Interactive effects of PTH and mechanical stress on nitric oxide and PGE2 production by primary mouse osteoblastic cells
Astrid D. Bakker,*
Manon Joldersma,*
Jenneke Klein-Nulend, and
Elisabeth H. Burger
Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam,
Vrije Universiteit, NL-1081BT Amsterdam, The Netherlands
Submitted 14 November 2002
; accepted in final form 9 May 2003
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ABSTRACT
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Parathyroid hormone (PTH) and mechanical stress both stimulate bone
formation but have opposite effects on bone resorption. PTH increased
loading-induced bone formation in a rat model, suggesting that there is an
interaction of these stimuli, possibly at the cellular level. To investigate
whether PTH can modulate mechanotransduction by bone cells, we examined the
effect of 10-9 M human PTH-(1-34) on fluid flow-induced
prostaglandin E2 (PGE2) and nitric oxide (NO) production
by primary mouse osteoblastic cells in vitro. Mechanical stress applied by
means of a pulsating fluid flow (PFF; 0.6 ± 0.3 Pa at 5 Hz) stimulated
both NO and PGE2 production twofold. In the absence of stress, PTH
also caused a twofold increase in PGE2 production, but NO release
was not affected and remained low. Simultaneous application of PFF and PTH
nullified the stimulating effect of PFF on NO production, whereas
PGE2 production was again stimulated only twofold. Treatment with
PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels. We
speculate that PTH prevents stress-induced NO production via the inhibition of
NOS, which will also inhibit the NO-mediated upregulation of PGE2
by stress, leaving only the NO-independent PGE2 upregulation by
PTH. These results suggest that mechanical loading and PTH interact at the
level of mechanotransduction.
parathyroid hormone; prostaglandin E2; nitric oxide synthase
MECHANICAL LOADING, resulting from daily activity, and
parathyroid hormone (PTH) are both able to modulate bone remodeling. PTH,
being a systemic hormone, stimulates overall bone turnover and increases bone
resorption and formation throughout the whole skeleton
(28). This is most clearly
shown in chemical and histomorphological studies in patients with
hyperparathyroidism, in whom both osteoclastic and osteoblastic activity are
increased as a result of the elevated PTH levels
(6,
34,
44). Mechanical loading, on
the other hand, serves to locally enhance osteoblastic bone formation but
inhibit local osteoclastic bone resorption
(11,
35,
36). PTH and mechanical
loading interact at the tissue level, as suggested by several studies in rats,
in which administration of PTH increased mechanical stress-induced bone
formation (5,
21) and reduced the effect of
disuse (21,
40). It is, however, still
unclear how mechanical stress and PTH interact at the cellular level.
Both PTH and mechanical loading can modulate the production of local
signaling molecules by cells of the osteoblastic lineage. In vitro studies
(12,
16,
32,
47) have shown that mechanical
stress stimulates the production of signaling molecules such as nitric oxide
(NO) and prostaglandin E2 (PGE2)
(2a), which are powerful local
modulators of osteoclast and osteoblast activity. PGE2 regulates
bone formation in several ways, such as by recruitment and stimulation of
cells of the osteoblastic lineage
(8,
43), and PGE2 as
well as NO have been shown to directly inhibit the activity and motility of
mature osteoclasts (23,
25). In animal studies, both
NO and prostaglandins were crucial for the anabolic effect of mechanical
stress (9,
39). PTH is also known to
stimulate PGE2 production by bone cells and bone organ cultures
(15,
17,
22). PTH does not seem to
modulate NO production (30) in
bone cells (33), although a
stimulatory effect of human (h)PTH-(1-34) on NO production by endothelial
cells has been reported
(13).
In the present study, bone cell cultures from adult mouse long bones were
subjected to mechanical stress, hPTH-(1-34), or both. Mechanical stress was
applied by subjecting the cells in monolayer to pulsating fluid flow (PFF).
This approach is based on the assumption that interstitial fluid flow in the
canaliculi of strained bone provides the stimulus for mechanoperception
(42). Mechanoperception is
considered to be primarily the task of osteocytes, which then send signals to
alter the activity of (pre)osteoblasts and/or osteoclasts
(3,
4,
37). In vitro, osteoblasts and
osteocytes show similar responses to strain, but osteocytes seem to respond
stronger (19). Both osteocytes
and osteoblasts express the PTH receptor and respond to PTH in a similar
manner in vitro (7,
29). In this study, we
investigated whether mechanical stress and PTH interact at the cellular level
in cells of the osteoblastic lineage. Because stress-induced PGE2
production is dependent on NO synthesis
(14,
18), both PGE2 and
NO production were studied as parameters of bone cell responsiveness. We
tested the hypothesis that application of PTH will modify the response of
osteoblastic cells to mechanical loading, measured as NO and PGE2
production.
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MATERIALS AND METHODS
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Isolation and culture of primary mouse bone cells. Mouse long bone
cells were obtained from the limbs of adult Swiss albino mice. The long bones
were aseptically harvested, the epiphyses were cut off, and bone marrow was
flushed out using a syringe and needle. The diaphyses were chopped into small
fragments, washed with PBS, and incubated with collagenase II (Sigma, St.
Louis, MO) at 2 mg/ml in Dulbecco's modified Eagle's medium (DMEM, GIBCO,
Paisley, UK) at 37°C in a shaking water bath to remove all adhering cells
from the bone chip surfaces. The bone fragments were washed with medium
containing 10% fetal calf serum (FCS) and transferred to 25-cm2
flasks (Nunc, Roskilde, Denmark). Bone fragments were cultured in DMEM
supplemented with 100 U/ml penicillin (Sigma), 50 µg/ml streptomycin
sulfate (GIBCO), 50 µg/ml gentamicin (GIBCO), 1.25 µg/ml fungi-zone
(GIBCO), 100 µg/ml of ascorbate (Merck, Darmstadt, Germany), and 10% FCS.
Upon reaching confluence, cells were harvested using 0.25% trypsin and 0.1%
EDTA in PBS, plated at 25 x 103 cells/well in six-well
culture dishes (Costar, Cambridge, MA), and cultured in 3 ml of medium as
described above, until the cell layer reached confluence again. Then the cells
were characterized as described below or used for PTH and/or PFF experiments
as follows. Cells were trypsinized from the six-well plates (day 0)
and seeded onto polylysine-coated (50 µg/ml; poly-L-lysine
hydrobromide, mol wt 15-30 x 104; Sigma) glass slides (2.5
x 6.5 cm) that fitted the parallel-plate flow chamber used for PFF.
Cells were plated at 5 x 105 cells/glass slide and were
cultured overnight in petri dishes with 12 ml of culture medium as described
above. The following day (day 1), culture medium was replaced by DMEM
containing 0.2% bovine serum albumin (BSA) and supplemented with antibiotics
and ascorbate as before. Similar medium, but supplemented with PTH or vehicle,
was used during PTH and/or PFF treatment on day 2.
Cell characterization. To test their osteoblastic phenotype,
expression of the bone-specific gene cbfa-1 was studied in the cell cultures
as well as their responsiveness to vitamin D. RNA was isolated from the
bone-derived cell cultures with TRIzol reagent according to the manufacturer's
instructions. cDNA synthesis was performed using 1 µg of total RNA in 50
µl of reaction mix consisting of the following final concentrations:
1x first-strand buffer (GIBCO), 500 µM dNTPs (GIBCO), 10 U of RNase
inhibitor (GIBCO), 8 mM dithiothreithol (GIBCO), 50 U of Superscript RT
(GIBCO), and 2 pmol primer p(dT)15 (Boehringer, Mannheim, Mannheim, Germany).
For the amplification of the cbfa1 product, 4 µl of cDNA were added to the
PCR reaction mixture, consisting of 1x Thermal Ace buffer (Invitrogen,
Carlsbad, CA), 0.2 mM dNTP, 6 mM sense primer, 6 mM antisense primer, and 1 U
of Thermal Ace polymerase (Invitrogen) in a final volume of 50 µl. The
cbfa1 upstream and downstream primer sequences were 5'-ATG CTT CAT TCG
CCT CAC AAA C-3' for the forward primer, and 5'-TTT GAT GCC ATA
GTC CCT CCT T-3' for the reverse primer, respectively. The samples were
preheated for 10 min at 95°C, followed by a three-step PCR procedure,
consisting of 45 s at 95°C, 15 s at 53°C, and 15 s at 74°C, for 45
cycles. The PCR products were subjected to electrophoresis on a 1.5% agarose
gel containing 0.5 µg/ml ethidium bromide. As a positive control, the
clonal osteoblastic mouse cell line MC3T3 was used.
Alternatively, bone cell cultures were incubated for 3 days in the presence
or absence of 10-8 M 1,25-dihydroxyvitamin D3
[1,25(OH)2D3] in medium supplemented with 0.2% BSA.
After a 3-day incubation, cells were harvested for determination of alkaline
phosphatase (ALP) activity and total protein content of the cell layer. ALP
activity was determined in the cell lysate by using p-nitrophenyl
phosphate (Merck) as a substrate at pH 10.3, according to the method as
described by Lowry (20). The
absorbance was read at 410 nm with a Dynatech MR7000 microplate reader
(Dynatech, Billing-hurst, UK). The amount of protein in the cell layer was
measured using a BSA protein assay reagent kit (Pierce, Rockford, IL), and the
absorbance was read at 570 nm. ALP values were expressed per amount of protein
in the cell layer. 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). Cells were stained by
immunofluorescence using a monoclonal vWF antibody, and a rodent endothelial
cell line served as a positive control.
PTH. hPTH (1-34) (Sigma) was dissolved in 0.005% HAc buffer
containing 0.1% BSA to a final concentration of 10-5 M
(stock solution) and stored at -80°C until further use. For determination
of the dose-response relationship as well as the time course of the effect of
PTH treatment, 10-11 to 10-7 M PTH
or vehicle alone was added to bone cell cultures for 24 h. Medium samples were
collected at 0.5, 1.5, 3, 6, 12, and 24 h after addition of PTH and assayed
for PGE2 production as described below.
PFF. PFF, at a 5-Hz pulse frequency, was generated by pumping 12
ml of culture medium in a pulsatile manner through a parallel-plate flow
chamber containing the bone cells, as described previously
(2). Mean fluid shear stress
was 0.6 Pa, with a pulse amplitude of 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, under similar conditions as the experimental cultures, i.e., at
37°C in a humidified atmosphere of 5% CO2 in air. To study the
interaction of mechanical loading and PTH treatment,
10-9 M PTH or vehicle was added to the medium before PFF
treatment or stationary incubation. Medium was collected after 5 and 30 min of
treatment and assayed for PGE2 content. NO release was only
measured after 5 min of treatment.
PGE2 and NO. PGE2 release
in the conditioned medium was measured by an enzyme immunoassay system
(Amersham, Buckinghamshire, UK), using an antibody raised against mouse
PGE2. Absorbance was read at 450 nm. NO was measured as nitrite
(NO2 -) accumulation in the conditioned medium with the
use of Griess reagent, consisting of 1% sulfanylamide, 0.1%
naphthylethylenediamine dihydrochloride, and 2.5 M
H3PO4. Serial dilutions of NaNO2 in
nonconditioned medium were used as standard curve. Absorbance was measured at
540 nm.
Total protein and DNA. Total protein and DNA were isolated from
the bone cells using TRIzol reagent (GIBCO) according to the manufacturer's
instructions. The amount of protein was determined using a bicinchoninic acid
protein assay reagent kit as described above. DNA content was determined by
measuring absorbance at 260 nm using an Ultrospec III spectrophotometer
(Amersham).
NOS activity assay. Cells were incubated for 45 min with culture
medium containing 10-9 M PTH or vehicle, washed with
PBS, and collected in 200 µl of homogenization buffer. NOS activity was
measured using an NOS-Detect Assay Kit (Stratagene), based on the conversion
of L-arginine to L-citrulline by NOS enzyme, following
the manufacturer's instructions. Briefly, radioactive L-arginine
was added to the cells as a substrate for NOS enzyme. After incubation,
reactions were stopped with a buffer containing EDTA, which chelates the
calcium required by NOS. Sample reactions were applied to spin cups containing
equilibrated resin, which binds L-arginine but not the radioactive
L-citrulline, it being ionically neutral at pH 5.5. Radioactivity
was measured in the eluate with a
-scintillation counter.
Statistical analysis. Mean values of data obtained from duplicate
cultures from at least three separate experiments were calculated and analyzed
using the Wilcoxon signed rank test or univariate analysis of variance (post
hoc: Bonferroni). Differences were considered significant if P was
<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 3-5 days of culture and reached confluence
within 13-17 days, when they were passaged. Expression levels of cbfa1 mRNA by
these bone cell cultures were comparable to those of the MC3T3-E1 mouse
osteoblastic cell line (Fig.
1). Treatment of the passaged cells with
10-8 M 1,25(OH)2D3 resulted in a
1.9-fold (range 1.4-2.7) mean increase in ALP activity. Together, these
findings demonstrate the osteoblastic characteristic of the cell population.
Immunostaining for the endothelial cell-specific vWF showed that all rodent
endothelial cells stained positive, whereas none of the primary mouse bone
cell cultures expressed vWF (data not shown). PFF treatment, in the presence
or absence of PTH, did not affect the total amount of protein or DNA,
demonstrating that no cells were lost as a result of the treatment with fluid
flow (data not shown).

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Fig. 1. Cbfa1 mRNA expression by three separate primary mouse bone cell cultures
and the osteoblastic cell line MC3T3. Reaction mixes were subjected to
electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide
staining. PMBC, primary mouse bone cell culture.
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To study the dose dependence of PTH-induced PGE2 production,
cells were incubated for 24 h with a range of PTH concentrations. We found
that 10-8 and 10-9 M PTH
significantly stimulated PGE2 production, but this effect was lost
at lower or higher PTH concentrations (Fig.
2A). Because a maximal 1.6-fold stimulation was found
with 10-9 M PTH (Fig.
2A), this concentration was used for all further
experiments. The time curve of PTH-stimulated PGE2 production
showed that stimulation of PGE2 production occurred primarily
during the first 6 h of the incubation period, with the highest production
rate during the first 30 min (Fig.
2B).

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Fig. 2. Effect of parathyroid hormone (PTH) on prostaglandin E2
(PGE2) production by mouse bone cells. A: dose-response
curve of PGE2 production in reaction to 24-h incubation with PTH.
Values, obtained from 3 separate experiments, are expressed as means ±
SE of PTH treated over control (T/C) ratios. Dashed line, T/C = 1 (no effect).
B: cumulative PGE2 production during 24-h treatment with
10-9 M PTH or vehicle. Values, obtained from 3 separate
experiments, are expressed as means ± SE. *Significant
effect of PTH; #significantly different from 10-8 M PTH,
P < 0.05. Treatment with PTH stimulated PGE2 production
in a dose-dependent and biphasic manner, with maximal stimulation after 6 h of
incubation with 10-9 M PTH.
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The effects of PTH and PFF were first studied separately by culturing cells
either under static conditions in the presence of 10-9 M
PTH or vehicle or by subjecting them to PFF. In the latter case, PTH vehicle
was added to the medium to allow comparison with subsequent combination
treatment. As expected from earlier studies, PFF treatment rapidly increased
NO production from 61.7 to 110.3 nmol/mg protein
(Fig. 3A). PFF
stimulated PGE2 production from 29.9 to 53.3 ng/mg protein
(Fig. 3B). PTH
treatment alone did not affect NO release
(Fig. 3A) but
significantly stimulated PGE2 production
(Fig. 3B) to 80.7
ng/mg protein.

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Fig. 3. Effect of PTH or pulsating fluid flow (PFF) on nitric oxide (NO) and
PGE2 production by mouse bone cells. A: NO production by
bone cells treated for 5 min with PFF or 10-9 M PTH.
B: PGE2 production by bone cells treated for 30 min with
PFF or 10-9 M PTH. Values were derived from 5
(A)or7(B) separate experiments and are expressed as means
± SE. Stat, stationary incubation without flow; vehicle, PTH vehicle.
*Significant effect of PTH or PFF, P < 0.05. PFF
significantly stimulated both NO and PGE2 production by the cells.
PTH (10-9 M) significantly stimulated PGE2
production but did not affect on NO production.
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Cell cultures were then subjected to a combined treatment of PTH and PFF to
study the interaction between PTH and mechanical stimulation. Surprisingly,
all treatments, i.e., PTH alone, PFF alone, and PTH combined with PFF,
resulted in an approximately twofold increase of PGE2 production,
indicating that the simultaneous treatment with PFF and PTH did not have an
additive or synergistic effect (Fig.
4B). The effect of PTH on PFF-induced NO production was
even more striking, as the 1.9-fold stimulation of NO production by PFF was
completely abolished in the presence of PTH
(Fig. 4A).

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Fig. 4. Effect of combined treatment with PFF and PTH on NO and PGE2
production by mouse bone cells. NO (A) and PGE2
(B) production by bone cells that were either kept under stationary
conditions in the presence or absence of 10-9 M PTH or
subjected to PFF for 30 min in the presence or absence of PTH. Medium was
assayed for NO release after 5 min and for PGE2 production after 5
and 30 min. Values are expressed as means ± SE of treatment over
control (T/C) values from 5 to 7 separate experiments. *Significant
effect of PFF, PTH, or PFF + PTH, P < 0.05. Although both PFF and
PTH significantly stimulated PGE2 production, combined treatment
with PFF and PTH did not result in an additive or synergistic effect on
PGE2 production. PFF-induced NO production was completely abolished
by PTH. Dashed line, T/C = 1 (no effect).
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In cells of the osteoblastic lineage, NO production induced by shear stress
results from the activation of endothelial cell NOS (ecNOS)
(16,
18,
47). Therefore, we tested
whether PTH treatment modulated NOS activity. Treatment of the bone cell
cultures with 10-9 M PTH dramatically reduced NOS
activity to values below the detection limit of the assay
(Fig. 5).

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Fig. 5. Effect of 45-min incubation with 10-9 M PTH on NO
synthase (NOS) activity in mouse bone cells. Values were obtained from
duplicate cultures from 6 mice and are expressed as means ± SE. cpm,
Counts per minute; n.d., not detectable. *Significant effect of
PTH, P < 0.05. Incubation with 10-9 M PTH
strongly inhibited NOS activity.
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DISCUSSION
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The purpose of the present study was to investigate whether PTH can
interfere in the signaling pathways used by osteoblastic cells during
transduction of mechanical signals. To this aim, we studied the effect of
hPTH-(1-34) treatment on fluid flow-induced NO and PGE2 production
by mouse primary bone cell cultures. We hypothesized that application of PTH
would modify the response of osteoblastic cells to mechanical loading,
measured as NO and PGE2 production. In line with earlier studies
(2,
12,
18), we found that fluid shear
stress rapidly stimulated NO and PGE2 production. Treatment with
PTH in the absence of shear stress stimulated PGE2 production but
not NO production, as was also found by others
(15,
17,
22,
30,
33). When PTH was added
shortly (
1 min) before fluid shear stress was applied, it completely
blocked the shear stress-induced NO production. We therefore tested whether
PTH modulates NOS enzyme activity and found that this activity is severely
inhibited by 10-9 M PTH. ecNOS, the isoform of NOS
enzyme that is sensitive to shear stress, is abundantly present in bone cells
(10,
16,
47). The present results
therefore suggest that the stimulating effect of shear stress on NO production
was prevented by an inhibiting effect of PTH on ecNOS, which blocked the
PFF-induced NO production.
To our knowledge, there is no literature describing any actions of PTH on
NOS in osteoblastic cells, but there are indications that PTH does affect NOS
in cells of the vascular wall. PTH is known to relax vascular smooth muscle
and to acutely lower blood pressure in rats when administered in high
concentrations (26), but
whether this action of PTH involves endothelium-derived NO is unresolved.
Evidence for a possible inhibiting effect of PTH on NOS was provided by a more
recent study, in which rats with chronic renal failure were
parathyroidectomized, resulting in lowered blood pressure, increased urinary
excretion of stable NO metabolites, enhanced vascular NOS activity, and ec-NOS
and inducible NOS expression in the thoracic aorta and remnant kidney
(41). Of course, one must be
cautious when interpreting these results, because many variables are affected
by chronic renal failure as well as by parathyroidectomy, and the observed
effect on NOS does not necessarily imply a direct action of PTH on NOS enzyme
activity. Using a highly sensitive porphyrinic microsensor, Kalinowski et al.
(13) found that administration
of hPTH-(1-34) stimulated NO release from a single endothelial cell in
culture. The stimulation of NO production by PTH was through the
PTH/PTH-related protein (PTHrP) receptors and mediated via the
calcium/calmodulin pathway
(13). We found no such
stimulation of NO production by PTH in our primary bone cell cultures when PTH
was administered in the absence of shear stress. This is in accord with
results of others, who observed no effect of PTH on NO release by primary
human osteoblastic cells, bone cells derived from neonatal mouse calvariae, or
by cells from the mouse osteoblast cell line MC3T3
(30,
33). This difference in NO
response to PTH between cultured bone cells and endothelial cells is
remarkable and suggests that NO-related signaling pathways may differ among
endothelial cells and bone cells.
NO is known to activate cyclooxygenase (COX) enzyme activity in
osteoblastic MC3T3-E1 cells
(14), and we showed earlier
(18) that inhibition of NOS
enzyme in mouse primary osteoblastic cells leads to a severe decrease of
stress-induced PGE2 production. This suggests that the effect of
mechanical loading on PGE2 production is dependent on NOS activity,
and inhibition of NOS enzyme by PTH could thus prevent activation of
prostaglandin synthesis by shear stress. The stimulation of PGE2
production by shear stress was therefore probably decreased during combined
treatment with shear stress and PTH. However, PGE2 production could
still have been stimulated by PTH through a NOS-independent pathway. We have
recently found that PFF-induced prostaglandin production is totally dependent
on activity of the inducible form of COX, COX-2
(2a). NO-dependent
PGE2 production by COX-2 might thus be inhibited during combined
treatment with shear stress and PTH, whereas prostaglandin production by COX-1
persists unaffected. Binding of PTH to its receptor activates the cAMP-protein
kinase A and -protein kinase C pathways, as well as calcium-signaling pathways
(1), and all of these pathways
have been shown to be involved in COX activation and/or COX mRNA induction
(27,
31,
32,
38). Thus we can explain how
combined treatment with PFF and PTH lead to PGE2 levels that were
similar to those found after treatment with PTH alone.
Our in vitro results do not explain how PTH and mechanical stress have
synergistic effects on bone formation, as was shown in animal studies
(5,
21); rather, they seem to
relate to the opposite effects of stress and PTH on bone resorption. Several
studies in animals have shown unequivocally that mechanical stress inhibits
bone resorption (11,
35,
36). PTH, on the other hand,
stimulates bone resorption as well as the subsequent formation of bone,
leading to a generally enhanced remodeling of bone, as demonstrated in
patients with hyperparathyroidism
(6,
34,
44). Thus mechanical stress
and PTH act in opposite directions in the regulation of bone resorption. In
this respect, it is important to note that NO is an inhibitor of bone
resorption. Administered to osteoclasts in vitro, NO causes the retraction of
the osteoclasts from their resorbing substrate and inhibition of resorbing
activity (23,
24). In vivo, administration
of NO has been reported to inhibit bone loss in animal studies as well as in
patients (45,
46). We have therefore
proposed that stress-induced NO production by osteoblastic cells serves to
inhibit osteoclastic bone resorption
(4,
37). Inhibition by PTH of
stress-induced NO production could then lead to enhanced resorption, in line
with the clinical observation of enhanced resorption in patients with
hyperparathyroidism. The opposite effects of PTH and mechanical loading on NO
production, as found in the present study, might thus relate to their opposite
effects on bone resorption. This subject needs further study, preferably using
an in vivo approach.
In sum, the present study suggests that PTH can directly interfere in the
transduction of mechanical signals by osteoblastic cells by inhibiting
stress-induced NO production. As the stress-induced rise in NO production
subsequently mediates an increase in PGE2 production, the
inhibitory effect of PTH on NO also leads to lack of an additive effect of PTH
and stress on PGE2 production. We speculate that the opposite
effects of stress and PTH on NO production, as found in this in vitro study,
may relate to their inhibiting vs. stimulating effect on bone resorption in
vivo.
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DISCLOSURES
|
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The Netherlands Organization for Scientific Research (NWO) supported the
work of A. Bakker (NWO Grant 903-41-193). The Netherlands Institute for
Dentistry supported the work of M. Joldersma. The European Community supported
the work of J. Klein-Nulend (Fifth Framework Grant QLK3-1999-00559).
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ACKNOWLEDGMENTS
|
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We thank Theodorus J. M. Bervoets, Marion A. van Duin, and Cornelis M.
Semeins for excellent technical assistance. We thank Marco Helder for
providing the primers that were used for the PCR on cbfa1.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. Klein-Nulend,
ACTA-Vrije Universiteit, Dept. of Oral Cell Biology, Van der Boechorststraat
7, NL-1081BT Amsterdam, The Netherlands (E-mail:
J.Klein_Nulend.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.
* These authors shared equally in first authorship. 
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