(23S)-25-Dehydro-1-Hydroxyvitamin D3-26,23-Lactone
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内分泌学杂志 2005年第4期
Teijin Institute for Bio-Medical Research (S.I.), Tokyo 191-8512, Japan; Division of Hematology/Oncology (S.I., N.K., S.V.R., G.D.R.), University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Department of Medicine (J.C., T.C.), University of Auckland, Auckland 92019, New Zealand; and Veterans Affairs Pittsburgh Healthcare System (G.D.R.), Pittsburgh, Pennsylvania 15240
Address all correspondence and requests for reprints to: G. David Roodman, M.D., Ph.D., Veterans Affairs Pittsburgh Healthcare System, Research and Development (151-U), University Drive, Pittsburgh, Pennsylvania 15240. E-mail: roodmangd@upmc.edu.
Abstract
Osteoclast (OCL) precursors from patients with Paget’s disease (PD) and normal OCL precursors transduced with the measles virus nucleocapsid protein gene (MVNP) are hyperresponsive to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and can form OCLs at physiologic concentrations of 1,25-(OH)2D3. This hyperresponsivity to 1,25-(OH)2D3 is due to increased expression of TATA box-associated factor II-17, a potential coactivator of the vitamin D receptor. Hyperresponsivity to 1,25-(OH)2D3 may permit OCL formation in PD patients with low levels of 1,25-(OH)2D3 and play a role in the pathogenesis of PD. Therefore, we tested the effects of a vitamin D antagonist, (23S)-25-dehydro-1-hydroxyvitamin D3-26,23-lactone (TEI-9647), to determine its potential to inhibit the enhanced OCL formation and bone resorption seen in patients with PD. TEI-9647, by itself, was not a vitamin D receptor agonist and did not induce OCL formation in vitro, even at 10–6 M. However, it dose-dependently (10–10 M to 10–6 M) inhibited osteoclast formation induced by concentrations of 1,25-(OH)2D3 (41 pg/ml, 10–10 M) detected in PD patients by bone marrow cells of patients with PD and MVNP-transduced colony-forming unit-granulocyte macrophage (CFU-GM) cells, which form pagetic-like OCL. Moreover, bone resorption by OCLs derived from MVNP-transduced CFU-GM treated with 10–9 M 1,25-(OH)2D3 was dose-dependently inhibited by TEI-9647 (10–9 M to 10–6 M). Furthermore, 10–7 M TEI-9647 by itself did not cause 1,25-(OH)2D3-dependent gene expression but almost completely suppressed expression of the TATA box-associated factor II-17 and 25-hydroxyvitamin D3-24-hydroxylase genes induced by 1,25-(OH)2D3 treatment of MVNP-transduced CFU-GM cells. These results demonstrate that TEI-9647 can suppress the excessive bone resorption and OCL formation seen in marrow cultures from patients with PD.
Introduction
OSTEOCLASTS (OCLS) FROM patients with Paget’s disease (PD) of bone are abnormal. Paget’s OCLs are increased in number and size and contain many more nuclei, compared with normal OCLs. OCL precursors from PD patients are hyperresponsive to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and form OCLs at low concentrations of 1,25-(OH)2D3 (1, 2). In addition, intranuclear inclusions are present in pagetic OCLs, which are characteristic of cells chronically infected with paramyxoviruses. This observation has led to the hypothesis that PD results in part from a slow virus infection (3). In previous studies, pagetic OCLs have been shown by immunocytochemistry to express respiratory syncytial virus and measles virus (4). Measles virus transcripts have also been detected by RT-PCR in marrow mononuclear cells in peripheral blood cells from patients with PD (5, 6).
Kurihara et al. (7) reported that normal OCL precursors transduced with the measles virus nucleocapsid gene formed OCLs that expressed many of the abnormal characteristics of pagetic OCLs. Moreover, they found that these cells were hyperresponsive to 1,25-(OH)2D3. Furthermore, the measles virus nucleocapsid gene induced the hyperresponsivity of OCL precursors to 1,25-(OH)2D3 through increased expression of TATA box associated factor II-17 (TAFII-17) (8). TAFII-17 is a potential coactivator of vitamin D receptor (VDR), which is also expressed in pagetic OCL precursors (8). Pagetic OCL precursors require 10–100 times less 1,25-(OH)2D3 to form OCLs than that required for normal bone marrow cells (7, 9, 10). Furthermore, Reddy et al. (11) reported that measles virus infection of mouse bone marrow cells resulted in formation of pagetic-like OCLs. These results suggest that measles virus nucleocapsid protein (MVNP)-transduced normal OCL precursors are a good model of pagetic OCL precursors and that in PD, bone resorption could be enhanced by physiologic levels of 1,25-(OH)2D3.
Recently Miura et al. (12) reported that a 1,25-(OH)2D3 analog, (23S)-25-dehydro-1-hydroxyvitamin D3-26,23-lactone (TEI-9647), inhibited monocyte differentiation of HL-60 cells induced by 1,25-(OH)2D3. TEI-9647 showed significant vitamin D antagonistic activity for 25-hydroxyvitamin D3-24-hydroxylase (25-OH-D3-24-hydroxylase) and p21 gene expression induced by 1,25-(OH)2D3 in HL-60 cells and human osteosarcoma cells (SaOS-2 and MG-63 cells) (12, 13, 14, 15). Moreover, they demonstrated that TEI-9647 prevented heterodimer complex formation between the VDR and retinoid X receptor and subsequent recruitment by VDR of coactivator proteins like steroid receptor coactivator-1 (13, 15). Thus, in this model system, TEI-9647 antagonized the genomic actions of 1,25-(OH)2D3 and interfered with VDR/VDR response element interactions with 1,25-(OH)2D3. However, its effects on OCL formation are unknown. Because the increased osteoclastic bone resorption in PD could in part result from increased OCL formation due to the hyperresponsivity of OCL precursors to 1,25-(OH)2D3, we tested the effects of TEI-9647 on OCL formation, bone resorption, and VDR-mediated transcriptional activity in pagetic and pagetic-like OCL precursors.
Here we report that TEI-9647 inhibited OCL formation and bone resorption by OCL formed by pagetic bone marrow cells and MVNP-transduced normal OCL precursors induced by 1,25-(OH)2D3. TEI-9647 also suppressed TAFII-17 gene expression and decreased TAFII-17 protein level induced by 1,25-(OH)2D3.
Materials and Methods
Chemicals
1,25-(OH)2D3 and the vitamin D antagonist, TEI-9647, were synthesized in our laboratory as described previously (12). Fetal bovine serum (FBS) was purchased from Gibco-BRL (Grand Island, NY). All other chemicals and media were purchased from Sigma Chemical Corp. (St. Louis, MO), unless otherwise noted.
Subjects and cell preparation
Bone marrow cells were aspirated under 2% Xylocaine anesthesia from the iliac crest of healthy normal volunteers or three patients with PD into heparinized MEM containing 5% FBS, as previously described (9). All PD patients had elevated alkaline phosphatase levels and had not received bisphosphonates for at least 3 months before testing. Bone marrow mononuclear cells were then isolated by separation on Hypaque-Ficoll gradients (density 1.077 g/ml), centrifuged at 400 x g for 30 min and then washed three times with MEM, as described previously (9). The Institutional Review Board of the University of Pittsburgh approved these studies.
MVNP gene transduction of human bone marrow cells
Human bone marrow mononuclear cells were cultured for 2 d in MEM containing 10% FBS that contained 10 ng/ml each of IL-3, IL-6, and stem cell factor (Amgen Immunex Research and Development Corp., Seattle, WA). The bone marrow cells were then cultured for an additional 48 h at 37 C in a humidified atmosphere of 5% CO2-air at a density of 1–2 x 105 cells/ml with supernatant (10% vol/vol) containing MVNP vector. Cultures were supplemented with 4 μg/ml polybrene, 20 ng/ml IL-3, 50 ng/ml IL-6, and 100 ng/ml stem cell factor, as described previously (7). MVNP-transduced cells were suspended at 106 cells/ml in MEM containing 1.2% methylcellulose, 30% FBS, 1% deionized BSA, and 100 pg/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Amgen) with 250 μg/ml G418. Transduced cells were plated in a volume of 1 ml in 35-mm culture dishes (Corning, New York, NY) and incubated at 37 C in a humidified atmosphere of 5% CO2-air for 7 d, for isolation of G418 resistance colony-forming unit-granulocyte macrophage (CFU-GM). The G418-resistant colonies were individually collected, using finally drawn pipettes, for use in OCL formation assays employing MVNP-transduced CFU-GM cells.
OCL formation induced by 1,25-(OH)2D3
Normal bone marrow mononuclear cells (106 cells/ml) were dispersed into MEM containing 20% horse serum and were seeded in 96-well multiplates (Becton Dickinson Labware, Frankin Lakes, NJ) at 100 μl/well. 1,25-(OH)2D3 (10–8 M), 10–11 M to 10–6 M TEI-9647, alone or in combination, was each added into a well. Half of the media was replaced two times a week, and the cultures were continued for 3 wk at 37 C in an incubator of 5% CO2-air. The OCLs that formed were then fixed with 2% formaldehyde and tested for cross-reactivity with the monoclonal antibody 23C6, which recognizes the OCL vitronectin receptor (generously provided by Michael Horton, Rayne Institute, Bone and Mineral Center, London, UK), using a Vectastain-ABC-AP kit (Vector Laboratories, Burlingame, CA), as described previously (9). Cells that cross-reacted with the 23C6 antibody and had three or more nuclei were scored as OCLs using an inverted microscope. In the case of MVNP-transduced CFU-GM-derived cells or pagetic bone marrow cells, 10–10 M 1,25-(OH)2D3 was used instead of 10–8 M 1,25-(OH)2D3, which was used for normal bone marrow cells.
Osteoclastic bone resorption on dentin slices
MVNP-transduced CFU-GM cells (2 x 106 cells/ml), as a model for pagetic OCL precursors, or normal marrow cells were dispersed into MEM media containing 20% horse serum and seeded on mammoth dentin slices (Wako Pure Chemical Industries Ltd., Osaka, Japan) in 96-well multiplates at 100 μl/well. Receptor activator of nuclear factor-B ligand (RANKL; 50 ng/ml) and macrophage-CSF (M-CSF; 50 ng/ml; Wako) were added into each well to induce OCL formation. Half of the media containing 20% horse serum, RANKL, and M-CSF was replaced two times a week, and the cultures were continued for 3 wk at 37 C in an atmosphere of 5% CO2-air. After 3 wk, the cells were incubated for 3 d in media lacking RANKL and M-CSF. Then the cultures were treated with 10–9 M or 10–8 M 1,25-(OH)2D3, 10–9 M to 10–6 M TEI-9647, or a combination of both for 10 d. After 10 d, OCLs on the dentin slices were removed by overnight incubation in 0.25% trypsin at 37 C, and the resorption lacunae were stained with hematoxylin. Pit area was quantified by image analysis.
Gene expression induced by 1,25-(OH)2D3 in MVNP-transduced bone marrow cells
MVNP-transduced CFU-GM cells (5 x 105 cells/ml) were cultured in MEM containing 10% FBS with 10–10 M to 10–8 M 1,25-(OH)2D3, 10–10 M to 10–7 M TEI-9647, or a combination of 10–8 M 1,25-(OH)2D3 and 10–10 M to 10–7 M TEI-9647 for 12 h. Total RNA extraction and RT-PCR were carried out as described previously (7). The gene-specific primers for TAFII-17 (GenBank accession no. U57693) were 5'-CATGCCATGGCTATGAACCAGTTTGGCCCCTCA-3' (sense) and 5'-ATACTGC AGTTATTTCTTGGTTGTTTTCCG-3' (antisense). The gene-specific primers for 25-OH-D3-24-hydroxylase (GenBank accession no. L13286) were 5'-ATTACCTGAGAATCAGAGGCCACG-3' (sense) and 5'GCCAAATGCAGTTTAAGCTCTGCT-3' (antisense). The conditions for amplification were as follows: a 5-min initiation step at 94 C; 35 cycles at 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min; and finally an extension step at 72 C for 7 min. PCR products were separated by 2% agarose gel electrophoresis and were revealed with ethidium bromide staining under UV light. The relative amounts of TAFII-17 mRNA and 25-OH-D3-24-hydroxylase mRNA were determined by densitometry and compared with ?-actin mRNA expression levels. The PCRs were performed during the linear phase of the reaction.
Western blot analysis of TAFII-17
To evaluate TAFII-17 protein levels, MVNP-transduced CFU-GM cells were cultured with 10–9 M 1,25-(OH)2D3, 10–7 M TEI-9647, or a combination of 10–9 M 1,25-(OH)2D3 and 10–10 M to 10–7 M TEI-9647 for 4 d, and then cell lysates were prepared as described previously (8). The protein concentration of the lysates was determined by the Bradford method, and the same amount of protein from each lysate was loaded onto SDS-PAGE 12% polyacrylamide gel. The blotted peptides on the gel were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). After blocking with 5% skim milk in Tris-HCl-buffered saline containing 0.1% Tween 20, the membranes were incubated with antirabbit-TAFII-17 antibody (generously provided by Dr. R. G. Roeder, Rockefeller University, New York, NY) at 1:2000 dilution in Tris-HCl-buffered saline containing 0.1% Tween 20 and 1% BSA for 1 h. The blot was incubated for 1 h with horseradish peroxidase-conjugated goat antirabbit IgG (Dako, Carpinteria, CA), and the bands were visualized with an enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL).
Modified mammalian two-hybrid assays
The TAFII-17 cDNA was digested with SmaI and SalI and inserted into the pM vector (pM-TAFII-17) (CLONTECH Laboratories Inc., Palo Alto, CA). The full-length cDNA for human VDR was released by EcoRI digestion from the pSG5 vector and fused in-frame into the pVP16 vector, which contains the activation domain of a herpesvirus (CLONTECH) (pVP16-hVDR) (13). To examine the interaction of TAFII-17 and VDR, 0.5 μg of pM-TAFII-17, 0.5 μg of pVP-16-hVDR, and 0.5 μg of pGVP2-GAL4BS together with 0.25 μg of plasmid containing ?-galactosidase cDNA were transfected into NIH3T3 cells using Lipofectamine (Gibco). Twenty-four hours later, vehicle, 10–8 M 1,25-(OH)2D3, 10–7 M TEI-9647, and a combination of 10–8 M 1,25-(OH)2D3 and 10–7 M TEI-9647 were added. After 24 h of incubation, the luciferase activity of the cell lysates was examined and standardized using ?-galactosidase activity.
Measurement of serum concentrations of vitamin D metabolites calcium, inorganic phosphate and alkaline phosphatase
Serum samples were collected from nine patients with PD and 10 age-matched healthy normal volunteers (3–5 ml/subject). The serum concentrations of vitamin D metabolites were measured as described previously (16). Total serum calcium (Ca), inorganic phosphate (Pi), and alkaline phosphatase (ALPase) were determined using commercially available kits (calcium C test, inorganic phosphorus P test, and ALPase B test, Wako).
Statistical analysis
Significance was evaluated using a two-sided, unpaired Student’s t test, with P < 0.01 considered significant.
Results
Serum concentrations of vitamin D metabolites in patients with PD
Our hypothesis is that increased OCL formation and bone resorption in PD is due in part to the hyperresponsivity of pagetic OCL precursors to 1,25-(OH)2D3. This hyperresponsivity to 1,25-(OH)2D3 results in OCL formation at physiologic levels of 1,25-(OH)2D3, something that occurs at very low levels in normals. However, measurements of 1,25-(OH)2D3 levels in PD patients have only rarely been reported and are conflicting (17, 18). Therefore, we determined the concentrations of vitamin D metabolites, Ca, and Pi in the serum of nine patients with PD and 10 age-matched normal healthy volunteers. The concentrations of vitamin D metabolites, 25-OH-D, 24,25-(OH)2D, and 1,25-(OH)2D, Ca, and Pi in the serum of patients with PD were very similar to their concentrations in the serum of age-matched normal healthy volunteers. No abnormality of vitamin D metabolism was detected in patients with PD (Table 1). In particular, the concentrations of 1,25-(OH)2D3 in the serum of patients with PD were 41.0 + 9.1 pg/ml serum (10–10 M) and were the same as that of age-matched normal healthy volunteers. ALPase activity in the serum of patients with PD was significantly higher than that of age-matched normal healthy volunteers (data not shown).
TABLE 1. Serum concentrations of vitamin D metabolites, calcium, and inorganic phosphate in patients with Paget’s disease and age-matched normal volunteers
Effects of TEI-9647 on OCL formation
We then confirmed our previous results that these levels of 1,25-(OH)2D3 could induce OCL formation in marrow cultures from PD patients and MVNP-transduced normal OCL precursors. Using bone marrow cells from patients with PD or MVNP-transduced CFU-GM, 10–11 M to 10–9 M 1,25-(OH)2D3 dose-dependently stimulated OCL formation. OCL formation in these cultures was significantly increased above control levels at 10–11 M 1,25-(OH)2D3 and reached maximum levels at 10–9 M 1,25-(OH)2D3. In contrast, although 1,25-(OH)2D3 10–10 M to 10–7 M dose-dependently stimulated OCL formation in normal marrow cultures, it was not significantly increased until 10–9 M 1,25-(OH)2D3 was added to the cultures and reached maximum levels at 10–7 M 1,25-(OH)2D3 (Fig. 1, A–C).
FIG. 1. Effect of TEI-9647 on OCL formation by normal bone marrow cells treated with 1,25-(OH)2D3 (A), patients with PD (B), and MVNP-transduced CFU-GM cells (C). Bone marrow mononuclear cells were dispersed into MEM containing 20% horse serum, and 105 cells were seeded in 96-well plates. The cultures were continued for 3 wk with vitamin D3 analogs (i.e. 10–11 M to 10–7 M 1,25-(OH)2D3, 10–11 M to 10–6 M TEI-9647, or a combination of 10–11 M to 10–6 M TEI-9647 and 1,25-(OH)2D3 (10–8 M in normal cells, 10–10 M in PD and MVNP-transduced CFU-GM cells). The media were replaced two times a week. Multinucleated cells that cross-reacted with the 23C6 antibody and had three or more nuclei were scored an OCLs. Data are expressed as the mean ± SD (n = 4). *, Significantly different from control cultures (media alone), P < 0.01; **, significantly different from cultures treated with 10–8 M 1,25-(OH)2D3, P < 0.01 (A); ***, significantly different from cultures treated with 10–10 M 1,25-(OH)2D3 treatment, P < 0.01 (B and C). MNC, Multinuclear cells.
We then tested the effects of TEI-9647 on OCL formation induced by 1,25-(OH)2D3 in bone marrow cell cultures from normal volunteers, patients with PD, or MVNP-transduced CFU-GM. TEI-9647 did not stimulate OCL formation in any of the cultures, even at high concentrations (10–6 M). In contrast, TEI-9647 dose-dependently blocked OCL formation induced by 10–8 M 1,25-(OH)2D3 in normal marrow cultures, which was significant at 10–9 M TEI-9647 or higher. TEI-9647 also dose-dependently blocked OCL formation induced by 10–10 M 1,25-(OH)2D3 in pagetic marrow cultures. TEI-9647 (10–10 M) significantly inhibited OCL formation induced by 10–10 M 1,25-(OH)2D3. TEI-9647 (10–6 M) completely blocked OCL formation (Fig. 1B). TEI-9647 also inhibited OCL formation by MVNP-transduced CFU-GM cells in a similar fashion as pagetic marrow cultures (Fig. 1C).
Effects of TEI-9647 on osteoclastic bone resorption
To determine whether TEI-9647 inhibits VDR-mediated osteoclastic bone resorption by pagetic-like OCLs, we examined the effects of TEI-9647 on bone resorption by OCLs that were formed on dentin slices by MVNP-transduced CFU-GM cells treated with RANKL and M-CSF and then activated by 10–9 M 1,25-(OH)2D3. Large OCLs were present with 10–9 M 1,25-(OH)2D3 treatment, compared with vehicle-treated cells (data not shown). Treatment with TEI-9647 did not change the number of OCLs on the dentin, compared with treatment with 10–9 M 1,25-(OH)2D3 (Fig. 2). Pit formation activated by 10–9 M 1,25-(OH)2D3 is shown in Fig. 2B. 1,25-(OH)2D3 (10–9 M) markedly enhanced osteoclastic bone resorption. TEI-9647 (10–9 M to 10–6 M) dose-dependently blocked osteoclastic bone resorption induced by 10–9 M 1,25-(OH)2D3, and 10–7 M TEI-9647 almost completely blocked bone resorption. The area resorbed by the OCLs was quantified by image analysis and is shown in Fig. 2C.
FIG. 2. Effects of TEI-9647 on bone resorption induced by 1,25-(OH)2D3. MVNP-transduced CFU-GM cells were dispersed into MEM containing 20% horse serum, and 105 cells were seeded in 96-well plate. RANKL (50 ng/ml) and M-CSF (50 ng/ml) were added into a well as stimulators of OCL formation. The cultures were continued for 3 wk. The media containing 20% horse serum, RANKL, and M-CSF were replaced two times per week. After 3 wk, media lacking RANKL and M-CSF were added and the cells incubated for 3 d. The OCLs that formed were then activated by the treatment with 10–9 M 1,25-(OH)2D3, 10–9 M to 10–6 M TEI-9647, or a combination of both for 10 d. After 10 d, OCLs on the dentin slices were stained for tartrate-resistant acid phosphatase (TRAP). A, Number of OCLs present on dentin slices. Data are expressed as the mean ± SD (n = 4). *, Significantly different from control cultures (media alone), P < 0.01. The OCLs were released by incubation with trypsin overnight, and the resorption lacunae were stained with hematoxylin. Pit area was quantified with an image analyzer. B, Pits on dentin slice. C, Pit area. Data are expressed as the mean ± SD (n = 4). *, Significantly different from control cultures (media alone), P < 0.01. MNC, Multinuclear cells.
Effect of TEI-9647 on TAFII-17 and 25-OH-D3-24-hydroxylase gene expression and TAFII-17 protein levels
We then performed time-course and dose-response experiments to assess the antagonistic actions of TEI-9647 on VDR-mediated gene transcription using MVNP-transduced CFU-GM cells as a model for pagetic OCL precursors. TEI-9647 (10–7 M) did not induce expression of the TAFII-17 and 25-OH-D3-24-hydroxylase genes but almost completely suppressed their expression induced by exposure to 10–9 M 1,25-(OH)2D3 for 6 and 12 h. TAFII-17 and 25-OH-D3-24-hydroxylase gene expression reappeared by 24 h after treatment with TEI-9647 (data not shown), demonstrating that TEI-9647 was not toxic to the cells. In dose-response experiments, at 12 h after treatment with 1,25-(OH)2D3 and TEI-9647, 10–10 M to 10–8 M 1,25-(OH)2D3 but not TEI-9647 dose-dependently increased TAFII-17 and 25-OH-D3-24-hydroxylase gene expression. As previously reported, TAFII-17 was expressed in the absence of added 1,25-(OH)2D3 in MVNP-transduced CFU-GM cells, but 25-OH-D3-24-hydroxylase was not (Fig. 3A). TEI-9647 dose-dependently suppressed TAFII-17 and 25-OH-D3-24-hydroxylase gene expression induced by 10–9 M 1,25-(OH)2D3, and 10–8 M TEI-9647 completely suppressed TAFII-17 and 25-OH-D3-24-hydroxylase gene expression. The relative effects of TEI-9647 on TAFII-17, and 25-OH-D3-24-hydroxylase mRNA expression in three independent experiments are shown in Fig. 3B.
FIG. 3. Effect of TEI-9647 on TAFII-17 gene expression and TAFII-17 protein levels. A, Effect of TEI-9647 on TAFII-17 and 25-OH-D3-24-hydroxylase gene expression induced by 1,25-(OH)2D3 in MVNP-transduced CFU-GM-cells. MVNP-transduced CFU-GM cells were cultured in MEM containing 10% FBS for 12 h with 10–10 M to 10–8 M 1,25-(OH)2D3, 10–7 M TEI-9647, or a combination of 10–9 M 1,25-(OH)2D3 and 10–10 M to 10–7 M TEI-9647. Total RNA was extracted and RT-PCR was carried out. PCR products were separated by 2% agarose gel electrophoresis and were revealed with ethidium bromide staining as described in Materials and Methods. Cont, Control (nontreated, media alone). B, Densitometric analysis of three independent PCR experiments examining the effects of TEI-9647 on TAFII-17 and 25-OH-D3-24-hydroxylase mRNA levels. C, Western blot analysis of TAFII-17 protein. To evaluate TAFII-17 protein levels, MVNP-transduced CFU-GM cells were cultured with 1,25-(OH)2D3, TEI-9647, or a combination of 1,25-(OH)2D3 and TEI-9647 for 4 d, and then cell lysates were prepared. Western blot analysis of TAFII-17 protein in the cell lysates was carried out as described in Materials and Methods. Seventeen- and 34-kDa bands both reacted with the anti-TAFII-17. The 34-kDa is a dimer of TAFII-17. D, Densitometric analysis of three independent Western blot analyses for the effects of TEI-9617 on TAFII-17 expression.
To determine whether TEI-9647 decreased protein levels of TAFII-17 induced by 1,25-(OH)2D3, we measured the relative levels of TAFII-17 in MVNP-transduced CFU-GM cells by Western blot analysis as shown in Fig. 3C. MVNP-transduced CFU-GM cells produced a 17- and 34-kDa protein, which reacted with TAFII-17 antibody in the absence of 1,25-(OH)2D3. The 34-kDa band is most likely a dimer of TAFII--17. Low concentration of 1,25-(OH)2D3 (10–9 M) increased the levels of TAFII-17. In contrast, 10–10 M to 10–7 M TEI-9647 dose-dependently decreased the levels of TAFII-17 enhanced by 10–9 M 1,25-(OH)2D3.
Effect of TEI-9647 on the interaction between VDR and TAFII-17
To confirm the role of TAFII-17 in VDR-mediated gene transcription, we investigated the interaction of VDR with TAFII-17. Protein-protein interaction between VDR and TAFII-17 was examined using the mammalian two-hybrid system. An expression vector in which human TAFII-17 was fused to GAL4DBD (pM-TAFII-17) was used as the bait construct, and the human VDR (pVP16-hVDR) was used as the prey vector (Fig. 4A). VDR-mediated gene transcription was induced when 10–8 M 1,25-(OH)2D3 was added with the VDR and TAFII-17 constructs, but it was significantly suppressed by treatment with 10–7 M TEI-9647 (Fig. 4B).
FIG. 4. Effect of TEI-9647 on the interaction between VDR and TAFII-17. A, Modified mammalian two-hybrid assay was used to examine the interaction between VDR and TAFII-17. The TAFII-17 cDNA was inserted at the EcoRI site of the pM vector, which contains a GAL4 DNA-binding domain (pM-TAFII-17). B, The interaction of VDR and TAFII-17 was examined by measuring the reporter activity in NIH3T3 cells transduced with pM-TAFII-17, pVP16-hVDR, and the reporter plasmid containing the GAL4 DNA-binding sites (pGVP2-GAL4BS). GAL4 protein is a transcriptional activator that binds to DNA. 1,25-(OH)2D3 (10–8 M), TEI-9647 (10–7 M), or both were added after transfection. After 24 h of incubation, the luciferase activity of the cell lysates was determined as described in Materials and Methods. Results are expressed as the mean ± SEM. *, P < 0.01, compared with cells treated with 10–8 M 1,25-(OH)2D3. Similar results were obtained in three independent experiments. No reporter activity was seen in control incubations lacking TAFII-17 or VDR constructs.
Discussion
We and others previously reported that OCL activity is markedly increased in patients with PD and that OCL formation is abnormal (9, 19). OCL precursors from patients with PD have increased sensitivity to 1,25-(OH)2D3 and can form OCLs at physiologic levels of 1,25-(OH)2D3, something that normal OCL precursors cannot do. As shown in Table 1, patients with PD have levels of 1,25-(OH)2D3 in their serum that do not differ from normals and are sufficient to induce osteoclastogenesis. These findings agree with previous work by Devlin et al. (17) and Foldes et al. (18), who showed similar levels of 1,25-(OH)2D3 in PD patients and normals. In addition, as shown in Fig. 2, C and D, these levels of 1,25-(OH)2D3 can enhance the bone resorption capacity of preformed OCLs that are similar to pagetic OCLs.
Therefore, we tested TEI-9647, a vitamin D analog that can block the effects of 1,25-(OH)2D3 on hematopoietic cell differentiation, for its capacity to block OCL formation in vitro by marrow samples from patients with PD and MVNP-transduced normal OCL precursors, as a surrogate model of pagetic OCL precursors. We previously reported that MVNP-transduced osteoclast precursors are very similar to pagetic osteoclasts precursors (7). As shown in Figure 1, 1,25-(OH)2D3 (10–11 M) induced high levels of OCL formation in PD cultures but minimally affected OCL formation in normal marrow cultures. TEI-9647 dose-dependently inhibited osteoclast formation in both normal and pagetic marrow cultures. OCL formation in PD cultures was more sensitive to TEI-9647. TEI-9647 (10–11 to 10–9 M) decreased OCL formation approximately 3-fold in PD cultures, whereas it decreased normal OCL formation only approximately 2-fold. Of interest, TEI-9647 also decreased basal OCL formation in both normal and pagetic marrow cultures. This decrease in basal OCL formation was not due to toxicity of TEI-9647 for hematopoietic cells. We have shown in preliminary studies that TEI-9647 does not block the growth of hematopoietic precursor cells such as CFU-GM and in the current study reversibly inhibited VDR-mediated gene transcription (Fig. 3A). Furthermore, TEI-9647 was not toxic to preformed OCL because OCL numbers were unchanged when OCLs were treated with TEI-9647 (Fig. 2B). Both 1,25-(OH)2D3-stimulated and basal OCL formation were not significantly inhibited in normal marrow cultures until the TEI-9647 concentration was 10–9 M, whereas pagetic marrow cultures were significantly inhibited by 10–10 M TEI-9647. TEI-9647 (10–8 M) decreased OCL formation almost to basal levels in marrow cultures from patients with PD who were treated with physiologic levels of 1,25-(OH)2D3 (10–10 M) (Fig. 1B). These data demonstrate that TEI-9647 inhibited OCL formation induced by 1,25-(OH)2D3 and that basal osteoclastogenesis in marrow cultures may in part be mediated by the low levels of 1,25-(OH)2D3 present in the horse serum in these cultures.
TEI-9647 also blocked VDR-mediated gene transcription. As shown in Fig. 3A, TEI-9647 markedly decreased TAFII-17 expression induced by 1,25-(OH)2D3 both at the mRNA and protein level. Furthermore, mammalian two hybrid assays demonstrated that TEI-9647 blocked the interaction between TAFII-17 and VDR. These results suggest that TEI-9647 or similar agents might in addition to decreasing OCL formation and activity could also affect the sensitivity of these precursors to 1,25-(OH)2D3. Kurihara et al. (8) reported that increased expression of TAFII-17 is in part responsible for the hypersensitivity of pagetic OCL precursors to 1,25-(OH)2D3. They showed that transfecting TAFII-17 into normal OCL precursors results in increased sensitivity of these precursors to 1,25-(OH)2D3. Furthermore, an antisense to TAFII-17 blocked OCL formation by pagetic osteoclast precursors induced by low levels of 1,25-(OH)2D3. TEI-9647 suppresses expression of TAFII-17 in MVNP-transduced normal OCL precursors, which have many of the characteristics of pagetic OCL, including hypersensitivity to 1,25-(OH)2D3 and high levels of TAFII-17. These results suggest that TEI-9647 may be a drug that can both block the enhanced OCL formation and bone resorption in patients with PD in response to 1,25-(OH)2D3 and possibility change the sensitivity of pagetic OCL precursors to 1,25-(OH)2D3.
References
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Address all correspondence and requests for reprints to: G. David Roodman, M.D., Ph.D., Veterans Affairs Pittsburgh Healthcare System, Research and Development (151-U), University Drive, Pittsburgh, Pennsylvania 15240. E-mail: roodmangd@upmc.edu.
Abstract
Osteoclast (OCL) precursors from patients with Paget’s disease (PD) and normal OCL precursors transduced with the measles virus nucleocapsid protein gene (MVNP) are hyperresponsive to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and can form OCLs at physiologic concentrations of 1,25-(OH)2D3. This hyperresponsivity to 1,25-(OH)2D3 is due to increased expression of TATA box-associated factor II-17, a potential coactivator of the vitamin D receptor. Hyperresponsivity to 1,25-(OH)2D3 may permit OCL formation in PD patients with low levels of 1,25-(OH)2D3 and play a role in the pathogenesis of PD. Therefore, we tested the effects of a vitamin D antagonist, (23S)-25-dehydro-1-hydroxyvitamin D3-26,23-lactone (TEI-9647), to determine its potential to inhibit the enhanced OCL formation and bone resorption seen in patients with PD. TEI-9647, by itself, was not a vitamin D receptor agonist and did not induce OCL formation in vitro, even at 10–6 M. However, it dose-dependently (10–10 M to 10–6 M) inhibited osteoclast formation induced by concentrations of 1,25-(OH)2D3 (41 pg/ml, 10–10 M) detected in PD patients by bone marrow cells of patients with PD and MVNP-transduced colony-forming unit-granulocyte macrophage (CFU-GM) cells, which form pagetic-like OCL. Moreover, bone resorption by OCLs derived from MVNP-transduced CFU-GM treated with 10–9 M 1,25-(OH)2D3 was dose-dependently inhibited by TEI-9647 (10–9 M to 10–6 M). Furthermore, 10–7 M TEI-9647 by itself did not cause 1,25-(OH)2D3-dependent gene expression but almost completely suppressed expression of the TATA box-associated factor II-17 and 25-hydroxyvitamin D3-24-hydroxylase genes induced by 1,25-(OH)2D3 treatment of MVNP-transduced CFU-GM cells. These results demonstrate that TEI-9647 can suppress the excessive bone resorption and OCL formation seen in marrow cultures from patients with PD.
Introduction
OSTEOCLASTS (OCLS) FROM patients with Paget’s disease (PD) of bone are abnormal. Paget’s OCLs are increased in number and size and contain many more nuclei, compared with normal OCLs. OCL precursors from PD patients are hyperresponsive to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and form OCLs at low concentrations of 1,25-(OH)2D3 (1, 2). In addition, intranuclear inclusions are present in pagetic OCLs, which are characteristic of cells chronically infected with paramyxoviruses. This observation has led to the hypothesis that PD results in part from a slow virus infection (3). In previous studies, pagetic OCLs have been shown by immunocytochemistry to express respiratory syncytial virus and measles virus (4). Measles virus transcripts have also been detected by RT-PCR in marrow mononuclear cells in peripheral blood cells from patients with PD (5, 6).
Kurihara et al. (7) reported that normal OCL precursors transduced with the measles virus nucleocapsid gene formed OCLs that expressed many of the abnormal characteristics of pagetic OCLs. Moreover, they found that these cells were hyperresponsive to 1,25-(OH)2D3. Furthermore, the measles virus nucleocapsid gene induced the hyperresponsivity of OCL precursors to 1,25-(OH)2D3 through increased expression of TATA box associated factor II-17 (TAFII-17) (8). TAFII-17 is a potential coactivator of vitamin D receptor (VDR), which is also expressed in pagetic OCL precursors (8). Pagetic OCL precursors require 10–100 times less 1,25-(OH)2D3 to form OCLs than that required for normal bone marrow cells (7, 9, 10). Furthermore, Reddy et al. (11) reported that measles virus infection of mouse bone marrow cells resulted in formation of pagetic-like OCLs. These results suggest that measles virus nucleocapsid protein (MVNP)-transduced normal OCL precursors are a good model of pagetic OCL precursors and that in PD, bone resorption could be enhanced by physiologic levels of 1,25-(OH)2D3.
Recently Miura et al. (12) reported that a 1,25-(OH)2D3 analog, (23S)-25-dehydro-1-hydroxyvitamin D3-26,23-lactone (TEI-9647), inhibited monocyte differentiation of HL-60 cells induced by 1,25-(OH)2D3. TEI-9647 showed significant vitamin D antagonistic activity for 25-hydroxyvitamin D3-24-hydroxylase (25-OH-D3-24-hydroxylase) and p21 gene expression induced by 1,25-(OH)2D3 in HL-60 cells and human osteosarcoma cells (SaOS-2 and MG-63 cells) (12, 13, 14, 15). Moreover, they demonstrated that TEI-9647 prevented heterodimer complex formation between the VDR and retinoid X receptor and subsequent recruitment by VDR of coactivator proteins like steroid receptor coactivator-1 (13, 15). Thus, in this model system, TEI-9647 antagonized the genomic actions of 1,25-(OH)2D3 and interfered with VDR/VDR response element interactions with 1,25-(OH)2D3. However, its effects on OCL formation are unknown. Because the increased osteoclastic bone resorption in PD could in part result from increased OCL formation due to the hyperresponsivity of OCL precursors to 1,25-(OH)2D3, we tested the effects of TEI-9647 on OCL formation, bone resorption, and VDR-mediated transcriptional activity in pagetic and pagetic-like OCL precursors.
Here we report that TEI-9647 inhibited OCL formation and bone resorption by OCL formed by pagetic bone marrow cells and MVNP-transduced normal OCL precursors induced by 1,25-(OH)2D3. TEI-9647 also suppressed TAFII-17 gene expression and decreased TAFII-17 protein level induced by 1,25-(OH)2D3.
Materials and Methods
Chemicals
1,25-(OH)2D3 and the vitamin D antagonist, TEI-9647, were synthesized in our laboratory as described previously (12). Fetal bovine serum (FBS) was purchased from Gibco-BRL (Grand Island, NY). All other chemicals and media were purchased from Sigma Chemical Corp. (St. Louis, MO), unless otherwise noted.
Subjects and cell preparation
Bone marrow cells were aspirated under 2% Xylocaine anesthesia from the iliac crest of healthy normal volunteers or three patients with PD into heparinized MEM containing 5% FBS, as previously described (9). All PD patients had elevated alkaline phosphatase levels and had not received bisphosphonates for at least 3 months before testing. Bone marrow mononuclear cells were then isolated by separation on Hypaque-Ficoll gradients (density 1.077 g/ml), centrifuged at 400 x g for 30 min and then washed three times with MEM, as described previously (9). The Institutional Review Board of the University of Pittsburgh approved these studies.
MVNP gene transduction of human bone marrow cells
Human bone marrow mononuclear cells were cultured for 2 d in MEM containing 10% FBS that contained 10 ng/ml each of IL-3, IL-6, and stem cell factor (Amgen Immunex Research and Development Corp., Seattle, WA). The bone marrow cells were then cultured for an additional 48 h at 37 C in a humidified atmosphere of 5% CO2-air at a density of 1–2 x 105 cells/ml with supernatant (10% vol/vol) containing MVNP vector. Cultures were supplemented with 4 μg/ml polybrene, 20 ng/ml IL-3, 50 ng/ml IL-6, and 100 ng/ml stem cell factor, as described previously (7). MVNP-transduced cells were suspended at 106 cells/ml in MEM containing 1.2% methylcellulose, 30% FBS, 1% deionized BSA, and 100 pg/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Amgen) with 250 μg/ml G418. Transduced cells were plated in a volume of 1 ml in 35-mm culture dishes (Corning, New York, NY) and incubated at 37 C in a humidified atmosphere of 5% CO2-air for 7 d, for isolation of G418 resistance colony-forming unit-granulocyte macrophage (CFU-GM). The G418-resistant colonies were individually collected, using finally drawn pipettes, for use in OCL formation assays employing MVNP-transduced CFU-GM cells.
OCL formation induced by 1,25-(OH)2D3
Normal bone marrow mononuclear cells (106 cells/ml) were dispersed into MEM containing 20% horse serum and were seeded in 96-well multiplates (Becton Dickinson Labware, Frankin Lakes, NJ) at 100 μl/well. 1,25-(OH)2D3 (10–8 M), 10–11 M to 10–6 M TEI-9647, alone or in combination, was each added into a well. Half of the media was replaced two times a week, and the cultures were continued for 3 wk at 37 C in an incubator of 5% CO2-air. The OCLs that formed were then fixed with 2% formaldehyde and tested for cross-reactivity with the monoclonal antibody 23C6, which recognizes the OCL vitronectin receptor (generously provided by Michael Horton, Rayne Institute, Bone and Mineral Center, London, UK), using a Vectastain-ABC-AP kit (Vector Laboratories, Burlingame, CA), as described previously (9). Cells that cross-reacted with the 23C6 antibody and had three or more nuclei were scored as OCLs using an inverted microscope. In the case of MVNP-transduced CFU-GM-derived cells or pagetic bone marrow cells, 10–10 M 1,25-(OH)2D3 was used instead of 10–8 M 1,25-(OH)2D3, which was used for normal bone marrow cells.
Osteoclastic bone resorption on dentin slices
MVNP-transduced CFU-GM cells (2 x 106 cells/ml), as a model for pagetic OCL precursors, or normal marrow cells were dispersed into MEM media containing 20% horse serum and seeded on mammoth dentin slices (Wako Pure Chemical Industries Ltd., Osaka, Japan) in 96-well multiplates at 100 μl/well. Receptor activator of nuclear factor-B ligand (RANKL; 50 ng/ml) and macrophage-CSF (M-CSF; 50 ng/ml; Wako) were added into each well to induce OCL formation. Half of the media containing 20% horse serum, RANKL, and M-CSF was replaced two times a week, and the cultures were continued for 3 wk at 37 C in an atmosphere of 5% CO2-air. After 3 wk, the cells were incubated for 3 d in media lacking RANKL and M-CSF. Then the cultures were treated with 10–9 M or 10–8 M 1,25-(OH)2D3, 10–9 M to 10–6 M TEI-9647, or a combination of both for 10 d. After 10 d, OCLs on the dentin slices were removed by overnight incubation in 0.25% trypsin at 37 C, and the resorption lacunae were stained with hematoxylin. Pit area was quantified by image analysis.
Gene expression induced by 1,25-(OH)2D3 in MVNP-transduced bone marrow cells
MVNP-transduced CFU-GM cells (5 x 105 cells/ml) were cultured in MEM containing 10% FBS with 10–10 M to 10–8 M 1,25-(OH)2D3, 10–10 M to 10–7 M TEI-9647, or a combination of 10–8 M 1,25-(OH)2D3 and 10–10 M to 10–7 M TEI-9647 for 12 h. Total RNA extraction and RT-PCR were carried out as described previously (7). The gene-specific primers for TAFII-17 (GenBank accession no. U57693) were 5'-CATGCCATGGCTATGAACCAGTTTGGCCCCTCA-3' (sense) and 5'-ATACTGC AGTTATTTCTTGGTTGTTTTCCG-3' (antisense). The gene-specific primers for 25-OH-D3-24-hydroxylase (GenBank accession no. L13286) were 5'-ATTACCTGAGAATCAGAGGCCACG-3' (sense) and 5'GCCAAATGCAGTTTAAGCTCTGCT-3' (antisense). The conditions for amplification were as follows: a 5-min initiation step at 94 C; 35 cycles at 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min; and finally an extension step at 72 C for 7 min. PCR products were separated by 2% agarose gel electrophoresis and were revealed with ethidium bromide staining under UV light. The relative amounts of TAFII-17 mRNA and 25-OH-D3-24-hydroxylase mRNA were determined by densitometry and compared with ?-actin mRNA expression levels. The PCRs were performed during the linear phase of the reaction.
Western blot analysis of TAFII-17
To evaluate TAFII-17 protein levels, MVNP-transduced CFU-GM cells were cultured with 10–9 M 1,25-(OH)2D3, 10–7 M TEI-9647, or a combination of 10–9 M 1,25-(OH)2D3 and 10–10 M to 10–7 M TEI-9647 for 4 d, and then cell lysates were prepared as described previously (8). The protein concentration of the lysates was determined by the Bradford method, and the same amount of protein from each lysate was loaded onto SDS-PAGE 12% polyacrylamide gel. The blotted peptides on the gel were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). After blocking with 5% skim milk in Tris-HCl-buffered saline containing 0.1% Tween 20, the membranes were incubated with antirabbit-TAFII-17 antibody (generously provided by Dr. R. G. Roeder, Rockefeller University, New York, NY) at 1:2000 dilution in Tris-HCl-buffered saline containing 0.1% Tween 20 and 1% BSA for 1 h. The blot was incubated for 1 h with horseradish peroxidase-conjugated goat antirabbit IgG (Dako, Carpinteria, CA), and the bands were visualized with an enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL).
Modified mammalian two-hybrid assays
The TAFII-17 cDNA was digested with SmaI and SalI and inserted into the pM vector (pM-TAFII-17) (CLONTECH Laboratories Inc., Palo Alto, CA). The full-length cDNA for human VDR was released by EcoRI digestion from the pSG5 vector and fused in-frame into the pVP16 vector, which contains the activation domain of a herpesvirus (CLONTECH) (pVP16-hVDR) (13). To examine the interaction of TAFII-17 and VDR, 0.5 μg of pM-TAFII-17, 0.5 μg of pVP-16-hVDR, and 0.5 μg of pGVP2-GAL4BS together with 0.25 μg of plasmid containing ?-galactosidase cDNA were transfected into NIH3T3 cells using Lipofectamine (Gibco). Twenty-four hours later, vehicle, 10–8 M 1,25-(OH)2D3, 10–7 M TEI-9647, and a combination of 10–8 M 1,25-(OH)2D3 and 10–7 M TEI-9647 were added. After 24 h of incubation, the luciferase activity of the cell lysates was examined and standardized using ?-galactosidase activity.
Measurement of serum concentrations of vitamin D metabolites calcium, inorganic phosphate and alkaline phosphatase
Serum samples were collected from nine patients with PD and 10 age-matched healthy normal volunteers (3–5 ml/subject). The serum concentrations of vitamin D metabolites were measured as described previously (16). Total serum calcium (Ca), inorganic phosphate (Pi), and alkaline phosphatase (ALPase) were determined using commercially available kits (calcium C test, inorganic phosphorus P test, and ALPase B test, Wako).
Statistical analysis
Significance was evaluated using a two-sided, unpaired Student’s t test, with P < 0.01 considered significant.
Results
Serum concentrations of vitamin D metabolites in patients with PD
Our hypothesis is that increased OCL formation and bone resorption in PD is due in part to the hyperresponsivity of pagetic OCL precursors to 1,25-(OH)2D3. This hyperresponsivity to 1,25-(OH)2D3 results in OCL formation at physiologic levels of 1,25-(OH)2D3, something that occurs at very low levels in normals. However, measurements of 1,25-(OH)2D3 levels in PD patients have only rarely been reported and are conflicting (17, 18). Therefore, we determined the concentrations of vitamin D metabolites, Ca, and Pi in the serum of nine patients with PD and 10 age-matched normal healthy volunteers. The concentrations of vitamin D metabolites, 25-OH-D, 24,25-(OH)2D, and 1,25-(OH)2D, Ca, and Pi in the serum of patients with PD were very similar to their concentrations in the serum of age-matched normal healthy volunteers. No abnormality of vitamin D metabolism was detected in patients with PD (Table 1). In particular, the concentrations of 1,25-(OH)2D3 in the serum of patients with PD were 41.0 + 9.1 pg/ml serum (10–10 M) and were the same as that of age-matched normal healthy volunteers. ALPase activity in the serum of patients with PD was significantly higher than that of age-matched normal healthy volunteers (data not shown).
TABLE 1. Serum concentrations of vitamin D metabolites, calcium, and inorganic phosphate in patients with Paget’s disease and age-matched normal volunteers
Effects of TEI-9647 on OCL formation
We then confirmed our previous results that these levels of 1,25-(OH)2D3 could induce OCL formation in marrow cultures from PD patients and MVNP-transduced normal OCL precursors. Using bone marrow cells from patients with PD or MVNP-transduced CFU-GM, 10–11 M to 10–9 M 1,25-(OH)2D3 dose-dependently stimulated OCL formation. OCL formation in these cultures was significantly increased above control levels at 10–11 M 1,25-(OH)2D3 and reached maximum levels at 10–9 M 1,25-(OH)2D3. In contrast, although 1,25-(OH)2D3 10–10 M to 10–7 M dose-dependently stimulated OCL formation in normal marrow cultures, it was not significantly increased until 10–9 M 1,25-(OH)2D3 was added to the cultures and reached maximum levels at 10–7 M 1,25-(OH)2D3 (Fig. 1, A–C).
FIG. 1. Effect of TEI-9647 on OCL formation by normal bone marrow cells treated with 1,25-(OH)2D3 (A), patients with PD (B), and MVNP-transduced CFU-GM cells (C). Bone marrow mononuclear cells were dispersed into MEM containing 20% horse serum, and 105 cells were seeded in 96-well plates. The cultures were continued for 3 wk with vitamin D3 analogs (i.e. 10–11 M to 10–7 M 1,25-(OH)2D3, 10–11 M to 10–6 M TEI-9647, or a combination of 10–11 M to 10–6 M TEI-9647 and 1,25-(OH)2D3 (10–8 M in normal cells, 10–10 M in PD and MVNP-transduced CFU-GM cells). The media were replaced two times a week. Multinucleated cells that cross-reacted with the 23C6 antibody and had three or more nuclei were scored an OCLs. Data are expressed as the mean ± SD (n = 4). *, Significantly different from control cultures (media alone), P < 0.01; **, significantly different from cultures treated with 10–8 M 1,25-(OH)2D3, P < 0.01 (A); ***, significantly different from cultures treated with 10–10 M 1,25-(OH)2D3 treatment, P < 0.01 (B and C). MNC, Multinuclear cells.
We then tested the effects of TEI-9647 on OCL formation induced by 1,25-(OH)2D3 in bone marrow cell cultures from normal volunteers, patients with PD, or MVNP-transduced CFU-GM. TEI-9647 did not stimulate OCL formation in any of the cultures, even at high concentrations (10–6 M). In contrast, TEI-9647 dose-dependently blocked OCL formation induced by 10–8 M 1,25-(OH)2D3 in normal marrow cultures, which was significant at 10–9 M TEI-9647 or higher. TEI-9647 also dose-dependently blocked OCL formation induced by 10–10 M 1,25-(OH)2D3 in pagetic marrow cultures. TEI-9647 (10–10 M) significantly inhibited OCL formation induced by 10–10 M 1,25-(OH)2D3. TEI-9647 (10–6 M) completely blocked OCL formation (Fig. 1B). TEI-9647 also inhibited OCL formation by MVNP-transduced CFU-GM cells in a similar fashion as pagetic marrow cultures (Fig. 1C).
Effects of TEI-9647 on osteoclastic bone resorption
To determine whether TEI-9647 inhibits VDR-mediated osteoclastic bone resorption by pagetic-like OCLs, we examined the effects of TEI-9647 on bone resorption by OCLs that were formed on dentin slices by MVNP-transduced CFU-GM cells treated with RANKL and M-CSF and then activated by 10–9 M 1,25-(OH)2D3. Large OCLs were present with 10–9 M 1,25-(OH)2D3 treatment, compared with vehicle-treated cells (data not shown). Treatment with TEI-9647 did not change the number of OCLs on the dentin, compared with treatment with 10–9 M 1,25-(OH)2D3 (Fig. 2). Pit formation activated by 10–9 M 1,25-(OH)2D3 is shown in Fig. 2B. 1,25-(OH)2D3 (10–9 M) markedly enhanced osteoclastic bone resorption. TEI-9647 (10–9 M to 10–6 M) dose-dependently blocked osteoclastic bone resorption induced by 10–9 M 1,25-(OH)2D3, and 10–7 M TEI-9647 almost completely blocked bone resorption. The area resorbed by the OCLs was quantified by image analysis and is shown in Fig. 2C.
FIG. 2. Effects of TEI-9647 on bone resorption induced by 1,25-(OH)2D3. MVNP-transduced CFU-GM cells were dispersed into MEM containing 20% horse serum, and 105 cells were seeded in 96-well plate. RANKL (50 ng/ml) and M-CSF (50 ng/ml) were added into a well as stimulators of OCL formation. The cultures were continued for 3 wk. The media containing 20% horse serum, RANKL, and M-CSF were replaced two times per week. After 3 wk, media lacking RANKL and M-CSF were added and the cells incubated for 3 d. The OCLs that formed were then activated by the treatment with 10–9 M 1,25-(OH)2D3, 10–9 M to 10–6 M TEI-9647, or a combination of both for 10 d. After 10 d, OCLs on the dentin slices were stained for tartrate-resistant acid phosphatase (TRAP). A, Number of OCLs present on dentin slices. Data are expressed as the mean ± SD (n = 4). *, Significantly different from control cultures (media alone), P < 0.01. The OCLs were released by incubation with trypsin overnight, and the resorption lacunae were stained with hematoxylin. Pit area was quantified with an image analyzer. B, Pits on dentin slice. C, Pit area. Data are expressed as the mean ± SD (n = 4). *, Significantly different from control cultures (media alone), P < 0.01. MNC, Multinuclear cells.
Effect of TEI-9647 on TAFII-17 and 25-OH-D3-24-hydroxylase gene expression and TAFII-17 protein levels
We then performed time-course and dose-response experiments to assess the antagonistic actions of TEI-9647 on VDR-mediated gene transcription using MVNP-transduced CFU-GM cells as a model for pagetic OCL precursors. TEI-9647 (10–7 M) did not induce expression of the TAFII-17 and 25-OH-D3-24-hydroxylase genes but almost completely suppressed their expression induced by exposure to 10–9 M 1,25-(OH)2D3 for 6 and 12 h. TAFII-17 and 25-OH-D3-24-hydroxylase gene expression reappeared by 24 h after treatment with TEI-9647 (data not shown), demonstrating that TEI-9647 was not toxic to the cells. In dose-response experiments, at 12 h after treatment with 1,25-(OH)2D3 and TEI-9647, 10–10 M to 10–8 M 1,25-(OH)2D3 but not TEI-9647 dose-dependently increased TAFII-17 and 25-OH-D3-24-hydroxylase gene expression. As previously reported, TAFII-17 was expressed in the absence of added 1,25-(OH)2D3 in MVNP-transduced CFU-GM cells, but 25-OH-D3-24-hydroxylase was not (Fig. 3A). TEI-9647 dose-dependently suppressed TAFII-17 and 25-OH-D3-24-hydroxylase gene expression induced by 10–9 M 1,25-(OH)2D3, and 10–8 M TEI-9647 completely suppressed TAFII-17 and 25-OH-D3-24-hydroxylase gene expression. The relative effects of TEI-9647 on TAFII-17, and 25-OH-D3-24-hydroxylase mRNA expression in three independent experiments are shown in Fig. 3B.
FIG. 3. Effect of TEI-9647 on TAFII-17 gene expression and TAFII-17 protein levels. A, Effect of TEI-9647 on TAFII-17 and 25-OH-D3-24-hydroxylase gene expression induced by 1,25-(OH)2D3 in MVNP-transduced CFU-GM-cells. MVNP-transduced CFU-GM cells were cultured in MEM containing 10% FBS for 12 h with 10–10 M to 10–8 M 1,25-(OH)2D3, 10–7 M TEI-9647, or a combination of 10–9 M 1,25-(OH)2D3 and 10–10 M to 10–7 M TEI-9647. Total RNA was extracted and RT-PCR was carried out. PCR products were separated by 2% agarose gel electrophoresis and were revealed with ethidium bromide staining as described in Materials and Methods. Cont, Control (nontreated, media alone). B, Densitometric analysis of three independent PCR experiments examining the effects of TEI-9647 on TAFII-17 and 25-OH-D3-24-hydroxylase mRNA levels. C, Western blot analysis of TAFII-17 protein. To evaluate TAFII-17 protein levels, MVNP-transduced CFU-GM cells were cultured with 1,25-(OH)2D3, TEI-9647, or a combination of 1,25-(OH)2D3 and TEI-9647 for 4 d, and then cell lysates were prepared. Western blot analysis of TAFII-17 protein in the cell lysates was carried out as described in Materials and Methods. Seventeen- and 34-kDa bands both reacted with the anti-TAFII-17. The 34-kDa is a dimer of TAFII-17. D, Densitometric analysis of three independent Western blot analyses for the effects of TEI-9617 on TAFII-17 expression.
To determine whether TEI-9647 decreased protein levels of TAFII-17 induced by 1,25-(OH)2D3, we measured the relative levels of TAFII-17 in MVNP-transduced CFU-GM cells by Western blot analysis as shown in Fig. 3C. MVNP-transduced CFU-GM cells produced a 17- and 34-kDa protein, which reacted with TAFII-17 antibody in the absence of 1,25-(OH)2D3. The 34-kDa band is most likely a dimer of TAFII--17. Low concentration of 1,25-(OH)2D3 (10–9 M) increased the levels of TAFII-17. In contrast, 10–10 M to 10–7 M TEI-9647 dose-dependently decreased the levels of TAFII-17 enhanced by 10–9 M 1,25-(OH)2D3.
Effect of TEI-9647 on the interaction between VDR and TAFII-17
To confirm the role of TAFII-17 in VDR-mediated gene transcription, we investigated the interaction of VDR with TAFII-17. Protein-protein interaction between VDR and TAFII-17 was examined using the mammalian two-hybrid system. An expression vector in which human TAFII-17 was fused to GAL4DBD (pM-TAFII-17) was used as the bait construct, and the human VDR (pVP16-hVDR) was used as the prey vector (Fig. 4A). VDR-mediated gene transcription was induced when 10–8 M 1,25-(OH)2D3 was added with the VDR and TAFII-17 constructs, but it was significantly suppressed by treatment with 10–7 M TEI-9647 (Fig. 4B).
FIG. 4. Effect of TEI-9647 on the interaction between VDR and TAFII-17. A, Modified mammalian two-hybrid assay was used to examine the interaction between VDR and TAFII-17. The TAFII-17 cDNA was inserted at the EcoRI site of the pM vector, which contains a GAL4 DNA-binding domain (pM-TAFII-17). B, The interaction of VDR and TAFII-17 was examined by measuring the reporter activity in NIH3T3 cells transduced with pM-TAFII-17, pVP16-hVDR, and the reporter plasmid containing the GAL4 DNA-binding sites (pGVP2-GAL4BS). GAL4 protein is a transcriptional activator that binds to DNA. 1,25-(OH)2D3 (10–8 M), TEI-9647 (10–7 M), or both were added after transfection. After 24 h of incubation, the luciferase activity of the cell lysates was determined as described in Materials and Methods. Results are expressed as the mean ± SEM. *, P < 0.01, compared with cells treated with 10–8 M 1,25-(OH)2D3. Similar results were obtained in three independent experiments. No reporter activity was seen in control incubations lacking TAFII-17 or VDR constructs.
Discussion
We and others previously reported that OCL activity is markedly increased in patients with PD and that OCL formation is abnormal (9, 19). OCL precursors from patients with PD have increased sensitivity to 1,25-(OH)2D3 and can form OCLs at physiologic levels of 1,25-(OH)2D3, something that normal OCL precursors cannot do. As shown in Table 1, patients with PD have levels of 1,25-(OH)2D3 in their serum that do not differ from normals and are sufficient to induce osteoclastogenesis. These findings agree with previous work by Devlin et al. (17) and Foldes et al. (18), who showed similar levels of 1,25-(OH)2D3 in PD patients and normals. In addition, as shown in Fig. 2, C and D, these levels of 1,25-(OH)2D3 can enhance the bone resorption capacity of preformed OCLs that are similar to pagetic OCLs.
Therefore, we tested TEI-9647, a vitamin D analog that can block the effects of 1,25-(OH)2D3 on hematopoietic cell differentiation, for its capacity to block OCL formation in vitro by marrow samples from patients with PD and MVNP-transduced normal OCL precursors, as a surrogate model of pagetic OCL precursors. We previously reported that MVNP-transduced osteoclast precursors are very similar to pagetic osteoclasts precursors (7). As shown in Figure 1, 1,25-(OH)2D3 (10–11 M) induced high levels of OCL formation in PD cultures but minimally affected OCL formation in normal marrow cultures. TEI-9647 dose-dependently inhibited osteoclast formation in both normal and pagetic marrow cultures. OCL formation in PD cultures was more sensitive to TEI-9647. TEI-9647 (10–11 to 10–9 M) decreased OCL formation approximately 3-fold in PD cultures, whereas it decreased normal OCL formation only approximately 2-fold. Of interest, TEI-9647 also decreased basal OCL formation in both normal and pagetic marrow cultures. This decrease in basal OCL formation was not due to toxicity of TEI-9647 for hematopoietic cells. We have shown in preliminary studies that TEI-9647 does not block the growth of hematopoietic precursor cells such as CFU-GM and in the current study reversibly inhibited VDR-mediated gene transcription (Fig. 3A). Furthermore, TEI-9647 was not toxic to preformed OCL because OCL numbers were unchanged when OCLs were treated with TEI-9647 (Fig. 2B). Both 1,25-(OH)2D3-stimulated and basal OCL formation were not significantly inhibited in normal marrow cultures until the TEI-9647 concentration was 10–9 M, whereas pagetic marrow cultures were significantly inhibited by 10–10 M TEI-9647. TEI-9647 (10–8 M) decreased OCL formation almost to basal levels in marrow cultures from patients with PD who were treated with physiologic levels of 1,25-(OH)2D3 (10–10 M) (Fig. 1B). These data demonstrate that TEI-9647 inhibited OCL formation induced by 1,25-(OH)2D3 and that basal osteoclastogenesis in marrow cultures may in part be mediated by the low levels of 1,25-(OH)2D3 present in the horse serum in these cultures.
TEI-9647 also blocked VDR-mediated gene transcription. As shown in Fig. 3A, TEI-9647 markedly decreased TAFII-17 expression induced by 1,25-(OH)2D3 both at the mRNA and protein level. Furthermore, mammalian two hybrid assays demonstrated that TEI-9647 blocked the interaction between TAFII-17 and VDR. These results suggest that TEI-9647 or similar agents might in addition to decreasing OCL formation and activity could also affect the sensitivity of these precursors to 1,25-(OH)2D3. Kurihara et al. (8) reported that increased expression of TAFII-17 is in part responsible for the hypersensitivity of pagetic OCL precursors to 1,25-(OH)2D3. They showed that transfecting TAFII-17 into normal OCL precursors results in increased sensitivity of these precursors to 1,25-(OH)2D3. Furthermore, an antisense to TAFII-17 blocked OCL formation by pagetic osteoclast precursors induced by low levels of 1,25-(OH)2D3. TEI-9647 suppresses expression of TAFII-17 in MVNP-transduced normal OCL precursors, which have many of the characteristics of pagetic OCL, including hypersensitivity to 1,25-(OH)2D3 and high levels of TAFII-17. These results suggest that TEI-9647 may be a drug that can both block the enhanced OCL formation and bone resorption in patients with PD in response to 1,25-(OH)2D3 and possibility change the sensitivity of pagetic OCL precursors to 1,25-(OH)2D3.
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