Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M304737200 on June 9, 2003

J. Biol. Chem., Vol. 278, Issue 34, 31756-31765, August 22, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/34/31756    most recent
M304737200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamamoto, H.
Right arrow Articles by Pike, J. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamamoto, H.
Right arrow Articles by Pike, J. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 Potently Stimulates Gene-specific DNA Binding of the Vitamin D Receptor in Osteoblasts*

Hironori Yamamoto {ddagger}, Nirupama K. Shevde, Anjali Warrier, Lori A. Plum, Hector F. DeLuca and J. Wesley Pike §

From the Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received for publication, May 6, 2003 , and in revised form, June 6, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD) is a highly potent analog of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) whose actions are mediated through the vitamin D receptor (VDR). In this report, we have replicated this increased potency of 2MD in vitro using osteoblastic cells and explored its underlying molecular mechanism. 2MD stimulates the expression of several vitamin D-sensitive genes including 25-hydroxyvitamin D3-24 hydroxylase (Cyp24), osteopontin and receptor activator of NF{kappa}B ligand and suppresses osteoprotegerin at concentrations two logs lower than that for 1,25(OH)2D3. 2MD is also more potent in stimulating transfected chimeric reporter genes under either Cyp24 or the osteocalcin promoter control. Enhanced potency is retained regardless of medium serum content. Interestingly, the uptake of both 1,25(OH)2D3 and 2MD into cells is similar, as is their rapid association with the VDR. This indicates that comparable levels of occupied VDR do not elicit equivalent levels of transactivation. Using chromatin immunoprecipitation (ChIP), however, we observed a strong correlation between DNA-bound receptor and the level of induced transcription suggesting a 2MD-induced increase in affinity of the VDR for DNA. Additional studies using a mammalian two-hybrid system and ChIP indicate that 2MD is also more potent in promoting interaction with RXR and the coactivators SRC-1 and DRIP205. Finally, protease digestion studies revealed a unique VDR conformation in the presence of 2MD. These studies suggest that the molecular mechanism of 2MD potency is due to its ability to promote enhanced levels of specific DNA binding by the VDR and could suggest possible explanations for the tissue- and gene-selective actions of 2MD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The physiological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)1 are mediated by the vitamin D receptor (VDR) (1). This predominantly nuclear protein is a member of the steroid receptor gene family of transcription factors and functions like other nuclear receptors to regulate the expression of genes involved in a wide variety of cellular activities. The major role of 1,25(OH)2D3 is to maintain calcium and phosphorus homeostasis through its direct actions on gene expression in the intestine, kidney, and bone (2). This hormone also regulates proliferation, differentiation, activity, and survival of a number of cell types, many of which are not involved in mineral homeostasis (3). The biological relevance of these latter actions remains to be determined, however, given the largely skeletal phenotype associated with null mutations of the VDR gene in both humans and in mice (4, 5). Ligand association with the VDR leads to rapid activation and subsequent accumulation of the receptor on vitamin D response elements (VDREs) located within the promoter regions of specific genes (1). VDRE binding is facilitated by the retinoid X receptor (RXR), which functions both as a heterodimeric DNA binding partner as well as a participant in transactivation (6). The role of RXR in VDR-mediated gene repression is unclear and appears to be somewhat controversial (7). Regardless, these initial molecular actions of 1,25(OH)2D3 and its receptor are essential to the subsequent alteration in the expression of genes involved in a variety of highly pleiotropic cellular responses.

A number of steroid hormone analogs, including those for 1,25(OH)2D3 have been synthesized and characterized (8). With respect to vitamin D3 analogs, many of these are highly potent activators of gene transcription despite the fact that their affinities for VDR are not different from that of the native hormone. While enhanced potency can arise in vivo as a result of an analog's pharmacokinetic properties, recent studies in vitro have suggested that at least for some analogs increased potency may be due to their ability to induce unique VDR conformations that favor enhanced interaction with either RXR or coactivators such as SRC-1 or DRIP205 (9–11). Interestingly, while altered receptor conformations are supported through proteolytic digestion studies in vitro, they are not supported by three-dimensional structural analyses (12, 13). Other 1,25(OH)2D3 analogs display disparate actions, exerting highly potent effects on cellular proliferation and differentiation or to suppress parathyroid hormone gene expression, yet demonstrating a restricted ability to induce calcium mobilization from bone or calcium uptake from the gut (14, 15). Since increased serum calcium promotes soft tissue degeneration and other equally detrimental effects, compounds with a low hypercalcemic potential are highly desirable therapeutically. The underlying mechanism(s) for this selectivity is not known, but is likely to be complex given the diverse molecular mechanisms that drive bone resorption, intestinal absorption, and renal reabsorption of calcium.

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD) is a highly potent 1,25(OH)2D3 analog that exhibits tissue-selective as well as gene-selective actions in vitro and in vivo (16, 17). This compound binds to the VDR with a Kd that is similar to that for 1,25(OH)2D3, although its interaction with serum vitamin D-binding protein (DBP) is exceedingly weak (Ref. 16).2 In vivo studies suggest that this analog appears to localize selectively to bone relative to intestine (16). 2MD strongly induces calcium resorption from bone in vivo, an activity that is likely due to its ability to induce receptor activator of NF{kappa}B ligand (RANKL) from supportive stromal cells and osteoblasts. RANKL is a key regulator of osteoclast formation, activity and survival (18, 19), and is potently induced by both 1,25(OH)2D3 and 2MD in cultured cells (17). Interestingly, treatment of ovariectomized adult rats with low doses of 2MD leads to a dramatic increase in bone mineral density (BMD) in aged, ovariectomized rats (17). While the mechanism of this dramatic anabolic activity of 2MD is not yet understood, in vitro studies suggest that 2MD exhibits an exceptionally potent and unique ability to enhance VDR-mediated osteoblast activity that leads to increased mineralization (17). In this article, we explore the underlying molecular basis for the increased potency of 2MD using osteoblast cell models. We find that 1,25(OH)2D3 and 2MD bind equivalently to the VDR under serum-free conditions. Despite this, localization of the receptor to VDREs lying adjacent to 1,25(OH)2D3-inducible promoters in intact cells occurs at concentrations of 2MD that are much lower than that for 1,25(OH)2D3. VDR DNA-binding correlates directly with the ability of the two ligands to activate transcription. A 2MD-mediated increase in VDR DNA affinity may result from enhanced VDR interaction with RXR and with coactivators such as SRC-1 and DRIP205, both of which could enhance the stability of the VDR on DNA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—1,25(OH)2D3 was obtained from Solvay (da Weesp, The Netherlands). 2MD was provided by Deltanoid Pharmaceuticals, Inc. (Madison, WI). Tritiated 1,25(OH)2D3 (166 Ci/mmol), L-[35S]methionine (1175 Ci/mmol), and [32P]dCTP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences. Tritiated 2MD (174 Ci/mmol) was prepared from an appropriate precursor3 and provided by Deltanoids Pharmaceuticals, Inc. Dexamethasone was obtained from Sigma Chemical Co. {alpha}-Modified Eagle's medium ({alpha}-MEM) and Dulbecco's modified Eagle's medium were purchased from Invitrogen. Oligonucleotide primers were obtained from IDT (Coralville, IA). Anti-VDR (H-81), RXR ({Delta}N197), and SRC-1 (M-341) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-RNA polymerase II antibodies (8WG16) were obtained Covance, Berkeley Antibody Company (Richmond, CA). LipofectAMINE Plus was obtained from Invitrogen. BioGel HTP was obtained from BioRad.

Plasmids—The human osteocalcin (OC) promoter reporter plasmid phOC-3900 luc, the human Cyp24 reporter plasmid phCyp24-luc (ph24OHase-luc) and pGal4(5x)-luc were previously reported (20, 21). pcDNA-hVDR, phVDR-VP16, and pCH110-{beta}gal have been also previously described (20, 22). pcDNA-hVDR(R417A/R420A) was prepared from pcDNA-hVDR using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The Gal4-(1–147) DNA-binding domain (DBD) fusion vectors pM-SRC-1NR (residues 621–765) and pM-GRIP-NR (residues 629–760) have also previously described (22, 23). Gal4-(1–147) DBD fusion vector pBIND (Promega, Madison, WI) was used to create pBIND-mRXR{alpha} (residues 292–467) and pBIND-hDRIP205NR (residues 527–970). pET-hVDR comprised of the full-length VDR containing a C-terminal His6 tag was constructed from the pET-29b vector obtained from Novagen (Darmstadt, Germany). pGEX-SRC-1NR (residues 621–765), pGEX-GRIP-NR (residues 629–760), pGEX-mRXR{alpha} (residues 203–467), and pGEX-DRIP205-NR (residues 527–970) were constructed using the pGEX parent plasmid obtained from Amersham Biosciences.

Cell Culture—MC3T3-E1 cells and ST2 cells were cultured in {alpha}-MEM medium supplemented with 10% fetal bovine serum (FBS) from BioWhittaker (Walkerville, MD). Vitamin D3 ligands were added in ethanol (0.1% maximum final concentration).

Osteoclast Formation—Spleen cells were obtained from 6-week-old C57B6 mice (Harlan-Sprague-Dawley, Indianapolis, IN), treated with lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 1 mM EDTA) and prepared as previously described (17). Coculture assays were carried out by plating ST2 cells (5 x 104/well) together with freshly isolated spleen cells (1 x 106/well) in 48-well plates. Cells were cultured in phenol red-free {alpha}-MEM with 10% charcoal dextran-treated FBS in the presence or absence of indicated compounds and the medium replaced on day 4 and day 7. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) activity on day 8–10 as previously described (24). Osteoclast number was determined by counting the total number of multinucleated (>3 nuclei), TRAP-positive cells/well.

Transfection Analysis—MC3T3-E1 cells were seeded into 24-well plates at a density of 5 x 104 cells/well and transfected 16–24 h later using LipofectAMINE Plus as described (23). Individual wells received 300 ng of DNA comprised of 250 ng of phOC-3900-luc or phCyp24-luc plasmid and 50 ng of pCH110-{beta}-gal. In the two-hybrid system, cells received 250 ng of pGal4(5x)-luc, 50 ng of pVDR-VP16, 50 ng of either pBIND-mRXR{alpha}, pM-SRC-1NR, pM-GRIP-NR, or pBIND-hDRIP205NR and 50 ng of pCH110-{beta}-gal. After transfection, the cells were normally cultured in medium supplemented with 10% FBS for 24 h with or without the indicated ligands. Cells were harvested 24 h after stimulation and lysates assayed for luciferase and {beta}-galactosidase ({beta}-gal) activities using standard methods. In some experiments, cells were transfected as above and treated with the indicated ligands in the absence of FBS for periods ranging from 15 min to 8 h. The medium was then changed to ligand-free medium containing 10% FBS. The cells were harvested 24 h post-transfection and analyzed as above. Luciferase units were normalized in all cases to {beta}-gal activity.

RNA Isolation and Analysis—MC3T3-E1 or ST2 cells were plated in 100-mm dishes in {alpha}-MEM supplemented with 10% FBS at densities of 5 x 105/ml and treated for up to 24 h with dexamethasone (10–7 M) without or with the indicated concentration of either 1,25(OH)2D3 or 2MD. Total RNA was isolated using the Triazol reagent from MRC (Cincinnati, OH), resolved using denaturing gel electrophoresis and transferred to Hybond-N+ membranes obtained from Amersham Biosciences. Double-stranded cDNA fragments encoding mouse RANKL (+270 to +940), mouse OPG (+346 to +1206), mouse Cyp24 (+435 to +707), mouse OPN (full-length cDNA), or mouse {beta}-actin (+806 to +1155) were labeled with [32P]dCTP (PerkinElmer Life Sciences) using a Megaprime labeling kit (Amersham Biosciences) and used as probes. Radioactive images were analyzed with a phosphorimager. Total RNA was also reverse transcribed using the SuperScript II RNase H Reverse transcriptase kit from Invitrogen and subjected to PCR analysis using standard methods (see also ChIP method).

Cell Uptake Assays—MC3T3-E1 cells (1 x 106/ml) were seeded into 24-well plates and cultured overnight in normal FBS-supplemented {alpha}-MEM. The medium was then replaced with medium containing the indicated concentration of FBS and either 0.1 nM 1,25(OH)2D3 (166 Ci/mmol) or 2MD (174 Ci/mmol) for periods ranging from 5 min to 2 h. Following removal of the medium, cells were washed three times with phosphate-buffered saline, dissolved in scintillation fluid, and evaluated for ligand uptake using a Packard Tricarb 2900TR Scintillation Spectrometer.

Hormone Binding Analysis—MC3T3-E1 cells (1 x 106/ml) were seeded into 100-mm plates and cultured overnight in normal FBS-supplemented {alpha}-MEM. The medium was then replaced with FBS-free medium, and 1,25(OH)2D3 (166 Ci/mmol) or 2MD (174 Ci/mmol) was added with or without a 100-fold molar excess of radioinert ligand for an additional 30 min. Following incubation, cells were washed, lysed in 50 mM Tris-HCl, pH 7.4, 0.3 M KCl, and 5 mM dithiothreitol containing 0.5% Nonidet P-40, and the soluble fraction subjected to hydroxylapatite (HAP) assay to assess total and nonspecific 1,25(OH)2D3- or 2MD-binding as previously described (25). The difference between total binding and nonspecific binding represents specific VDR binding. Total cellular uptake of both ligands in these experiments was also assessed as described above.

GST Pull-down Assays—pET-VDR was transformed into BL21(DE3) codon Plus RIL cells (Stratagene). Transformed cells were cultured with 100 µM isopropyl-1-thio-{beta}-D-galactopyranoside at 23 °C for 6 h and soluble VDR protein purified to homogeneity from lysates using sequential Ni-NTA and SP-Sepharose column chromatography. Lysates from pGST-RXR{alpha}-, pGST-SRC-1NR-, pGST-GRIP-NR-, and pGST-DRIP205-NR-transformed Escherichia coli were incubated with glutathione-Sepharose 4B for 2 h at 4 C, and then washed extensively to prepare an immobilized GST fusion reagent of mRXR{alpha}, hSRC-1, hGRIP, or hDRIP205. Purified VDR and immobilized GST fusion proteins were incubated with 10–8 to 10–12 M 1,25(OH)2D3 or 2MD in GST binding buffer (20 mM Tris-HCl, pH 7.9, 180 mM KCl, 0.2 mM EDTA, 0.05% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) containing 1 mg/ml bovine serum albumin for 30 min at room temperature. After five washes, dissolved samples were resolved by SDS-PAGE, transferred to PVDF membranes and subjected to Western blot analysis as previously described (20) using the anti-VDR monoclonal antibody 9A7 (26). Blots were visualized using the ECL method.

Chromatin Immunoprecipitation (ChIP) Assays—Chromatin immunoprecipitation was performed as described previously (27, 28). Briefly, MC3T3-E1 cells were cultured in FBS-free {alpha}-MEM and treated with 1,25(OH)2D3 or 2MD for the times or concentrations indicated. Following a wash with phosphate-buffered saline, cells were subjected to cross-linking with 1% formaldehyde. Cells were extracted in 5 mM Pipes pH 8.0, 85 mM KCl, 0.5% Nonidet P-40 and then in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1. Chromatin pellets were sonicated to an average of 300–500-bp fragments of DNA, centrifuged, and then diluted into ChIP buffer (16.7 mM Tris-HCl, pH 8.1, 150 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA). Immunoprecipitations were performed overnight at 4 °C with the indicated antibodies and then collected following a 1-h incubation with salmon sperm DNA-and bovine serum albumin-pretreated Zysorbin (Zymed Laboratories Inc., San Francisco, CA). Precipitates were then washed sequentially and the cross-links reversed with a 6-h incubation at 65 °C in 1% SDS and 0.1 M NaHCO3. DNA fragments were purified using Qiagen QIAquick Spin Kits (Valencia, CA) and subjected to PCR techniques using primers designed to amplify fragments of murine Cyp24 promoter region (–236 to –51) and murine OPN promoter region (–854 to –658). All PCR analyses for each primer set were carried out in a predetermined linear range of DNA amplification. PCR products were resolved on 2% agarose gels and visualized using ethidium bromide staining.

Protease Digestion Studies—The plasmids pcDNA-hVDR or pcDNA-hVDR(R417A/R420A) were utilized to prepared in vitro-translated, [35S]methionine-labeled hVDR or hVDR(R417A/R420A) using the TNT/T7-coupled Reticulocyte Lysate System from Promega (Madison, WI). Appropriate aliquots were treated with the indicated concentrations of 1,25(OH)2D3 or 2MD for 10 min and then digested with trypsin (0.12 µg/sample) at room temperature for the indicated time periods as described (9). Digestion was terminated with denaturing buffer, whereupon the samples were resolved by SDS-PAGE. Dried gels were subjected to autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
2MD Is a 1,25(OH)2D3-like Superagonist in Cell Culture— Previous studies revealed that 2MD is a highly potent compound that exhibits both tissue and gene selectivity in vivo (16). Several of these characteristics have also been demonstrated in cell culture (17). For example, 2MD is at least 100-fold more active than 1,25(OH)2D3 in inducing a collection of vitamin D-sensitive genes in the mouse osteoblastic cell line ST2, including Cyp24, OPN, and RANKL, and in suppressing the expression of OPG (Fig. 1A). Separate additional experiments indicated that while peak activity of 1,25(OH)2D3 and 2MD was reached at 10–7 M and 10–9 M, respectively, there was no increase in the efficacy of 2MD over that of 1,25(OH)2D3. Importantly, this increased potency of 2MD over 1,25(OH)2D3 extends to its ability to stimulate osteoclast formation in cocultures of ST2 cells and mouse osteoclast precursors (Fig. 1B), an effect due to increased expression of RANKL and decreased expression of OPG (19). These observations verify that 2MD is a highly potent 1,25(OH)2D3 agonist in cultured bone cells. This is an intriguing finding in view of the fact that the affinity of the VDR for 2MD (Kd) is not different from that of the native hormone (16).



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 1.
2MD is a highly potent regulator of endogenous gene expression in osteoblastic MC3T3-E1 cells. A, dose response modulation of Cyp24, OPN, RANKL, OPG, and {beta}-actin mRNA levels by 1,25(OH)2D3 and 2MD in MC3T3-E1 cells. Cells were treated in the presence of dexamethasone with the indicated concentrations of either vehicle (NT), 1,25(OH)2D3, or 2MD. Total RNA was isolated 36 h later and analyzed by Northern blot analysis. Autoradiographic signals were quantitated using densitometry, normalized to {beta}-actin levels and plotted as fold changes compared with untreated controls. These results are representative of several different experiments. B, osteoclast formation in cocultures of ST2 stromal cells and mouse spleen cells. Cells were cultured in the presence of dexamethasone and the indicated concentrations of either 1,25(OH)2D3 or 2MD for 10 days. Cells were fixed, stained for tartrate-resistance acid phosphatase, and quantitated by counting the total number of TRAP+, multinucleated (>3 nuclei) cells. 1,25(OH)2D3-(upper left panel) or 2MD-(upper right panel) induced osteoclasts photographed at x20. Quantitation of osteoclast number as a function of ligand dose (lower panels). The results are the means ± S.E. of triplicate wells for each condition. Similar results were obtained in three independent experiments.

 

Transcriptional Models Recapitulate 2MD Potency—To establish a functional model for evaluating the potency of 2MD, we introduced vitamin D-sensitive Cyp24 and OC promoter-reporter gene plasmids into pre-osteoblastic ST2 or MC3T3-E1 cells by transient transfection and assessed dose-dependent transcriptional activity in response to 2MD or 1,25(OH)2D3. As can be seen in Fig. 2, both 1,25(OH)2D3 and 2MD dose-dependently induced transcriptional activation of each reporter gene in MC3T3-E1 cells; similar results were obtained in ST2 cells (data not shown). The transcriptional potency of 2MD was at least two logs greater than for 1,25(OH)2D3, however, reflecting that observed for endogenous gene expression. These results suggest that the enhanced potency of 2MD is transcriptional in nature and provide a model for determining the underlying molecular basis for this increased potency.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2.
2MD stimulates transcription of Cyp24 and OC promoter-dependent reporter genes with high potency in osteoblast cells. MC3T3-E1 cells were seeded into 24-well plates and transfected 24 h later with pCH110-{beta}gal and either phOC-3900-luc (A) or phCyp24-luc (B). Cells were treated with vehicle (NT) or the indicated concentrations of either 1,25(OH)2D3 or 2MD in 10% serum-supplemented medium, and cell lysates were assessed for {beta}-gal and luciferase activities 24 h later. Each point represents the average of quadruplicate analyses ± S.E. normalized for {beta}-gal activity. Similar results were obtained for three independent experiments.

 

Enhanced Potency of 2MD Is Not Due to Serum—Ligand-induced transcriptional responses in cells are influenced by cell culture conditions and can be significantly affected by serum. Serum also contains DBP, an avid binder of and presumed primary although not exclusive carrier in the blood for most vitamin D metabolites (24). In contrast to 1,25(OH)2D3, 2MD binds only weakly to DBP, an effect that could lead to an increase in its functional concentration relative to 1,25(OH)2D3. We explored this possibility by transfecting the OC promoter into MC3T3-E1 cells and then treating the cells with increasing concentrations of 1,25(OH)2D3 or 2MD for 1 h in serum-free medium. The medium was then replaced with ligand-free medium supplemented with 10% FBS and transcriptional activity assessed ~24 h later. The results indicate that 2MD continues to exhibit an approximately two-log increase in potency (Fig. 3A), revealing that its superagonist activity is not due to serum. Similar results were obtained with the hCyp24 promoter (data not shown). Interestingly, the level of transcription induced under serum-free conditions in 1 h or less exposure to functionally equivalent levels of 1,25(OH)2D3 (10–7 M) and 2MD (10–9 M) is at least 50% of that achieved following continual treatment with the two ligands for 24 h in the presence of serum (Fig. 3B). Irrespective of this, the data suggest that the potency of 2MD is not derived from its lack of interaction with serum components.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3.
The potency of 2MD is not affected by serum content. A, MC3T3-E1 cells were transfected with phOC-3900-luc and pCH110-{beta}Gal and then treated with either vehicle (NT), 1,25(OH)2D3 (10–7 M), or 2MD (10–9 M) for 1 h in the absence serum. Ligand-containing medium was then removed and replaced with normal serum-supplemented, ligand-free medium. Cells were lysed and {beta}-gal and luciferase activities were assessed 24 h later. Each point represents the average of quadruplicate analyses ± S.E. normalized for {beta}-gal activity. Similar results were obtained for at least three independent experiments. B, MC3T3-E1 cells were transfected with phOC-3900-luc and pCH110-{beta}Gal and then treated with either 1,25(OH)2D3 (10–7 M) or 2MD (10–9 M) for periods ranging from 0 to 8 h in the absence serum. Ligand-containing medium was then removed and replaced with normal serum-supplemented ligand-free medium. All lysates were analyzed for {beta}-gal and luciferase activity 24 h following the original transfection. Control wells were transfected as above and received either vehicle, 1,25(OH)2D3 (10–7 M) or 2MD (10–9 M) for 24 h in the presence of normal serum-supplemented medium. The luciferase activities of quadruplicate wells were averaged, normalized for {beta}-gal activity and plotted as fold-induction versus untreated controls ± S.E. Cells exposed to ligand for 24 h were treated identically. These data are representative of at least two experiments.

 

Cellular Uptake of 1,25(OH)2D3 and 2MD Does Not Determine Potency—We next tested the possibility that 2MD might enter cells more readily than 1,25(OH)2D3 and thus saturate intracellular VDR at much lower medium ligand concentrations. Cells were incubated with 0.1 nM tritiated 1,25(OH)2D3 or 2MD (a concentration which leads to transcriptional activation with 2MD but not with 1,25(OH)2D3) under various serum conditions for periods ranging from 0 to 15 min, then washed, and the cellular content of ligand determined by scintillation counting. As seen in Fig. 4A, incubation of cells in the absence of FBS leads to a rapid and equivalent accumulation of both ligands within the cells. Surprisingly, this accumulation peaked within 10 min (Fig. 4A), and then decreased to a steady state nadir by 15–30 min and remained relatively stable for the next several hours (data not shown). Interestingly, increasing concentrations of FBS in these experiments significantly reduced the uptake of both ligands into the cells (Fig. 4, A and B). This effect was most evident for 1,25(OH)2D3 at the highest level of serum added (5-fold), although the difference does not seem able to account for the 2 log increase in potency. Despite this, the observation that virtually identical levels of both 1,25(OH)2D3 and 2MD accumulate in cells in the absence of serum indicates that the increased potency of 2MD is not due to altered cellular uptake.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 4.
Uptake of 1,25(OH)2D3 and 2MD into MC3T3-E1 cells is similar. A, time course of radioligand uptake. MC3T3-E1 cells were seeded into 24-well plates and incubated with 0.1 nM 1,25(OH)2D3 (166 Ci/mmol) or 2MD (174 Ci/mmol) for 0–15 min in the presence of 0 (open or closed circles), 1 (open or closed squares), or 10% (open or closed triangles, 5 min time point only) serum-supplemented medium. At term, cells were washed 3x with phosphate-buffered saline, dissolved in scintillation fluid, and evaluated for radiolabeled ligand content. B, FBS reduces the uptake of both 1,25(OH)2D3 and 2MD. MC3T3-E1 cells were incubated with medium containing the indicated concentrations of serum and either 0.1 nM 1,25(OH)2D3 (166 Ci/mmol) or 2MD (174 Ci/mmol) for 5 min. Cells were then treated as in A. All assays were carried out in triplicate, and the data represent the mean ± S.E. Similar results were obtained for at least three independent experiments.

 

Binding of 1,25(OH)2D3 and 2MD to the VDR Is Similar In Intact Cells—Based upon the above findings, we tested an alternative possibility that VDR might exhibit an increase in affinity for 2MD relative to 1,25(OH)2D3 in intact cells. MC3T3-E1 cells were incubated in the absence of serum for 30 min (37 °C) with increasing concentrations (0.05, 0.1, and 0.5 nM) of either 1,25(OH)2D3 or 2MD in the absence or presence of 200-fold molar excess of each ligand. Total and VDR-bound ligands were then assessed. As observed in Fig. 5, neither total cellular uptake (Fig. 5A) nor VDR binding (Fig. 5B) was substantially different between 1,25(OH)2D3 and 2MD at the concentrations tested. Although this experiment does not represent a complete saturation curve for VDR, ~5500 VDR mol/cell were detected at the 0.5 nM ligand concentration, roughly 60% of the MC3T3-E1 cell's complement of saturable VDR.4 These results indicate that when measured in intact cells, the relative affinities of the VDR for 2MD and 1,25(OH)2D3 are probably not substantially different from that observed in broken-cell preparations and thus do not provide an explanation for the increased potency of 2MD. Rather, they indicate that this characteristic of 2MD resides in its enhanced ability to stimulate VDR-mediated transcription.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 5.
Specific binding of 1,25(OH)2D3 and 2MD to the VDR is similar in intact cells. A, total cellular uptake of 1,25(OH)2D3 or 2MD. Total cellular uptake of radiolabeled 1,25(OH)2D3 or 2MD corresponding to B below was determined as in Fig. 4. The 100-fold molar excess of unlabeled ligand did not alter radiolabeled ligand uptake (not depicted) indicating a non-saturable process. Each estimate represents the mean ± S.E. for a triplicate assay. B, specific 1,25(OH)2D3- or 2MD-binding activity in intact cells. MC3T3E1 cells were seeded into 100-mm plates and at confluency incubated with 0.05, 0.1 or 0.5 nM 1,25(OH)2D3 (166 Ci/mmol) or 2MD (174 Ci/mmol) in FBS-free medium with or without a 100-fold molar excess of radioinert ligand for 30 min. Following incubation, cells lysates were subjected to hydroxylapatite (HAP) assay to assess total and nonspecific 1,25(OH)2D3- or 2MD-binding. Specific binding is depicted (nonspecific binding was less than 20% of total binding activity). Each point represents the mean ± S.E. for triplicate assays. These data are representative of three similar experiments.

 

2MD Potently Stimulates Binding of VDR to Active Promoters in Intact Cells—Two possible mechanisms might account for the increased transcriptional potential of 2MD: 1) increased VDR affinity for DNA via some undefined mechanism or 2) increased transcriptional activity of individual receptor molecules. We therefore utilized ChIP assays to examine the relative abilities of the two ligands to stimulate VDR binding to the VDREs located in OPN and Cyp24 promoters in intact MC3T3-E1 cells. The degree to which the OPN or Cyp24 DNA promoter fragment can be detected by PCR is directly related to the association of the VDR with these promoters. A dose response curve for VDR localization to the OPN promoter at 2 h is observed in Fig. 6. Clearly, although the overall appearance of the VDR on this promoter is similar, the ligand concentrations required for maximal binding are quite different: while maximal binding is achieved with 0.1 nM 2MD, at least 10 nM is required for 1,25(OH)2D3. Not surprisingly, this accumulation of VDR on the promoters was also associated with recruitment of RNA pol II (Fig. 6). To ensure that the kinetics of VDR binding was not different, we treated cells with 0.1 nM 1,25(OH)2D3 or 2MD and assessed DNA localization on both the OPN and Cyp24 promoter every 15 min up to 135 min. While VDR localized to both promoters following treatment with 2MD (maximal at 45 min), VDR binding was undetectable with 1,25(OH)2D3 at any of the time points evaluated (data not shown). In contrast, treatment of cells with functionally equivalent concentrations of the two ligands (1 nM 2MD and 100 nM 1,25(OH)2D3) led to qualitatively and quantitatively similar results (Fig. 6 and data not shown). These studies indicate that the increased potency of 2MD is likely due to enhanced VDR localization on promoter DNA, although whether this is the result of increased VDR DNA binding or increased VDR complex stability cannot be distinguished by this experiment.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 6.
2MD potently stimulates binding of VDR to active promoters in intact cells. MC3T3-E1 cells were seeded into 100-mm plates and treated with increasing concentrations of 1,25(OH)2D3 or 2MD in serum-free medium. After 15 min, the medium was changed to that containing 10% FBS. 2 hr later, cells were harvested and subjected to ChIP analysis as described in "Materials and Methods" using anti-VDR, anti-RNA pol II, or in the absence of antibody. Isolated DNA was subjected to PCR (28 cycles) using primers designed to amplify a 196-bp fragment of the OPN promoter from –854 to –658 that contained the VDRE at –764 to –748. Upper panel, amplified ethidium bromide-stained DNA fragments. Lower panel, densitometric quantitation of the PCR fragments. Precipitation was anti-VDR or anti-RNA pol II antibody concentration-dependent, and similar results were obtained using the anti-VDR monoclonal antibody 9A7. Immunoprecipitation in the absence of antibody or in the presence of an irrelevant antibody was identical. PCR amplification of the coding region of either gene did not produce visible products. The results are typical of at least three experiments performed under similar conditions.

 

Increased VDR DNA Binding May Be Due to Enhanced Interaction with RXR{alpha}—The VDR is believed to bind to DNA as a ligand-induced RXR heterodimer (30, 31). Thus, the possibility exists that binding of 2MD to the VDR enhances receptor association with RXR leading in turn to VDRE DNA binding. To explore this hypothesis, we contrasted the ability of 1,25(OH)2D3 and 2MD to promote VDR interaction with RXR{alpha} using both an in vitro GST pull-down assay and a mammalian two-hybrid assay in MC3T3-E1 cells. GST-RXR{alpha} fusion protein was retained on glutathione-Sepharose beads and incubated with highly purified human VDR in the presence of increasing concentrations of either 1,25(OH)2D3 or 2MD. Following a 30-min incubation at RT, the beads were washed, and solubilized VDR resolved by SDS-PAGE and subjected to Western blot analysis. Fig. 7A reveals that while both 1,25(OH)2D3 and 2MD promote the association of VDR with RXR, the concentrations of the two ligands necessary for interaction are relatively high and do not appear to be different. In the mammalian two-hybrid system, MC3T3-E1 cells were cotransfected together with RXR{alpha}-Gal4 DBD, a full-length VDR-VP16 fusion construct and a luciferase reporter gene containing tandem Gal4 response elements and then incubated with increasing concentrations of either 2MD or 1,25(OH)2D3 for 1 h. Fresh medium containing serum was then added and the cells were allowed to incubate for an additional 24-h period. Fig. 7B documents that while both ligands induce an interaction between VDR and RXR, 2MD is at least two logs more potent than 1,25(OH)2D3, a potency which appears to correspond to that seen with the transfected OC and Cyp24 gene promoters. Enhanced association of the VDR with RXR in the presence of 2MD is also supported in vivo by ChIP analysis (Fig. 7C). Again, while 0.1 nM 1,25(OH)2D3 was ineffectual, the same concentration of 2MD strongly induced association of RXR as well as VDR to the Cyp24 gene promoter. These observations indicate that 2MD potently induces VDR/RXR interaction in intact cells but not in vitro, suggesting that the effects of 2MD require either additional factors or perhaps post-translational modifications of the receptor(s) that do not occur in vitro.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 7.
2MD is a potent inducer of VDR/RXR interaction. A, biochemical interaction between VDR and RXR in vitro. GST-RXR{alpha} immobilized on glutathione-Sepharose beads was incubated with purified hVDR (100 ng) in the presence of increasing concentrations of 1,25(OH)2D3 or 2MD. Complexes were collected after 30 min, washed extensively, solubilized in denaturing buffer, and subjected to Western blot analysis using the anti-VDR 9A7 antibody. The data are representative of two experiments. B, 2MD induces interaction of VDR with RXR using mammalian two-hybrid assay. MC3T3-E1 cells were transfected with phVDR-VP16, pBIND-mRXR{alpha} and pGal4(5x)-luc and treated and evaluated as described under "Materials and Methods" using a 1-h ligand treatment in the absence of serum. Each point represents the mean ± S.E. of quadruplicate transfections. The experiment is representative of several identical analyses as well as evaluations carried out in the presence of serum. C, 2MD recruits RXR to the OPN promoter in intact cells via ChIP analysis. MC3T3-E1 cells were treated with either vehicle, 1,25(OH)2D3 (0.1 nM) or 2MD (0.1 nM). After 45 min, cells were harvested and subjected to ChIP analysis using anti-VDR, anti-RXR or no antibodies. DNA input is indicated. Two different anti-VDR antibodies and two anti-RXR antibodies produced similar results. PCR analysis was performed for 28 cycles using primers designed to amplify a mouse Cyp24 promoter fragment from –236 to –51 with a VDRE at –163 to –148. These data are representative of at least three similar experiments.

 

Increased VDR DNA Binding May Be Influenced by Interaction With Coactivator—In order to examine the possibility that additional factors might affect VDR/RXR DNA binding or influence the stability of the complex, we assessed whether 2MD promoted association of the VDR with coregulators such as DRIP205 or the p160 coactivators such as SRC-1 and SRC-2/GRIP. The interaction between receptor and these coactivators is believed to occur via the AF-2 region of the receptor and LXXLL motifs located within coactivators (32). As in the RXR assessment, both the GST pull-down assay and the mammalian two-hybrid assay were employed. The results seen in Fig. 8A indicate that as with GST-RXR/VDR interaction, the two ligands induce GST-SRC-1 and GST-DRIP205 interactions with the VDR in vitro with similar potency profiles. The results with GST-GRIP were identical (data not shown). In the two-hybrid system, however, 2MD again manifested a two log increase in potency over 1,25(OH)2D3 in promoting VDR interaction with both SRC-1 (Fig. 8B) and DRIP205 (Fig. 8C) and similar findings were observed with GRIP/SRC-2 (data not shown). ChIP analysis supported this finding. Thus, while 0.1 nM 1,25(OH)2D3 was ineffectual, the same concentration of 2MD strongly induced association of not only VDR and RXR to the Cyp24 gene promoter, but SRC-1 as well (Fig. 8D). These results support the idea that 2MD promotes DNA binding though enhanced formation of a complex that involves not only RXR but LXXLL-containing p160 and DRIP205 coactivators as well.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 8.
2MD is a potent inducer of VDR/SRC-1 and VDR/DRIP205 interaction. A, biochemical interaction of VDR with either SRC-1 or DRIP205 in vitro. GST-SRC-1 or GST-DRIP205 immobilized on glutathione-Sepharose beads were incubated for 30 min with purified hVDR (100 ng) in the presence of increasing concentrations of 1,25(OH)2D3 or 2MD. Complexes were collected after 30 min, washed extensively, dissolved in denaturing buffer, and subjected to Western blot analysis using the anti-VDR 9A7 antibody. These data are representative of several similar experiments. B and C, 1,25(OH)2D3- or 2MD-induced interaction of VDR and either SRC-1 or DRIP205 using mammalian two-hybrid assay. MC3T3-E1 cells were transfected with phVDR-VP16, pM-SRC-1NR, or pBIND-hDRIP205NR and pGal4(5x)-luc and the cells treated and evaluated as described under "Materials and Methods" using a 1-h ligand treatment in the absence of serum. Each point represents the mean ± S.E. of quadruplicate transfections. The data are representative of several similar experiments carried out either in the absence or in the presence of serum. B, SRC-1; C, DRIP205. D, 2MD recruits SRC-1 to the murine Cyp24 promoter in intact cells. MC3T3-E1 cells were treated with either vehicle, 1,25(OH)2D3 (0.1 nM) or 2MD (0.1 nM). Cells were harvested 45 min later and subjected to ChIP analysis using anti-VDR or anti-SRC-1 or in the absence of antibody. PCR analysis was performed for 28 cycles using primers designed to amplify a mouse Cyp24 promoter fragment from –236 to –51 with a VDRE at –163 to –148. These data are representative of several similar experiments.

 

2MD Induces a Unique VDR Conformation—Enhanced interaction of VDR with either RXR or SRC-1 seen in intact cells in response to 2MD is likely to be mediated through a unique receptor conformation. We therefore prepared in vitro translated full-length VDR and subjected it to limited proteolytic digestion using trypsin. Previous studies with the VDR (as well as other nuclear receptors) have revealed that ligand-specific cleavage products can be demonstrated following SDS-PAGE (9, 33). Fig. 9A demonstrates that incubation of receptor with either 1,25(OH)2D3 or 2MD protects the VDR from complete degradation over time, and results in the production of two receptor fragments of 28 and 34 kDa (see arrows). These fragments are believed to comprise the C-terminal hormone-binding domain of the VDR. No difference in ligand potency was evident (data not shown). Treatment with 2MD, however, leads to the appearance of a unique third fragment of ~32 kDa (Fig. 9A, see arrows). This result indicates that the conformation of the VDR differs depending upon the nature of the activating ligand. We also examined the tryptic digestion pattern of a hVDR that contained mutations in helix 12 of VDR AF-2. These mutations do not alter hormone binding activity, but completely abrogate its transcriptional capabilities (34). More importantly, proteolytic cleavage products can be demonstrated more readily with this VDR product (35). High concentrations of both 1,25(OH)2D3 and 2MD protect this mutant VDR from complete degradation (Fig. 9B). More importantly, while both the 34 and the 28 kDa bands are evident with the 1,25(OH)2D3-treated samples, it is the 28 and 32 kDa bands that are most apparent in the 2MD-treated samples. These data provide strong structural evidence that the VDR exhibits a conformation in the presence of 2MD that is different from that with 1,25(OH)2D3.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 9.
2MD induces a unique VDR conformation. [35S]methionine-labeled hVDR or mutant hVDR(R417A/R420A) was incubated with the indicated concentrations of either 1,25(OH)2D3 or 2MD for 10 min followed by treatment with trypsin (1 µg) for the indicated time (A) or 10 min (B). Digestions were terminated using denaturing buffer and the proteins resolved using SDS-PAGE and autoradiographed. Input VDR is indicated. Arrows denote receptor fragments in kDa. Upper panel, wild-type hVDR; lower panel, hVDR(R417A/R420A). The results are typical of several similar experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
2MD is a 1,25(OH)2D3 analog in which the 20-carbon methyl group resides in the S rather than the normal R configuration and a methylene group is located at carbon 2 rather than carbon 19 (16). Like 1,25(OH)2D3, 2MD displays an affinity (Kd) for the VDR of ~1 x 10–10 M, although its affinity for serum DBP is weak. These characteristics are likely to influence its pharmacokinetic properties and thus its actions in vivo. Biologically, 2MD is an extremely potent analog with significant anabolic actions in bone both in vivo and in vitro (16, 17). These observations suggest that 2MD may be both tissue- and cell-specific in its actions. In this report, we focused upon the molecular mechanisms that underlie the enhanced potency of 2MD. This potency is manifested in cell culture at the level of endogenous gene expression as well as transcription, and occurs despite the fact that both 2MD and 1,25(OH)2D3 bind equally well to the VDR. Indeed, this property is a characteristic of many chemically related classes of vitamin D analogs. We show herein using intact cell chromatin localization and cofactor recruitment assays as well as intact cell protein interaction assays and in vitro assays that the potency of 2MD is due to its ability to induce a unique conformation within the VDR that is manifested by increased VDRE binding. This increased binding is likely the result of enhanced interaction with RXR but also with receptor comodulators such as the p160 coactivator SRC-1 and/or DRIP205. These findings provide a molecular explanation for the increased potency of 2MD and together with previous studies may explain the potency of other analogs of vitamin D that exhibit similar properties.

Reported modifications of 1,25(OH)2D3 on carbon 2 have been infrequent (36, 37). Perhaps the most notable is 2{beta} (3-hydroxypropyl)-1,25(OH)2D3 (ED-71), a compound which like 2MD also promotes bone formation in vivo, although its effects are much weaker (36). Modifications in other regions of the 1,25(OH)2D3 molecule, particularly the side-chain, are more common. Of considerable interest are those that contain altered stereochemistry at carbon 20. The (20S)or20-epi analogs of the native hormone are numerous, and many of them display significantly increased potency despite an otherwise similar affinity for VDR (38). As with other analogs, the effects of this modification on receptor activity have been investigated rather extensively, although often with conflicting results. Earlier studies, for example, indicate that (20S) analogs enhance DNA binding by promoting VDR interaction with RXR in vitro (33). Additional results also suggested a hypothesis that (20S) analog-induced coactivator interactions can enhance VDR DNA-binding and/or alter transactivation due to differences in receptor-ligand contacts within the LBD of the VDR. Yang and Freedman were unable to confirm enhanced DNA binding and RXR interaction with VDR in vitro, but did demonstrate increased interaction with the coactivator DRIP205 (10). This interaction appeared to increase the extent of activity in a cell-free transcriptional assay, although this system utilized non-chromatin templates that are to a large degree independent of hormonal regulation. It is unclear at present whether either of the above observations is correct, but they do indicate the difficulties of relying exclusively on biochemical interaction assays in vitro. Accordingly, we focused upon VDR complex formation on specific chromatin DNA in intact cells using ChIP assays. We also evaluated the interaction of the VDR with its heterodimeric partner RXR as well as with potential facilitators of transactivation such as SRC-1 and DRIP205 using a mammalian two-hybrid system.

Despite the differences in potency between 1,25(OH)2D3 and 2MD both in the presence and absence of serum, the uptake of the two ligands into cells and their capacity to occupy significant levels of cellular VDR in the 0.05–0.5 nM range were very similar. Since only 2MD was capable of transcription at these concentrations, we conclude that 1,25(OH)2D3- and 2MD-liganded receptors differ in their apparent transcriptional capabilities. To explore this further, we assessed the ability of the two ligands to induce VDR binding to the OPN and Cyp24 promoters in cells using ChIP. This assay measures the relative amount of VDR bound to an endogenous gene promoter in intact cells in response to ligand activation. The results revealed a direct dose-dependent correlation between DNA-bound receptor content and transcriptional activation, with 2MD exhibiting enhanced potency relative to 1,25(OH)2D3. This correlation makes it unlikely that 2MD-activated receptors exhibit a "hyperactive" receptor state. Interestingly, when VDR ligand affinity and VDR DNA occupancy are both considered, it is not the apparent potency of 2MD but rather the "impotency" of 1,25(OH)2D3 that underlies the differences between the two ligands. Presumably, this characteristic of the 1,25(OH)2D3 ligand was evolutionarily advantageous.

Tryptic digestion of nuclear receptors has resulted in specific, ligand-dependent proteolytic patterns indicative of different receptor conformations (39, 40). That these conformational differences exist with various ligands have been substantiated particularly well for the estrogen receptor in x-ray diffractions studies (41, 42). We observed such an altered pattern of digestion of the VDR, suggesting that 2MD promotes a receptor conformation different from that with 1,25(OH)2D3. This conformation was most evident when a form of the VDR that contained mutations in two of the residues responsible for the formation of the transactivation helix-12 was examined. These patterns did not reveal a difference in dose response between the two ligands, however, suggesting that potency differences are manifested only in the cell. Interestingly, the pattern of tryptic digestion was reminiscent of that obtained by Peleg and co-workers (9, 33) following VDR protection with 20-epi-1,25(OH)2D3. Indeed, in our hands the proteolytic digestion pattern of VDR with 20-epi-1,25(OH)2D3 was the same as that with 2MD (data not shown). Thus, it seems likely that the altered VDR pattern seen in our studies with 2MD is due to the (20S) configuration in this analog and not to its 2-methylene substitution. Additional studies using different proteases may be necessary to establish whether 2MD can promote a conformation different from that with 20-epi-1,25(OH)2D3. Interestingly, the three-dimensional structure of the VDR LBD does not reveal differences in structure at the atomic level when VDR is associated with 1,25(OH)2D3 or 20-epi-1,25(OH)2D3 (12, 13). Our current crystallography studies of the VDR comparing 1,25(OH)2D3 with 2MD suggest similar conclusions.5 It is possible that ligand-dependent differences will become apparent when the structure of the full-length receptor or the VDR/RXR heterodimer is solved.

Differences in VDR structure in the presence of 2MD may be responsible for the enhanced interactions of VDR with RXR as well as with the coactivators SRC-1 or DRIP205 that are observed in the two-hybrid system. Since potency is not manifested in vitro, it suggests that formation of a multiprotein DNA complex is essential for increased DNA affinity, either as a result of increased DNA binding or as a function of enhanced complex stability. Little is known of the processes that terminate nuclear receptor-mediated transactivation or of the signals that initiate the event, although it is known that receptor ubiquitination and 26 S proteosome-mediated degradation are involved (43, 44). A key question is whether off signals are initiated though the loss of receptor ligand or whether other signals are responsible for triggering transcriptional termination. If ligand dissociation is integral to transcriptional termination, increased stability could derive from a decreased off-rate of 2MD. This would, however, necessitate a comparable decrease in the association rate in view of the similarity in Kd between 1,25(OH)2D3 and 2MD. Such a possibility has been suggested recently for a progesterone receptor mutant (45). Regardless of the mechanism, it is clear that 2MD promotes enhanced affinity of the VDR for multiple components.

2MD is both tissue as well as gene selective, eliciting preferential activity in bone relative to the gut and stimulating bone formation that is not normally associated with 1,25(OH)2D3 or its analogs (16). Why bone is a focal point of 2MD action in vivo is unknown currently, although evidence suggests that its anabolic activity may be due to its ability to promote osteoblast mineralization in vitro (17). Many vitamin D analogs appear to exhibit characteristics of tissue and/or gene selectivity. Perhaps most notable is the inability of many analogs to provoke calcium homeostatic actions in the intestine, bone, and kidney that lead to an elevation in serum calcium levels while simultaneously modulating cellular events such as proliferation and differentiation or regulating the expression of specific genes (46). The physiologic basis for this selectivity is not understood; it may be pharmacokinetic in some cases (47) and cellular in others (9, 10, 33). The mechanistic basis for selectivity could involve many aspect of VDR action. Many VDREs, for example, differ in structure from canonical direct repeats, and may well influence VDR binding and coactivator recruitment (48). This has not been proven, however. In addition, VDREs are often located immediately adjacent to binding sites for other transcription factors, suggesting the possibility of ligand-dependent selectivity for protein-protein interactions that could involve not only the adjacent transcription factors but coactivators or repressors as well (49, 50). With respect to the comodulator class of transcription factors, many have been identified (51). They differ in concentration from cell to cell, are often promoter-specific in their actions, and their individual activities are modulated through diverse signaling pathway (51, 52). Thus, ligand-selective recruitment of these coregulators could also occur, providing additional mechanisms of cell and gene selectivity. Thus, while our findings focus primarily on the potency of 2MD, they may well provide avenues to explore additional interesting features of this analog's actions.

In conclusion, we have demonstrated that the potency of 2MD is due to its ability to promote increased affinity of the VDR for specific DNA. Whether this increase is due to enhanced binding or increased stability of the transcriptional complex remains to be determined. Nevertheless, these features of 2MD action may provide hints as to how 1,25(OH)2D3 analogs such as 2MD might exert tissue- and gene-selective actions in vivo. Future studies focus on these biological properties of 2MD.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK-52453 (to J. W. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Dept. of Clinical Nutrition, School of Medicine, University of Tokushima, Tokushima 770-8503, Japan. Back

§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-262-8229; Fax: 608-263-7609; E-mail: pike{at}biochem.wisc.edu.

1 The abbreviations used are: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 2MD, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3; DBP, vitamin D-binding protein; VDR, vitamin D receptor; VDRE, vitamin D response element; ChIP, chromatin immunoprecipitation; RANKL, receptor activator of NF{kappa}B ligand; RXR, retinoid X receptor; OPG, osteoprotegerin; Cyp24, 25 hydroxyvitamin D3-24 hydroxylase; OC, osteocalcin; OPN, osteopontin; HAT, histone acetyltransferase; TRAP, tartrate-resistant acid phosphatase; LBD, ligand-binding domain; Pipes, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; FBS, fetal bovine serum; {beta}-gal, {beta}-galactosidase. Back

2 H. F. DeLuca, unpublished observations. Back

3 P. K. Grzywacz, R. R. Scizinski, and H. F. DeLuca, unpublished data. Back

4 J. W. Pike, unpublished data. Back

5 J. Vanhooke, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank members of the Pike and DeLuca laboratories for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Haussler, M. R., Whitfield, G. K., Haussler, C. A., Hsieh, J. C., Thompson, P. D., Selznick, S. H., Dominguez, C. E., and Jurutka, P. W. (1998) J. Bone Miner. Res. 13, 325–349[CrossRef][Medline] [Order article via Infotrieve]
  2. Jones, G., Strugnell, S. A., and DeLuca, H. F. (1998) Physiol. Rev. 78, 1193–1231[Abstract/Free Full Text]
  3. Beckman, M. J., and Deluca, H. F. (1998) Prog. Med. Chem. 35, 1–56[Medline] [Order article via Infotrieve]
  4. Hughes, M. R., Malloy, P. J., Kieback, D. G., Kesterson, R. A., Pike, J. W., Feldman, D., and O'Malley, B. W. (1988) Science 242, 1702–1705[Abstract/Free Full Text]
  5. Li, Y. C., Pirro, A. E., Amling, M., Delling, G., Baron, R., Bronson, R., and Demay, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9831–9835[Abstract/Free Full Text]
  6. Thompson, P. D., Remus, L. S., Hsieh, J. C., Jurutka, P. W., Whitfield, G. K., Galligan, M. A., Encinas, Dominguez. C., Haussler, C. A., and Haussler, M. R. (2001) J. Mol. Endocrinol. 27, 211–227[Abstract]
  7. Mackey, S. L., Heymont, J. L., Kronenberg, H. M., and Demay, M. B. (1996) Mol. Endocrinol. 10, 298–305[Abstract/Free Full Text]
  8. Pike, J. W., Yamamoto, H., and Shevde, N. K. (2002) Adv. Ren. Replace Ther. 9, 168–174[CrossRef][Medline] [Order article via Infotrieve]
  9. Peleg, S., Sastry, M., Collins, E. D., Bishop, J. E., and Norman, A. W. (1995) J. Biol. Chem. 270, 10551–10558[Abstract/Free Full Text]
  10. Yang, W., and Freedman, L. P. (1999) J. Biol. Chem. 274, 16838–16845[Abstract/Free Full Text]
  11. Carlberg, C. (2003) J. Cell. Biochem. 88, 274–281[CrossRef][Medline] [Order article via Infotrieve]
  12. Rochel, N., Wurtz, J. M., Mitschler, A., Klaholz, B., and Moras, D. (2000) Mol. Cell 5, 173–179[CrossRef][Medline] [Order article via Infotrieve]
  13. Tocchini-Valentini, G., Rochel, N., Wurtz, J. M., Mitschler, A., and Moras, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5491–5496[Abstract/Free Full Text]
  14. Bikle, D. D. (1992) Endocr. Rev. 13, 765–788[Abstract/Free Full Text]
  15. Norman, A. W. (1995) J. Cell. Biochem. 22, S218–S222
  16. Sicinski, R. R., Prahl, J. M., Smith, C. M., and DeLuca, H. F. (1998) J. Med. Chem. 41, 4662–4674[CrossRef][Medline] [Order article via Infotrieve]
  17. Shevde, N. K., Plum, L. A., Clagett-Dame, M., Yamamoto, H., Pike, J. W., and DeLuca, H. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 13487–13491[Abstract/Free Full Text]
  18. Hofbauer, L. C., Khosla, S., Dunstan, C. R., Lacey, D. L., Boyle, W. J., and Riggs, B. L. (2000) J. Bone Miner. Res. 15, 2–12[CrossRef][Medline] [Order article via Infotrieve]
  19. Morony, S., Capparelli, C., Lee, R., Shimamoto, G., Boone, T., Lacey, D. L., and Dunstan, C. R. (1999) J. Bone Miner. Res. 14, 1478–1485[CrossRef][Medline] [Order article via Infotrieve]
  20. Jin, C. H., Kerner, S. A., Hong, M. H., and Pike, J. W. (1996) Mol. Endocrinol. 10, 945–957[Abstract/Free Full Text]
  21. Zou, A., Elgort, M. G., and Allegretto, E. A. (1997) J. Biol. Chem. 272, 19027–19034[Abstract/Free Full Text]
  22. Chang, C., Norris, J. D., Gron, H., Paige, L. A., Hamilton, P. T., Kenan, D. J., Fowlkes, D., and McDonnell, D. P. (1999) Mol. Cell. Biol. 19, 8226–8239[Abstract/Free Full Text]
  23. Pathrose, P., Barmina, O., Chang, C. Y., McDonnell, D. P., Shevde, N. K., and Pike J. W. (2002) J. Bone Miner. Res. 17, 2196–2205[CrossRef][Medline] [Order article via Infotrieve]
  24. Shevde, N. K., Bendixen, A. C., Dienger, K. M., and Pike, J. W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7829–7834[Abstract/Free Full Text]
  25. Pike, J. W., and Haussler, M. R. (1983) J. Biol. Chem. 258, 8554–8560[Abstract/Free Full Text]
  26. Pike, J. W., Marion, S. L., Donaldson, C. A., and Haussler, M. R. (1983) J. Biol. Chem. 258, 1289–1296[Free Full Text]
  27. Weinman, A. S., and Farnham, P. J. (2002) Methods 26, 37–47[CrossRef][Medline] [Order article via Infotrieve]
  28. Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000) Cell 103, 843–852[CrossRef][Medline] [Order article via Infotrieve]
  29. Cook, N. E., and Haddad, J. G. (1984) Endocr. Rev. 10, 294–307[CrossRef]
  30. Sone, T., Kerner, S., and Pike, J. W. (1991) J. Biol. Chem. 266, 23296–23305[Abstract/Free Full Text]
  31. Cheskis, B., and Freedman, L. P. (1996) Biochemistry 12, 3309–3318
  32. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) Nature 387, 733–736[CrossRef][Medline] [Order article via Infotrieve]
  33. Liu, Y. Y., Nguyen, C., and Peleg, S. (2000) Mol. Endocrinol. 14, 1776–1787[Abstract/Free Full Text]
  34. Kraichely, D. M., Collins, J. J., III, DeLisle, R. K., and MacDonald, P. N. (1999) J. Biol. Chem. 274, 14352–14358[Abstract/Free Full Text]
  35. Vaisanen, S., Ryhanen, S., Saarela, J. T., and Maenpaa, P. H. (1999) Eur. J. Biochem. 261, 706–713[Medline] [Order article via Infotrieve]
  36. Kubodera, N., Tsuji, N., Uchiyama, Y., and Endo, K. (2003) J. Cell. Biochem. 88, 286–289[CrossRef][Medline] [Order article via Infotrieve]
  37. Kittaka, A., Kurihara, M., Peleg, S., Suhara, Y., and Takayama, H. (2003) Chem. Pharm. Bull. (Tokyo) 51, 357–358
  38. Binderup, L., Binderup, E., and Godtfredsen, W. O. (1997) in Vitamin D (Feldman, D., Glorieux, F., and Pike, J. W., eds) pp. 1027–1043, Academic Press, San Diego
  39. Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M. J., and O'Malley, B. W. (1992) J. Biol. Chem. 267, 19513–19520[Abstract/Free Full Text]
  40. Beekman, J. M., Allan, G. F., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1993) Mol. Endocrinol. 7, 1266–1274[Abstract/Free Full Text]
  41. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927–937[CrossRef][Medline] [Order article via Infotrieve]
  42. Pike, A. C., Brzozowski, A. M., Walton, J., Hubbard, R. E., Bonn, T., Gustafsson, J. A., and Carlquist, M. (2000) Biochem. Soc. Trans. 28, 396–400[Medline] [Order article via Infotrieve]
  43. Nawaz, Z., Lonard, D. M., Dennis, A. P., Smith, C. L., and O'Malley, B. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1858–1862[Abstract/Free Full Text]
  44. Masuyama, H., and MacDonald, P. N. (1998) J. Cell. Biochem. 71, 429–440[CrossRef][Medline] [Order article via Infotrieve]
  45. James, A. J., Agoulnik, I. U., Harris, J. M., Buchanan, G., Tilley, W. D., Marcelli, M., Lamb, D. J., and Weigel, N. L. (2002) Mol. Endocrinol. 16, 2692–2705[Abstract/Free Full Text]
  46. Brown, A. J., and Slatopolsky, E. S. (1997) in Vitamin D (Feldman, D., Glorieux, F., and Pike, J. W., eds) pp. 1027–1043, Academic Press, San Diego
  47. Kissmeyer, A. M., and Binderup, L. (1991) Biochem. Pharmacol. 41, 1601–1606[CrossRef][Medline] [Order article via Infotrieve]
  48. Herdick, M., Bury, Y., Quack, M., Uskokovic, M. R., Polly, P., and Carlberg, C. (2000) Mol. Pharmacol. 57, 1206–1217[Abstract/Free Full Text]
  49. Ozono, K., Liao, J., Kerner, S. A., Scott, R. A., and Pike, J. W. (1990) J. Biol. Chem. 265, 21881–21888[Abstract/Free Full Text]
  50. Dwivedi, P. P, Hii, C. S., Ferrante, A., Tan, J., Der, C. J., Omdahl, J. L., Morris, H. A., and May, B. K. (2002) J. Biol. Chem. 277, 29643–29653[Abstract/Free Full Text]
  51. McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999) Endocr. Rev. 20, 321–344[Abstract/Free Full Text]
  52. Rowan, B. G., Weigel, N. L., and O'Malley, B. W. (2000) J. Biol. Chem. 275, 4475–4483[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 JCBHome page
T. Mizoguchi, A. Muto, N. Udagawa, A. Arai, T. Yamashita, A. Hosoya, T. Ninomiya, H. Nakamura, Y. Yamamoto, S. Kinugawa, et al.
Identification of cell cycle-arrested quiescent osteoclast precursors in vivo
J. Cell Biol., February 23, 2009; 184(4): 541 - 554.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. A. Zella, N. K. Shevde, B. W. Hollis, N. E. Cooke, and J. W. Pike
Vitamin D-Binding Protein Influences Total Circulating Levels of 1,25-Dihydroxyvitamin D3 but Does Not Directly Modulate the Bioactive Levels of the Hormone in Vivo
Endocrinology, July 1, 2008; 149(7): 3656 - 3667.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
R. D. Nerenz, M. L. Martowicz, and J. W. Pike
An Enhancer 20 Kilobases Upstream of the Human Receptor Activator of Nuclear Factor-{kappa}B Ligand Gene Mediates Dominant Activation by 1,25-Dihydroxyvitamin D3
Mol. Endocrinol., May 1, 2008; 22(5): 1044 - 1056.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
J. A. Fretz, N. K. Shevde, S. Singh, B. G. Darnay, and J. W. Pike
Receptor Activator of Nuclear Factor-{kappa}B Ligand-Induced Nuclear Factor of Activated T Cells (C1) Autoregulates Its Own Expression in Osteoclasts and Mediates the Up-Regulation of Tartrate-Resistant Acid Phosphatase
Mol. Endocrinol., March 1, 2008; 22(3): 737 - 750.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. B. Meyer, L. A. Zella, R. D. Nerenz, and J. W. Pike
Characterizing Early Events Associated with the Activation of Target Genes by 1,25-Dihydroxyvitamin D3 in Mouse Kidney and Intestine in Vivo
J. Biol. Chem., August 3, 2007; 282(31): 22344 - 22352.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Kim, M. Yamazaki, L. A. Zella, N. K. Shevde, and J. W. Pike
Activation of Receptor Activator of NF-{kappa}B Ligand Gene Expression by 1,25-Dihydroxyvitamin D3 Is Mediated through Multiple Long-Range Enhancers.
Mol. Cell. Biol., September 1, 2006; 26(17): 6469 - 6486.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
J. A. Fretz, L. A. Zella, S. Kim, N. K. Shevde, and J. W. Pike
1,25-Dihydroxyvitamin D3 Regulates the Expression of Low-Density Lipoprotein Receptor-Related Protein 5 via Deoxyribonucleic Acid Sequence Elements Located Downstream of the Start Site of Transcription
Mol. Endocrinol., September 1, 2006; 20(9): 2215 - 2230.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
L. A. Zella, S. Kim, N. K. Shevde, and J. W. Pike
Enhancers Located within Two Introns of the Vitamin D Receptor Gene Mediate Transcriptional Autoregulation by 1,25-Dihydroxyvitamin D3
Mol. Endocrinol., June 1, 2006; 20(6): 1231 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. B. Meyer, M. Watanuki, S. Kim, N. K. Shevde, and J. W. Pike
The Human Transient Receptor Potential Vanilloid Type 6 Distal Promoter Contains Multiple Vitamin D Receptor Binding Sites that Mediate Activation by 1,25-Dihydroxyvitamin D3 in Intestinal Cells
Mol. Endocrinol., June 1, 2006; 20(6): 1447 - 1461.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Q. Shen and S. Christakos
The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional Regulation of Osteopontin
J. Biol. Chem., December 9, 2005; 280(49): 40589 - 40598.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
T. Matsumoto, T. Miki, H. Hagino, T. Sugimoto, S. Okamoto, T. Hirota, Y. Tanigawara, Y. Hayashi, M. Fukunaga, M. Shiraki, et al.
A New Active Vitamin D, ED-71, Increases Bone Mass in Osteoporotic Patients under Vitamin D Supplementation: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial
J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5031 - 5036.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
S. Nagpal, S. Na, and R. Rathnachalam
Noncalcemic Actions of Vitamin D Receptor Ligands
Endocr. Rev., August 1, 2005; 26(5): 662 - 687.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
G. Eelen, L. Verlinden, N. Rochel, F. Claessens, P. De Clercq, M. Vandewalle, G. Tocchini-Valentini, D. Moras, R. Bouillon, and A. Verstuyf
Superagonistic Action of 14-epi-Analogs of 1,25-Dihydroxyvitamin D Explained by Vitamin D Receptor-Coactivator Interaction
Mol. Pharmacol., May 1, 2005; 67(5): 1566 - 1573.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
H. F DeLuca
Overview of general physiologic features and functions of vitamin D
Am. J. Clinical Nutrition, December 1, 2004; 80(6): 1689S - 1696S.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
L. Peng, P. J. Malloy, and D. Feldman
Identification of a Functional Vitamin D Response Element in the Human Insulin-Like Growth Factor Binding Protein-3 Promoter
Mol. Endocrinol., May 1, 2004; 18(5): 1109 - 1119.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Jiang, P. Li, A. J. Fornace Jr., S. V. Nicosia, and W. Bai
G2/M Arrest by 1,25-Dihydroxyvitamin D3 in Ovarian Cancer Cells Mediated through the Induction of GADD45 via an Exonic Enhancer
J. Biol. Chem., November 28, 2003; 278(48): 48030 - 48040.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/34/31756    most recent
M304737200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamamoto, H.
Right arrow Articles by Pike, J. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamamoto, H.
Right arrow Articles by Pike, J. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement