The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, S.-K.
Right arrow Articles by Lorenzo, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S.-K.
Right arrow Articles by Lorenzo, J. A.
The Journal of Immunology, 2002, 169: 2374-2380.
Copyright © 2002 by The American Association of Immunologists

1,25 (OH)2 Vitamin D3-Stimulated Osteoclast Formation in Spleen-Osteoblast Cocultures Is Mediated in Part by Enhanced IL-1{alpha} and Receptor Activator of NF-{kappa}B Ligand Production in Osteoblasts1

Sun-Kyeong Lee2, Judy Kalinowski, Sandra Jastrzebski and Joseph A. Lorenzo

Division of Endocrinology, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the ability of 1,25 (OH)2 vitamin D3 (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose dependently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor activator of NF-{kappa}B ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1{alpha} protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1{alpha}, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-{kappa}B ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1{alpha} protein expression in WT osteoblasts. However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1{alpha} protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1{alpha} production and the ability of Vit D to support osteoclastogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-1 is a proinflammatory cytokine that is a potent stimulator of bone resorption and inhibitor of bone formation (1). A variety of cells in the bone microenvironment, including monocyte/macrophages, osteoblasts, and osteoclasts, can synthesize IL-1 (2). Production of IL-1 has been linked to the disease osteoporosis because inhibitors of IL-1 activity block the bone loss in mice that occurs with ovariectomy (3). In addition, mice, which are insensitive to IL-1 because they lack the bioactive type 1 IL-1R (IL-1R1),3 fail to lose bone mass after ovariectomy (4). In humans, the presence of osteoporotic fractures was linked to a polymorphism in the gene locus for IL-1R antagonist (IL-1Ra) in two studies (5, 6), but not in a third (7).

Two biologically active isoforms of IL-1 are known. IL-1{alpha} and IL-1{beta} have similar biologic activities and structure and both bind the same receptors. A third IL-1 isoform, IL-1Ra, is a competitive inhibitor of IL-1{alpha} and {beta}. It binds the bioactive IL-1R1 without stimulating downstream events (8). Two receptors for IL-1 have been described (9). IL-1R1 binds both IL-1{alpha} and {beta} and appears to be the principal mediator of IL-1 actions. The type 2 IL-1R (IL-1R2) has a short cytoplasmic tail and does not transmit a biologic signal. Instead, it is believed to be a decoy receptor, which binds IL-1{alpha} and {beta} to prevent their binding to IL-1R1. Estrogen can regulate IL-1R1 and IL-1R2 on osteoclasts, and this response may be involved in the effects that estrogen has on bone (10). Binding of IL-1 to cells requires interaction between the IL-1R accessory protein and IL-1R1 or IL-1R2 (11).

Osteoclasts form in vitro through the interactions of hemopoietic cells, which contain osteoclast precursors, and support cells of mesenchymal origin (stromal cells or osteoblasts; Ref. 12). The latter produce factors that are required for the maturation and terminal differentiation of osteoclast precursor cells. It appears that physical interactions between osteoclast precursor cells and mesenchymal support cells are necessary for osteoclasts to form in response to signals from most resorption stimuli (13).

Among the cytokines that are produced by mesenchymal support cells and have been identified as regulating osteoclast development are receptor activator of NF-{kappa}B ligand (RANKL) and osteoprotegerin (OPG; Ref. 12). RANKL is a TNF-like protein, which binds to a receptor on osteoclast precursor cells, named receptor activator of NF-{kappa}B (RANK). OPG is a soluble decoy receptor for RANKL that is released from cells, binds RANKL, and prevents it from interacting with RANK. Stimulators of resorption enhance RANKL production in mesenchymal support cells, and some also inhibit OPG (14, 15). Regulation of RANKL and OPG in bone is believed to be a critical mechanism for the precise management of osteoclastogenesis and bone resorption.

In the current study, we examined the role of IL-1 in the osteoclastogenic response to 1,25 (OH)2 vitamin D3 (Vit D) by examining the effects that Vit D had on cocultures of spleen cells, which are primarily hemopoietic in origin, and primary murine osteoblasts, which derive from mesenchymal cells. In these cultures either, both, or neither the hemopoietic and mesenchymal cells were from wild-type (WT) or IL-1R1-deficient mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

IL-1R1-deficient (knockout; KO) mice were a gift from Dr. J. Peschon (Immunex, Seattle, WA; Refs. 4 and 16). Animals were in a mixed C57BL/6 x 129Sv background. WT controls were also C57BL/6 x 129Sv mice that were derived from the same heterozygous breeding pair, which have been used to generate the homozygous KO mice.

Materials

Recombinant human IL-1{alpha} and IL-1Ra were from R&D Systems (Minneapolis, MN) and ELISA kits for murine IL-1{alpha} was from Endogen (Woburn, MA). Vit D was a gift from Roche Pharmaceuticals (Nutley, NJ). Unless otherwise stated, all other reagents were from Sigma-Aldrich (St. Louis, MO).

Primary osteoblastic cells

Calvaria were removed from 3-day-old neonatal mice, dissected free of loose connective tissue, and rinsed in PBS as previously described (17). Cells were liberated by five sequential 15-min incubations of calvaria with bacterial collagenase (Collagenase P; Boehringer Mannheim, Indianapolis, IN), 0.1% trypsin, and 0.8 mM Na2EDTA in Ca2+, Mg2+ free PBS. Cells were collected by centrifugation after each digestion and washed with DMEM (Life Technologies, Gaithersburg, MD) and 10% heat-inactivated FBS (HIFBS; HyClone, Logan, UT). Cells obtained from digestions three to five were pooled and used as primary osteoblasts. Cells were first cultured to confluence in 100-mm dishes with DMEM and 10% HIFBS in a humidified incubator with 5% CO2, and then plated at 2 x 104 cells per well of a 24-well plate. For coculture experiments, cells were incubated overnight to allow the osteoblasts to attach before spleen cells were added to the wells.

Spleen cells

Spleens from 8- to 10-wk-old male mice were macerated with a sterile needle. Cells were collected by allowing the splenic tissue to settle at unit gravity before removing the supernatant, which contained the liberated cells. Cells were pelleted by centrifugation and washed three times with PBS. The spleen cells (2 x 106 cells/well of a 24-well plate) were then cocultured with osteoblasts for 5–6 days. Osteoclast-like cells (OCL) were identified by their characteristic multinucleation and tartrate-resistant acid phosphatase (TRAP) staining. Osteoblast layer was trypsinized before cells were fixed with 2.5% glutaraldehyde in PBS for 30 min and TRAP stained with a commercial kit (Sigma-Aldrich).

To determine cells that possess calcitonin receptors, cultures were incubated with 125I-labeled salmon calcitonin (1 x 106 dpm/ml; Amersham Pharmacia Biotech, Piscataway, NJ) in medium for 2 h at room temperature, washed two times in PBS, and developed by autoradiography (18). Specificity of the 125I-labeled calcitonin binding was determined by adding excess cold salmon calcitonin (100-fold, 10-7 M; Bachem, Torrence, CA) to the reaction mixture before incubating it with the cells. Slides were stained with Giemsa.

Bone marrow cells

Bone marrow cells from C57BL/6 mice (Charles River Farms, Wilmington, MA) were isolated by a modification of previously published methods (19, 20, 21, 22, 23, 24, 25, 26). Mouse bone marrow cells from femur, tibia, and humerus were flushed, collected into tubes, washed twice with {alpha}-MEM, and cultured (1 x 106 cells/cm2) in {alpha}-MEM containing 10% HIFBS. Cultures were fed every 3 days with fresh medium. IL-1Ra (100 ng/ml), indomethacin (10-6 M), and/or Vit D (10-9 or 10-8 M) was added to cultures as indicated in each experiment. Cells were fixed on day 6 of culture with 2.5% glutaraldehyde in PBS for 30 min at room temperature before being stained for TRAP. Enzyme histochemistry for TRAP was performed with a commercial kit (Sigma-Aldrich).

PCR amplification

Total RNA was extracted from the cells with TRI REAGENT (Molecular Research Center, Cincinnati, OH). Total RNA was converted to cDNA by reverse transcriptase (Superscript II; Life Technologies) using random hexamer primers. Aliquots of the first-strand cDNA were amplified by PCR.

PCR amplification was done as previously described (19) using gene-specific primers and Taq polymerase (AmpliTaq; Applied Biosystems, Norwalk, CT). The PCR mixture (without enzyme) was overlaid with mineral oil and heated to 94°C for 5 min. During the last minute, AmpliTaq was added (hot start) and amplification was allowed to proceed in a thermal cycler (Applied Biosystems). Temperature cycling was as follows: denaturation at 94°C for 1 min, primer annealing at 65°C for 2 min, and extension at 72°C for 3 min for 10 cycles. In subsequent cycles, the primer annealing temperature was decreased stepwise (step-down method) by 5°C every five cycles. After the last cycle, the mixture was incubated at 72°C for 7 min. To verify that amplification was in the linear range for each PCR analysis, we performed PCR amplification between 24 and 36 cycles at three cycle intervals, and measured product yield as previously described (19). Specific amplimer sets are designed from published cDNA sequences: murine RANKL (27) spanning all five exons (antisense: 5'-TCCCGATGTTTCATGATGC-3', sense: 5'-TGTACTTTCGAGCGCAGATG-3'), murine OPG (28) spanning exons II to V (antisense: 5'-TCAAGTGCTTGAGGGCATAC-3'; sense: 5'-TGGAGATCGAATTCTGCTTG-3'), murine {beta}-actin (29) (antisense: 5'-CTCTTTGATGTCACGCACGATTTC-3'; sense: 5'-GTGGGCCGCTCTAGGCACCAA-3'), murine IL-1{alpha} (30) (antisense: 5'-AGGTCGGTCTCACTACCTGTGATGAGTTTTGG-3'; sense: 5'-AAGATGTCCAACTTCACCTTCAAGGAGAGCCG-3'), murine IL-1{beta} (31) (antisense: 5'-CAGGACAGGTATAGATTCTTTCCTTT-3', sense: 5'-ATGGCAACTGTTCCTGAACTCAACT-3'), and murine G3PDH (32) (antisense: 5'-CATGTAGGCCATGAGGTCCACCAC-3', sense: 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3').

ELISAs

Cells were incubated for the indicated times with stimuli before the medium was removed and replaced with 250 µl of {alpha}-MEM without serum per well in a 12-well plate. Cells were then frozen and thawed three times, and the cell extracts removed for assay by commercial ELISA according to the manufacturer’s recommendations, because most IL-1{alpha} remains in the cytosol of producing cells in its precursor form (33). Data were expressed as picograms of protein per milliliters of cell extract. We also assayed the levels of IL-1{alpha} and {beta} in the conditioned medium from both cocultures and osteoblasts, but were unable to detect either protein under any condition.

Statistics

Differences between groups were assessed by ANOVA and then by the Bonferroni post hoc test if significant differences were identified. All experiments were repeated at least once with similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Coculture of osteoblasts and spleen cells alone produced relatively few OCLs (<10 per well; Fig. 1Go), which were defined as multinucleated and having positive staining for TRAP. In contrast, treatment of the cultures with Vit D caused a dose-dependent increase in the number of OCL in cultures that contained both WT osteoblasts and spleen cells (Fig. 1Go). In cultures that contained IL-1R1 deficient (KO) osteoblasts, Vit D (10-8M) stimulated osteoclast formation, but their numbers were only 10% of those in cocultures that contained WT osteoblasts (Fig. 2Go). To confirm that the OCL that formed in these cultures had characteristics of authentic osteoclasts, we demonstrated that they contained large numbers of calcitonin receptor as determined by specific binding of 125I-labeled calcitonin to the OCL (Fig. 3Go).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. Dose-response effects of Vit D on osteoclast formation in WT osteoblast-spleen cell cocultures. Cells from WT mice were cultured for 6 days with vitamin D. OCL were identified as TRAP+ multinucleated (>3 nuclei) giant cells. Values are mean ± SEM for six determinations per group. *, Significant effect of vitamin D, p < 0.01

 


View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 2. Effects of IL-1 responsiveness on the ability of Vit D to induce osteoclast formation in mixed osteoblast-spleen cell cocultures. Cells were from either WT or IL-1R1-deficient (KO) mice and were cultured for 6 days with vitamin D (10-8 M). OCL were identified as TRAP+ multinucleated (>3 nuclei) giant cells. Values are mean ± SEM for four determinations per group. *, Significantly different from respective group using WT osteoblasts, p < 0.01

 


View larger version (136K):
[in this window]
[in a new window]
  		FIGURE 3. OCL formed in osteoblast-spleen cell cocultures have specific calcitonin receptors. Cells from WT osteoblasts and WT spleen cells were incubated with Vit D (10-8 M) for 7 days. At the conclusion of the experiment, cells were then incubated with radiolabeled calcitonin and without (A and B) or with 100-fold excess cold calcitonin (C and D). Cells were stained for TRAP and then developed for autoradiography. A and C are brightfield. B and D are darkfield. Silver grains, indicating radiolabeled calcitonin binding, appear as black dots in brightfield images and white dots in darkfield images. Note white reticular pattern in B, overlying OCL which is not present in D, demonstrating specific OCL calcitonin binding.

 
To further characterize the effects of 1,25 (OH)2 vitamin D3 on this system, we measured mRNA levels of RANKL, OPG, and IL-1{alpha} in the cocultures (Fig. 4Go). Relatively little RANKL and IL-1{alpha} and high levels of OPG mRNA were present in control culture. Vit D treatment increased RANKL and IL-1{alpha} mRNA and decreased OPG mRNA expression dose dependently. Treatment with Vit D (10-8 M) also increased RANKL and inhibited OPG mRNA levels in the cocultures that contained KO osteoblasts (Fig. 5Go). However, the increase in RANKL expression was 17- to 19-fold in cocultures that contained WT osteoblasts, but only 3.5-fold in cocultures that contained IL-1R1 KO osteoblasts. Inhibition of OPG mRNA expression with Vit D treatment in all cocultures was similar and almost complete.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 4. Dose-response effects of Vit D on RANKL, OPG, and IL-1{alpha} mRNA expression in osteoblast-spleen cell cocultures. Cells were from WT mice, were cultured for 6 days without or with Vit D, and then extracted for RNA. Levels of RANKL, OPG, IL-1{alpha}, and G3PDH mRNA were determined by RT-PCR. Values below each band are the ratio of the band intensity normalized to that of G3PDH.

 


View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 5. Effects of IL-1 responsiveness on the ability of Vit D to regulate RANKL and OPG mRNA expression in osteoblast-spleen cell cocultures. Cells were from either WT or IL-1R1-deficient (KO) mice, were cultured for 7 days without or with Vit D (10-8 M), and then extracted for RNA. Levels of RANKL, OPG, and {beta}-actin mRNA were determined by RT-PCR. Values below each band are the ratio of the band intensity normalized to that of {beta}-actin.

 
Levels of intracellular IL-1{alpha} protein were also measured in the cocultures (Fig. 6Go). In cocultures that were not treated with Vit D, little IL-1{alpha} protein was detectable in the cells. In contrast, in cocultures that were stimulated with Vit D (10-8 M), there was a marked increase in IL-1{alpha} protein in those cocultures that contained WT osteoblasts, and this response was similar with either WT or KO spleen cells. In contrast, little intracellular IL-1{alpha} protein was detected in Vit D-stimulated cocultures that contained KO osteoblasts and either WT or KO spleen cells.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 6. Effects of Vit D on intracellular IL-1{alpha} protein levels in osteoblast-spleen cell cocultures. Cells were from either WT or IL-1R1-deficient (KO) mice, and were cultured for 7 days without or with Vit D (10-8 M). Levels of intracellular IL-1{alpha} were determined by ELISA. Values are mean ± SEM for four determinations per group. *, Significantly different from respective control group, p < 0.01

 
These results suggested that differences existed in the osteoclastogenic signal that were produced by osteoblasts from WT and KO mice, and that these differences were related to the ability of Vit D to stimulate IL-1 production. To further examine these differences, we measured the effects of Vit D treatment on cultures of either WT or IL-1R1 KO osteoblasts. In these experiments, cultures were treated with doses of IL-1 (10 ng/ml) and/or Vit D (10-8 M), which produced maximal responses. In WT osteoblasts, only low levels of RANKL mRNA were present in controls and both Vit D and IL-1 increased the levels of RANKL mRNA by 3- or 4-fold, respectively (Fig. 7Go). There was an additive effect of the combination of Vit D and IL-1 on RANKL mRNA expression, which produced a 7-fold increase in RANKL mRNA expression. In KO osteoblasts, the response to Vit D was preserved but, as expected, there was no effect of IL-1{alpha} treatment on RANKL mRNA levels.



View larger version (73K):
[in this window]
[in a new window]
  		FIGURE 7. Effects of Vit D and IL-1{alpha} on RANKL, OPG, IL-1{alpha}, and IL-1{beta} mRNA expression in osteoblast cultures. Cells were from either WT or IL-1R1-deficient (KO) mice, were cultured for 5 days without or with Vit D (10-8 M), and then extracted for RNA. Levels of RANKL, OPG, IL-1{alpha}, IL-1{beta}, and {beta}-actin mRNA were determined by RT-PCR. Values below each band are the ratio of the band intensity normalized to that of {beta}-actin.

 
In contrast to the spleen cell osteoblast cocultures, there was less regulation of OPG mRNA expression in WT osteoblast or KO osteoblast cultures that were treated with either Vit D or IL-1. Vit D treatment produced a partial inhibition of the OPG mRNA levels, which was enhanced in WT osteoblast cultures that were treated with both Vit D and IL-1. IL-1 alone had no effect on OPG mRNA expression in WT osteoblasts. In KO osteoblasts, only Vit D treatment partially inhibited OPG mRNA expression. In addition, Vit D had no effect on IL-1{alpha} mRNA expression in WT osteoblast cultures, while IL-1 treatment induced a small signal. Combined treatment with both Vit D and IL-1 increased IL-1{alpha} mRNA expression by 3-fold over that seen in WT osteoblast cultures that were treated with IL-1 alone. There was no detectable IL-1{alpha} mRNA expression in any group of KO osteoblasts cells. There was also little regulation of the relatively low level of IL-1{beta} mRNA by treatment of the cultures with Vit D, IL-1, or their combination in either WT osteoblasts or KO osteoblasts.

Expression of IL-1{alpha} protein in osteoblast cultures was similar to the effects on mRNA expression. IL-1{alpha} was not detectable in WT osteoblast controls or cultures that were treated with Vit D (Fig. 8Go). However, there was a small but significant production of IL-1{alpha} in WT osteoblasts that were treated with IL-1. In WT osteoblast cultures that were treated with both Vit D and IL-1, there was a synergistic increase in intracellular IL-1{alpha} protein, with levels being 100-fold greater than those seen in cultures that were treated with IL-1 alone. IL-1{alpha} protein was not detectable in control KO osteoblast cultures, and treatment with Vit D, IL-1, or their combination failed to produce detectable IL-1{alpha} protein levels in KO osteoblast cells.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 8. Effects of Vit D and IL-1{alpha} on intracellular IL-1{alpha} protein levels in osteoblast cultures. Primary murine osteoblastic cells from either WT or IL-1R1-deficient (KO) mice were cultured for 5 days without or with Vit D (10-8 M), IL-1, or their combination. Levels of intracellular IL-1{alpha} were determined by ELISA. Values are mean ± SEM for four determinations per group. *, Significantly different from respective control group, p < 0.01. **, Significantly different from cells treated with IL-1{alpha} alone, p < 0.01

 
To examine the role of IL-1 production in the effects of Vit D on OCL formation in another in vitro model of osteoclastogenesis, we treated WT murine bone marrow cultures with or without Vit D and with or without IL-1Ra, a competitive inhibitor of IL-1{alpha} and IL-1{beta} (Fig. 9Go). As with cocultures that contained IL-1R1 KO osteoblastic cells, IL-1Ra inhibited osteoclast formation that was stimulated by Vit D (10-9 M) in the bone marrow cultures. Because increased osteoclast formation by IL-1 has been shown to be mediated by PGE2 (34, 35), we investigated whether prostaglandin synthesis was involved in the responses of bone marrow cultures to Vit D. Bone marrow cells were stimulated with or without Vit D and with or without indomethasin, a selective inhibitor of prostaglandin synthesis. We found that indomethacin had no effect on OCL formation in Vit D-stimulated bone marrow cultures (data not shown).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 9. Effects of Vit D and IL-1Ra on osteoclast formation in bone marrow cell cultures. Cells from C57BL/6 mice were cultured for 6 days with vitamin D (10-9 M) and/or IL-1Ra (100 ng/ml). OCL were identified as TRAP+ multinucleated (>3 nuclei) giant cells. Values are mean ± SEM for six determinations per group. *, Significant effect of vitamin D; +, significant effect of IL-1Ra, p < 0.01

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results demonstrate that the ability of Vit D to stimulate OCL formation in mixed spleen cell osteoblast cocultures is dependent on the ability of the osteoblasts in the cultures to respond to IL-1. This effect appears dependent on changes in the expression of RANKL and OPG as increases in OCL formation correlated with increased expression of RANKL mRNA and inhibition of OPG mRNA levels in the cocultures.

In these cocultures, we found both additive and synergistic interactions between Vit D and IL-1 in their ability to stimulate RANKL and IL-1{alpha} production by osteoblasts. Additive effects occurred between IL-1 and Vit D with regards to their ability to stimulate RANKL and inhibit OPG mRNA levels. Synergistic effects were demonstrated in the ability of IL-1 and Vit D to stimulate IL-1{alpha} protein and mRNA. As previously demonstrated by Pfeilschifter et al. (36), IL-1 enhanced osteoclast formation in Vit D-treated human marrow cultures. PGs have previously been shown to mediate some of their effects on osteoclast formation through IL-1 (34, 35). However, it is unlikely that Vit D induced PG, which in turn, induced IL-1 in our models because the specific PG synthesis inhibitor, indomethacin, had no effect on Vit D or IL-1-mediated actions in our studies.

It is likely that endogenous IL-1 production in our cultures is involved in the ability of Vit D to stimulate an osteoclastic response because Vit D enhanced the ability of IL-1 to stimulate IL-1{alpha} production and the combination of IL-1 and Vit D enhanced RANKL and inhibited OPG production in primary murine osteoblasts. In addition, we found that IL-1Ra, which specifically blocks IL-1 activity, inhibited Vit D-mediated osteoclastogenesis in WT murine bone marrow cultures.

We have previously demonstrated a tight correlation between increases in RANKL mRNA, decreases in OPG mRNA, and increases in OCL formation with parathyroid hormone in murine marrow cell cultures (19); and it appears that similar relationships exist for Vit D stimulation of OCL formation in murine spleen cell osteoblast cocultures.

The ability of Vit D to regulate IL-1 production in primary osteoblast cultures appears restricted to IL-1{alpha} because we found no effect of Vit D on IL-1{beta} mRNA (Fig. 7Go) when added to the medium either directly or with IL-1{alpha}. In addition, Vit D alone appeared not to stimulate IL-1{alpha} production. Instead, it markedly augmented the ability of exogenously added IL-1 to increase endogenous production of this cytokine in primary murine osteoblast cultures.

There have been varying effects of Vit D on IL-1 production in other models. In the human monocyte cell line U-937, Vit D had no effect alone on IL-1 production, but augmented the ability of T lymphocyte-produced cytokines, PMA, or LPS to stimulate IL-1 (37, 38, 39). However, LPS directly supported survival and fusion of preosteoclast in inflammatory bone loss and this phenomenon was independent of IL-1 and RANKL action (40). In the murine macrophage cell line P388D1 and in human peripheral monocytes (PBMs), Vit D was found to be a direct stimulator of IL-1 (41, 42). In addition, Vit D stimulated IL-1 in PBM and ST-2 cell cocultures (43, 44). However, another group found no effect of Vit D alone on IL-1 production by PBM, but did find it to augment IL-1 production that was stimulated by hydroxyapetite and collagen (45). In contrast to our results, which found no effects of Vit D on IL-1{beta} expression in primary osteoblasts, Wang et al. (46) demonstrated that Vit D increased IL-1{beta} mRNA in osteoblastic cells from 5–12-wk-old mice, but not in osteoblastic cells from 10–12-mo-old mice. Hence, the age of the mice from which the osteoblasts are derived may affect their ability to be regulated by Vit D.

In some models, Vit D inhibits IL-1 activity. Muller et al. (47) found that Vit D inhibited the ability of LPS-stimulated human PBM to produce IL-1{alpha}. Vit D also inhibited IL-1 production by a human leukemia cell line OCIM2 (48), human monocyte/macrophages (49), and human corneal epithelial cells (50).

We found that the regulation of OPG mRNA by Vit D in primary osteoblast cultures was less in cells that were incubated alone than in cells that were cocultured with spleen cells. This result is similar to our previous finding of decreased OPG regulation in primary murine osteoblasts with parathyroid hormone treatment compared with the responses of whole murine marrow cultures (19). These observations suggest that interaction between osteoblasts and hemopoietic cells modulate the OPG responses of osteoblasts. It has previously been demonstrated that there are bidirectional interactions between mesenchymal and hemopoietic cells with Vit D stimulation (43, 44), and similar effects may regulate OPG production.

The ability of IL-1 and Vit D to stimulate RANKL production appears to involve different pathways because inhibitors of STAT3 block responses to IL-1 but not to Vit D (51). NF-{kappa}B, which is a mediator of the response of cells to IL-1 (52), is regulated by Vit D in human MRC-5 fibroblasts (53), and this effect may be involved in the interactions that we observed between these agents. The ability of IL-1 to stimulate IL-6 in MC3T3-E1 osteoblastic cells was also enhanced by treatment with Vit D through a mechanism that was dependent on the IL-1R1 (54).

Direct actions of IL-1 on osteoclast precursor and osteoclast have recently been demonstrated (55). However, it is unlikely that such effects were involved in the responses that we observed because we found no difference in OCL formation between cocultures that used WT and KO spleen cells (Fig. 2Go). Instead, it seems that the differences in OCL formation in the cocultures depended solely on the ability of osteoblasts to express the IL-1R1. It has been shown that IL-1 stimulates bone resorption through a primary action on osteoblast that was induced by IL-1 to transmit a signal that stimulates osteoclastic bone resorption (56).

It is tempting to speculate on the role that the interaction of IL-1 and Vit D may have in vivo. IL-1 production occurs in bone (57) and is believed involved in the role that estrogen plays in postmenopausal bone loss (58). In addition, hemopoietic cells including activated macrophages and T lymphocytes can synthesize Vit D (59). Hence, there may be a paracrine system in the bone-bone marrow microenvironment involving Vit D and IL-1 that regulates both their own expression and their effects on bone cells and skeletal function.


   Footnotes
 
1 This work was supported by Grant PO1-AR38933 from the U.S. Public Health Service. Back

2 Address correspondence and reprint requests to Dr. Sun-Kyeong Lee, Division of Endocrinology, University of Connecticut Health Center, AM047, MC 1850, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: slee{at}neuron.uchc.edu Back

3 Abbreviations used in this paper: IL-1R1, type 1 IL-1R; OCL, osteoclast-like cell; WT, wild type; KO, knockout; OPG, osteoprotegerin; IL-1Ra, IL-1R antagonist; IL-1R2, type 2 IL-1R; HIFBS, heat-inactivated FBS; TRAP, tartrate-resistant acid phosphatase; PBM, peripheral monocyte; RANK, receptor activator of NF-{kappa}B; RANKL, RANK ligand; Vit D, 1,25 (OH)2 vitamin D3. Back

Received for publication October 11, 2001. Accepted for publication June 24, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dinarello, C. A.. 1994. The biological properties of interleukin-1. Eur. Cytokine Netw. 5:517.[Medline]
  2. Horowitz, M. C., J. Lorenzo. 1996. Local regulators of bone: IL-1, TNF, and lymphotoxin, interferon {gamma}, IL-8, IL-10, IL-4, the LIF/IL-6 family and additional cytokines. J. Bilezikian, and L. Raisz, and G. Rodan, eds. Principals of Bone Biology 687. Academic, San Diego.
  3. Kimble, R. B., A. B. Matayoshi, J. L. Vannice, V. T. Kung, C. Williams, R. Pacifici. 1995. Simultaneous block of interleukin-1 and tumor necrosis factor is required to completely prevent bone loss in the early postovariectomy period. Endocrinology 136:3054.[Abstract]
  4. Lorenzo, J. A., A. Naprta, Y. Rao, C. Alander, M. Glaccum, M. Widmer, G. Gronowicz, J. Kalinowski, C. C. Pilbeam. 1998. Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy. Endocrinology 139:3022.[Abstract/Free Full Text]
  5. Keen, R. W., K. L. Woodford-Richens, J. S. Lanchbury, T. D. Spector. 1998. Allelic variation at the interleukin-1 receptor antagonist gene is associated with early postmenopausal bone loss at the spine. Bone 23:367.[Medline]
  6. Langdahl, B. L., E. Lokke, M. Carstens, L. L. Stenkjær, E. F. Eriksen. 2000. Osteoporotic fractures are associated with an 86-base pair repeat polymorphism in the interleukin-1-receptor antagonist gene but not with polymorphisms in the interleukin-1{beta} gene. J. Bone Miner. Res. 15:402.[Medline]
  7. Bajnok, E., I. Takacs, P. Vargha, G. Speer, Z. Nagy, P. Lakatos. 2000. Lack of association between interleukin-1 receptor antagonist protein gene polymorphism and bone mineral density in Hungarian postmenopausal women. Bone 27:559.[Medline]
  8. Dinarello, C. A.. 1991. Interleukin-1 and interleukin-1 antagonism. Blood 77:1627.[Abstract/Free Full Text]
  9. Dower, S. K., J. E. Sims, D. P. Cerretti, T. A. Bird. 1992. The interleukin-1 system: receptors, ligands and signals. Chem. Immunol. 51:33.[Medline]
  10. Sunyer, T., J. Lewis, P. Osdoby. 1997. Estrogen decreases the steady state levels of the IL-1 signaling receptor (type I) while increasing those of the IL-1 decoy receptor (type II) mRNAs in human osteoclast-like cells. J. Bone Miner. Res. 12:(Suppl.1):S135. (Abstr.).
  11. Wesche, H., C. Korherr, M. Kracht, W. Falk, K. Resch, M. U. Martin. 1997. The interleukin-1 receptor accessory protein (IL-1RAcP) is essential for IL-1-induced activation of interleukin-1 receptor-associated kinase (IRAK) and stress-activated protein kinases (SAP kinases). J. Biol. Chem. 272:7727.[Abstract/Free Full Text]
  12. Suda, T., N. Takahashi, N. Udagawa, E. Jimi, M. T. Gillespie, T. J. Martin. 1999. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20:345.[Abstract/Free Full Text]
  13. Takahashi, N., T. Akatsu, N. Udagawa, T. Sasaki, A. Yamaguchi, J. M. Moseley, T. J. Martin, T. Suda. 1988. Osteoblastic cells are involved in osteoclast formation. Endocrinology 123:2600.[Abstract]
  14. Ross, F. P.. 2000. RANKing the importance of measles virus in Paget’s disease. J. Clin. Invest. 105:555.[Medline]
  15. Hofbauer, L. C., S. Khosla, C. R. Dunstan, D. L. Lacey, W. J. Boyle, B. L. Riggs. 2000. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J. Bone Miner. Res. 15:2.[Medline]
  16. Glaccum, M. B., K. L. Stocking, K. Charrier, J. L. Smith, C. R. Willis, C. Maliszewski, D. J. Livingston, J. J. Peschon, P. J. Morrissey. 1997. Phenotypic and functional characterization of mice that lack the type I receptor for IL-1. J. Immunol. 159:3364.[Abstract]
  17. Vargas, S. J., A. Naprta, M. Glaccum, S. K. Lee, J. Kalinowski, J. A. Lorenzo. 1996. Interleukin-6 expression and histomorphometry of bones from mice deficient in receptors for interleukin-1 or tumor necrosis factor. J. Bone Miner. Res. 11:1736.[Medline]
  18. Hurley, M. M., S. K. Lee, L. G. Raisz, P. Bernecker, J. Lorenzo. 1998. Basic fibroblast growth factor induces osteoclast formation in murine bone marrow cultures. Bone 22:309.[Medline]
  19. Lee, S. K., J. A. Lorenzo. 1999. Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: correlation with osteoclast-like cell formation. Endocrinology 140:3552.[Abstract/Free Full Text]
  20. Takahashi, N., T. Akatsu, T. Sasaki, G. C. Nicholson, J. M. Moseley, T. J. Martin, T. Suda. 1988. Induction of calcitonin receptors by 1{alpha},25 dihydroxyvitamin D3 in osteoclast-like multinucleated cells formed from mouse bone marrow cells. Endocrinology 123:1504.[Abstract]
  21. Udagawa, N., N. Takahashi, T. Akatsu, T. Sasaki, A. Yamaguchi, H. Kodama, T. J. Martin, T. Suda. 1989. The bone marrow-derived stromal cell lines MC3T3–G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells. Endocrinology 125:1805.[Abstract]
  22. Lee, S. K., S. R. Goldring, J. A. Lorenzo. 1995. Expression of the calcitonin receptor in bone marrow cell cultures and in bone: a specific marker of the differentiated osteoclast that is regulated by calcitonin. Endocrinology 136:4572.[Abstract]
  23. Takahashi, N., H. Yamana, S. Yoshiki, G. D. Roodman, G. R. Mundy, S. J. Jones, A. Boyde, T. Suda. 1988. Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122:1373.[Abstract]
  24. Akatsu, T., T. Tamura, N. Takahashi, N. Udagawa, S. Tanaka, T. Sasaki, A. Yamaguchi, N. Nagata, T. Suda. 1992. Preparation and characterization of a mouse osteoclast-like multinucleated cell population. J. Bone Miner. Res. 7:1297.[Medline]
  25. Akatsu, T., N. Takahashi, K. Debari, I. Morita, N. Nagata, O. Takatani, T. Suda. 1989. Prostaglandins promote osteoclast-like cell formation by a mechanism involving cyclic adenosine 3',5'-monophosphate in mouse bone marrow cell cultures. J. Bone Miner. Res. 4:29.[Medline]
  26. Shuto, T., T. Kukita, M. Hirata, E. Jimi, T. Koga. 1994. Dexamethasone stimulates osteoclast-like cell formation by inhibiting granulocyte-macrophage colony-stimulating factor production in mouse bone marrow cultures. Endocrinology 134:1121.[Abstract]
  27. Hsu, H., D. L. Lacey, C. R. Dunstan, I. Solovyev, A. Colombero, E. Timms, H.-L. Tan, G. Elliott, M. J. Kelley, I. Sarosi, et al 1999. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. USA 96:3540.[Abstract/Free Full Text]
  28. Simonet, W. S., D. L. Lacey, C. R. Dunstan, M. Kelley, M. S. Chang, R. Luthy, H. Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al 1997. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309.[Medline]
  29. Alonso, S., A. Minty, Y. Bourlet, M. Buckingham. 1986. Comparison of the three actin-coding sequences in the mouse: evolutionary relationships between the actin genes of warm-blooded vertebrates. J. Mol. Evol. 23:11.[Medline]
  30. Lomedico, P. T., U. Gubler, C. P. Hellmann, M. Dukovich, J. G. Giri, Y. C. Pan, K. Collier, R. Semionow, A. O. Chua, S. B. Mizel. 1984. Cloning and expression of murine interleukin-1 cDNA in Escherichia coli. Nature 312:458.[Medline]
  31. Gray, P. W., D. Glaister, E. Chen, D. V. Goeddel, D. Pennica. 1986. Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1-{beta}. J. Immunol. 137:3644.[Abstract]
  32. Sabath, D. E., H. E. Broome, M. B. Prystowsky. 1990. Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned T-helper lymphocyte. Gene 91:185.[Medline]
  33. Dinarello, C. A., S. M. Wolff. 1993. The role of interleukin-1 in disease. New Engl. J. Med. 328:106.[Free Full Text]
  34. Akatsu, T., N. Takahashi, N. Udagawa, K. Imamura, A. Yamaguchi, K. Sato, N. Nagata, T. Suda. 1991. Role of prostaglandins in interleukin-1-induced bone resorption in mice in vitro. J. Bone Miner. Res. 6:183.[Medline]
  35. Amano, S., K. Naganuma, Y. Kawata, K. Kawakami, S. Kitano, S. Hanazawa. 1996. Prostaglandin E2 stimulates osteoclast formation via endogenous IL-1{beta} expressed through protein kinase A. J. Immunol. 156:1931.[Abstract]
  36. Pfeilschifter, J., C. Chenu, A. Bird, G. R. Mundy, G. D. Roodman. 1989. Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclast like cells in vitro. J. Bone Miner. Res. 4:113.[Medline]
  37. Amento, E. P., A. K. Bhalla, A. K. Kurnick, J. T. Kardin, T. L. Clemens, S. A. Holick, M. R. Holick, S. M. Krane. 1884. 1{alpha}25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U-937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor. J. Clin. Invest. 73:731.
  38. Bhalla, A. K., T. Paavonen, M. M. Williams, P. J. Delves, P. M. Lydyard. 1991. Regulation of interleukin-1 and tumour necrosis factor gene expression in myelomonocytic cell lines by 1,25-dihydroxyvitamin D3. Immunology 72:61.[Medline]
  39. Fagan, D. L., J. L. Prehn, J. S. Adams, S. C. Jordan. 1991. The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1{beta} messenger RNA levels by 1,25-dihydroxyvitamin D3. Mol. Endocrinol. 5:179.[Abstract]
  40. Suda, K., J.-T. Woo, M. Takami, P. M. Sexton, K. Nagai. 2002. Lipopolysaccharide supports survival and fusion of preosteoclast independent of TNF-{alpha}, IL-1 and RANKL. J. Cell. Physiol. 190:101.[Medline]
  41. Hodler, B., V. Evequoz, U. Trechsel, H. Fleisch. 1985. Influence of vitamin D3 metabolites on the production of interleukin 1, 2 and 3. Immunobiology 170:256.[Medline]
  42. Bhalla, A. K., E. P. Amento, S. M. Krane. 1986. Differential effects of 1,25-dihydroxyvitamin D3 on human lymphocytes and monocyte/macrophages: inhibition of interleukin-2 and augmentation of interleukin-1 production. Cell. Immunol. 98:311.[Medline]
  43. Haynes, D. R., G. J. Atkins, M. Loric, T. N. Crotti, S. M. Geary, D. M. Findlay. 1999. Bidirectional signaling between stromal and hemopoietic cells regulates interleukin-1 expression during human osteoclast formation. Bone 25:269.[Medline]
  44. Atkins, G. J., D. R Haynes, S. M. Geary, M. Loric, T. N. Crotti, D. M. Findlay. 2000. Coordinated cytokine expression by stromal and hematopoietic cells during human osteoclast formation. Bone 26:653.[Medline]
  45. Hogasen, A. K., L. Nordsletten, A. O. Aasen, J. A. Falch. 1998. 17{beta}-oestradiol and 1{alpha}, 25-dihydroxycholecalciferol modulate constitutive and bone matrix-induced interleukin-1{beta} (IL-1{beta}) production by peripheral blood mononuclear cells isolated from postmenopausal women. Scand. J. Clin. Lab. Invest. 58:97.[Medline]
  46. Wang, X., Z. Schwartz, P. Yaffe, A. Ornoy. 1999. The expression of transforming growth factor-{beta} and interleukin-1{beta} mRNA and the response to 1,25 (OH)2D3, 17{beta}-estradiol, and testosterone is age dependent in primary cultures of mouse-derived osteoblasts in vitro. Endocrine 11:13.[Medline]
  47. Muller, K., P. M. Haahr, M. Diamant, K. Rieneck, A. Kharazmi, K. Bendtzen. 1992. 1,25-dihydroxyvitamin D3 inhibits cytokine production by human blood monocytes at the post-transcriptional level. Cytokine 4:506.[Medline]
  48. Peleg, S., H. Qiu, S. Reddy, D. Harris, Q. Van, E. H. Estey, M. Talpaz, Z. Estrov. 1997. 1,25-dihydroxyvitamin D3 and its analogues inhibit acute myelogenous leukemia progenitor proliferation by suppressing interleukin-1{beta} production. J. Clin. Invest. 100:1716.[Medline]
  49. Muller, K., K. Bendtzen. 1996. 1,25-dihydroxyvitamin D3 as a natural regulator of human immune functions. J. Invest. Dermatol. Symp. Proc. 1:68.
  50. Suzuki, T., Y. Sano, C. Sotozono, S. Kinoshita. 2000. Regulatory effects of 1{alpha},25-dihydroxyvitamin D3 on cytokine production by human corneal epithelial cells. Curr. Eye Res. 20:127.[Medline]
  51. O’Brien, C. A., I. Gubrij, S. C. Lin, R. L. Saylors, S. C. Manolagas. 1999. STAT3 activation in stromal osteoblastic cells is required for induction of the receptor activator of NF-{kappa}B ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone. J. Biol. Chem. 274:19301.[Abstract/Free Full Text]
  52. Jimi, E., T. Ikebe, N. Takahashi, M. Hirata, T. Suda, T. Koga. 1996. Interleukin-1{alpha} activates all NF-{kappa}B-like factor in osteoclast-like cells. J. Biol. Chem. 271:4605.[Abstract/Free Full Text]
  53. Harant, H., B. Wolff, I. J. Lindley. 1998. 1{alpha},25-dihydroxyvitamin D3 decreases DNA binding of nuclear factor-{kappa}B in human fibroblasts. FEBS Lett. 436:329.[Medline]
  54. Lacey, D. L., L. E. Grosso, S. A. Moser, J. Erdmann, H.-L. Tan, R. Pacifici, D. T. Villareal. 1993. IL-1-induced murine osteoblast IL-6 production is mediated by the type 1 IL-1 receptor and is increased by 1,25 dihydroxyvitamin D3. J. Clin. Invest. 91:1731.
  55. Jimi, E., I. Nakamura, L. T. Duong, T. Ikebe, N. Takahashi, G. A. Rodan, T. Suda. 1999. Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells. Exp. Cell Res. 247:84.[Medline]
  56. Thomson, B. M., J. Saklatvala, T. J. Chambers. 1986. Osteoblasts mediate interleukin1 stimulation of bone resorption by rat osteoclasts. J. Exp. Med. 164:104.[Abstract/Free Full Text]
  57. Lorenzo, J. A., S. L. Sousa, S. E. Van Den Brink-Webb, J. H. Korn. 1990. Production of both interleukin-1{alpha} and {beta} by newborn mouse calvaria cultures. J. Bone Miner. Res. 5:77.[Medline]
  58. Pacifici, R.. 1998. Cytokines, estrogen, and postmenopausal osteoporosis–the second decade. Endocrinology 139:2659.[Free Full Text]
  59. Reichel, H., H. P. Koeffler, A. W. Norman. 1990. Production of 1{alpha}, 25-dihydroxyvitamin D3 by hematopoietic cells. Prog. Clin. Biol. Res. 332:81.[Medline]



This article has been cited by other articles:


Home page
 Endocr. Rev.Home page
J. Lorenzo, M. Horowitz, and Y. Choi
Osteoimmunology: Interactions of the Bone and Immune System
Endocr. Rev., June 1, 2008; 29(4): 403 - 440.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Ha, J.-H. Lee, H.-N. Kim, H.-M. Kim, H. B. Kwak, S. Lee, H.-H. Kim, and Z. H. Lee
{alpha}-Lipoic Acid Inhibits Inflammatory Bone Resorption by Suppressing Prostaglandin E2 Synthesis
J. Immunol., January 1, 2006; 176(1): 111 - 117.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. L. Roccisana, N. Kawanabe, H. Kajiya, M. Koide, G. D. Roodman, and S. V. Reddy
Functional Role for Heat Shock Factors in the Transcriptional Regulation of Human RANK Ligand Gene Expression in Stromal/Osteoblast Cells
J. Biol. Chem., March 12, 2004; 279(11): 10500 - 10507.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
H. Chagraoui, M. Tulliez, T. Smayra, E. Komura, S. Giraudier, T. Yun, N. Lassau, W. Vainchenker, and F. Wendling
Stimulation of osteoprotegerin production is responsible for osteosclerosis in mice overexpressing TPO
Blood, April 15, 2003; 101(8): 2983 - 2989.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, S.-K.
Right arrow Articles by Lorenzo, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S.-K.
Right arrow Articles by Lorenzo, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS