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J Am Soc Nephrol 12:1468-1474, 2001
© 2001 American Society of Nephrology

Relative Potencies of 1,25-(OH)2D3 and 19-Nor-1,25-(OH)2D2 on Inducing Differentiation and Markers of Bone Formation in MG-63 Cells

JANE L. FINCH, ADRIANA S. DUSSO, TRICIA PAVLOPOULOS and EDUARDO A. SLATOPOLSKY

Renal Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri.

Correspondence to Dr. Eduardo A. Slatopolsky, Department of Internal Medicine, Renal Division, 660 South Euclid, Washington University School of Medicine, St. Louis, MO 63110. Phone: 314-362-7208; Fax: 314-362-7875; E-mail: eslatopo{at}imgate.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. 19-Nor-1,25-(OH)2D2, an analog of 1,25-(OH)2D3, is used to treat secondary hyperparathyroidism because it suppresses parathyroid hormone synthesis and secretion with lower calcemic and phosphatemic activities. 19-Nor-1,25-(OH)2D2 is approximately 10 times less active than 1,25-(OH)2D3 in promoting bone resorption, which accounts in part for the low potency of this analog in increasing serum calcium and phosphorus. Concern that 19-nor-1,25-(OH)2D2 also could be less potent than 1,25-(OH)2D3 on bone formation led to a comparison of the potency of both compounds on osteoblasts. In the human osteoblast-like cell line MG-63, 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 had a similar potency in upregulating vitamin D receptor content and suppressing proliferation. Both sterols caused a similar reduction in DNA content and proliferating cell nuclear antigen protein expression. Time-course and dose-response studies on 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 induction of the marker of bone formation, osteocalcin, showed overlapping curves. The effects on alkaline phosphatase (ALP) activity also were studied in MG-63 cells that had been co-treated with either sterol and transforming growth factor-ß, an enhancer of 1,25-(OH)2D3—induced ALP activity in this cell line. Transforming growth factor-ß alone had no effect, whereas 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 increased ALP activity similarly. These studies demonstrate that 19-nor-1,25-(OH)2D2 has the same potency as 1,25-(OH)2D3 not only in inducing vitamin D receptor content, osteocalcin levels, and ALP activity but also in controlling osteoblastic growth. Therefore, it is unlikely that 19-nor-1,25-(OH)2D2 would have deleterious effects on bone remodeling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
19-Nor-1,25-(OH)2D2, an analog of 1,25-(OH)2D3, is being used for the treatment of secondary hyperparathyroidism because of its ability to suppress parathyroid hormone (PTH) synthesis and secretion while at the same time having low calcemic and phosphatemic activities (1,2,3). Previous studies from our laboratory demonstrated that the decreased calcemic and phosphatemic activities of 19-nor-1,25-(OH)2D2 are due in part to decreased bone resorption (4). Because 1,25-(OH)2D3 is not only a potent stimulator of bone resorption but also a promoter of bone formation, these studies examined whether 19-nor-1,25-(OH)2D2 also is less active than 1,25-(OH)2D3 in bone-forming activity.

1,25-(OH)2D3 promotes bone formation by inhibiting proliferation of osteoblast precursors and promoting their differentiation into mature osteoblasts (5,6). Most biologic responses to 1,25-(OH)2D3 in vitamin D—target tissues are mediated through the vitamin D receptor (VDR). Because 1,25-(OH)2D3 is known to upregulate its own receptor (7,8,9), we compared the ability of 19-nor-1,25-(OH)2D2 to mimic 1,25-(OH)2D3 in the regulation of VDR content in osteoblasts before assessing the relative potencies of these two sterols in suppressing osteoblast proliferation and inducing the expression of markers of bone-forming activity in osteoblasts. Studies were conducted with the use of the well-characterized human osteoblastic cell line MG-63, which was derived from a human osteosarcoma and is capable of expressing the phenotype of the most differentiated osteoblast (10,11). The antiproliferative properties of 1,25-(OH)2D3 on MG-63 cells (12) led us to compare the effects of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 on MG-63 proliferation by measuring DNA content and proliferating cell nuclear antigen (PCNA). PCNA, a processivity factor for DNA polymerase-{delta} and an important regulator of cell cycle progression (13,14), is increased in the nuclei of cells that are undergoing mitosis.

To assess the ability of both sterols to induce the expression of markers of osteoblast bone formation, we compared the potencies of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 in increasing osteocalcin (OC) levels and alkaline phosphatase (ALP) activity. In MG-63 cells, 1,25-(OH)2D3 treatment increases OC secretion and ALP activity (15,16,17), the latter only when cells also are exposed to transforming growth factor-ß (TGF-ß) (18). By itself, TGF-ß has little effect on ALP activity in these cells. Although the exact mechanism by which TGF-ß enhances 1,25-(OH)2D3-mediated ALP expression is unclear, it is thought that TGF-ß induces these cells to an intermediate stage of differentiation in which they are held (11). In the present studies, the comparison of the potencies of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 in inducing ALP activity were performed in MG-63 cells that had been co-treated with TGF-ß.

The results of these in vitro studies demonstrate that 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 have a similar potency in controlling the proliferation of osteoblasts and their differentiation into mature bone-forming cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
MG-63 cells were grown to confluence in 75-cm2 tissue culture flasks in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) in a humidified atmosphere containing 5% CO2 in air at 37°C. Cells were subcultured weekly with trypsin-ethylenediaminetetraacetate. For experimental protocols, cells were plated in Eagle's minimum essential medium with 10% FBS at a concentration of 100,000 cells/10-cm2 well. Twenty-four h later, the medium was removed and the cells were washed three times with phosphate-buffered saline (PBS). The medium was replaced with fresh medium containing 2% FBS and vehicle, 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2. ALP measurements were performed both under basal conditions and after co-treatment with either sterol and TGF-ß (10 ng/ml). Dose-response experiments were carried out for 72 h with concentrations of 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 ranging from 10-11 to 10-6 M. Time-course experiments were performed from 0 to 72 h with the use of 10-7 M 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2.

Western Blot Analysis for VDR and PCNA
For Western blot analysis, cells were maintained in culture as described above and plated at a density of 3 x 106 cells/10-cm-diameter culture dish. After 24 h, cells were washed three times with PBS and fresh media containing vehicle or 10-7 M 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 was added. Cell lysates were prepared with the use of a modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8), and 1 x protease inhibitor cocktail (Roche Analytics, Newark, NJ). Lysates were analyzed for total protein with the use of the Bradford Method (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (40 µg) were resolved by polyacrylamide gel electrophoresis in 4 to 20% gradient gels (Fisher Scientific, St. Louis, MO) and wet-transferred to 0.45-µm nitrocellulose membranes in 20% methanol in Tris-glycine at 100 volts for 1 h. Gel loading and transfer efficiencies were examined by staining total protein in the nitrocellulose membranes with Ponceau red. After blocking, membranes were exposed to either a monoclonal antibody against PCNA (PC10 mouse anti-PCNA; Zymed Laboratories Inc., San Francisco, CA) or VDR (a kind gift from Dr. Paul N. McDonald, Case Western Reserve University, Cleveland, OH) at 4°C overnight. Blots then were incubated with horseradish peroxidase—conjugated second antibody (Pierce, Rockford, IL) for 1 h at room temperature. Specific bands were detected with the use of the Supersignal Substrate Working Solution (Pierce) and enhanced autoradiographic film (Amersham, Arlington Heights, IL). Gels were scanned, and the densitometric analysis was performed with the use of the Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD).

OC Measurement
At the end of each experiment, medium was frozen at -20°C for later OC determinations and cells again were washed three times with PBS. A buffer containing TRIZMA-HCl 100 mM and NaCl 100 mM (pH 7.5; 500 ml) was added, and the cells were sonicated on ice for 45 s. Sonicates were frozen at -20°C for later DNA analysis. For OC levels, media samples were assayed in duplicate by specific competitive RIA with the use of a goat anti-human—OC antibody, human OC as the standard and 125I-labeled OC (19). Antibody/OC complexes were separated from free iodinated tracer with an anti-goat IgG. The assay buffer consisted of 132 mM NaCl, 10 mM NaH2PO4, 25 mM Na-ethylenediaminetetraacetate, 0.1% Tween 20 (wt/vol), and 0.1% bovine serum albumin (pH 7.4).

ALP Measurement
At the end of each experiment, cells were washed three times with PBS. Triton X-100 (0.05%) was added, and the cells were sonicated on ice for 45 s. ALP was analyzed immediately by autoanalyzer (Miraplus; COBAS, Indianapolis, IN) with the use of p-nitrophenyl phosphate as substrate. The remainder of the sonicate was frozen at -20°C for later DNA analysis.

DNA Assay
Cell sonicates were prepared from MG-63 cells that had been subjected to the experimental conditions described previously. DNA was measured with the use of the fluorometric quantitation of ethidium bromide complexes according to the method of LePecq and Paoletti (20).

Materials
TGF-ß was purchased from R & D Systems (Minneapolis, MN). 1,25-(OH)2D3 was a kind gift of Dr. Milan Uskokovic (Hoffmann-La Roche, Nutley, NJ). 19-Nor-1,25-(OH)2D2 was supplied by Dr. Hector DeLuca (University of Wisconsin, Madison, WI). Unless otherwise stated, all other reagents were purchased from Sigma Chemical Company (St. Louis, MO).

Statistical Analyses
Results are expressed as mean ± SEM. ANOVA was used to examine statistical significance in dose-response or time-course protocols for either 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 treatment. The Bonferroni correction identified the dose or time points responsible for the statistical significance revealed by ANOVA tests. Comparisons between 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 at a given time point or dose were performed with the use of the unpaired t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
These studies compared the relative potencies of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 in suppressing proliferation and inducing differentiation in human osteoblasts. Because most biologic responses to 1,25-(OH)2D3 are mediated by the VDR, we first compared the abilities of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 to upregulate VDR content in MG-63 cells.

Effect of 1,25-(OH)2D3 and 19-Nor-1,25-(OH)2D2 on VDR Content
VDR protein was measured in whole-cell extracts from MG-63 cells that had been treated for 48 h with 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 at a concentration of 10-7 M. The top panel of Figure 1 shows a typical Western blot for the VDR. Recombinant human VDR was used as a positive control to assess for the specificity of the antibody used in these studies. This antibody, 9A7, is a rat monoclonal antibody that recognizes the DNA binding domain of the VDR molecule. The double bands, one co-migrating with recombinant human VDR, may represent differentially phosphorylated VDR isoforms. The bottom panel of Figure 1 shows the corresponding densitometric analysis. 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 treatment induced a marked increase in VDR protein compared with cells that were treated with vehicle alone (1,25-(OH)2D3, 4423 ± 194 integrated optical density [IOD]; 19-nor-1,25-(OH)2D2, 4073 ± 242 IOD versus vehicle, 2991 ± 160 IOD). The compilation of results from two independent experiments indicated an increase in VDR of 71.2 and 42.9% by 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2, respectively.



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  		Figure 1. The top panel is a representative enhanced chemiluminescence (ECL) Western blot analysis of the VDR of 40 µg of total protein from whole-cell extracts from MG-63 cells treated for 48 h with vehicle or 10-7 M of either 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2. The bottom panel represents the densitometric analysis of VDR content using Gel-Pro Analyzer. The results are two experiments done in duplicate (n = 4) and are expressed as integrated optical density/area.

 

Effect of 1,25-(OH)2D3 and 19-Nor-1,25-(OH)2D2 on Cell Proliferation
The effect of various concentrations of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 on total DNA is shown in Figure 2. ANOVA analysis showed that treatment of MG-63 cells for 72 h with concentrations of 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 ranging from 10-11 to 10-6 M resulted in a significant dose-dependent reduction in DNA content for both sterols (1,25-(OH)2D3, P < 0.0001; 19-nor-1,25-(OH)2D2, P < 0.0001). The dose-response curves for both compounds were virtually identical. Bonferroni comparison revealed that the decrease in DNA induced by both sterols reached statistical significance at 10-10 M. At a dose of 10-6 M, DNA decreased from 3.46 ± 0.19 µg/well in control cells to 1.77 ± 0.03 µg/well ({downarrow} 48.8%, P < 0.001) in 1,25-(OH)2D3-treated cells and to 1.81 ± 0.02 µg/well ({downarrow} 47.7%, P < 0.001) after 19-nor-1,25-(OH)2D2 treatment.



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  		Figure 2. Comparative effects of increasing doses of 1,25-(OH)2D3 (solid line) and 19-nor-1,25-(OH)2D2 (broken line) on DNA content. MG-63 cells were treated with concentrations from 10-11 M to 10-7 M 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 for 72 h. DNA is expressed as µg/well. Data points and error bars represent mean ± SEM (n = 7).

 

On the basis of the reduction in DNA observed after 72 h of treatment with 10-7 M of 1,25-(OH)2D3 or 19-nor,1,25-(OH)2D2, we examined the expression of PCNA in whole-cell extracts from MG-63 cells 48 h after treatment with either sterol because a halt in proliferation rate precedes an actual reduction in DNA content. The top panel of Figure 3 shows a typical Western blot for PCNA, and the bottom panel depicts the corresponding densitometric analysis. 1,25-(OH)2D3 treatment decreased PCNA from 6919 ± 127 IOD in vehicle-treated cells to 5750 ± 199 IOD. 19-Nor-1,25-(OH)2D2 decreased PCNA to 4813 ± 124 IOD. Compilation of results from two independent experiments indicates a differential reduction in PCNA expression by 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 of 19.8% and 31.3%, respectively.



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  		Figure 3. The top panel is a representative ECL Western blot analysis of PCNA of 40 µg of total protein from whole-cell extracts from MG-63 cells treated for 48 h with vehicle or 10-7 M of either 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2. The bottom panel represents the densitometric analysis of proliferating cell nuclear antigen (PCNA) content using Gel-Pro Analyzer. The results are two experiments done in duplicate (n = 4) and are expressed as integrated optical density/area.

 

Effect of 1,25-(OH)2D3 and 19-Nor-1,25-(OH)2D2 on Bone-Forming Activity
Osteocalcin. Media OC levels were measured in confluent cultures of MG-63 cells. The effect of increasing doses of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 on OC secretion by MG-63 cells is shown in Figure 4A. While undetectable in control cells, ANOVA analysis demonstrated a dose-dependent increase in OC in cells that were treated for 72 h with various concentrations of 1,25-(OH)2D3 (P < 0.0001) or 19-nor-1,25-(OH)2D2 (P < 0.0001). Bonferroni comparison revealed that OC increased significantly from 0.107 ± 0.058 ng/µg DNA with 10-11 M 1,25-(OH)2D3 to 8.65 ± 0.49 ng/µg DNA (P < 0.001) when cells were treated with 10-7 M 1,25-(OH)2D3. A similar increase was seen with 19-nor-1,25-(OH)2D2 (10-11 M, 0.096 ± 0.065 ng/µg DNA; 10-7 M, 9.19 ± 0.27 ng/µg DNA; P < 0.001). This increase in OC with both compounds reached significance at a concentration of 10-10 M (P < 0.05). Unpaired t-test analysis indicated that 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 increased OC levels to the same degree at every concentration tested.



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  		Figure 4. (A) Comparative effects of increasing doses of 1,25-(OH)2D3 ({blacksquare}) and 19-nor-1,25-(OH)2D2 ({blacksquare}) on media osteocalcin (OC). MG-63 cells were treated with concentrations from 10-11 M to 10-7 M of 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 for 72 h. Media osteocalcin is expressed as ng/µg DNA. Bars and error bars represent mean ± SEM (n = 6). (B) Time course for the effects of increasing doses of 1,25-(OH)2D3 ({blacksquare}) and 19-nor-1,25-(OH)2D2 ({blacksquare}) on media OC. MG-63 cells were treated with 10-7 M 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 for 0 to 72 h. Media OC levels are expressed as ng/µg DNA. Bars and error bars represent mean ± SEM (n = 6).

 

The time course for the effect of these two compounds on OC secretion is shown in Figure 4B. OC was measured at 24, 48, and 72 h in media from confluent MG-63 cells that were treated with 10-7 M 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2. ANOVA analysis demonstrated a significant increase in OC over time in cells that were treated with 1,25-(OH)2D3 (P < 0.0001) or 19-nor-1,25-(OH)2D2 (P < 0.0001). OC was undetectable in control cells at all time points and was barely detectable after 24 h of treatment with either 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2. Bonferroni comparison revealed a significant increase in OC at 48 h (1,25-(OH)2D3, 2.37 ± 0.14 [P < 0.01]; 19-nor-1,25-(OH)2D2, 2.31 ± 0.04 ng/µg DNA [P < 0.01]) and 72 h (1,25-(OH)2D3, 6.17 ± 0.48 [P < 0.001]); 19-nor-1,25-(OH)2D2, 6.45 ± 0.31 ng/µg DNA [P < 0.001]) with either sterol. Both compounds had identical potency on OC secretion at each time point tested.

Alkaline Phosphatase. The demonstration that 1,25-(OH)2D3—induced ALP activity in MG-63 cells is greatly enhanced by co-treatment with TGF-ß (21) led us to study the effects of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 in confluent cultures of MG-63 cells that were co-treated with either sterol and 10 ng/ml TGF-ß. The effect of increasing doses of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 on ALP activity in MG-63 cells after a 72-h incubation period is shown in Figure 5A. Treatment with TGF-ß alone had no effect on ALP activity compared with control cells (TGF-ß, 0.22 ± 0.09 U/ml.µg DNA; control, 0.40 ± 0.18 U/ml.µg DNA). ANOVA analysis showed a significant dose-dependent increase in ALP activity by 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 at doses ranging from 10-9 M to 10-6 M (P < 0.0001 for both compounds). ALP activity rose in a dose-dependent manner, increasing to 7.73 ± 0.84 U/ml.µg DNA with 10-6 M 1,25-(OH)2D3 (P < 0.001) and to 7.43 ± 0.83 U/ml.µg DNA with 10-6 M 19-nor-1,25-(OH)2D2 (P < 0.001). The increase in ALP activity reached significance at 10-7 M for both 1,25-(OH)2D3 (P < 0.05) and 19-nor-1,25-(OH)2D2 (P < 0.01). The profiles for both dose-response curves were the same. Unpaired t-test analysis showed no statistically significant difference between either compound at any dose tested.



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  		Figure 5. (A) Comparative effects of increasing doses of 1,25-(OH)2D3 ({blacksquare}) and 19-nor-1,25-(OH)2D2 ({blacksquare}) on alkaline phosphatase (ALP) activity. MG-63 cells were treated with various concentrations (10-9 to 10-6 M) of 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 for 72 h. ALP activity is expressed as U/ml.µg DNA. The second bar (T) represents the effect of transforming growth factor-ß alone. Bars and error bars represent mean ± SEM (n = 6). (B) Time course for the effects of increasing doses of 1,25-(OH)2D3 ({blacksquare}) and 19-nor-1,25-(OH)2D2 ({blacksquare}) on ALP activity. MG-63 cells were treated with 10-7 M of 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 for 8 to 72 h. ALP activity is expressed as U/ml.µg DNA. Bars and error bars represent mean ± SEM (n = 6).

 

The time course for the effect of these two compounds on ALP activity is shown in Figure 5B. Confluent cells were treated with 10-7 M 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2, and ALP activity was measured at various times from 8 to 72 h. ANOVA analysis demonstrated a significant increase in ALP over time in cells that were treated with 1,25-(OH)2D3 (P < 0.0001) or 19-nor-1,25-(OH)2D2 (P < 0.0001). Treatment with TGF-ß alone had no effect on ALP activity at any time point tested (data not shown). ALP activity was increased by both 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 in as little as 8 h (1,25-(OH)2D3, 3.13 ± 0.98 U/ml.µg DNA; 19-nor-1,25-(OH)2D2, 3.50 ± 1.19 U/ml.µg DNA) and continued to increase in a similar manner for up to 72 h (1,25-(OH)2D3, 9.39 ± 0.353 [P < 0.001]; 19-nor-1,25-(OH)2D2, 11.71 ± 1.57 U/ml.µg DNA [P < 0.01]). Unpaired t test revealed no significant differences in ALP activity between the two compounds at any time point tested.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of 1,25-(OH)2D3 on bone remodeling is complex because of the dual effects of the sterol, increasing both bone formation and resorption. Although osteoclasts are responsible for resorbing bone, both actions of 1,25-(OH)2D3 on bone remodeling are mediated through the osteoblast (22,23,24). Previous studies from our laboratory showed the bone-resorbing activity of 19-nor-1,25-(OH)2D2 to be approximately 10 times less than that of 1,25-(OH)2D3 (4), suggesting the possibility of an impaired action of 19-nor-1,25-(OH)2D2 on the osteoblast. The importance of maintaining a balance between bone formation and resorption raised serious concerns about possible deleterious effects of long-term 19-nor-1,25-(OH)2D2 therapy on bone formation. The present study assessed directly the effects of 19-nor-1,25-(OH)2D2 on osteoblast bone-forming activity.

Because 1,25-(OH)2D3 promotes bone formation by inhibiting the proliferation of osteoblast precursors and promoting their differentiation into a more mature phenotype (5,6), we compared the effects of 19-nor-1,25-(OH)2D2 and 1,25-(OH)2D3 on cell proliferation and two markers of bone formation in the human osteoblast-like cell line, MG-63.

VDR content and intracellular levels of vitamin D metabolites are the main determinants of the magnitude of the response to vitamin D therapy by a target tissue (25,26). Studies in uremic rats that were treated with 1,25-(OH)2D3 or 19-nor-1,25-(OH)2D2 for 8 wk revealed 19-nor-1,25-(OH)2D2 to be less potent than 1,25-(OH)2D3 in upregulating the intestinal VDR (3). In short-term studies, however, 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 had similar effects on the VDR content (27). These opposing effects of 19-nor-1,25-(OH)2D2 on regulating intestinal VDR levels led us to compare the effectiveness of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 regulation of the VDR protein in MG-63 cells before assessing the relative potency of both sterols on osteoblast function. Both sterols were equally effective in increasing VDR content in this cell line.

1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 also are equally potent in the control of osteoblast proliferation as evidenced by similar decreases in DNA content induced by both sterols in MG-63 cells. However, when PCNA expression was used to assess mitotic activity, 19-nor-1,25-(OH)2D2 was more potent than 1,25-(OH)2D3. The cause of this discrepancy was not investigated further. Although PCNA expression correlates with mitotic activity, growth arrest could occur despite high PCNA if PCNA activity is blocked by the cyclin-dependent kinase inhibitor p21. In addition to its intrinsic ability to inhibit mitosis, p21 binds PCNA, preventing DNA replication (28). The demonstration that 1,25-(OH)2D3 induces p21 gene transcription (29) leads us to postulate that a differential ability of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 to induce p21 expression in MG-63 cells could render both sterols equally effective in controlling proliferation despite different PCNA content.

1,25-(OH)2D3 induces MG-63 cells to differentiate into a more osteoblastic phenotype. A certain order of gene expression exists during the differentiation of osteoblasts (21). ALP expression is elevated mainly after the decrease in proliferation, whereas OC expression is a marker of the most differentiated osteoblast. Co-treatment of MG-63 cells with 1,25-(OH)2D3 and TGF-ß is thought to promote the differentiation of these cells to a more intermediate stage of differentiation and retard their further differentiation (11,30). In these studies, 19-nor-1,25-(OH)2D2 and 1,25-(OH)2D3 were equally potent in increasing both markers of bone formation, ALP and OC, in a time- and dose-dependent manner, indicating that both of these compounds can induce equally the differentiation of MG-63 cells to a mature osteoblastic phenotype.

These in vitro results suggest that 19-nor-1,25-(OH)2D2 may be as active as 1,25-(OH)2D3 in stimulating bone formation in vivo. This is not surprising considering that the affinity of 19-nor-1,25-(OH)2D2 for the VDR is only three times less than that of 1,25-(OH)2D3 (H. F. DeLuca, University of Wisconsin, Madison, WI, personal communication, July 1999) and our findings in the present study showing that in MG-63 cells, 19-nor-1,25-(OH)2D2 can mimic 1,25-(OH)2D3 in its ability to upregulate VDR content.

In contrast to the findings in the present study showing identical potencies of both vitamin D compounds in stimulating the expression of markers of bone formation in osteoblasts, we previously showed 19-nor-1,25-(OH)2D2 to be approximately 10 times less active in promoting bone resorption than 1,25-(OH)2D3 (4). In vitro studies of mouse bone marrow cultures revealed 19-nor-1,25-(OH)2D2 to be 70% less bone resorptive than 1,25-(OH)2D3 (31). Because 1,25-(OH)2D3-induced bone resorption also is mediated through the osteoblastic VDR, the differential effects of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 on bone formation and resorption seem to be gene specific and not cell specific.

Potential mechanisms to explain the disparity between 19-nor-1,25-(OH)2D2 and 1,25-(OH)2D3 regulation of genes involved in bone formation and resorption include intrinsic properties of the D2 and D3 moieties (32,33,34,35,36) or the 19-nor modification, either of which might lead to a different rate of catabolism for 19-nor-1,25-(OH)2D2 compared with 1,25-(OH)2D3 in osteoblasts and altered VDR binding to nuclear co-activators. More rapid catabolism of 19-nor-1,25-(OH)2D2 compared with 1,25-(OH)2D3 could explain the observed differences between bone formation and resorption. There is evidence that the analog 22-oxa-calcitriol, which is cleared rapidly from circulation, produces a transient increase in calcium transport but a prolonged suppression of PTH (37). Although the rate of degradation of 19-nor-1,25-(OH)2D2 in osteoblasts is not known, a faster rate of catabolism, while having little effect on genes that need only a short exposure to the 19-nor-1,25-(OH)2D2-VDR complex, could decrease significantly the transcription of genes that require a longer exposure.

There also is evidence in the literature that vitamin D analogs can bind differently than 1,25-(OH)2D3 to the VDR, producing a conformational change in the VDR molecule that favors the selective recruitment of nuclear transcriptional co-activators and resulting in a variable degree of VDR-mediated transcription (38,39,40,41). The binding of 19-nor-1,25-(OH)2D2 to the VDR could prevent the recruitment of nuclear co-activators required for bone resorption while still being capable of bringing to the transcription initiation complex those co-activators required for genes involved in bone formation.

In summary, these in vitro studies demonstrate that 19-nor-1,25-(OH)2D2 has the same potency as 1,25-(OH)2D3 not only in upregulating the VDR and in controlling osteoblastic growth but also in inducing osteoblastic OC levels and ALP activity. This makes it unlikely that this analog would have deleterious effects on bone remodeling.

The results from this in vitro study, however, should not be completely equated to those expected in vivo. The prolonged administration of vitamin D or its analogs to patients with secondary hyperparathyroidism could result in an excessive decrease in PTH levels and a subsequent reduction in bone resorption and formation leading to adynamic bone disease. In addition, there are instances of adynamic bone disease occurring in patients with elevated PTH values (42) that could result from a resistance to vitamin D therapy. Clearly, an assessment of the effects of prolonged 19-nor-1,25-(OH)2D2 therapy on bone in these patients is mandatory.


   Acknowledgments
 
This work was supported in part by a grant provided by Abbott Pharmaceutical Company and by Research in Renal Diseases, Washington University. The authors express their appreciation to Dr. Alex Brown for valuable suggestions and critical review of this manuscript.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication April 27, 2000. Accepted for publication December 28, 2000.




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