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*
Department of Medicine, Division of Nephrology and Hypertension, and
Department of Anatomy & Cell Biology, University of Florida College of
Medicine, Gainesville, Florida
Renal Division, Department of Internal Medicine, Washington University
School of Medicine, St. Louis, Missouri.
Correspondence to Dr. Alex J. Brown, Department of Internal Medicine, Renal Division, 660 South Euclid, Washington University School of Medicine, St. Louis, MO 63110. Phone: 314-362-8232; Fax: 314-362-8237; E-mail: abrown{at}imgate.wustl.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment of secondary hyperparathyroidism involves correction of the
hyperphosphatemia, usually with calcium-based phosphate binders that retard
intestinal absorption of dietary phosphate, and restoration of the
1,25(OH)2D3 levels by replacement therapy. However, as a
result of its potent calcemic actions in the intestine and bone,
1,25(OH)2D3 often produces hypercalcemia in renal
patients, especially in those who are receiving oral calcium. This limitation
of 1,25(OH)2D3 therapy has led to the development of
vitamin D analogs that retain the direct action of
1,25(OH)2D3 to suppress PTH gene expression but with
less calcemic activity. Several analogs have been developed for the treatment
of secondary hyperparathyroidism. These include
19-nor-1,25(OH)2D2 (19-norD2)
(3,4),
22-oxacalcitriol (OCT)
(5,6),
and 1 
(OH)D2
(7). 19-norD2 and
OCT have been shown to exert a selective action on PTH in animal models of
renal failure, e.g., suppression of PTH levels with less
hypercalcemia
(3,5).
The mechanisms by which these analogs exert this selectivity on the parathyroid glands are under investigation. The selective action of OCT on PTH seems to be due to its altered pharmacokinetics (8,9). Its low affinity for the serum vitamin D binding protein (DBP) leads to rapid clearance but greater tissue accessibility. The transient appearance of OCT in target tissues after injection elicits only short-lived effects on intestinal calcium absorption and bone mobilization but a prolonged suppression of PTH gene expression (10).
The mechanism for the selectivity of 19-norD2 is less clear but is not due to altered pharmacokinetics (11). Furthermore, the decreased intestinal calcium absorption and bone mobilization activities measured in vivo are due to an acquired resistance that is observed only after several days of chronic treatment (12). These observations led us to determine whether the low bone mobilizing activity of 19-norD2 observed in vivo could be reproduced in the mouse bone marrow culture system (13). We report that 19-norD2 is equipotent to 1,25(OH)2D3 in differentiating osteoclast precursors to a mature phenotype but has a decreased capacity to induce the mature osteoclasts to resorb bone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse Marrow Cultures
Mouse marrow osteoclasts were generated as described previously
(13,14).
Briefly, 8- to 20-g Swiss-Webster mice were killed by cervical dislocation.
This protocol was approved by the Animal Studies Committee of the Washington
University School of Medicine. Femur and tibia were dissected free from
adherent tissue; marrow was removed by cutting both bone ends, inserting a
syringe with a 25-gauge needle, and flushing the marrow using MEM plus
10% fetal bovine serum (MEM D10). The marrow was washed twice with
MEM D10 and plated at a density of 1 x 106
cells/cm2 in tissue culture plates in MEM D10 containing
various concentrations of 1,25(OH)2D3 or
19-norD2. Cultures were fed every other day by replacing half the
media per plate and adding fresh vitamin D compounds. After 5 d in culture,
osteoclasts appeared. These were detected by staining for tartrate-resistant
acid phosphatase (TRAP) activity using a kit from Sigma. TRAP-positive (TRAP+)
cells were segregated into two groups: multinuclear (2 to 10 nuclei) and giant
(>10 nuclei), as described previously
(15).
In Vitro Bone Resorption Assay
Resorption assays were performed as described previously
(14) using sperm whale teeth
sliced into 100-µm thick specimens with a surface area of 1 cm
(2). The slices were washed by
agitation in 50 ml of sterile PBS, transferred to 24-well plates, and soaked
overnight in MEM D10 before exposure to mouse marrow cultures. Mouse
marrow was cultured for 5 d on tissue culture plates in the presence of
various concentrations of 1,25(OH)2D3 or
19-norD2. The medium was replenished on day 3 with fresh compound
at the indicated concentration. Cells were then scraped free from the tissue
culture plates using a disposable plastic cell scraper (Costar, Cambridge,
MA), washed with MEM D10 three times, and plated onto dentine slices in
24-well plates at a concentration of 1 x 106 cells per well.
The indicated concentrations of 1,25(OH)2D3 and
19-norD2 in MEM D10 were added, and the cells were incubated
on dentine wafers for 5 d with no change of medium. After 5 d, cells and
debris were removed from the dentine slices by washing with 2% sodium dodecyl
sulfate. The slices were dehydrated through an ethanol series, air dried,
sputter coated with gold, and examined using a Hitachi H-400 scanning electron
microscope (Nissei Sangyo America, Ltd., Rolling Meadows, IL) operated at 15
kV. Photos of the bone slices were taken at 100 x with no tilt angle.
Overlays that divided the micrographs into grid spaces of 50 µm were placed
over the photos, and the percentage of grid intersections falling over a pit
was determined. The total area analyzed was 680,000 µm
(2). Thus, the percentage of
grid intersections over pits gave an estimation of the percentage of the total
area of the slice that was resorbed. For quantification, a single pit was
counted as any contiguous area of bone resorption, even if it contained more
than one scalloped area. Area and pit number were determine in three randomly
selected fields from each separate dentine slice. Between three and six
dentine slices were assayed per experimental condition.
Vitamin D Receptor Content
The vitamin D receptor (VDR) content in the bone marrow cell cultures was
determined by an intact cell assay previously described in detail
(16). Briefly, mouse bone
marrow cells were incubated as described above with radioinert
1,25(OH)2D3 or 19-norD2 for 5 d. The cells
were washed three times with PBS containing 1% bovine serum albumin (BSA) to
remove the radioinert vitamin D compounds, and then incubated for 2 h at
37°C in serum-free medium containing 1 nM
1,25(OH)2-[26,27-3H]D3 with or without 250 nM
radioinert 1,25(OH)2D3. Under these conditions, vitamin
D compounds bound to the VDR at the end of the 5-d incubation period were
competed away by the excess of tritiated ligand. At the end of the incubation,
the medium was aspirated and the cells were placed on ice. After the
monolayers were washed once with cold PBS containing 1% BSA and twice with
cold PBS, they were sonicated into TEDKI buffer (10 mM Tris-HCl, pH 7.4, 1.5
mM ethylenediaminetetraacetate, 5 mM dithiothreitol, 300 mM KCl, and 300 µM
phenylmethylsulfonyl fluoride). Aliquots of the sonicate were removed for
protein analysis, and the remainder was mixed the charcoal-coated dextran.
After 15 min on ice, the samples were centrifuged for 15 min at 2000 x
g. Tritium in the supernatant was determined by scintillation
counting. Specific binding was calculated as the difference between total
binding (tritiated 1,25(OH)2D3 only) and nonspecific
binding (tritiated 1,25(OH)2D3 plus radioinert
1,25(OH)2D3). VDR content is expressed as specific
binding (fmol) per milligram of cell protein.
Catabolism of 1,25(OH)2D3 and
19-norD2
The rates of catabolism of 1,25(OH)2D3 and
19-norD2 by mouse bone marrow cultures were determined by plating
the freshly isolated bone marrow cells in culture dishes in MEM D10
medium containing 10 nM
1,25(OH)2-[26,27-3H]D3 or
19-nor-[11-3H]D2 at a specific activity of 0.05
µCi/pmol. Medium containing the tritiated vitamin D compounds was replaced
after 48 h. Samples of the medium (100 µl) were taken on day 3 (immediately
after the medium change), day 4, and day 5 to determine the amount of
tritiated compound remaining. The medium aliquots were mixed with 100 µl of
acetonitrile containing 500 ng of radioinert compound to act as carrier and to
allow assessment of recovery. Two milliliters of acetonitrile:water (1:1) was
added, and the samples were centrifuged at 2000 x g for 10 min.
The supernatants were applied to C18-silica cartridges. The cartridges were
washed with 5 ml of water, and the vitamin D compounds were eluted with 4 ml
of acetonitrile and dried under a stream of nitrogen. The amount of the
tritiated vitamin D compound remaining was determined by resolving the samples
on normal phase HPLC using methylene chloride:isopropanol (97:3) as the
running solvent. Fractions eluting near the 1,25(OH)2D3
or 19-norD2 peaks were collected, dried, and quantified by
scintillation counting. Recovery was assessed by measuring the UV absorbance
of the peak of the carrier vitamin D compound.
Statistical Analyses
Results are expressed as mean ± SEM. Samples were compared by ANOVA
using the program SigmaStat (Jandel, San Rafael, CA). P values <
0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The relative potencies of 19-norD2 and 1,25(OH)2D3 to induce osteoclast maturation were determined by treating mouse bone marrow cultures for 5 d with various concentrations of the two compounds. The concentration-dependence curves for the formation of multinucleated TRAP+ cells (Figure 2A) and TRAP+ giant cells (Figure 2B) were biphasic and peaked at 10 nM with both vitamin D compounds. There was no significant difference in the responses to 1,25(OH)2D3 and 19-norD2 at 1, 10, and 100 nM, indicating that the two compounds had similar potencies in stimulating osteoclastogenesis.
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Effect of 19-norD2 on Osteoclastic Bone Resorption
Having found little difference in the ability of
1,25(OH)2D3 or 19-norD2 to stimulate the
formation of TRAP+ cells, we next tested whether there were differences in the
capacity of the two compounds to stimulate bone resorption. Mouse marrow in
tissue culture plates was stimulated for 5 d with 10 nM 19-norD2.
On day 6, the cells were scraped and loaded onto dentine slices and then
incubated in the absence or presence of 10 nM
1,25(OH)2D3 or 19-norD2. After 5 d, the
dentine slices were stripped of cells and the number of resorption pits, the
size of the pits, and the total area resorbed were determined by scanning
electron microscopy as described in the Materials and Methods section
(Figure 3). In the absence of
either vitamin D compound during the incubation with dentine slices, no pits
were observed despite that cultures placed on the dentine slices initially
contained mature osteoclasts. Conversely, substantial resorption was observed
when either vitamin D compound was present during the resorption phase.
However, the amount of total area resorbed was 63% less in the
19-norD2-treated cultures than in those treated with
1,25(OH)2D3 during the resorption phase. This seemed to
be due to a fewer number of pits formed in the presence of 19-norD2
(Figure 3).
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These findings suggest that 19-norD2 may be unable to activate mature osteoclasts to resorb bone. Therefore, mouse bone marrow cultures were differentiated by treatment with either 19-norD2 or 1,25(OH)2D3 and then treated with 19-norD2 during the resorption phase. The results, shown in Figure 4, indicate that osteoclasts that formed in response to 1,25(OH)2D3 treatment during the differentiation phase were able to resorb bone in response to 19-norD2 added during the resorption phase. The amount of resorption under these conditions was not significantly different from that produced by treatment with 1,25(OH)2D3 during both phases. However, when 19-norD2 was present during both the differentiation and resorption phases, the amount of resorption was significantly less (Figure 4). Furthermore, osteoclasts that formed in response to 19-norD2 treatment during the differentiation phase resorbed bone at a high rate when treated with 1,25(OH)2D3 during the resorption phase. Thus, the reduced bone resorption was observed only when the cultures were treated with 19-norD2 during both the differentiation and resorption phases.
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Concentration-dependence curves for activation of resorption were determined by first differentiating the osteoclast precursor by treatment with various amounts of 19-norD2 or 1,25(OH)2D3 and then incubating the differentiated cultures on dentine slices in the presence of the same concentrations of the same compound for 5 additional days. The highest resorption was obtained at 10 nM of both compounds, but the maximum amount achieved with 19-norD2 was approximately 30% of that achieved with 1,25(OH)2D3 (Figure 5A). The lower resorption by 19-norD2 was reflected in the reduced number of pits (Figure 5B); the area per pit was not different for the two compounds (Figure 5C).
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To test possible mechanisms that may be responsible for the lower bone resorbing activity of 19-norD2, we measured the rates of catabolism of 1,25(OH)2D3 or 19-norD2 in the mouse marrow cultures. Relatively little degradation of either molecule was detected during the first 3 d of culture (data not shown). Fresh medium containing tritiated 19-norD2 or 1,25(OH)2D3 was added on day 3, and the amount of each compound remaining was determined by HPLC analysis as described in the Materials and Methods section. Significant catabolism of both 1,25(OH)2D3 and 19-norD2 occurred between days 3 and 5 (Figure 6). After 5 d, 78% of the 19-norD2 and 63% of the 1,25(OH)2D3 had been degraded. The slightly higher rate of degradation of 19-norD2 may contribute to its lower bone resorbing activity but is unlikely to be the primary factor responsible for the disparate effects of 19-norD2 and 1,25(OH)2D3 on bone resorption.
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Active vitamin D compounds are known to stabilize the VDR in target cells, leading to VDR upregulation and potentiation of the activity of the ligand. Therefore, we determined whether the lower activity of 19-norD2 with time could be due to defective stabilization of the VDR. Bone marrow cultures treated for 5 d with 10 nM 19-norD2 or 1,25(OH)2D3 had the same VDR levels as determined by whole-cell binding assay (Figure 7). Thus, the disparate activities of 19-norD2 and 1,25(OH)2D3 could not be explained by differences in VDR levels.
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To determine which structural differences in 19-norD2 are responsible for the decreased bone resorbing activity in vitro, we also tested the activities of 1,25(OH)2D2 and 19-norD3 in this system. Bone marrow cultures were treated with 10-nM concentrations of the compounds during both the differentiation and activation phases, and the area resorbed was determined. Although total resorption was lower in cultures that were treated with 19-norD3, the reduction was not significant (Figure 8). The data suggest that both the 19-nor modification and the D2 side chain are necessary for the reduced activity of 19-norD2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we showed that 19-norD2 is dramatically less effective than 1,25(OH)2D3 in stimulating bone resorption in an in vitro mouse marrow culture system. In principle, this reduction in bone resorption could be accomplished by limiting the number of mature osteoclasts present or by limiting the bone resorptive activity per osteoclast. One way to limit the bone resorptive activity of osteoclasts is to reduce their survival. However, the number of surviving mature osteoclasts was not different in cultures treated with 19-norD2 or 1,25(OH)2D3 (Figures 1 and 2). We believe, therefore, that 19-norD2 is fully capable of inducing survival factors that are required by the osteoclasts.
It was more difficult to distinguish between potential effects on differentiation versus activation. By one measure of differentiation, the formation of multinucleated TRAP+ cells, we did not detect statistical differences in the number of cells that were differentiated by the 19-norD2 and 1,25(OH)2D3, and we did not detect distinct morphologic differences. However, in resorption studies, we found that the reduced activity of 19-norD2 was evident only when the analog was present during both the differentiation and activation phases. Cultures differentiated with 19-norD2 could be activated by 1,25(OH)2D3 to resorb bone as well as cultures treated with 1,25(OH)2D3 in both phases, suggesting that the limitation in the action of 19-norD2 could be rapidly reversed by 1,25(OH)2D3. However, cultures differentiated with 1,25(OH)2D3 could resorb bone normally when treated with 19-norD2 during the activation phase, suggesting that the reduced activity of 19-norD2 could not be manifested within the 5-d activation phase. At least one explanation for these findings is that the reduced resorption by 19-norD2 requires a prolonged exposure to the analog. This would be consistent with our in vivo observation that calcium mobilization from bone within 24 h of injection is similar for 19-norD2 and 1,25(OH)2D3, whereas after 7 daily injections, there is a clear difference in the calcemic response by the bone (12).
The dose-response curves for 1,25(OH)2D3 and 19-norD2 for TRAP+ cell formation and bone resorption showed maximum stimulation for each compound at 10 nM. This indicated that the diminished action of 19-norD2 on bone resorption probably was not due simply to a shift in the concentration-dependence curve. In fact, the VDR affinity of 19-norD2 is only slightly lower (one third less) than that of 1,25(OH)2D3 (3).
The mechanism(s) responsible for the lower in vitro bone resorbing activity of 19-norD2 is not clear. One possible explanation, a more rapid catabolism of 19-norD2, was investigated. The analog was degraded slightly more rapidly than 1,25(OH)2D3, and although this may contribute to the lower activity of 19-norD2, it is unlikely that this could explain fully the disparity in bone resorption elicited by the two compounds. The activities of vitamin D analogs can also be greatly influenced by their affinities for the serum DBP. This protein can significantly retard cellular uptake of vitamin D compounds that bind with high affinity. This mechanism cannot explain the low in vitro bone resorbing activity of 19-norD2 because its DBP affinity is only slightly lower (one third less) than that of 1,25(OH)2D3.
There is also evidence that some vitamin D analogs bind to the VDR differently than 1,25(OH)2D3, producing a functionally altered conformation in the receptor that can influence its activity and/or the recruitment of coactivators (18,19,20,21). Conformational changes in the VDR upon binding of 19-norD2 has not been examined.
The bone resorbing action of 1,25(OH)2D3 does not
involve a direct stimulation of the osteoclast but is mediated indirectly by a
factor released by the osteoblast. Recent studies have identified this
osteoclast differentiation factor as osteoprotegerin ligand (OPGL)
(22,23,24),
a protein on the surface of the osteoblast that is induced by
1,25(OH)2D3
(25,26).
Interaction of OPGL with a receptor (receptor activator of NF-
B [RANK])
on osteoclast precursors induces maturation. In addition, binding of OPGL to
RANK on mature osteoclasts is involved in
1,25(OH)2D3-mediated activation of bone resorption.
Other factors can also influence osteoclast formation and activity. One of
these, osteoprotegerin, binds to osteoclast differentiation factor and
prevents its interaction with the receptor on the osteoclast
(27,28).
OPG expression is downregulated by 1,25(OH)2D3. It is
the ratio of OPGL/OPG that determines both the rate of maturation of
osteoclast precursors and the degree of activation of osteoclastic bone
resorption.
A potential mechanism to explain our results is that 19-norD2 produces a lower OPGL/OPG ratio. This lower ratio may still produce a full effect on differentiation (at least cell fusion and induction of TRAP expression) but is unable to activate fully the mature osteoclasts. In fact, Lacey et al. (22) found that maturation of osteoclast precursors requires lower levels of OPGL than are needed for activation of resorption. In the present study, when the cultures were switched from 1,25(OH)2D3 (in the differentiation phase) to 19-norD2 (during the activation phase), the OPGL/OPG ratio may have changed so slowly that the rate of bone resorption was not markedly less than with 1,25(OH)2D3 present during the activation phase. Only when the OPGL/OPG ratio was low at the onset of the activation phase, i.e., when 19-norD2 was present during the differentiation phase, and remained low was there a substantial reduction in bone resorption. This model also suggests that when cells were differentiated with 19-norD2 and then activated by 1,25(OH)2D3, the OPGL/OPG ratio increased rapidly enough to activate resorption nearly as effectively as when the cultures were treated with 1,25(OH)2D3 in both phases. This hypothesis, which is based our current understanding of the mechanism by which vitamin D compounds mediate the maturation and activation of osteoclasts, remains to be tested.
In summary, we have demonstrated an impairment of bone resorption in vitro by 19-norD2. Although the mechanism for the disparate effects of the analog and 1,25(OH)2D3 are not known, the mouse bone marrow culture model will facilitate the determination of the molecular basis for the blunted calcemic actions of 19-norD2 on bone. In addition, structure/activity studies in this model may define modifications to vitamin D analogs that reduce their calcemic effect on bone and enhance their selectivity, providing safer, more effective therapeutic agents.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D2 (Paricalcitol) safely and
effectively reduces the levels of intact parathyroid hormone in patients on
hemodialysis. J Am Soc Nephrol9
: 1427-1432,1998[Abstract]
,25-dihydroxyvitamin D3 are associated with enhanced
activation of the vitamin D receptor. J Biol Chem270
: 10551-10558,1995,25-dihydroxyvitamin D3, prostaglandin E2, or
parathyroid hormone in the microenvironment of bone. Biochem
Biophys Res Commun 246:337
-341, 1998[Medline]
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),
1,25(OH)2D3 (
), or 19-norD2 ([UNK]) for
7 d with culture media changes and re-addition of hormone on days 3 and 6. On
day 7, the cultures were fixed and stained for TRAP activity, and the TRAP+
multinucleated (2 to 10 nuclei) and giant (> 10 nuclei) cells were
determined. n = 8 per condition. *P < 0.05
versus control, #P < 0.05 versus
1,25(OH)2D3.
