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Endocrinology Vol. 143, No. 5 1625-1636
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Cellular and Molecular Events Associated with the Bone-Protecting Activity of the Noncalcemic Vitamin D Analog Ro-26-9228 in Osteopenic Rats

Sara Peleg, Milan Uskokovic, Ago Ahene, Brian Vickery and Zafrira Avnur

Department of Endocrine Neoplasia and Hormonal Disorders, University of Texas, M. D. Anderson Cancer Center (S.P.), Houston, Texas 77030; and Department of Musculoskeletal Research, Roche Bioscience (M.U., A.A., B.V., Z.A.), Palo Alto, California 94305

Address all correspondence and requests for reprints to: Sara Peleg, Ph.D., Department of Endocrine Neoplasia and Hormonal Disorders, Box 435, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: . speleg{at}mail.mdanderson.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have examined several analogs of 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an animal model of osteoporosis (ovariectomized rats) to identify a compound with a greater therapeutic range than 1,25-(OH)2D3 for treatment of this bone disease. Here, we report that one analog, Ro-26-9228, had a bone-protecting effect but did not induce hypercalcemia at a wide concentration range. Analysis of biochemical markers and the bone histomorphometry of analog-treated rats suggested that Ro-26-9228 acted by inhibiting bone resorption and increasing the number of differentiated osteoblasts. To determine the basis for the segregation between hypercalcemia and bone-protecting action, we examined gene expression in tissues that regulate calcium homeostasis. We found that 1,25-(OH)2D3 induced 24-hydroxylase mRNA expression in the duodena of ovariectomized rats, but Ro-26-9228 did not. Furthermore, in the duodena of intact animals, 1,25-(OH)2D3 induced a significant increase in calbindin D 9K and plasma membrane calcium pump 1 mRNAs, but Ro-26-9228 had no effect on these mRNAs. On the other hand, the osteoblast-specific gene products osteocalcin and osteopontin were significantly up-regulated in trabecular bone by both the natural hormone and Ro-26-9228. Further investigation of gene-regulatory events in trabecular bone revealed that both 1,25-(OH)2D3 and Ro-26-9228 up-regulated TGF ß1 and ß2 mRNAs. We concluded that the unique properties of Ro-26-9228 include preferential gene regulation in osteoblasts over duodenum and effective induction of growth factors in bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOPOROSIS IS CHARACTERIZED by low bone mass and microarchitectural deterioration of bone tissue leading to enhanced bone fragility and increasing risk of fracture (1). Postmenopausal osteoporosis, defined as bone density more than 2.5 SD below mean values of young adults, is the most common metabolic disorder of bone and affects more than 26 million women in the United States. Based on the bone density criterion, 30% of Caucasian postmenopausal women in the United States are classified by the World Health Organization as osteoporotic (2). The risk of fracture increases exponentially as bone density decreases; and because bone density decreases with age, postmenopausal women experience an exponential increase in fracture incidence with increasing age (3).

Bone is a metabolically active tissue with a high potential for developing pathologic reductions in mass, over many cycles of remodeling (formation and resorption) (4). Endocrine regulation of bone remodeling involves PTH, calcitonin, insulin, GH, 1{alpha},25-dihydroxyvitamin D3 (1,25-(OH)2D3), glucocorticoids, sex steroids, and thyroid hormones (5). Local skeletal autocrine and paracrine regulation is mediated by IGF-1, bone morphogenetic proteins, TGF-ß, fibroblast growth factor, and ILs (6). Loss of bone mass in osteoporosis involves both the organic (e.g. collagen) and mineral (e.g. calcium) constituents of normal bone. However, the biochemical and molecular mechanisms responsible for osteoporotic bone loss remain largely unknown. Effective therapies for the prevention and treatment of osteopenia and osteoporosis require agents that can safely induce clinically significant net bone formation in addition to merely preventing further bone loss.

The efficacy and safety of several vitamin D metabolites and analogs, including the active hormonal form of vitamin D3 (1,25-(OH)2D3) have been tested in women with postmenopausal osteoporosis. Each of these metabolites and analogs has a narrow therapeutic window for the prevention and treatment of osteoporosis (7). Therefore, although beneficial skeletal effects have been observed, the hypercalcemic potency of these agents has been difficult to control. Hypercalcemia has been controlled on a chronic basis only by frequent monitoring of serum calcium and subsequent dose adjustments and by restricting dietary calcium to levels far below those recommended by national and international health policy organizations (8).

To identify an effective therapeutic and preventive agent for osteoporosis without these limitations, we have considered the use of vitamin D analogs that may have low calcemic activity, with significant ability to prevent bone loss. Extensive research on vitamin D analogs, in the past few years, has shown that structural modifications in these compounds may result in differential activation of the VDR and that this is associated with changes in VDR-mediated actions in vivo (9). For example, epimerization at carbon 20 of the side chain of 1,25-(OH)2D3 dramatically increases transcriptional activation of the VDR, by enhancing dimerization of VDR with RXR and interaction of VDR with the VDR-interacting protein, DRIP 250 (10, 11). Compounds with this side-chain modification are very potent inhibitors of cell growth and are also very calcemic in vivo (12). The noncalcemic analog OCT makes the VDR interact selectively with coactivators of transcription (13). The analog (23S)-25-dehydro-1{alpha}-hydroxyvitamin D3–26,23-lactone (TEI-9647) is the first synthetic antagonist that inhibits 1,25-(OH)2D3-mediated transcriptional activities of the VDR in vitro as well as calcium-regulating activities of 1,25-(OH)2D3 in vivo (14, 15). The realization that the VDR may be differently activated by the hormone and by its synthetic analogs led us to explore the possibility that some structural modifications in 1,25-(OH)2D3 may have tissue-selective or gene-selective activities, not unlike the selective ER modulators (16). For that purpose, we screened numerous vitamin D analogs, using an animal model of osteopenia to identify compounds that increased bone mineral density (BMD) without inducing hypercalcemia; and we identified a promising compound, Ro-26-9228 (Fig. 1Go). In the experiments described below, we found that Ro-26-9228 had a significant therapeutic window for treatment of osteoporosis in ovariectomized (OVX) rats. The gene expression profile of Ro-26-9228-treated animals and its transcriptional activities in culture system suggested that this compound might be a selective modulator of vitamin D-responsive genes.



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  		Figure 1. Structures of 1,25-(OH)2D3 and its analog Ro-26-9228.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatment
The Roche Bioscience Animal Use and Care Committee approved all experimental procedures. Adult female rats (Sprague Dawley) were subjected to bilateral ovariectomy or sham surgery by the vendor (Charles River Laboratories, Inc., Wilmington, MA). After shipment and acclimatization for at least 1 wk, the animals were given 1,25-(OH)2D3 or Ro-26-9228 at the specified concentrations by oral gavage of 1 ml/kg. Stock solutions of the drugs were dissolved in ethanol to a concentration of 100 mg/ml. Before dosing, the compound solutions were rotary-evaporated at 37 C to remove the ethanol and then dissolved in vehicle (Miglyol 812, a medium-chain triglyceride with the antioxidants 0.01% butylated hydroxyanisole and 0.01% butylated hydroxytoluene). Dose volume was adjusted weekly by using the group average body weight. Dosing was started 19 d after surgery and continued for the times indicated in the figure legends.

Bone density determinations by dual-energy x-ray absorption
Twenty-four hours after the final treatment, the animals were killed by carbon dioxide inhalation and frozen for BMD determinations. Each animal was thawed and then placed supine on a scanning block, with the right hind leg perpendicular to the body and the tibia perpendicular to the femur. Bone density was measured in the distal femur metaphysis with a dual-energy absorptiometer (QDR-1000W or QDR-1500 Bone Densitometer; Hologic, Inc., Waltham, MA) and a high-resolution software package.

Serum and urine collection and biochemical analysis
Blood (1.5 ml) was collected from each animal, by orbital puncture, under ether anesthesia, on the final day of dosing. The blood was transferred to serum separator tubes and centrifuged at 2000 rpm for 15 min, and the serum was aliquoted. A colorimetric assay, using Biomek Robotics (Beckman Coulter, Inc., Fullerton, CA) and reagents from Sigma (St. Louis, MO), were used to measure serum calcium and creatinine levels. For urine collection, the animals were placed in metabolic cages for 24 h, after 3 wk of dosing. Urine calcium and creatinine were measured by the colorimetric assay described above. An ELISA kit (Pyrilinks Kit 8014; Metra Biosystems, Palo Alto, CA) was used to measure urinary pyridinoline.

Bone histomorphometry and histology
OVX rats were treated with 1,25-(OH)2D3 or Ro-26-9228 for 3 wk, starting on d 19 post surgery, and then killed, and bone samples were collected. The proximal third of each tibia (n = 6–10) was processed for methyl/methacrylate embedding. Approximately one-eighth of the anterior face of each tibia was removed to facilitate embedding. The tissues were fixed in 4% buffered paraformaldehyde, dehydrated with a series of alcohol and xylene changes, and infiltrated with methyl/methacrylate resin with a catalyst and hardener, by using a Shandon Lipshaw Hypercenter Tissue Processor (Shandon Scientific Ltd., Cheshire, UK). After 3–7 d of embedding in glass vials at 37 C, 5-µm longitudinal sections of each proximal tibia metaphysis were cut with a Leitz (Rockleigh, NJ)-Reichart 2065 microtome, deplasticized, and stained with a modified Goldner’s trichrome stain and toluidine blue counterstain. The standard sampling area was defined in the secondary spongiosa of the metaphysis. To identify this area, 1 mm distal to the junction of the epiphyseal growth plate and the metaphysis, a constant reference point was placed at the upper edge of the image field perpendicular to, and on the long axis of, the bone. The trabecular bone was examined in this and five adjacent fields by using a 20x objective.

Preparation of RNA from tissues and analysis of gene expression
Duodenal mucosa and tibias were collected 7 or 24 h after a single oral dose of the compounds and quickly frozen in liquid nitrogen. Total RNA was extracted by the guanidine/phenol/chloroform procedure (17). Northern blot analysis was used to detect 24-hydroxylase, calbindin D 9K, and plasma membrane calcium pump 1 (PMCA-1) mRNAs in duodenum and osteopontin and osteocalcin in tibia metaphysis, a region containing mostly trabecular bone and therefore undergoing active bone remodeling in our experimental model. Glyceraldehyde-3-phosphate dehydrogenase (G3P) mRNA was used as a control to normalize vitamin D-regulated mRNA expression in individual samples.

Total RNA (10 µg/lane) from each animal (6–10/group) was denatured in formamide/formaldehyde loading buffer and separated by formaldehyde/agarose gel electrophoresis. The RNA was transferred onto nylon membranes overnight and cross-linked by baking in a vacuum oven. The membranes were prehybridized in 50% formamide buffer containing salmon sperm DNA and dextran sulfate. For preparation of probes, the fragments encoding the relevant cDNAs were labeled with [32P]deoxycytidine triphosphate with a random priming labeling kit (Amersham Pharmacia Biotech, Arlington Heights, IL) to a specific activity of 3–5 x 108 cpm/µg. The 32P-labeled heat-denatured probes were hybridized in the same buffer at 42 C overnight. Then, unbound probe was discarded, and nonspecific hybridization was removed by stringent washing (the temperature and salt concentration of the wash buffer were adjusted for each probe). The membranes were exposed to x-ray film for 3 h to 3 d, and signals were recorded by densitometric scanning. Each membrane was rehybridized up to 3 times.

For the detection of TGF-ß1 and TGF-ß2 mRNA, we performed semiquantitative RT-PCR, using the following primers: The forward primer for rat TGF-ß1 was 5'-GAAGCGCATCGAAGCCATCCGTGG-3', and the reverse primer was 5'-GCAGTTCTTCTCTGTGGAGCTG-3'. The forward primer for TGF-ß2 was 5'-AAATGGATACACGAACCCAA-3', and the reverse primer was 5'-GCTGCATTTGCAAGACTTTAC-3'. The forward primer for rat G3P was 5'-CATGTAAGGCCATGAGGTCCACCAC-3', and the reverse primer was 5'-CATGTAGGCCATGAGGTCCACCAC-3'. Primer sequences were obtained from GenBank.

RT-PCR was performed as follows: Five micrograms of total RNA was heat-denatured with 1 µg random primer for 10 min at 70 C and then placed on ice, and first-strand buffer (Life Technologies, Inc., Rockville, MD), dithiothreitol, and 25 mM deoxynucleotide triphosphates were added. This mixture was incubated 10 min at 25 C and 2 min at 42 C, 1 µl of reverse transcriptase (40 U, SuperScript II; Life Technologies, Inc.) was added, and the reaction was continued for 50 min at 42 C. The semiquantitative PCRs were performed by using 3 vol of cDNA (2.5%, 5%, and 10% of the reverse transcriptase reaction mixture) and the appropriate set of primers, in a final vol of 50 µl containing the reaction buffer and Taq polymerase (Boehringer Ingelheim GmbH, Mannheim, Germany). The amplification was performed for 30 cycles of 1 min at 95 C, 1 min at 55 C, and 2 min at 72 C, with a final step of 6 min at 72 C, in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). A fraction of the reaction mixture was analyzed by agarose gel electrophoresis and detected by ethidium bromide staining under UV light. The PCR signals were quantified, relative to the G3P product, by densitometric scanning of the ethidium bromide-stained gels.

Pharmacokinetics
Female rats (Sprague Dawley) were injected with the analog Ro-26-9228 via the tail vein. Blood samples from each rat were collected, from the retroorbital sinus or by cardiac puncture, into syringes containing lithium heparin at 0, 5, 15, and 30 min and 1, 2, 4, 6, 8, 10, 12, and 24 h after dosing. Three rats were used for each time point. Blood samples were cooled on ice immediately and centrifuged in a refrigerated centrifuge, at 10 C, to obtain plasma. Plasma samples (0.3 ml) were added to 50 µl of an internal standard (0.2 µg/ml D10-Ro-26-9228 in 1:1 isopropanol and water), mixed with 0.25 ml water, and extracted with 1 ml of 20:80 (vol:vol) ethyl acetate:hexane. The samples were agitated for 10 min and then centrifuged at 5 C for 10 min. The aqueous (bottom) layer was then frozen on dry ice; and the upper, organic layer was decanted into a clean tube. The organic layer was evaporated and then reconstituted in 70% (vol:vol) methanol in water. The reconstituted solution was subjected to liquid chromatography/mass spectrometry with a 1090 HPLC pump and a cooled autosampler compartment (both from Hewlett-Packard Co., Palo Alto, CA), and a Finnigan TSQ 7000 mass spectrometer (Finnigan-MAT, San Jose, CA) operated in positive-atmospheric-pressure chemical ionization mode. The mobile phase consisted of a premixed solution containing 20:75:5 ammonium formate (50 mM, pH 4.2) :methanol: acetonitrile. The flow rate was 0.25 ml/min. The column used was a C8 Hypersil column, from Column Engineering (Ontario, CA), with dimensions of 2 mm x 150 mm x 5 µm. The column temperature was kept at 40 C. The retention time of Ro-26-9228 was about 7 min. The amount of Ro-26-9228 in the plasma was expressed as the ratio of the peak height ratio to the internal standard, and the concentration of Ro-26-9228 was interpolated from a standard curve constructed with known concentrations of Ro-26-9228.

Binding to vitamin D binding protein (DBP)
Human DBP (Biodesign, Kennebunk, ME) was reconstituted in deionized water and diluted with HEPES buffer (pH 7.4) containing 5 g/liter BSA and 0.1 ml/liter Tween 80, to final DBP concentrations of 1000, 500, 250, 100, 50, 25, and 12.5 µg/ml. Twenty microliters of [3H]Ro-26-9228 (0.118 µCi/ml) or [3H]1,25-(OH)2D3 (0.126 µCi/ml) was added to 2 ml of either DBP solution or its buffer, at 37 C, to a final concentration of 0.5 pg/ml. The mixtures were incubated at 37 C for 30 min, and then 0.2-ml aliquots of each incubation mixture were transferred to six tubes. These tubes were set on ice for 5 min, and then 0.2-ml aliquots of dextran-coated charcoal [10 mg/ml prepared in PBS containing 0.5 g/liter BSA (pH 8.6)] were added. The mixtures were vortexed and kept in ice for 10 min. The charcoal-containing tubes were centrifuged for 20 min at 3000 rpm at 4 C, the supernatants were decanted into scintillation vials, and the radioactivity was measured in a Beckman Coulter, Inc. scintillation counter. Specific binding to DBP was determined by subtracting radioactivity in the HEPES/BSA buffer-containing tubes from radioactivity recovered from the DBP-containing tubes.

Receptor binding and competition assays
To assess the relative affinities of 1,25-(OH)2D3 and Ro-26-9228 for the rat VDR (rVDR), whole-cell homogenates from confluent cultures of rat osteosarcoma ROS-17/2.8 cells were prepared in KTED [10 mM Tris-HCl (pH 7.4), 1.5 mM EDTA, 0.3 M KCl, and 1 mM dithiothreitol] (18). To assess the relative affinities of 1,25-(OH)2D3 and the analog for the human VDR, whole-cell homogenates were prepared from COS-1 cells transfected with recombinant human VDR expression vector, as described previously (18). The homogenates were aliquoted into tubes containing 0.2 pmol [3H]1,25-(OH)2D3 and increasing concentrations of nonradioactive ligand. The mixtures were incubated on ice for 3–4 h; after which, free ligand was separated from bound ligand by hydroxyapatite. The bound ligand was released from the hydroxyapatite by ethanol extraction, and the radioactivity was measured by scintillation counting. The results of the competition assays were plotted as the inverse value of percent maximal binding against competitor concentration, by the method of Wecksler and Norman (19).

Transfections and transcriptional assays
Caco-2 human colon carcinoma cells and MG-63 human osteosarcoma cells were obtained from the ATCC (Manassas, VA) and maintained in DMEM and 10% FBS. Forty-eight hours before transfection, the cells were plated in 35-mm dishes at a density of 3 x 105 cells/dish. The cells were transfected by the diethylaminoethyl-dextran method, with 2 µg/dish of a reporter construct containing the human osteocalcin vitamin D-responsive element (VDRE) linked to the thymidine kinase promoter and the GH reporter gene (18). Medium was collected 2 d after transfection, and GH was measured by using an RIA as described by the manufacturer (Nichols Institute Diagnostics, San Juan Capistrano, CA).

Statistics
The treatment and control groups (n = 6–10 per group) were compared by one-way ANOVA followed by Fisher’s least significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment of OVX osteopenic rats with Ro-26-9228 restored bone loss without inducing hypercalcemia
Three-month-old OVX female rats were given either 1,25-(OH)2D3 (0.1–0.4 µg/kg) or Ro-26-9228 (0.1–14 µg/kg) daily, starting 3 wk after ovariectomy. Three weeks later, the BMD of the femur metaphyses was measured by dual-energy x-ray absorption. Figures 2AGo and 3AGo show that estrogen depletion in OVX animals significantly decreased BMD. The average decrease in BMD, at this site, in the OVX animals was 18% at the beginning of the dosing period (3 wk after ovariectomy) and 21% at the end of the experiment (6 wk after ovariectomy). Both 1,25-(OH)2D3 and Ro-26-9228 significantly increased BMD in the OVX animals. The lowest concentration at which 1,25-(OH)2D3 induced a significant increase in BMD was 0.2 µg/kg, and the lowest concentration at which Ro-26-9228 induced a significant increase in BMD was 3 µg/kg. To determine the mechanism for this increase in BMD, we measured urine levels of type I collagen degradation products (pyridinoline cross-links). Figures 2BGo and 3BGo show that pyridinoline levels increased significantly in OVX animals and that both 1,25-(OH)2D3 and Ro-26-9228 inhibited pyridinoline excretion. The analog had a significant effect on this parameter of bone remodeling at as little as 0.2 µg/kg; and at 9 and 14 µg/kg, it decreased pyridinoline excretion below the levels of sham-operated control animals. 1,25-(OH)2D3 significantly inhibited pyridinoline excretion at as little as 0.1 µg/kg; and at 0.4 µg/kg, it decreased pyridinoline excretion below the levels of sham-operated control animals.



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  		Figure 2. Changes in calcium metabolism in OVX osteopenic rats treated with 1,25-(OH)2D3. Three-month-old female rats were sham-operated (sham) or OVX. Three weeks later, the OVX rats were given the indicated amounts of 1,25-(OH)2D3 daily. A, BMD values at the femur metaphysis after 3 wk of treatment. For measurement of collagen degradation products (pyridinoline cross-links) (B) and urine calcium (D), urine was collected over 24 h, and pyridinoline was measured with an enzyme-linked immunoabsorbant assay. Urinary pyridinoline values and urinary calcium values were normalized against urine creatinine levels. C, Serum calcium. The number of animals per group was 10. *, P < 0.05, compared with OVX rats.

 


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  		Figure 3. Changes in calcium metabolism in OVX osteopenic rats treated with Ro-26-9228. Three-month-old female rats were sham or OVX and then given the indicated doses of Ro-26-9228 daily. BMD (A), pyridinoline cross-links (B), serum calcium (C), and urinary calcium (D) were measured as described in Fig. 2Go and in Materials and Methods. *, P < 0.05, compared with OVX rats.

 
To determine whether these changes in bone remodeling occurred concurrently with hypercalcemia and hypercalciuria, urine calcium (Figs. 2DGo and 3DGo) and serum calcium (Figs. 2CGo and 3CGo) were also measured. These measurements showed that toxic hypercalciuria (more than 1 mg/dl) was detected in 1,25-(OH)2D3-treated animals, with as little as 0.1 µg/kg, and hypercalcemia was detected at 0.2 µg/kg. In contrast, Ro-26-9228 induced a significant increase in urine calcium only at 9 µg/kg; and even at 14 µg/kg, urine calcium in the analog-treated animals did not reach 1 mg/dl. Likewise, no increase in serum calcium was detected in this experiment, even at 14 µg/kg. Therefore, these results indicate that the hormone inhibited bone resorption and increased BMD at concentrations that were already compromising calcium homeostasis in the OVX rats, whereas the analog inhibited bone resorption and increased BMD at concentrations that did not disrupt calcium homeostasis.

Table 1Go summarizes results from 4 similar experiments assessing the actions of Ro-26-9228 on BMD and calcium homeostasis. The minimum dose of Ro-26-9228 that induced a significant increase in BMD was 1 µg/kg, hypercalciuria greater or equal to 1 mg/dl was reached at more than 14 to less than 27 µg/kg, and only 17–27 µg/kg of the analog induced a significant increase in serum calcium. In addition, the maximum efficacy of the analog’s effect on BMD within the safe concentration range was 60–70%, whereas the efficacy of 1,25-(OH)2D3 to restore BMD at 0.2 µg/kg (a concentration at which a significant increase in BMD occurred but was associated with hypercalciuria and hypercalcemia) was 33–54%. Because most of the bone loss occurred in these animals within the first 3 wk after ovariectomy, the increases in BMD in 1,25-(OH)2D3- and analog-treated animals at the end of these experiments (6 wk after ovariectomy) reflect both prevention of bone loss and restoration of bone in the OVX rats. Overall, these experiments indicated that 1,25-(OH)2D3 has a narrow therapeutic window, because it increased BMD at doses overlapping those that induced significant hypercalcemia. In contrast, the analog had a significant therapeutic window, because doses that induced hypercalcemia were 3–11 times greater than doses that induced a significant increase in BMD. The finding that Ro-26-9228 (and 1,25-(OH)2D3) reduced the levels of urinary pyridinoline strongly suggests that their bone-protecting action is attributable, in part, to their antiresorptive activity.


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  		Table 1. Safety and efficacy of Ro-26-9228 in osteopenic rats1

 
Effects of 1,25-(OH)2D3 and Ro-26-9228 on bone structure
To further characterize the changes in bone induced by Ro-26-9228 and to compare them with changes induced by 1,25-(OH)2D3, we examined the histology and histomorphometry of trabecular bone from OVX rats treated for 3 wk with 1,25-(OH)2D3 or Ro-26-9228. Figure 4Go shows Goldner’s staining with a tuloidine blue counterstain of sections prepared from undecalcified trabecular bone taken from Ro-26-9228-treated animals and OVX animals. Figure 4BGo show that the analog increased the area of trabecular bone to a level greater than that of OVX animals (Fig. 4AGo). These changes were confirmed quantitatively (data not shown).



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  		Figure 4. Effect of Ro-26-9228 on the histology of trabecular bone from osteopenic rats. Shown are 5-µm sections from the tibial metaphyses of OVX and Ro-26-9228-treated 3-month-old OVX rats. The sections were prepared as described in Materials and Methods and stained with a modified Goldner’s trichrome and toluidine blue counterstain. The photographs were taken at a magnification of 200x.

 
Additional histomorphometric studies of trabecular bone from these animals (Fig. 5Go) revealed qualitative and quantitative differences in the cellular components of bone from 1,25-(OH)2D3- and Ro-26-9228-treated animals. The analog increased the ratio of osteoblast cell surface area to bone surface area and the number of osteoblasts per square millimeter at a dose as low as 1 µg/kg. At the same time, the analog decreased the number of lining cells, suggesting that there was significant induction of an osteoblastic reaction. In contrast, 1,25-(OH)2D3 moderately (but significantly) decreased osteoblast number without changing the number or density of the lining cells. Although this is a qualitative difference in the action of the two compounds on osteoblasts and osteoblast precursors, it is not possible to determine, from these analyses, whether Ro-26-9228 promotes osteoblast proliferation, differentiation, or both.



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  		Figure 5. Differential changes in the cellular components of bone from OVX rats treated with 1,25-(OH)2D3 or Ro-26-9228. Dosing was for 3 wk, starting 19 d after ovariectomy. Histomorphometry was performed on longitudinal sections of the proximal tibia metaphysis, as described in Materials and Methods. The samples were from OVX rats (n = 9), OVX rats treated with 0.1 µg/kg 1,25-(OH)2D3 (n = 7), OVX rats treated with 0.2 µg/kg 1,25-(OH)2D3 (n = 5), OVX rats treated with 1 µg/kg Ro-26-9228 (n = 6), and OVX rats treated with 5 µg/kg Ro-26-9228 (n = 8). *, P < 0.05, compared with OVX rats.

 
These analyses also showed that 1,25-(OH)2D3 increased osteoclast number and density, whereas the analog did not. This difference between 1,25-(OH)2D3 and the analog may reflect the fact that the concentrations of 1,25-(OH)2D3 used in these experiments increased BMD and induced hypercalcemia, whereas the concentrations of analog used increased BMD without hypercalcemia. It is possible that the calcemic effect of 1,25-(OH)2D3 is attributable, in part, to an increase in osteoclast number and activity. We cannot exclude the possibility that doses of the analog that induce hypercalcemia (higher than 17 µg/kg) may also increase osteoclast number and activity.

Regulation of gene expression by 1,25-(OH)2D3 and Ro-26-9228 in the duodena of OVX rats
That Ro-26-9228 increased BMD without inducing hypercalcemia at a wide concentration range, whereas 1,25-(OH)2D3 increased BMD at the same dose range that induced hypercalcemia could be attributable to general differences in the pharmacokinetic properties of the two compounds, tissue-specific catabolism (20), or tissue-specific activation of the VDR. We hypothesized that, in any case, the low calcemic activity of Ro-26-9228 might be reflected by poor ability to induce gene expression in the duodenum, the major site for 1,25-(OH)2D3-dependent calcium absorption (21). To test this possibility, we examined expression of three genes: 24-hydroxylase, a target gene for the VDR whose expression reflects transcriptional activation of the VDR (22, 23); calbindin D 9K, which has been implicated in modulation of calcium transport and whose expression is regulated by 1,25-(OH)2D3 directly by transcriptional activation of the VDR and indirectly by mRNA stabilization (21, 24, 25); and PMCA-1, which also has been implicated in the regulation of calcium absorption by vitamin D, but whose expression is not as tightly regulated by 1,25-(OH)2D3 as that of the other two genes, and for whom the evidence for this regulation being transcriptional is only partly substantiated (26).

Sham-operated female rats or OVX rats, 3 wk after ovariectomy, were treated with a single dose of 1,25-(OH)2D3 or the analog Ro-26-9228. Because the genes we examined were primarily VDR-responsive genes, the dose chosen was based on competition assays with the rVDR, using ROS 17/2.8 cells to determine the approximate concentration required to occupy 50% of VDR ligand binding sites (IC50). Our binding assays (Fig. 6Go) revealed that the average IC50 was 1.1 nM for 1,25-(OH)2D3 and 12 nM for Ro-26–9288. When these values are adapted to body weight, the equivalent doses are approximately 0.2–0.4 µg/kg for 1,25-(OH)2D3 and 5 µg/kg for Ro-26-9228.



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  		Figure 6. Relative affinities of 1,25-(OH)2D3 and Ro-26-9228 for the rVDR. Competition assays were performed with homogenates from confluent cultures of the rat osteosarcoma cells ROS 17/2.8. Aliquots of cell homogenates were incubated with [3H]1,25-(OH)2D3, without or with increasing concentrations of the indicated ligands, as described in Materials and Methods. Each plot represents one competition assay. The IC50s (ligand concentrations required to compete 50% of [3H]1,25-(OH)2D3-occupied binding sites) are the average from three competition assays.

 
Figure 7AGo shows that intestinal 24-hydroxylase mRNA was not detected in the sham-operated and OVX rats; but 7 h after drug administration, the levels had increased significantly in the 1,25-(OH)2D3-treated OVX rats but not in the analog-treated OVX rats. Twenty-four hours after drug administration, 24-hydroxylase mRNA was not detected in either the 1,25-(OH)2D3- or Ro-26-9228-treated duodenal mucosa (data not shown). These results confirm previous studies demonstrating the short half-life of the 24-hydroxyylase mRNA in the duodenum and suggest that 1,25-(OH)2D3 and the analog differently regulate expression of the 24-hydroxylase gene, which is primarily dependent on VDR-mediated transcription.



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  		Figure 7. Differential regulation of vitamin D-responsive genes in the duodena of 1,25-(OH)2D3- and Ro-26-9228-treated female rats. Three-month-old female rats were treated with a single oral dose of 1,25-(OH)2D3 (0.2 µg/kg) or Ro-26-9228 (5 µg/kg). Total RNA was extracted from duodenal mucosa and analyzed by Northern blotting. A, Expression of 24-hydroxylase mRNA in sham, OVX, and OVX rats, 7 h after treatment with 1,25-(OH)2D3 or Ro-26-9228. Expression of calbindin D 9K mRNA (B) and PMCA-1 (C) was measured 24 h after vehicle, 1,25-(OH)2D3, and Ro-26-9228 treatment of female rats. The results were normalized against expression of G3P mRNA and are expressed as the means ± SD of six individually tested samples from each group.

 
The expression of calbindin D 9K and PMCA-1 mRNAs was down-regulated in the OVX rats, and a single administration of either 1,25-(OH)2D3 or Ro-26-9228 did not change their levels (data not shown). Based on studies by others (27, 28), we considered the possibility that ovarian steroid hormones are important in the regulation of calbindin D 9K and PMCA-1 mRNAs. Therefore, we repeated the experiments, with intact female rats that received a single dose of 0.2 µg/kg 1,25-(OH)2D3 or 5 µg/kg Ro-26-9228. Figure 7Go, B and C, shows that 1,25-(OH)2D3 significantly increased the steady-state levels of PMCA-1 and calbindin D 9K mRNAs, but Ro-26-9228 had no effect on these mRNAs. This increase was observed at 24 h (but not at 7 h) after treatment, confirming the previous observation that the up-regulation of these mRNAs is slower and their half-lives are longer than that of 24-hydroxyalse (22, 29). We concluded that because the duodena of both OVX and intact female rats were not responsive to amounts of Ro-26-9228 that should saturate 50–100% of the VDR binding sites, the analog’s low calcemic activity may be attributable, in part, to inability to induce VDR-responsive gene expression in the duodenum.

Osteoblast-specific gene regulation by 1,25-(OH)2D3 and Ro-26-9228 in trabecular bone of OVX rats
To determine whether the analog has a significant effect on expression of genes specific to differentiated osteoblasts, we examined the regulation of the osteocalcin and ostepontin mRNAs, which in rats and humans are up-regulated by 1,25-(OH)2D3 through the VDR (30, 31, 32). We studied a region containing primarily trabecular bone (tibia metaphysis), in which active bone remodeling is expected to occur in the drug-treated OVX animals. Figure 8BGo shows that the basal levels of osteocalcin mRNA increased significantly after ovariectomy, and 1,25-(OH)2D3 and Ro-26-9228 induced additional, small increases. The osteopontin mRNA levels did not change after ovariectomy, and both 1,25-(OH)2D3 and Ro-26-9228 induced a significant increase in osteopontin mRNA. Therefore, Ro-26-9228 and 1,25-(OH)2D3 had similar effects on VDR-mediated gene regulation in bone, but Ro-26-9228 had little effect on VDR-mediated gene regulation in the duodenum. Therefore, we hypothesize that the VDR-mediated action of Ro-26-9228 is probably tissue-specific and that its preferred target cells in the bone are the osteoblasts.



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  		Figure 8. Regulation of osteoblast-specific gene expression in trabecular bone from OVX rats treated for 24 h with 1,25-(OH)2D3 or Ro-26-9228. Total RNA was extracted from the proximal third of the tibia, and the RNA was subjected to Northern blot analysis as described in Materials and Methods. The results were normalized against expression of G3P mRNA and are expressed as the means ± SD of 10 individually tested samples from each group.

 
Effects of Ro-26-9228 and 1,25-(OH)2D3 on the expression of growth factors in trabecular bone of OVX rats
Bone contains heterogeneous cell population that produces many cytokines and growth factors that act in autocrine and paracrine fashions. 1,25-(OH)2D3 is known to alter the expression of some of these factors, and therefore may have an indirect effect on growth and function of bone cells, by regulating the production of cytokines and growth factors (33, 34). To determine whether 1,25-(OH)2D3 and Ro-26-9228 differently regulate the expression of the genes that encode TGF-ßs, which are important in bone formation and are direct targets for transcriptional regulation by vitamin D, we measured steady-state levels of TGF-ß mRNA by semiquantitative RT-PCR. We found that ovariectomy did not significantly change the levels of TGF-ß1 and TGF-ß2 mRNAs. A single dose of 1,25-(OH)2D3 (0.2 µg/kg) or Ro-26-9228 (5 µg/kg) significantly up-regulated these mRNAs, but Ro-26-9228 was more effective than 1,25-(OH)2D3 (Fig. 9Go). These results suggest that Ro-26-9228 and 1,25-(OH)2D3 may promote bone formation by up-regulating these growth factors and further confirm that the bone is a preferred site for the analog’s action.



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  		Figure 9. Regulation of TGF-ß1 and TGF-ß2 gene expression in trabecular bone from OVX rats treated for 7 h with 1,25-(OH)2D3 or Ro-26-9228. Total RNA was extracted from the proximal third of the tibia, and 5 µg from each RNA sample was subjected to semiquantitative RT-PCR as described in Materials and Methods. The results were normalized against expression of G3P mRNA and are expressed as the means ± SD of six individually tested samples from each group.

 
Pharmcokinetic properties of Ro-26-9228
Low calcemic activity of vitamin D analogs has been often linked to their short half-lives and is often correlated with poor abilities to bind the serum DBP (35). To determine whether such properties of Ro-26-9228 contributed to its low calcemic activity, we determined the terminal half-life of Ro-26–9288 after iv administration in vivo and found that is was 1.6 h, whereas the reported terminal half-life for 1,25-(OH)2D3 in rats after iv administration is 2.2 h (36). Next, we compared the binding of 1,25-(OH)2D3 and Ro-26-9228 to DBP in vitro and found that, at physiologic concentration of DBP (200 µg/ml), the binding of Ro-26-9228 was 33% that of 1,25-(OH)2D3. These measurements suggest that binding to DBP and half-life are not major causes of the low calcemic activity of this analog. However, we cannot exclude the possibility that selective retention or differences in the analog’s catabolism in duodenum and bone contribute to its apparent tissue selectivity.

Cell-specific transactivation of the VDR by Ro-26-9228
To determine whether the apparent tissue preference of Ro-26-9228 can be reproduced in a culture system and, more importantly, whether it occurs in human cell lines, we performed transactivation assays with the human osteosarcoma cell line MG-63 and the human colon carcinoma cell line Caco-2. MG-63 cells have the characteristics of differentiated osteoblasts (37, 38); and Caco-2 cells, although not from duodenum, have been reported to have transcriptionally active VDR and the cellular mechanism required for vitamin D-regulated calcium transport (from mucosa to serosa) (39). Both cell types have detectable levels of immunoreactive VDR, and this amount increased within 1 h of ligand treatment in both cell types (data not shown). The cells were transfected with a reporter gene containing the minimal VDRE from the human osteocalcin gene attached to a thymidine kinase-GH fusion gene. The ED50s for transactivation of this reporter gene by 1,25-(OH)2D3 and Ro-26-9228 in Caco-2 cells were 2 nM and 120 nM, respectively (Fig. 10Go, A and B). In contrast, the ED50s for MG-63 cells were 3 nM for 1,25-(OH)2D3 and 5 nM for Ro-26-9228. The affinity of the analog for the human VDR, as revealed from competition assays (Fig. 10CGo, IC50 = 6.2 nM) was approximately 10-fold lower than the affinity of 1,25-(OH)2D3 for the human VDR (IC50 = 0.56 nM). These results suggest that Ro-26-9228 modulates VDR-mediated transcription in a cell-specific manner. The discrepancy in the affinity of Ro-26-9228 for the human VDR and the VDR-mediated transcriptional potency of the analog in Caco-2 cells suggests that transcriptional activation of the VDR by Ro-26-9228 is suppressed in these intestinal cells.



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  		Figure 10. Differential activation of the VDR by Ro-26-9228 in culture. Subconfluent cultures of Caco-2 (A) and MG-63 (B) cells were transfected with the osteocalcin VDRE-thymidine kinase/GH fusion gene and treated with the indicated concentrations of 1,25-(OH)2D3 or Ro-26-9228 in duplicate. Medium samples were collected, 48 h later, and reporter gene expression was assessed by an RIA. The range of maximum amounts of GH produced by MG-63 cells and by Caco-2 cells in response to treatment was 1–1.4 ng/ml and 3–4.5 ng/ml, respectively. The data were plotted as percentages of maximal induction of GH by 1,25-(OH)2D3. The ED50 values in each panel are the means ± SE from three to five experiments. C, Homogenates from confluent cultures of COS-1 cells transfected with the human VDR expression vector were incubated with [3H]1,25-(OH)2D3 and with increasing concentrations of the indicated ligands. The plots show the results of one representative competition assay. The IC50s are the averages calculated from three competition assays.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the study described here, we examined whether it is possible to fine-tune the calcium-regulating activities of 1,25-(OH)2D3 by structurally modifying it so that its ability to prevent bone loss exceeds its ability to increase circulating calcium. This biological profile was achieved by synthesizing Ro-26-9228, a vitamin D analog that has structural features that alter catabolism (unsaturation at carbons 16, 23, and 24) (40, 41, 42, 43) and that change the mode of interaction with the VDR (20-epi configuration and elongation of the side chain, and replacement of the 1{alpha} hydroxyl group with a fluoride, Fig. 1Go) (40, 44). The latter modification has also been shown to compensate for loss of binding affinity for DBP that side-chain modifications might cause (40).

1,25-(OH)2D3 and its analog Ro-26-9228 seemed to restore bone loss by inhibiting bone resorption, because both compounds significantly inhibited the excretion of collagen degradation products (pyridinoline cross-links). However, it is difficult to determine how 1,25-(OH)2D3 does that, because most studies of 1,25-(OH)2D3’s action in bone have shown that it promotes several molecular events that facilitate bone resorption, including the induction of osteoclast differentiation through increased expression of the osteoclast differentiation factor (ODF/RANKL) (45) and up-regulation of cytokines (ILs 1ß and 6) that facilitate bone resorption (46, 47, 48). One explanation could be that 1,25-(OH)2D3 or its derivatives indirectly inhibit bone resorption by inhibiting PTH expression (49, 50) or by stimulating TGF-ß expression (33, 51). The complex role of 1,25-(OH)2D3 in orchestrating positive and negative events that modulate bone remodeling probably contributes to its narrow therapeutic range in bone disease and should be a subject for future investigations. Interestingly, Ro-26-9228’s action in this animal model of osteopenia seemed to be somewhat different. Although there was biochemical evidence that Ro-26-9228 also had antiresorptive activity, i.e. it inhibited pyridinoline excretion, the results of bone histomorphometry studies suggested that Ro-26-9228 has a strong osteoblastic proliferative-differentiating effect that 1,25-(OH)2D3 does not. Therefore, Ro-26-9228, unlike 1,25-(OH)2D3, may have both antiresorptive action and anabolic activity in osteopenic rats.

1,25-(OH)2D3 had strong antiresorptive activity, but this action occurred simultaneously with hypercalciuria and hypercalcemia. This may suggest that the increases in urine and serum calcium that were induced by 1,25-(OH)2D3 in this model came primarily from enhanced intestinal absorption of calcium and less so from bone resorption. By extension, the fact that the analog had antiresorptive activity and induced net bone gain without increasing urine and serum calcium suggests that its ability to enhance intestinal calcium absorption is less than that of 1,25-(OH)2D3. Our experiments showed that Ro-26-9228 did not up-regulate expression of 24 hydroxylase, calbindin D 9K, or PMCA-1, which are vitamin D-responsive genes in the duodenum, whereas 1,25-(OH)2D3 did, and at a 25-fold lower concentration. We considered the possibility that the time courses for regulation of these genes by 1,25-(OH)2D3 and the analog were different, as has been shown for the regulation of 24-hydroxylase by 1,25-(OH)2D3 and by the analogs OCT and ED-71 (52, 53). Therefore, we examined their expression at two times, 7 and 24 h after a single dosing. However, we found that, in the duodenum, Ro-26-9228 failed to induce either a short-lived mRNA (the 24 hydroxylase) or a long-lived one (calbindin D 9K) at either time. We concluded that, although the regulation of these gene products may not directly cause calcium absorption, the apparent inability of the analog to regulate gene expression in the duodenum suggests that this main site for vitamin D-regulated calcium absorption is not responsive to Ro-26-9228 and that this contributes to its low calcemic activity.

Importantly, Ro-26-9228 increased the steady-state mRNA levels of vitamin D-responsive genes in the bone, 7 h (TGF-ß1 and TGF-ß2) or 24 h (osteocalcin and osteopontin) after treatment with the same concentration that failed to induce gene expression in the duodenum. In contrast, a concentration of 1,25-(OH)2D3 that saturated less than 50% of the VDR binding sites and 25 times lower than that of Ro-26-9228 induced gene expression in both target tissues.

The promoters of five of the tested genes (osteocalcin, osteopontin, TGF-ß2, 24-hydroxylase, and calbindin D 9K) contained VDREs, which are binding sites for VDR-ligand complexes. Therefore, we hypothesize that the different tissue sensitivity of Ro-26-9228 is attributable to either preferential activation of the VDR in osteoblasts or suppression of VDR-mediated transcription in the duodenum. Our experiments with the human osteoblast-like line MG-63 and the human colon carcinoma line Caco-2 provided evidence for differential activation of the VDR-analog complexes in culture. Whether the poor transcriptional potency of the analog in the colon carcinoma line and its significant transcriptional potency in the osteosarcoma line reflect the apparent preference of Ro-26-9228 for osteoblasts in rats remains to be further investigated.

Ro-26-9228 may be the first vitamin D analog that selectively modulates the VDR. This may occur by mechanisms that directly alter VDR activation. However, we cannot exclude the possibility that the apparent tissue preference of Ro-26-9228 and the lack of tissue preference for 1,25-(OH)2D3 may reflect differences in their relative stabilities in those target tissues or in the compounds’ catabolic pathways, such as rapid clearance of the analog from duodenum or slow catabolism of the analog in the bone, or both. Determining how this tissue-selective activation occurs and using it to develop the next generation of such compounds for various therapeutical uses may require research approaches such as those being applied to the estrogen receptors and their selective modulators (16, 54, 55, 56). The results from our laboratory have shown it is possible to reproduce the cell preference in culture; and so, the way is clear for detailed investigation of the molecular mechanisms for the action of selective VDR modulators.


   Acknowledgments
 
We thank Dr. S. Christakos for the generous gift of the calbindin D 9K and the PMCA-1 cDNA probes, Dr. G. Karsenty for the osteocalcin cDNA probe, and Dr. J. W. Pike for the human VDR expression plasmid. We also wish to thank L. Chen and C. Nguyen for their technical assistance, and Dr. M. Goode for her helpful comments on this manuscript.


   Footnotes
 
Abbreviations: BMD, Bone mineral density; DBP, vitamin D binding protein; 20-epi, epimerization at carbon 20; G3P, glyceraldehyde-3-phosphate dehydrogenase; IC50, concentration required to occupy 50% of binding site; 1,25-(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; OVX, ovariectomized; PMCA-1, plasma membrane calcium pump 1; rVDR, rat VDR; VDRE, vitamin D-responsive element.

Received September 17, 2001.

Accepted for publication January 15, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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