antibody raised against
rabbit 6-keto-PGF1
. The detection limit was 3.0 pg/ml.
Absorbance at 450 nm was determined with the Dynatech MR7000 microplate reader.
PGF2.
PGF2
release in the conditioned medium
was measured by a 3H assay system (Amersham)
using a PGF2
-specific antibody. The detection limit was
30 pg/ml. Radioactivity was measured in a
-scintillation counter (EG
and G Wallac, Turku, Finland).
Total RNA, DNA, and protein. RNA, DNA, and protein were isolated from the bone cells by means of TRIzol reagent (GIBCO) according to the manufacturer's instructions. The amount of protein was determined with a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL). Absorbance at 570 nm was determined with the Dynatech MR7000 microplate reader. The RNA and DNA content was determined by measurement of absorbance at 260 nm with an Ultrospec III spectrophotometer (Amersham).
cDNA synthesis and RT-PCR. cDNA synthesis was performed with 1 µg of total RNA. After incubation of the RNA at 65°C for 2 min, cDNA synthesis was performed with the following final reaction concentrations: 500 µM dNTPs (GIBCO), 10 units RNase inhibitor (GIBCO), 8 mM dithiothreithol (GIBCO), 50 units of Superscript RT (GIBCO) and 2 pmol of primer p(dT)15 (Boehringer, Mannheim, Germany); 1 µl of cDNA was used in semiquantitative PCRs. Semiquantitative (ratiometric) RT-PCR to detect COX-2 mRNA expression was performed with the use of GAPDH as an internal control. Oligonucleotide primers specific for COX-1 and COX-2 were obtained from Oxford Biomedical Research (Oxford, UK). The COX-1 upstream and downstream primer sequences were 5'-CGGGATCCATGAGTCGAAGGAGTCTCTC-3' and 5'-GCTCTAGACTCAATGACAATTTTGATGG-3', respectively; the COX-2 upstream and downstream primer sequences were 5'-CGGGATCCTGCCAGCTCCACCG-3' and 5'-GCTCTAGAACAAACTGAGTGAGTCC-3', respectively; GAPDH primers were obtained from Clontech (Palo Alto, CA), and the upstream and downstream primer sequences of GAPDH were 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3', respectively. COX-1, COX-2, and GAPDH primers yielded products of 756, 724, and 983 bp, respectively. First, the linear phase of amplification was determined. Therefore, threefold dilutions of cDNA were used with the following PCR conditions: 5 min of denaturation at 95°C, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were loaded on a 1.5% agarose gel containing 0.5 µg/ml of ethidium bromide and were subjected to electrophoresis. Optical densities were quantitated by a video densitometer using Molecular Analyst/PC Software version 1.5 (Bio-Rad Laboratories). The log net intensities were plotted against the dilution factors of the cDNA to determine the linear phase of amplification. Dilutions that yielded PCR products lying within the linear range were used for the final PCR reactions, which were performed in duplicate to calculate the ratios between the intensities of COX-1, COX-2, and GAPDH PCR products.
Statistical analysis. Mean values of data obtained from duplicate or triplicate cultures from individual donors were calculated and analyzed using a Wilcoxon's signed-ranks test. Differences were considered significant if P < 0.05.
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RESULTS |
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Cells started to grow out of the collagenase-stripped bone chips
along the bottom of the culture flask after 1-2 wk of culture. The
cell layer reached confluency within 2-8 wk, when they were passaged. Treatment of the passaged cells with 108 M
1,25-dihydroxyvitamin D3 for 2 days resulted in a 3.3-fold (ranging from 1.8- to 15.4-fold) mean increase in osteocalcin production and a 1.8-fold (ranging from 1.1- to 4.7-fold) mean increase
in alkaline phosphatase activity, demonstrating the osteoblastic characteristics of the cell population. Immunostaining for the endothelial cell-specific vWF was performed to preclude the possibility of endothelial cells contaminating the bone cell cultures. Umbilical cord-derived endothelial cells, which served as a positive control for
the immunostaining, were positive for vWF (data not shown). The
bone-derived cell cultures, however, did not express vWF, demonstrating
that endothelial cells did not contaminate them (data not shown).
Application of PFF for 1 h to the bone-derived cells did not result in visible changes in cell shape or alignment of the cells in the direction of the flow (data not shown). PFF treatment, in either the presence or absence of E2, had no effect on the total amount of DNA, demonstrating that no cells were removed by the fluid shear stress (data not shown). Preincubation with E2 for 2 days had no effect on the total amount of DNA, indicating that E2 did not affect proliferation.
One hour of PFF treatment in the absence of E2 increased
PGE2 production in six of seven donors. PGE2
production remained undetectable in one donor (Fig.
1A). PFF treatment increased
PGI2 production in six of seven donors. One donor did not
respond to PFF treatment (Fig. 1B). PGF2
production remained undetectable in five of seven donors. Two donors
responded to PFF with an increased PGF2
production (Fig.
1C). Wilcoxon's signed-ranks tests showed that 1 h of
PFF treatment resulted in a significant increase (3.0-fold,
P < 0.03) in PGE2 production expressed as
pg/µg DNA and a significant increase (2.0-fold, P < 0.03) in PGI2 production expressed as picograms per
microgram of DNA.
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Under stationary conditions, 1011 M E2
treatment slightly increased PGE2 production in four of
seven donors but decreased PGE2 production in one donor.
The production of PGE2 remained undetectable in one donor,
whereas one donor did not respond to E2 treatment (Fig.
2A). E2 treatment
increased PGI2 production in five of seven donors but
decreased PGI2 production in two donors (Fig.
2B). E2 treatment increased PGF2
production in two of seven donors, whereas PGF2
remained
undetectable in five donors (Fig. 2C). Wilcoxon's
signed-ranks tests using all data showed that E2 did not
significantly affect PGE2, PGI2, or
PGF2
production under stationary conditions.
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E2 treatment enhanced PFF-stimulated PGE2
production in five of seven donors (Fig.
3A). E2 slightly
decreased PFF-stimulated PGE2 production in one donor,
whereas PGE2 production remained undetectable in one donor
(Fig. 3A). E2 treatment enhanced PFF-stimulated PGI2 production in four of seven donors but inhibited
PFF-stimulated PGI2 production in three donors (Fig.
3B). E2 treatment increased PFF-stimulated
PGF2 production in two of seven donors (Fig. 3C). PGF2
production remained undetectable in
five donors (Fig. 3C). Wilcoxon's signed-ranks tests showed
that E2 significantly increased PFF-stimulated
PGE2 (1.9-fold, P < 0.05), but not
PGI2 or PGF2
production.
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One hour of PFF treatment resulted in a 2.9-fold mean increase
(P < 0.05) in COX-2, but not COX-1, mRNA expression
(Fig. 4). E2 treatment did
not significantly change basal COX-1 or COX-2 mRNA expression (Fig.
5) or PFF-dependent COX-1 or COX-2
expression (Fig. 6).
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DISCUSSION |
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In a previous study (13), we showed that human bone cells respond to PFF with increased prostaglandin production and upregulation of COX-2, but not COX-1, mRNA expression. In the present study, we investigated whether estrogen enhancement of adaptive bone formation occurs at the level of mechanosensing by bone cells. Therefore, we pretreated human bone cells for 2 days with E2 or vehicle and measured their response to PFF. Pretreatment with E2 enhanced the response to PFF as measured by PGE2 production, but an effect on PFF-dependent COX-1 or COX-2 mRNA expression could not be demonstrated. Pretreatment with E2 by itself had no effect on basal PGE2 production or COX mRNA levels.
As in previous studies with human bone cells (13, 28), basal prostaglandin production varied considerably between donors. Because we could test only a limited number of donors, we used the Wilcoxon's signed-ranks test, which allowed us to test within-subject differences rather than the between-subject variation. The reason for the variability in basal prostaglandin production could not be determined in this study and may be related to different lifestyles, medical histories, or genetic backgrounds.
Jagger et al. (12) suggested that estrogen enhances
components in the osteogenic response to signals generated by bone
cells after mechanical loading, rather then stimulating the stress or strain-sensing system itself. However, the present study suggests that
estrogen does indeed act on the sensitivity of the bone cells for
mechanical loading and enhances early loading-induced cell-to-cell signaling by bone cells. A direct comparison between the study by
Jagger et al. and the present study is difficult, because different model systems were used. The in vivo model in the study by Jagger et
al. is particularly useful to determine relatively late effects on
actual bone formation, whereas our in vitro model in the present study
is best suitable to investigate relatively early effects at a cellular
level. A direct comparison is also difficult because different
parameters for the response to mechanical loading were used, namely
c-fos and IGF-1 mRNA expression in the study by Jagger et
al. and prostaglandin production in the present study. Of these three,
prostaglandin production has been tied most directly to adaptive bone
formation in response to loading (9). In the study by
Jagger et al. c-fos and IGF-1 mRNA upregulation were not
affected by estrogen. The present study shows that E2 at
1011 M further enhances the effect of mechanical loading
on prostaglandin production. Thus the parameters used for measuring
bone cell responsiveness to mechanical loading seem to be crucial for
the conclusions that are drawn from the results. Finally, Jagger et al.
investigated relatively young, predominantly modeling, rat bone.
However, because bone loss under estrogen-deficient conditions is
associated with increased bone remodeling rather than modeling
(27), we used bone cells that were obtained as outgrowth
from predominantly remodeling bone from elderly women. We showed
earlier, using finite element analysis, that the remodeling process
generates local strain fields within the bone tissue and that active
osteoclasts are found on the surface of areas with low strain, whereas
active osteoblasts are found on the surface of areas with high strain (25). We hypothesized that local strain stimulates
osteocytes to produce factors such as prostaglandins and nitric oxide
that stimulate osteoblast activity and inhibit osteoclast recruitment, respectively (9, 34). In the present in vitro study, we
found that E2 enhances strain-induced production of
PGE2, a potent stimulator of bone formation. Therefore, we
hypothesize that, in vivo and under estrogen-deficient conditions,
osteocytes are less sensitive to local strain during remodeling and
produce fewer anabolic products such as PGE2, leading to a
net bone loss.
In the present study, we found a stimulating effect on PFF-stimulated
PGE2 production already with only a (physiologically) low
concentration of 1011 M E2. Whether other
concentrations of E2 affect PFF-induced prostaglandin production is unknown, because the limited number of cells obtained from one donor did not allow us to perform dose-response experiments.
Under stationary conditions, 1011 M E2 did
not significantly affect PGE2 production or COX-1 or COX-2
mRNA expression. Apparently, therefore, E2 by itself did
not modulate COX enzyme activity. The rapid stimulation of
PGE2 release by PFF treatment results most likely from the
activity of COX-1, the constitutive form of COX (14, 32).
Cissel et al. (6) demonstrated that E2 increases bradykinin-stimulated release of arachidonic acid (AA), the
substrate for COX. However, basal release of AA was not affected by
E2 (5). Similarly, in the present study,
E2 may have stimulated PFF-induced, but not the basal
release of AA, resulting in a higher availability of substrate for the
PFF-activated COX-1 enzyme and therefore increased PFF-stimulated
PGE2 production. Future studies have to be performed to
unravel the exact mechanism of the effect of E2 on
PFF-induced prostaglandin production.
Frost (10) has suggested that estrogen deficiency shifts the "set point" for bone mass adaptation to mechanical loads. One of the factors determining the set point is the sensitivity of bone cells to mechanical stimuli. This hypothesis implies that, if bone cells are more mechanosensitive, lower mechanical forces suffice to maintain skeletal bone mass. Under estrogen-deficient conditions, the response of bone cells to mechanical stimulation is reduced, leading to a net negative bone balance. Our in vitro results are in accord with this hypothesis, even if they relate only to the very early steps in the cascade of events leading to bone mass adaptation and maintenance.
In summary, 1011 M E2 enhanced the fluid
shear stress-induced prostaglandin production by cultured bone cells
from elderly women. However, 10
11 M E2 did
not affect basal levels of prostaglandins or COX-1 or COX-2 mRNA
expression and did not affect shear stress-induced COX-2 mRNA
expression. These results suggest that, in vivo, estrogen may modulate
the mechanoresponsiveness of bone tissue by sensitizing the bone cells
to mechanical loading through a pathway that is independent of COX expression.
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ACKNOWLEDGEMENTS |
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We thank Paul Lips (Academic Hospital Vrije Universiteit, Dept. of Endocrinology) for his assistance in obtaining the bone biopsies. We thank the staff of the Endocrine Laboratory (Dr. Corry Popp-Snijders, Head) for performing the osteocalcin assays. We thank Albert Lind (University of Amsterdam, Medical School, Dept. of Physiology) for his excellent technical help in the von Willebrand factor immunostaining.
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FOOTNOTES |
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The Netherlands Institute for Dentistry supported the work of M. Joldersma.
Address for reprint requests and other correspondence: M. Joldersma, ACTA-Vrije Universiteit, Dept. of Oral Cell Biology, Van der Boechorststraat 7, 1081 BT Amsterdam The Netherlands, (E-mail: m.joldersma.ocb.acta{at}med.vu.nl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 August 2000; accepted in final form 31 October 2000.
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