Estrogen enhances mechanical stress-induced prostaglandin production by bone cells from elderly women

Manon Joldersma1, Jenneke Klein-Nulend1, Anna M. Oleksik2, Ide C. Heyligers3, and Elisabeth H. Burger1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 17beta -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; 17beta -estradiol; fluid shear stress; human bone cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PGF2alpha . 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 17beta -estradiol on fluid flow-stimulated prostaglandin production and COX-1 and COX-2 mRNA expression by human bone cells in vitro.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-8 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 10-11 M 17beta -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 PGF2alpha 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-PGF1alpha , this metabolite was measured by an EIA system with the use of an antibody raised against rabbit 6-keto-PGF1alpha . The detection limit was 3.0 pg/ml. Absorbance at 450 nm was determined with the Dynatech MR7000 microplate reader.

PGF2alpha . PGF2alpha release in the conditioned medium was measured by a 3H assay system (Amersham) using a PGF2alpha -specific antibody. The detection limit was 30 pg/ml. Radioactivity was measured in a beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-8 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). PGF2alpha production remained undetectable in five of seven donors. Two donors responded to PFF with an increased PGF2alpha 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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Fig. 1.   Effect of pulsating fluid flow (PFF) on prostaglandin (PG)E2, PGI2, and PGF2alpha production by bone cells from individual women. After 1 h of PFF treatment or stationary incubation (control), medium was collected and assayed for PGE2 (A), PGI2, measured as 6-keto-PGF1alpha , which is the stable metabolite (B), and PGF2alpha (C), and cells were harvested for DNA isolation. Each point represents the mean of duplicate or triplicate cultures from 1 donor. Lines connect data of parallel cultures of the same donor treated with () or without (open circle ) PFF. PG values were normalized for the amount of DNA. Means of pooled data of each group are expressed as horizontal bars. Data of individual donors were used in the Wilcoxon's signed-ranks test. *Significant effect of PFF, P < 0.03. After 1 h of PFF, PGE2, and PGI2 but not PGF2alpha levels were increased compared with stationary control levels.

Under stationary conditions, 10-11 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 PGF2alpha production in two of seven donors, whereas PGF2alpha 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 PGF2alpha production under stationary conditions.


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Fig. 2.   Effect of 17beta -estradiol (E2) on PGE2, PGI2, and PGF2alpha production by bone cells from individual women. After 2 days of preincubation with 10-11 M E2 or vehicle (control), culture medium was replaced by similar fresh medium. After 1 h of incubation, medium was collected and assayed for PGE2 (A), PGI2 (B), and PGF2alpha (C), and cells were harvested for DNA isolation. Each point represents the mean of duplicate or triplicate cultures from 1 donor. Lines connect data of parallel cultures of the same donor treated with (triangle ) or without (open circle ) E2. PG values were normalized for the amount of DNA. Means of pooled data of each group are expressed as horizontal bars. Data of individual donors were used in the Wilcoxon signed-ranks test. E2 did not affect PGE2, PGI2, or PGF2alpha levels.

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 PGF2alpha production in two of seven donors (Fig. 3C). PGF2alpha 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 PGF2alpha production.


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Fig. 3.   Effect of PFF in combination with E2 on PGE2, PGI2, and PGF2alpha production by bone cells from individual women. Before pulsating fluid flow experiments, cells were preincubated for 2 days in culture medium containing 10-11 M E2 or vehicle. After 1 h of PFF treatment in the presence of E2 (PFF+E2) or vehicle (PFF), medium was collected and assayed for PGE2 (A), PGI2 (B), and PGF2alpha (C), and cells were harvested for DNA isolation. Each point represents the mean of duplicate or triplicate cultures from 1 donor. Lines connect data of parallel cultures of the same donor treated with PFF in the presence of E2 (black-triangle) or vehicle (). Prostaglandin values were normalized for the amount of DNA. Means of pooled data of each group are expressed as horizontal bars. Numbers in parentheses are pg/µg DNA. Data of individual donors were used in a Wilcoxon's signed-ranks test. *Significant effect of E2, P < 0.05. E2 increased PFF-stimulated PGE2, but not PGI2 or PFF-dependent PGF2alpha .

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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Fig. 4.   Semiquantitative (ratiometric) RT-PCR analysis of the effect of PFF on COX-1 and COX-2 mRNA expression in bone cells from individual women. After 1 h of PFF treatment or stationary incubation (control), total RNA from duplicate or triplicate cultures from 5 (COX-1) or 6 donors (COX-2) was isolated, cDNA was prepared and used in semiquantitative (ratiometric) RT-PCR. A: representative example of semiquantitative RT-PCR. cDNA samples were obtained from 1 donor. According to titration analysis, ×10 diluted cDNA was used to obtain COX-2 and GAPDH PCR products lying in the linear range of amplification. COX-1 PCR products were derived from undiluted cDNA. Ten µl of PCR product were loaded on the ethidium bromide-stained agarose gel. COX-1, COX-2, and GAPDH primers yielded products of 756, 724, and 983 bp, respectively. COX-1, COX-1 mRNA; COX-2, COX-2 mRNA; GAPDH, GAPDH mRNA; con, control. B: after densitometry, relative levels of COX-1 and COX-2 mRNA were calculated. For each donor, the amount of COX mRNA was normalized against the corresponding level of GAPDH mRNA. Then, control values were set at 1, and experimental values were expressed as treatment over control (T/C) ± SE. Data were used in a Wilcoxon's signed-ranks test. *Significant effect of PFF, P < 0.05. After 1 h of PFF, COX-2, but not COX-1, mRNA expression was increased compared with stationary control levels.



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Fig. 5.   Semiquantitative (ratiometric) RT-PCR analysis of the effect of E2 on COX-1 and COX-2 mRNA expression in bone cells from individual women. After 2 days of preincubation with 10-11 M E2 or vehicle (control), culture medium was replaced by similar fresh medium. After 1 h of incubation, total RNA from duplicate or triplicate cultures from 4 (COX-1) or 5 donors (COX-2) was isolated, cDNA was prepared and used in semiquantitative RT-PCR. A: representative example of semiquantitative RT-PCR. cDNA samples were obtained from 2 donors. According to titration analysis, ×3 diluted cDNA was used to obtain COX-2 PCR products lying in the linear range of amplification. COX-1 PCR products were derived from undiluted cDNA and GAPDH PCR products from ×10 (corresponding to COX-1) or ×30 (corresponding to COX-2) diluted cDNA. Ten µl of PCR product were loaded on the ethidium bromide-stained agarose gel. COX-1, COX-2, and GAPDH primers yielded products of 756, 724, and 983 bp, respectively. E2, E2 treatment. B: relative levels of COX-1 and COX-2 mRNA were calculated as described in Fig. 4. Data were used in a Wilcoxon's signed-ranks test. E2 did not affect COX-1 or COX-2 mRNA expression.



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Fig. 6.   Semiquantitative (ratiometric) RT-PCR analysis of the effect of PFF in combination with E2 on COX-1 and COX-2 mRNA expression in bone cells from individual women. Before PFF experiments, cells were preincubated for 2 days in culture medium containing 10-11 M E2 or vehicle. After 1 h of PFF treatment in the presence of E2 (PFF+E2) or vehicle (PFF), total RNA from duplicate or triplicate cultures from 4 (COX-1) or 5 donors (COX-2) was isolated, cDNA was prepared and used in semiquantitative RT-PCR. A: representative example of semiquantitative RT-PCR. cDNA samples were obtained from 2 donors. According to titration analysis, ×3 diluted cDNA was used to obtain COX-1 and COX-2 PCR products lying in the linear range of amplification. GAPDH PCR products were derived from ×10 diluted cDNA. Ten µl of PCR product were loaded on the ethidium bromide-stained agarose gel. COX-1, COX-2, and GAPDH primers yielded products of 756, 724, and 983 bp, respectively. PFF+E2, combined PFF and E2 treatment. B: relative levels of COX-1 and COX-2 mRNA were calculated as described in Fig. 4. Data were used in a Wilcoxon's signed-ranks test. E2 did not affect PFF-stimulated COX-2 mRNA expression or PFF-dependent COX-1 mRNA expression.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-11 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 10-11 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, 10-11 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, 10-11 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ajubi, NE, Klein-Nulend J, Nijweide PJ, Vrijheid-Lammers T, Alblas MJ, and Burger EH. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes---a cytoskeleton-dependent process. Biochem Biophys Res Commun 225: 62-68, 1996[ISI][Medline].

2.   Chambers, TJ, Evans M, Gardner TN, Turner-Smith A, and Chow JWM Induction of bone formation in rat tail vertebrae by mechanical loading. Bone Miner 20: 167-178, 1993[ISI][Medline].

3.   Cheng, MZ, Zaman G, Rawlinson SCF, Suswillo RFL, and Lanyon LE. Mechanical loading and sex hormone interactions in organ cultures of rat ulna. J Bone Miner Res 11: 502-511, 1996[ISI][Medline].

4.   Chow, JWM, Fox SW, Lean JM, and Chambers TJ. Role of nitric oxide and prostaglandins in mechanically induced bone formation. J Bone Miner Res 13: 1039-1044, 1998[ISI][Medline].

5.   Cissel, DS, Birkle DL, Whipkey DL, Blaha JD, Graeber GM, and Keeting PE. 1,25-dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells. J Cell Biochem 57: 599-609, 1995[ISI][Medline].

6.   Cissel, DS, Murty M, Whipkey DL, Blaha JD, Graeber GM, and Keeting PE. Estrogen pretreatment increases arachidonic acid release by bradykinin stimulated normal human osteoblast-like cells. J Cell Biochem 60: 260-270, 1996[ISI][Medline].

7.   DeWitt, DL. Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochim Biophys Acta 1083: 121-134, 1991[ISI][Medline].

8.   Feyen, JH, van der Wilt G, Moonen P, Di Bon A, and Nijweide PJ. Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs. Prostaglandins 28: 769-781, 1984[Medline].

9.   Forwood, MR. Inducible cyclo-oxygenase (COX-2) mediates the induction of bone formation by mechanical loading in vivo. J Bone Miner Res 11: 1688-1693, 1996[ISI][Medline].

10.   Frost, HM. Vital biomechanics: Proposed general concepts for skeletal adaptations for mechanical usage. Calcif Tissue Int 42: 145-156, 1988[ISI][Medline].

11.   Hla, T, and Maciag T. Cyclooxygenase gene expression is down-regulated by heparin-binding (acidic fibroblast) growth factor-1 in human endothelial cells. J Biol Chem 266: 24059-24063, 1991[Abstract/Free Full Text].

12.   Jagger, CJ, Chow JWM, and Chambers TJ. Estrogen suppresses activation but enhances formation phase of osteogenic response to mechanical stimulation in rat bone. J Clin Invest 98: 2351-2357, 1996[Abstract/Free Full Text].

13.   Joldersma, M, Burger EH, Semeins CM, and Klein-Nulend J. Mechanical stress induces COX-2 mRNA expression in bone cells from elderly women. J Biomech 33: 53-61, 2000[ISI][Medline].

14.   Klein-Nulend, J, Burger EH, Semeins CM, Raisz LG, and Pilbeam CC. Pulsating fluid flow stimulates prostaglandin release and inducible prostaglandin G/H synthase mRNA expression in primary mouse bone cells. J Bone Miner Res 12: 45-51, 1997[Medline].

15.   Klein-Nulend, J, Semeins CM, Ajubi NE, Nijweide PJ, and Burger EH. Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts---correlation with prostaglandin upregulation. Biochem Biophys Res Commun 217: 640-648, 1995[ISI][Medline].

16.   Klein-Nulend, J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA, Nijweide PJ, and Burger EH. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9: 441-445, 1995[Abstract/Free Full Text].

17.   Kohrt, WM, Snead DB, Slatopolsky E, and Birge SJ. Additive effects of weight-bearing exercise and estrogen on bone mineral density in older women. J Bone Miner Res 11: 1303-1311, 1995.

18.   Lin, AH, Bienkowski MJ, and Gorman RR. Regulation of prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by platelet-derived growth factor. J Biol Chem 264: 17379-17383, 1989[Abstract/Free Full Text].

19.   Lowry, OH. Micromethods for the assay of enzyme. II. Specific procedures. Alkaline phosphatase. Methods Enzymol 4: 371, 1955.

20.   Ma, Y, Jee WSS, Yuan Z, Wei W, Chen H, Pun S, Liang H, and Lin C. Parathyroid hormone and mechanical usage have a synergistic effect in rat tibial diaphyseal cortical bone. J Bone Miner Res 14: 439-448, 1999[ISI][Medline].

21.   Nolan, RD, Partridge NC, Godfrey HM, and Martin TJ. Cyclo-oxygenase products of arachidonic acid metabolism in rat osteoblasts in culture. Calcif Tissue Int 35: 294-297, 1983[ISI][Medline].

22.   O'Banion, MK, Sadowski HB, Winn V, and Young DA. A serum- and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein. J Biol Chem 266: 23261-23267, 1991[Abstract/Free Full Text].

23.   Pilbeam, CC, Kawaguchi H, Hakeda Y, Voznesensky O, Alander CB, and Raisz LG. Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells. J Biol Chem 268: 25643-25649, 1993[Abstract/Free Full Text].

24.   Rodan, GA. Mechanical loading, estrogen deficiency, and the coupling of bone formation to bone resorption. J Bone Miner Res 6: 527-530, 1991[ISI][Medline].

25.   Smit, TH, and Burger EH. Is BMU-coupling a strain-regulated phenomenon? A finite element analysis. J Bone Miner Res 15: 301-307, 2000[ISI][Medline].

26.   Smith, EL, Garavito RM, and DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 271: 33157-33160, 1996[Free Full Text].

27.   Steiniche, T, Hasling C, Charles P, Eriksen EF, Mosekilde L, and Melsen F. A randomized study on the effects of estrogen/gestagen or high dose oral calcium on trabecular bone remodeling in postmenopausal osteoporosis. Bone 10: 313-320, 1989[ISI][Medline].

28.   Sterck, JGH, Klein-Nulend J, Lips P, and Burger EH. Response of normal and osteoporotic human bone cells to mechanical stress in vitro. Am J Physiol Endocrinol Metab 274: E1113-E1120, 1998[Abstract/Free Full Text].

29.   Turner, CH, Forwood MR, and Otter MW. Mechanotransduction in bone: do bone cells act as sensors of fluid flow? FASEB J 8: 875-878, 1994[Abstract/Free Full Text].

30.   Turner, RT, Evans GL, Cavolina JM, Halloran B, and Morey-Holton E. Programmed administration of parathyroid hormone increases bone formation and reduces bone loss in hindlimb-unloaded ovariectomized rats. Endocrinology 139: 4086-4091, 1998[Abstract/Free Full Text].

31.   Weinbaum, S, Cowin SC, and Zeng Y. A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech 27: 339-360, 1994[ISI][Medline].

32.   Westbroek, I, Ajubi NE, Alblas MJ, Semeins CM, Klein-Nulend J, Burger EH, and Nijweide PJ. Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow. Biochem Biophys Res Commun 268: 414-419, 2000[ISI][Medline].

33.   Westerlind, KC, Wronski TJ, Ritman EL, Luo ZP, An KN, Bell NH, and Turner RT. Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain. Proc Natl Acad Sci USA 94: 4199-4204, 1997[Abstract/Free Full Text].

34.   Wimalawansa, SJ. Restoration of ovariectomy-induced osteopenia by nitroglycerin. Calcif Tissue Int 66: 56-60, 2000[ISI][Medline].


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