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The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7877
Address all correspondence and requests for reprints to: Dr. L. F. Bonewald, Department of Medicine, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284. E-mail: bonewald{at}uthscsa.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, the effects of 5-lipoxygenase (5-LO)-derived arachidonic acid metabolites on bone metabolism have not been well studied. Studies so far show that 5-LO metabolites (namely 5-HETE and the peptido-leukotrienes LTC4, LTD4 and LTE4), stimulate the formation and activity of osteoclasts in vitro (7). Leukotriene B4 (LTB4) has been shown to stimulate bone resorption both in vitro and in vivo (8, 9). However, few studies have assessed the effects of these metabolites on osteoblastic bone formation. Recent preliminary data suggest that mice lacking the functional gene for 5-LO have increased cortical bone thickness compared with wild-type mice (10, 11), lose less bone due to ovariectomy compared with wild-type animals (12), and have significantly different mechanical properties of bone compared with wild-type animals (13). These observations suggest that increased bone formation may occur in the absence of the 5-LO enzyme and suggest the possibility that 5-LO metabolites might act as negative regulators of bone formation. Therefore, the aim of the present study was to investigate the effects of the 5-LO metabolites, 5-HETE, the leukotriene LTB4, and, as representative of the peptido-leukotrienes, LTD4, on bone formation in vitro.
In this study, we examined the effects of these 5-LO metabolites on mineralized bone-like nodule formation in fetal rat calvarial cell cultures stimulated by dexamethasone and recombinant human bone morphogenetic protein-2 (rhBMP-2) and intact mouse calvarial organ culture stimulated by rhBMP-2. Alkaline phosphatase activity and cell proliferation was also assessed. We show that the capacity of osteoblasts to differentiate and to form bone was inhibited in both the bone nodule formation assay and the calvarial organ culture assay in the presence of 5-LO metabolites.
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Materials and Methods |
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Fetal rat calvarial cell culture
Pregnant (timed) Sprague-Dawley rats and ICR Swiss White mice
were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). All
animals were maintained in accordance with the NIH Guide for the Care
and Use of Laboratory Animals.
The isolation of fetal rat calvarial cells with osteoblast-like
characteristics was essentially performed as described by Bellows
et al. (14) and Harris et al. (15). Briefly,
first passage cells were maintained in 
-modified essential media
(-MEM) (Gibco BRL-Life Technologies, Gaithersburg, MD) supplemented
with 7% FBS (Summit Biotechnology, Ft. Collins, CO) until confluence
at which time cells were trypsinized, resuspended in freeze mix
solution, and stored under liquid nitrogen for future use. For all
experimental procedures, cells were rapidly thawed, washed, and
resuspended in -MEM supplemented with 7% FBS, then plated
150-cm2 tissue culture flasks (Costar Corp., Cambridge, MA)
and allowed to reach confluence (3–4 days). At this time, cells were
trypsinized, collected by centrifugation, and resuspended in -MEM
supplemented with 7% FBS and plated at a density of 104
cells per well in 48-well tissue culture dishes (Costar). Cells were
grown to confluence and experimental procedures initiated with a media
change to -MEM supplemented with 5% FBS, 100 µg/ml ascorbic acid
(Sigma), and 5 mM ß-glycerophosphate (Sigma).
Antibiotics, penicillin (100 U/ml), and streptomycin (100 µg/ml)
(Cellgro, Mediatech, Inc., Herndon, VA) and gentamicin (30 µg/ml)
(Gibco), were included in all media. Dexamethasone (0.01
µM) and rhBMP-2 (100 ng/ml) were added at each media
change (every 4 days), and the leukotrienes and 5-HETE were added, at
the doses indicated, every 2 days. Three replicate wells were used for
each treatment within each experiment and for each assay (ALP, cell
proliferation, and nodule number). Each experiment was performed twice.
The data shown are representative results of a single experiment.
Fetal rat calvarial bone-like nodule assay
Fetal rat calvarial cells were treated as above until the
mineralized nodules were evident. Nodule number and area was assessed
at the end of each experiment. At this time, cultures were fixed in
10% formalin and stained for mineral by Von Kossa’s method as
previously described (16). Nodules were assessed with respect to number
and total nodule area using JAVA automated imaging system (Jandel
Scientific, San Rafael, CA). Nodules were visualized using a
fluorescent light box (Kaiser Corp., Germany) and Macro TV Zoom lens
18–108 mm f2.5 (Olympus Optical Co., Tokyo, Japan) attached to a Model
DXC-151 Sony video camera (Sony Corp., Tokyo, Japan). Video images were
captured using a frame grabber board (Targa+, Truevision, Santa Clara,
CA) with an IBM compatible 486/33 MHz computer.
Cell proliferation, alkaline phosphatase activity and total protein
determination
Cell counts and alkaline phosphatase activity was determined at
the end of each experiment. Cell counts were performed electronically
using a Coulter counter (Coulter Electronics Ltd., Hertfordshire, UK).
Cells were harvested using 0.2% collagenase and trypsin to solubilize
the matrix produced by these cells which can interfere with electronic
cell counting (17). Cells were harvested by centrifugation and
resuspended in PBS. The cells were gently passed through a 21-gauge
needle, thus forming a single cell suspension. An aliquot was taken for
cell counts.
Specific alkaline phosphatase activity and total protein content were determined as previously described (18). Alkaline phosphatase was expressed as nmol p-nitrophenol phosphate per µg protein/min. Standard curves for alkaline phosphatase and protein were constructed using p-nitrophenol and human IgG, respectively.
Bone formation - mouse calvarial organ culture
To determine whether metabolites of the 5-LO pathway could
affect bone formation, a modified version of the neonatal mouse
calvarial assay, as described by Gowen et al. (19) was used.
The calvaria from 4-day-old Swiss White mice were excised and cut in
half along the sagittal suture. Each half of the calvaria was placed on
a stainless steel grid in a 12-well tissue culture dish (Costar). Each
well contained 1 ml of BGJ media (Sigma) supplemented with 0.1% BSA
(Sigma), to which the relevant factors were added. Preliminary studies
were conducted to determine whether bone formation was
histomorphologically evident in these calvaria over a 7-day period.
Unstimulated calvaria (from littermates) showed an increase in bone
formation in this culture system as evidenced by increasing total bone
area. This was due to new bone formation since old bone (at day 0, day
of excision) remained relatively constant over the 7-day period; day 0,
1, 2, 4, 7 had 13.8 ± 0.7, 17.4 ± 2.6, 18.8 ± 2.0,
19.6 ± 2.5 and 22.8 ± 1.5 x
10-3mm2 total bone area, respectively (n
= 4 in each group). These data show that there was an increase of
approximately 50% in total bone area over the 7 days of culture.
Histomorphometric analysis is performed using the Osteomeasure System
(Osteometrics Inc., Atlanta, GA). A representative image generated
using the Osteometrics software program of a calvaria section viewed at
20x magnification is shown in Fig. 1
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The enclosed blank area represents new bone area; the
enclosed shaded area represents old bone area; each black
dot represent a single osteoblast, the total sum of which
represents osteoblasts on both the endosteal and periosteal surfaces of
the calvaria; the bars with arrows represent calvaria width
determinations (10 equally spaced linear measures across the field of
view were averaged to obtain the width). First, the total bone area in
the field of view is outlined followed by outlining the old bone using
the "void" function in the program. These outlines could be
determined by the differential color intensity obtained with
hematoxylin and eosin staining. The sum of the old and new bone areas
equals the total bone area. The total and new bone area (expressed as
mm2), and calvaria thickness (mm) was determined in the
section posterior to the coronal suture within the parietal bone.
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Statistical analysis
All results are expressed as the mean ± SEM.
Data were analyzed using one-way ANOVA (SigmaStat, Jandel Corp.).
P values of < 0.05 were considered significant.
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Results |
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Figure 2 shows representative wells from
a 48-well plate stained with von Kossa to identify mineralized
bone-like nodules in fetal rat calvarial cell cultures treated with
0.01 µM dexamethasone (13 day culture) or rhBMP-2
(100ng/ml) (12-day culture) and cotreated with either LTD4,
LTB4, or 5-HETE. Clearly, dexamethasone and rhBMP-2
treatment stimulated nodule formation in fetal rat calvarial cell
cultures compared with control (untreated) cells. Cotreatment with
LTD4 had no effect compared with the inhibitory effect of
either LTB4 or 5-HETE. This is graphically depicted in
Figs. 3 and 4, which show total nodule
number and total nodule area in cultures treated with dexamethasone
(Fig. 3) or rhBMP-2 (Fig. 4). 5-HETE
inhibited dexamethasone-stimulated nodule formation by 64%, whereas
LTB4 inhibited formation by 24% (Fig. 3A). Nodule area was
similarly affected (Fig. 3B). The inhibitory effect of 5-HETE was more
pronounced in fetal rat calvarial cell cultures treated with rhBMP-2 as
shown in Fig. 4, A and B. 5-HETE showed a dose-dependent-decrease in
rhBMP-2-stimulated nodule formation (Fig. 4A), completely inhibiting
nodule formation at 1 µM. LTB4 was less
potent at the same dose and LTD4 had no effect. Total
nodule area (Fig. 4B) was similarly affected.
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shows the effects of
5-HETE, LTB4, and LTD4 on dexamethasone (13 day
cultures) and rhBMP-2-treated (12-day cultures) fetal rat calvarial
cell cultures. Dexamethasone (0.01 µM) and rhBMP-2 (100
ng/ml) stimulated alkaline phosphatase activity in fetal rat calvarial
cell cultures (Fig. 5, A and B, respectively). 5-HETE and
LTB4 were able to partially inhibit
dexamethasone-stimulated increase in alkaline phosphatase activity in
these cultures at the highest concentration (1 µM),
whereas LTD4 had no effect (Fig. 5A). 5-HETE displayed a
dose-dependent inhibitory effect on alkaline phosphatase activity in
rhBMP-2-treated cultures, and LTB4 at the same doses did
not (Fig. 5B). LTD4, as with dexamethasone, had no effect
on alkaline phosphatase activity in rhBMP-2-treated fetal rat calvarial
cell cultures.
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shows cell proliferation in the
same fetal rat calvarial cell cultures as Fig. 5
. Cells were treated
with dexamethasone (Fig. 6A) or rhBMP-2 (Fig. 6B) and cotreated with
either 5-HETE, LTB4, or LTD4. There was a small
but significant decrease in cell growth in the dexamethasone-treated
cultures that were cotreated with 1 µM 5-HETE compared
with dexamethasone-treated cultures alone. However, this growth rate
was not significantly different compared with control cultures,
indicating that there was no toxic effect at this dose of 5-HETE. There
was no significant difference in cell number in rhBMP-2-treated
cultures compared with either control or cultures that were cotreated
with the 5-LO metabolites (Fig. 6B). Initial time course experiments
showed no significant differences in cell number at days 0, 4, 8, or 10
(not shown). Therefore, as with the other measurements, measurements
were taken for cell counts at harvest for nodule assessment. At
confluence, day 0 (before treatment) cell numbers averaged 27980
± 735 (mean ± SEM) compared with cultures at the end
of a treatment period, 159,338 ± 1131 (Fig. 6).
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shows
representative sections from untreated calvaria; calvaria treated with
5 ng/ml rhBMP-2; calvaria cotreated with rhBMP-2 and 1 µM
LTB4 and calvaria cotreated with rhBMP-2 and 1
µM 5-HETE. Recombinant hBMP-2 stimulated bone formation
compared with control calvaria. This stimulation was clearly inhibited
by cotreatment with LTB4 or 5-HETE. Quantitative
histomorphometric analysis is shown in Table 1. Both total and new bone areas were
significantly increased with rhBMP-2 treatment compared with untreated
control calvaria. New bone formation was reduced to control levels by
cotreatment with LTB4 or 5-HETE. However, total bone was
reduced to below control levels with 5-HETE cotreatment. This could be
attributed to the inhibition of new bone area and the significant
decrease in old bone area (control vs. rhBMP-2+5-HETE,
8.56 ± 0.80 vs. 4.85 ± 1.45 x
10-3mm2, respectively, P <
0.05). This was also reflected in the calvaria thickness. The number of
osteoblasts was not significantly different between each treatment
group. However, the morphology of the osteoblasts was clearly
different. Figure 8 shows a high power
image (125x) of the osteoblasts that surround the same calvaria
depicted in Fig. 7. Osteoblasts that surround the control and
rhBMP-2-treated calvaria are clearly cuboidal rather than flat, as is
the case with the calvaria cotreated with LTB4 or 5-HETE.
The morphology of the osteoblasts may be a reflection of their relative
level of activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous work examining the role of leukotrienes in bone metabolism has mainly focused on their effects on osteoclasts. There is clear evidence from in vitro and in vivo data that the leukotriene LTB4 and the peptido-leukotrienes, LTC4, LTD4, and LTE4, have profound effects on formation and resorptive activity in osteoclasts (7, 8, 9). When LTB4 was injected over the calvaria of mice, there was a significant increase in osteoclast numbers per unit surface area of bone (9). Meghji and co-workers (20) had also found LTB4 to be a more potent activator of bone resorption in the mouse calvarial assay. These studies indicated that 5-LO metabolites stimulate the recruitment, formation and activation of osteoclasts.
The 5-LO metabolites, 5-HETE and LTB4, inhibit differentiation of osteoblasts in both rhBMP-2 and dexamethasone-treated FRC cultures. 5-HETE was more effective in inhibiting bone-like nodule formation compared with LTB4 in both dexamethasone and rhBMP-2 treated cultures. However, the degree of inhibition in dexamethasone-treated cultures was not to the same extent as that seen with the rhBMP-2-treated cultures. Glucocorticoids have many putative and controversial functions in vivo and in vitro (21). Although glucocorticoids stimulate bone-like nodule formation in vitro (14), their use is associated with severe bone loss in vivo (22). Dexamethasone may stimulate differentiation of a different subset of osteoblasts compared with those stimulated by rhBMP-2. The dexamethasone-treated nodules appeared small with a clear demarcation between the mineralized and nonmineralized are compared with rhBMP-2-treated cultures that showed extensive mineralized areas (personal observation).
The cellular morphology of osteoblasts lining the surface of the control and rhBMP-2-treated calvaria were large and cuboidal indicative of active matrix producing cells. This is in stark contrast to the cells surrounding the calvaria of rhBMP-2-treated calvaria that were cotreated with either LTB4 or 5-HETE, which had a flat stromal cell appearance, likely indicative of their inactivity. This further supports the evidence that 5-LO metabolites inhibit the differentiated function of the osteoblasts.
Few studies have directly addressed the effects of 5-LO metabolites on osteoblast function. Previous reports have shown that LTB4 inhibited proliferation of normal osteoblastic rat calvarial cells and the osteoblastic cell lines SaOS-2 and G292 (23). However, in the present study, rhBMP-2 and dexamethasone-stimulated cell proliferation was not significantly affected by the coaddition of LTB4 or LTD4. Only at the highest dose of 5-HETE was there a small but significant difference in cell proliferation in the dexamethasone-treated cultures. LTB4 has also been shown to increase intracellular calcium release in osteoblasts derived from neonatal mice calvaria (24). The effect of mechanical stress combined with the use of leukotriene inhibitors was shown to enhance bone formation and block bone resorption (25). These studies all support the hypothesis that 5-LO metabolites are negative regulators of bone formation.
5-LO metabolites may be responsible for decreased osteoblast function or decreased bone formation in conditions of elevated 5-LO metabolite production such as the acute phase inflammatory response and rheumatoid arthritis. The leukotrienes and peptido-leukotrienes have been implicated in a number of chronic inflammatory conditions such as rheumatoid arthritis, asthma, psoriasis, periodontal disease and inflammatory bowel disease (26). The prolonged stimulation of production of different eicosanoids alludes to their destructive nature in chronic conditions. These compounds are also destructive to bone. An acute phase inflammatory response, induced by an sc injection of magnesium silicate (27) or talc powder (28), has been shown to result in decreased bone formation in rats. Patients diagnosed with rheumatoid arthritis, who had not received steroid hormone therapy, were shown to have bone formation rates less than half that of healthy controls (29). The authors concluded that a negative remodeling balance existed in these patients. As 5-LO metabolites are increased in these conditions, the present data suggest that these metabolites may be partially responsible.
Steroid hormone treatment exacerbates the effects of chronic inflammatory conditions on bone loss. Glucocorticoids are natural steroid hormones that play important roles in development, homeostasis, and immunomodulation. They are also potent antiinflammatory agents and are commonly used in the treatment of chronic inflammatory conditions (30). However, rapid bone loss occurs with steroid hormone treatment (22, 31) along with decreased serum osteocalcin levels (32). Opposing effects of glucocorticoids on 5-LO have been reported. The antiinflammatory effect of glucocorticoids was attributed to the reduction of 5-LO metabolites (31). However, other investigators have reported no effect or a stimulation of leukotriene synthesis in neutrophils, primarily due to increased activity of the 5-lipoxygenase activating protein (33). Riddick and co-workers demonstrated that dexamethasone stimulated release of 5-LO products from both monocytes and an acute monocytic leukemia cell line (34). This increase was not attributed to changes in the availability of arachidonic acid. Conditioning with dexamethasone increased the immunoreactive protein and steady-state levels of messenger RNA encoding for both 5-LO and FLAP. This suggests a mechanism whereby osteoclast/osteoblast activity may be affected secondarily to effects on the immune system by glucocorticosteroids.
Current therapies used for chronic inflammatory diseases include inhibitors of 5LO and LTB4 receptor antagonists (35, 36). Clinical trials show that asthmatic patients treated with 5-LO inhibitors required significantly less steroid treatment (37). The prolonged use of these compounds may result in a decrease in bone loss in these patients, but this has yet to be assessed.
The results of the work presented here clarify the role of metabolites of the 5-LO pathway on bone cells. It is clear from the previous studies in vivo demonstrating increased osteoclast numbers following injection of LTB4 over the calvaria of mice (9) and the in vitro studies described here that these metabolites serve as negative regulators of bone metabolism. These metabolites function by inhibiting bone formation and stimulating osteoclast recruitment and activation. The regulation and production of these metabolites in other cell systems may lend insights into their regulation, production and functions within the bone microenvironment.
Received December 12, 1997.
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