Antagonistic Action of Novel 1alpha ,25-Dihydroxyvitamin D3-26,23-lactone Analogs on Differentiation of Human Leukemia Cells (HL-60) Induced by 1alpha ,25-Dihydroxyvitamin D3*

Daishiro MiuraDagger , Kenji Manabe§, Keiichi Ozono, Mariko Saito, Qingzhi Gao§, Anthony W. Normanparallel **, and Seiichi Ishizuka§

From the Dagger  Safety Research Department and § Department of Bone and Calcium Metabolism, Teijin Institute for Bio-Medical Research, 4-3-2 Asahigaoka, Hino, Tokyo 191-8512, Japan,  Department of Environmental Medicine Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo-Cho Izumi, Osaka 594-1101, Japan, and parallel  Department of Biochemistry and Division of Biomedical Sciences, University of California, Riverside, California 92521

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

We examined the effects of two novel 1alpha ,25-dihydroxyvitamin D3-26,23-lactone (1alpha ,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha ,25-dihydroxyvitamin D3 (1alpha ,25(OH)2D3) and its analogues. We found that the 1alpha ,25-lactone analogues (23S)-25-dehydro-1alpha -hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha -hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10-6 M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha ,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10-7 M) was found to be an effective antagonist of both 1alpha ,25(OH)2D3 (10-8 M) mediated induction of p21WAF1,CIP1 in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha ,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha ,25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is widely accepted that the fundamental biological activities of the hormonal form of vitamin D3, 1alpha ,25-dihydroxyvitamin D3 (1alpha ,25(OH)2D3),1 are to stimulate intestinal calcium absorption and to increase bone calcium mobilization (1, 2). In recent years, however, many new biological functions different from those mentioned above have been reported (3); these include inhibition of cell proliferation and induction of cell differentiation (4), modulation of immunological responses (5), stimulation of insulin secretion (6, 7), and neurobiological functions (8, 9). 1alpha ,25(OH)2D3 is believed to mediate biological responses as a consequence of its interaction with both a nuclear receptor (VDR) to regulate gene transcription (10, 11) and with a putative cell membrane receptor to generate rapid nongenomic effects (12), including the opening of voltage-gated calcium and chloride channels (13), and activation of mitogen-activated protein kinase (14).

To better understand the interactions of the ligand/VDR interacting with a vitamin D-responsive element (DRE) located on the promoter of regulated genes, it would be helpful to identify analogues of 1alpha ,25(OH)2D3 that can modulate or antagonize these interactions. However, to date the only known antagonist of 1alpha ,25(OH)2D3 is the analogue 1beta ,25(OH)2D3, which blocks rapid nongenomic responses but is without effect on the classical nuclear VDR (15).

(23S,25R)-1alpha ,25-dihydroxyvitamin D3-26,23-lactone ((23S, 25R)-1alpha ,25(OH)2 D3-26,23-lactone) was found by Ishizuka et al. (16-19) as a major metabolite of 1alpha ,25(OH)2D3 both in vivo and in vitro. They reported that the naturally occurring (23S, 25R)-1alpha ,25(OH)2D3-26,23-lactone has unique biological features in comparison with 1alpha ,25(OH)2D3. First of all, the VDR binding affinity of the naturally occurring 1alpha ,25-lactone is very low (17, 20). Nonetheless it can stimulate collagen synthesis in osteoblasts (21, 22) and inhibit formation of osteoclast-like multinucleated cells from bone marrow mononuclear cells and bone resorption induced by 1alpha ,25(OH)2D3 (21, 23, 24). It can also stimulate proteoglycan synthesis and type II collagen synthesis in chondrocytes from rabbit costal growth cartilage (25).

Normally proliferating human promyelocytic leukemia cells (HL-60) show promyelocytic features and no differentiated functions (for example, nitro blue tetrazolium (NBT)-reducing activity, monocyte-specific esterase activity, and cell surface marker expression, used as differentiation markers) are detected. However, their differentiation can be induced in vitro by various compounds including all-trans retinoic acid (ATRA), 9-cis-retinoic acid (9-cis-RA) (into granulocytes), 1alpha ,25(OH)2D3, or 12-O-tetradecanoylphorbol-13-acetate (TPA) (into monocyte/macrophages) (4, 26-28). It is well known that HL-60 cells have the VDR, and its cell differentiation is induced by 1alpha ,25(OH)2D3 through a VDR/DRE-mediated pathway (29); as such this is a useful system to study genomic actions of 1alpha ,25(OH)2D3 and related analogues.

We have recently synthesized various analogues of 1alpha ,25-lactone to investigate which functionality of the 1alpha ,25-lactone structure is responsible for its unique biological functions. In this study we report the discovery of the antagonistic biological activities of two novel 1alpha ,25-lactone analogues ((23S)-25-dehydro-1alpha (OH)D3-26,23-lactone) (TEI-9647) and ((23S)-25-dehydro-1alpha (OH)D3-26,23-lactone) (TEI-9648). These analogues were found to block both 1alpha ,25(OH)2D3-mediated HL-60 cell differentiation and also activation of the luciferase reporter in COS-7 cells that had been transfected with the cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Chemicals-- 25-Hydroxyvitamin D3 (25(OH)D3), 1alpha ,25(OH)2D3, 1beta ,25-dihydroxyvitamin D3 (1beta ,25(OH)2D3), 1alpha ,25(OH)2D3-26,23-lactone, and its analogues (TEI-9616, TEI-9647, and TEI-9648) were synthesized in our laboratory as described previously (20, 30). The chemical structures of 1alpha ,25(OH)2D3-26,23-lactone and its analogues are shown in Fig. 1. The 20-epi-22-oxa-24a,26a,27a-trihomo-1alpha ,25-dihydroxyvitamin D3 (KH-1060) was synthesized in our laboratory. TPA and 9-cis-RA were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). ATRA was obtained from Sigma. NBT was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). May-Grünwald-Giemsa solution, Kernechrot solution and esterase staining kit were obtained from Muto Pure Chemicals Co., Ltd. (Tokyo, Japan). Anti-CD11b antibody (PE conjugated anti-human CD11b antibody) and anti-CD71 antibody (fluorescein isothiocyanate-conjugated anti-human CD71 antibody) were purchased from Pharmingen (San Diego, CA). [26,27-methyl-3H]1alpha ,25(OH)2D3 (specific activity, 179 Ci/mmol) and [26,27-methyl-3H]25(OH)D3 (specific activity, 17 Ci/mmol) were purchased from Amersham International plc (Little Chalfont, Buckinghamshire, United Kingdom). [1-3H]1alpha ,25(OH)2D3 (specific activity, 16.2 Ci/mmol) was synthesized in our laboratory.

Cell and Cell Culture-- HL-60 cells were obtained from Japanese Cancer Research Resources Bank. Cells were passaged twice a week to maintain exponential proliferating phase. RPMI 1640 (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum (FBS) (Bioserum, lot number 01307-01) was used as culture medium.

The monkey kidney epithelial cell line, COS-7, was maintained in Dulbecco's modified Eagle medium (Nissui Pharmaceutical Co., Tokyo) with 10% dextran-charcoal-stripped fetal bovine serum (JRH Bioscience, Dexton, KS).

Binding Affinity to VDR and to Vitamin D-binding Protein (DBP)-- A competitive receptor binding assay for 1alpha ,25(OH)2D3 and 1alpha ,25-lactone analogues was performed using VDR from HL-60 cells as described previously (31, 32). Exponentially proliferating HL-60 cells were disrupted by sonication in TEDK buffer (50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 5 mM dithiothreitol, 300 mM KCl). After ultracentrifugation at 105,000 × g for 60 min, supernatant was collected and used as VDR fraction. [26,27-methyl-3H]1alpha ,25(OH)2D3 (specific activity, 179 Ci/mmol, 15,000 dpm, 15.7 pg) and various amounts of 1alpha ,25-lactone analogues to be tested were dissolved in 50 ml of absolute ethanol in 12 × 75-mm polypropylene tubes (Sarstedt, Nümbrecht, Germany). 1 ml of the HL-60 cell VDR fraction and 1 mg of gelatin were added to each tube in an ice bath. The assay tubes were incubated in a shaking water bath for 1 h at 25 °C and then chilled in an ice bath. 1 ml of 40% polyethylene glycol 6000 in distilled water was added to each tube, which was then mixed vigorously and centrifuged at 2,260 × g for 60 min at 4 °C. After the supernatant was decanted, the bottom of the tube containing the pellet was cut off into a scintillation vial containing 10 ml of dioxane-based scintillation fluid consisting of 10% naphthalene and 0.5% Omnifluor (DuPont) in 1,4-dioxane. The radioactivity was measured with Beckman liquid scintillation counter (model LS6500) using an external standard. In the assay, using chick intestinal VDR, 0.2 mg of protein/ml of chick VDR was used instead of HL-60 cell VDR fraction. A competitive binding assay of DBP in FBS for 25(OH)D3 and 1alpha ,25-lactone analogues was performed as described previously (33).

Cytohistochemical Assay-- Cell morphology, NBT-reducing activity and monocytic cell-specific esterase (alpha -naphthylbutyrate (alpha -NB) used as a substrate) activity was used as cell differentiation markers. HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Exponentially proliferating cells were collected, suspended in fresh medium, and seeded in culture vessels. 24-well culture plates (Falcon, Becton Dickinson and Co., Franklin Lakes, NJ) were used. Cell concentration at seeding was adjusted to 2 × 104 cells/ml and seeding volume was 1 ml/well. 1alpha ,25(OH)2D3 and 1alpha ,25-lactone analogues dissolved in ethanol were added to the culture medium at 0.1% volume and cultured with cells for 4 days at 37 °C in a humidified atmosphere of 5% CO2/air without medium change. The same amount of vehicle was added to the control culture. NBT reduction assay was performed according to the method of Collins et al. (34). Briefly, cells were collected and washed with PBS. After washing, cells were suspended in serum-free medium, and NBT/TPA solution (dissolved in PBS) was added. Final concentrations of NBT and TPA were 0.1% and 100 ng/ml, respectively. Then, cell suspensions were incubated at 37 °C for 25 min. After incubation, cells were collected by centrifugation and resuspended in FBS. Cytospin smears were prepared, and the counterstaining of nucleus was done with Kernechrot solution. At least 500 cells per preparation were observed.

alpha -NB esterase activity was measured as follows: cell seeding, treatment, and collection were performed according to the method described above. Cells were resuspended in FBS and then cytospin smears were prepared. Esterase activity of cells was examined after staining with an esterase staining kit. For cell morphology examination, cytospin smears were stained with May-Grünwald-Giemsa solution.

Cell Surface Marker Expression-- Cells were treated with compounds and collected according to the same methods described above. Collected cells were suspended in PBS, and antibodies were added. After incubation on ice for 30 min, cells were collected and washed with PBS. Cells were resuspended in PBS, and the cell surface marker expression was measured with fluorescent-activated cell sorter (FACS) (Becton Dickinson and Co.).

Reverse Transcription PCR of p21WAF1,CIP1 and beta -Actin-- RNA of HL-60 cells was extracted and purified using CLONsep total RNA isolation kit (CLONTECH Laboratories, Inc., Palo Alto, CA). 2 mg of total RNA were reverse-transcribed with 50 units of murine leukemia virus reverse transcriptase (Takara Biomedicals, Shiga, Japan) in 20 ml containing 1 mM deoxyribonucleoside triphosphates, 5 mM MgCl2, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 20 units of RNase inhibitor (RNasin, Promega Corp., Madison, WI), 2.5 mM oligo(dT) primer. Samples were diluted to 100 ml with buffer containing 2 mM MgCl2, 10 mM Tris-HCl, pH 8.3, 50 mM KCl. 100 pmol of each primer and 2.5 units Taq DNA polymerase (Takara Biomedicals, Shiga, Japan) were added, and samples were covered with mineral oil and then subjected to PCR amplification in a programmed thermal cycler. PCR primer was selected with OLIGOTM (National Bioscience), referring to the mRNA sequence registered in GenBankTM. For p21WAF1,CIP1 amplification, the PCR primers were 5' to 3' AGGAGGCCCGTGAGCGATGGAAC and ACAAGTGGGGAGGAGGAAGTAGC. PCR cycles were as follows: 1 min at 94 °C for denaturation, 1 min at 59 °C for annealing, 1 min at 72 °C for polymerization, 26 cycles. For beta -actin amplification, the PCR primers were 5' to 3' GATATCGCCGCGCTCGTCGTCGAG and CAGGAAGGAAGGCTGGAAGAGTGC. PCR cycles were as follows: 1 min at 94 °C for denaturation, 1 min at 61 °C for annealing, 1 min at 72 °C for polymerization, 20 cycles. PCR products were analyzed by 2% agarose gel electrophoresis (about 400-base pair product was obtained in p21WAF1,CIP1 PCR and about 800-base pair product was obtained in beta -actin PCR).

Luciferase Reporter Gene Assay-- The promoter region of the rat 24-hydroxylase gene (-291/+9), which also contains two DREs (a gift from Dr. Y. Ohyama, Hiroshima University, Japan) (35) were cloned into a luciferase reporter vector pGV-B2 (Toyo Ink Co. Ltd., Tokyo, Japan). The DNA sequences of these plasmids were confirmed using an ABI 373A DNA sequencer (PE Applied Biosystems, Tokyo, Japan). The luciferase activities of the cell lysates were measured with a luciferase assay kit (Toyo Ink Co. Ltd.) according to the manufacturer's manual. Transactivation measured by luciferase activities was standardized by the galactosidase activities of the same cells determined by a beta -galactosidase enzyme assay system (Promega).

These plasmids, together with the hVDR expression vector, pSG5hVDR (a gift from Dr. M. R. Haussler, University of Arizona) were introduced into cells by DEAE-dextran. 16 h after the transfection, 10-8 M 1alpha ,25(OH)2D3, 10-7 M TEI-9647 or both, or vehicle was added. 48 h after the addition, cells were harvested in the cell lysate solution provided by luciferase assay kit (Toyo Ink). Luciferase activity was adjusted by internal beta -galactosidase activity.

Metabolism of [1-3H]1alpha ,25(OH)2D3 in HL-60 Cells as Modulated by Lactone Analogs-- HL-60 cells (106 cells/ml in a 100 mm-diameter dish, 10 ml) were cultured in RPMI 1640 medium supplemented with 10% FBS and 10-8 M [1-3H]1alpha ,25(OH)2D3 (specific activity 16.2 Ci/mmol, 1.62 µCi), and 10-7 M 1alpha ,25-lactone analogues, and then cultured for the indicated time. [1-3H]1alpha ,25(OH)2D3 metabolites were extracted with chloroform-methanol (1:1, v/v), and analyzed using Zorbax Sil column (4.6 × 250-mm) eluted with 15% isopropanol in n-hexane at a flow rate of 1 ml/min. Fractions were collected each 30 s for 50 min. Radioactivity in the effluent mixed with 8 ml of toluene-based scintillation fluid was measured by a Beckman model LS6500 liquid scintillation counter. On this system standard 1alpha ,25(OH)2D3, 24-oxo-1alpha ,25(OH)2D3, 24,25,26,27-tetranor-1alpha ,25(OH)2D3, (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone and 1alpha ,24R,25(OH)3D3 eluted at 16.3, 19.3, 23.8, 29.0, and 29.3 min, respectively.

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Fig. 1 indicates the structures of the naturally occurring 1alpha ,25-lactone and its three analogues. TEI-9616 is a 25-dehydroxylated version of the naturally occurring (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone. TEI-9647 and TEI-9648 are both 25-dehydrated lactones of the (23S,25R)- and (23R,25R)-1alpha ,25(OH)2D3-26,23-lactone, respectively. Formally, TEI-9647 and TEI-9648 are 23-diastereoisomers of one another.


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Fig. 1.   Structures of 1alpha ,25(OH)2D3-26,23-lactone analogues. The (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone is a naturally occurring metabolite derived from 1alpha ,25(OH)2D3 (55).

The receptor binding affinities of 1alpha ,25-lactone and its analogues to VDR prepared from HL-60 cells are shown in Fig. 2 and summarized in Table I. The VDR binding affinities of TEI-9647 and TEI-9648 were 10 and 8%, respectively, as compared with 1alpha ,25(OH)2D3. Their binding affinities to VDR of HL-60 cells were 120-140 times stronger than that of the naturally occurring (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone. In contrast, the binding affinities of TEI-9616 and the naturally occurring (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone to the VDR of HL-60 cells were about 237 (0.48%) and 1,400 (0.07%) times weaker than that of 1alpha ,25(OH)2D3. Similar results were obtained using chick intestinal VDR.


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Fig. 2.   Typical results from the VDR and DBP binding assays for 1alpha ,25(OH)2D3-26,23-lactone analogues. A, HL-60 cell VDR binding assay with 1alpha ,25(OH)2D3 and 1alpha ,25(OH)2D3-26,23-lactone analogues in comparison with [3H]1alpha ,25(OH)2D3. B, chick intestinal VDR binding assay with 1alpha ,25(OH)2D3 and 1alpha ,25(OH)2D3-26,23-lactone analogues in comparison with [3H]1alpha ,25 (OH)2D3. C, competitive binding assay of DBP in FCS for 1alpha ,25(OH)2D3 and 1alpha ,25(OH)2D3-26,23-lactone analogues in comparison with [3H]25(OH)D3. Analogs are 25(OH)D3 (), 1alpha ,25(OH)2D3 (open circle ), TEI-9647 (triangle ), TEI-9648 (black-triangle), TEI-9616 () and (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone (black-square). Points are means of triplicate determinations. All values are within 5% of mean.

                              
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Table I
Biological characteristics of 1alpha ,25(OH)2D3-26,23-lactone analogs
The relative activity for each analog was calculated from their respective EC50 results (obtained from a series of experiments similar to that shown in Fig. 2) and then normalized to the result obtained for 1alpha ,25(OH)2D3, which was set to 100%.

(23S,25R)-1alpha ,25(OH)2D3-26,23-lactone bound to the plasma DBP 6.2 times stronger than 1alpha ,25(OH)2D3. However, the DBP binding affinities of TEI-9616, TEI-9647, and TEI-9648 are 2.4, 9.3, and 2.5%, respectively, as compared with 1alpha ,25(OH)2D3 (Table I).

Our preliminary data indicated that the natural (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone has very weak HL-60 cell differentiation inducing activity (36). From this data and results of the VDR and DBP affinity studies, we predicted that the 1alpha ,25-lactone analogues might be more potent in HL-60 cell differentiation than the natural (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone. We did find that TEI-9616 is a more potent HL-60 cell differentiation agent than the natural (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone; in contrast, neither TEI-9647 nor TEI-9648 could induce cell differentiation even after treatment at 10-6 M (data not presented).

In agreement with other studies (37), concentrations of 10-9 to 10-7 M 1alpha ,25(OH)2D3 dose dependently induced differentiation of HL-60 cells; a concentration of 10-8 M of 1alpha ,25(OH)2D3 differentiated >50% of the cells into NBT-reducing activity positive cells during a 96-h culture period (data not presented). Fig. 3 presents the morphological and histocytochemical changes in HL-60 cells after treatment with TEI-9647 or TEI-9648 in the absence or presence of 10-8 M 1alpha ,25(OH)2D3. Although undifferentiated HL-60 cells showed promyelocytic features, cells differentiated by 1alpha ,25(OH)2D3 displayed a monocytic appearance (Fig. 3A). However, TEI-9647 and TEI-9648 did not mediate the appearance of any monocyte-like morphological changes even after treatment at 10-6 M for 96 h. Surprisingly, the HL-60 cell morphological changes induced by 10-8 M 1alpha ,25(OH)2D3 were markedly inhibited in the presence of 10-6 M TEI-9647 or TEI-9648 (Fig. 3A). Monocytic differentiation markers, such as NBT-reducing activity and alpha -NB esterase activity, are known to be up-regulated by 1alpha ,25(OH)2D3. Therefore, we examined the effect of TEI-9647 and TEI-9648 to mediate the up-regulation of differentiation markers induced by 1alpha ,25(OH)2D3. TEI-9647 or TEI-9648 alone could not induce activation of NBT-reducing activity or alpha -NB esterase activity. In contrast, they both markedly suppressed the up-regulation induced by 1alpha ,25(OH)2D3 (Fig. 3, B and C).


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Fig. 3.   Effects of TEI-9647 and TEI-9648 on 1alpha ,25(OH)2D3-induced HL-60 cell differentiation. HL-60 cells were treated with TEI-9647 or TEI-9648 in the absence (-) or presence (+10-8 M) of 1alpha ,25(OH)2D3 for 96 h, and cytohistochemical assays were done as described under "Experimental Procedures." A, morphological changes examined with May-Grünwald-Giemsa stained preparations; B, NBT-reducing activity; C, alpha -NB esterase activity.

Next we examined separately the inhibitory effects of TEI-9647 and TEI-9648 on 1alpha ,25(OH)2D3 action in more detail using NBT-reducing activity as a cell differentiation marker. TEI-9647 dose dependently inhibited the cell differentiation induced by 10-8 M 1alpha ,25(OH)2D3 (Fig. 4A); it caused 40% suppression at 10-9 M and almost complete inhibition was observed at 10-7 M. Complete suppression was observed at 10-6 M TEI-9647. TEI-9648 showed a similar dose-dependent response curve, but its suppressive effect was consistently weaker than that of TEI-9647 (Fig. 4B). In contrast, neither the naturally occurring (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone nor TEI-9616 displayed any ability to inhibit HL-60 cell differentiation, even at 10-6 M (data not presented).


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Fig. 4.   Effects of TEI-9647 and TEI-9648 on 1alpha ,25(OH)2D3-induced HL-60 cell differentiation as examined with NBT-reducing activity. HL-60 cells were treated with TEI-9647 (A) or TEI-9648 (B) in the absence (-) or presence (+10-8 M) of 1alpha ,25(OH)2D3 for 96 h, and NBT-reducing activity was examined. Rectangles and bars show mean ± S.D. of triplicates, respectively.

A particularly potent analogue of 1alpha ,25(OH)2D3 is KH-1060, which has been shown to have a 5,000-10,000-fold more potent cell differentiating activity than 1alpha ,25(OH)2D3 (3, 38). As shown in Fig. 5, both TEI-9647 and TEI-9648 could dose dependently (10-8-10-6 M) antagonize the HL-60 cell differentiating actions of KH-1060 (3 × 10-11 M).


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Fig. 5.   Effects of TEI-9647 and TEI-9648 on KH-1060-induced HL-60 cell differentiation. HL-60 cells were treated with TEI-9647 or TEI-9648 in the absence (O) or the presence of KH-1060 (3 × 10-11 M) for 96 h followed by determination of NBT-reducing activity. The bars show the mean of duplicate determination.

Fig. 6 shows the consequences of TEI-9647 and TEI-9648 on the changes of cell surface marker expression. In HL-60 cells, 1alpha ,25(OH)2D3 simultaneously mediates an increase in CD11b expression and a decrease in CD71 expression. Neither TEI-9647 nor TEI-9648 alone could induce such changes of cell surface marker expression (Fig. 6, left side). In contrast, TEI-9647 and TEI-9648 dose dependently blocked the reciprocal changes of CD11b and CD71 expression associated with HL-60 cell differentiation induced by 1alpha ,25-(OH)2D3. TEI-9647 completely blocked the increase in CD11b and the decrease in CD71 expression at 10-7 M (Fig. 6). Similar results were observed after treatment with TEI-9648, but its potency seemed to be weaker (data not presented).


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Fig. 6.   Effects of TEI-9647 on 1alpha ,25(OH)2D3-induced HL-60 cell differentiation as examined with cell surface marker expression. HL-60 cells were treated with TEI-9647 in the absence (-) or presence (+10-8 M) of 1alpha ,25(OH)2D3 for 96 h, and cell surface marker expression was examined by the FACS analysis. A, changes of CD11b expression; B, changes of CD71 expression. 15,000 cells were analyzed in each analysis.

ATRA and 9-cis-RA are also known to promote cell differentiation of HL-60 cells but into granulocytes. TPA can also induce HL-60 cell differentiation into macrophage-like cells. We examined whether TEI-9647 and TEI-9648 could inhibit HL-60 cell differentiation induced by these compounds. In data not presented, we found that neither TEI-9647 nor TEI-9648 could cause inhibition even after treatment at 10-6 M.

Collectively, Figs. 3-6 document the stereospecific ability of TEI-9647 and TEI-9648 to inhibit the ability of both 1alpha ,25(OH)2D3 and KH-1060 to mediate the complex process of HL-60 cell differentiation via the VDR. To assess the potential antagonistic action of these two lactone analogues on specific 1alpha ,25(OH)2D3/VDR-activated genes, two separate assays were conducted.

Fig. 7 presents the results of p21WAF1,CIP1 reverse transcription PCR after examining the effect of TEI-9647 on 1alpha ,25(OH)2D3 regulated gene expression. The gene expression of p21WAF1,CIP1 was clearly up-regulated by 10-8 M of 1alpha ,25(OH)2D3; whereas 10-7 M of TEI-9647 alone did not induce up-regulation of gene expression. Impressively, TEI-9647 clearly suppressed p21WAF1,CIP1 gene expression induced by 1alpha ,25(OH)2D3.


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Fig. 7.   Effect of TEI-9647 on p21WAF1,CIP1 gene expression. HL-60 cells were treated in the absence (-) or presence (+) of 10-7 M TEI-9647 or 10-8 M 1alpha ,25(OH)2D3 for the indicated time. Total RNA was extracted, and reverse transcription PCR of p21WAF1,CIP1 or beta -actin was done as described under "Experimental Procedures."

Fig. 8 reports the antagonistic action of TEI-9647 on 1alpha ,25(OH)2D3/VDR-DRE-mediated expression of the 25(OH) D3-24-hydroxylase gene after plasmid transfection in COS-7 cells as evaluated by a luciferase reporter assay. In the absence of TEI-9647, 1alpha ,25(OH)2D3, 10-8 M effected a ~600-fold increase in 24-hydroxylase reporter gene expression after 48 h. TEI-9647 acting alone had no discernible effect on the 24-hydroxylase gene expression. Impressively 10-7 M TEI-9647 inhibited by ~50% the 24-hydroxylase activity induced by 1alpha ,25(OH)2D3 at 10-8 M.


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Fig. 8.   Antagonistic action of TEI-9647 on 1alpha ,25(OH)2D3 (VDR)-mediated expression of the 25(OH)D3-24-hydroxylase gene in a luciferase reporter assay. COS-7 cells were transfected with the luciferase expression vector driven by the DRE of the rat 25(OH)D3-24-hydroxylase gene along with plasmids containing the cDNA of human VDR and beta -galactosidase (beta-gal). Then the cells were treated for 48 h with 1alpha ,25(OH)2D3 or TEI-9647 alone or in combination, and the luciferase activity of the lysate was determined and adjusted for the beta -galactosidase activity. The bars indicate mean ± S.D. of three independent experiments.

The results presented in Figs. 7 and 8 suggest that TEI-9647 is a specific antagonist for VDR/DRE activation of gene expression. However, an alternative interpretation for these results is that the TEI-9647 enhances the catabolism of 1alpha ,25(OH)2D3 in the cell culture system over 24 h (Fig. 7) to 48 h. (Fig. 8). Thus, the apparent inhibition of the 1alpha ,25(OH)2D3 agonist effect could have been due to a reduction in the effective concentration of the secosteroid. However, in light of the results presented in Fig. 9, this seems not to be a valid concern. Fig. 9 evaluates the catabolism of [1-3H]1alpha ,25(OH)2D3 induced by the 1alpha ,25-lactone analogues in HL-60 cells. When TEI-9647, TEI-9648, TEI-9616, or (23S,25R)-1alpha ,25(OH)2D3-lactone were separately added to the [1-3H]1alpha ,25(OH)2D3, there was a consistent reduction in the rate of catabolism of the [1-3H]1alpha ,25(OH)2D3. These results suggest that the antagonistic actions of TEI-9647 and TEI-9648 occur despite blocking of the metabolism of 1alpha ,25(OH)2D3.


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Fig. 9.   Evaluation of the catabolism of [3H]1alpha ,25(OH)2D3-1alpha ,25(OH)2D3 induced by 1alpha ,25(OH)2D3-26,23-lactone analogues in HL-60 cells. HL-60 cells (106 cells/ml in a 100-mm diameter dish) were cultured in RPMI 1640 medium supplemented with FBS and [1-3H]1alpha ,25(OH)2D3-1alpha ,25(OH)2D3 10-8 M and the indicated 1alpha ,25(OH)2D3-26,23-lactone analogues and then cultured for the indicated time. The procedures of cell culture, metabolite extraction, and high pressure liquid chromatography separation is described under "Experimental Procedures." open circle , [1-3H]1alpha ,25(OH)2D3 only in cell-free culture; , [1-3H]1alpha ,25(OH)2D3 in HL-60 cells; triangle , [1-3H]1alpha ,25(OH)2D3 + TEI-9647 10-7 M; black-triangle, [1-3H]1alpha ,25(OH)2D3 + TEI-9648 10-7 M; , [1-3H]1alpha ,25(OH)2D3 + TEI-9616 10-7 M; black-square, [1-3H]1alpha ,25(OH)2D3 + (23S,25R)-1alpha ,25(OH)2D3-lactone 10-7 M.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the process of characterizing the relative importance of the 26,23-lactone ring present on the naturally occurring metabolite (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone, we chemically synthesized two lactone analogues that had a 25-dehydrated(TEI-9647 and TEI-9648). Although the two 25-dehydrated diastereoisomers bound to the VDR 115-140-fold better than the natural (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone, neither analogue was able to function as an agonist for the VDR with respect to stimulation of HL-60 cell differentiation (Figs. 3-5) or to activation of the 25(OH)D3-24-hydroxylase promoter, which had been transfected into COS-7 cells (Fig. 8). Intriguingly both 25-dehydro analogues were able to antagonize the action of 1alpha ,25(OH)2D3 or KH-1060 on effecting VDR-mediated HL-60 cell differentiation. In contrast, neither analogue blocked the actions of ATRA and 9-cis-RA on HL-60 cell differentiation, suggesting that the inhibitory actions of TEI-9647 and TEI-9648 may be 1alpha ,25(OH)2D3/VDR-specific.

The antagonist activity of TEI-9647 and TEI-9648 has been confirmed in two other systems. In COS-7 cells (which are devoid of both the VDR and the 25(OH)D3-24-hydroxylase), after co-transfection with the cDNA for both the VDR and the promoter of the 24-hydroxylase, TEI-9647 was found to antagonize 1alpha ,25(OH)2D3 action (Fig. 8). Secondly, TEI-9647 antagonized gene expression of p21WAF1,CIP1 regulated by 1alpha ,25(OH)2D3 (Fig. 7). Collectively these results described the first example of a stereospecific vitamin D secosteroid, which functions as an antagonist of the nuclear receptor for 1alpha ,25(OH)2D3.

It is well known that 1alpha ,25(OH)2D3 is initially deactivated and metabolized to 1alpha ,24R,25-trihydroxyvitamin D3 (1alpha , 24R,25(OH)3D3) by C-24 hydroxylation through a side-chain oxidation pathway resulting in C-23-C-24 cleavage, ultimately yielding 24,25,26,27-tetranor-1alpha ,23-dihydroxyvitamin D3 (24,25,26,27-tetranor-1alpha ,23(OH)2D3) in HL-60 cells (39-41). Moreover, 1alpha ,25(OH)2D3 causes the expression of the 25(OH)D3-24-hydroxylase gene through VDR/DRE-mediated genomic action in various cells (42, 43). Because of a concern that the apparent inhibition of 1alpha ,25(OH)2D3 might be a consequence of enhanced catabolism of 1alpha ,25(OH)2D3, we have investigated the effect of the 1alpha ,25-lactone analogues on the metabolism of 10-8 M [3H]1alpha ,25(OH)2D3 in HL-60 cells (Fig. 9). In a control experiment in the absence of antagonist, 1alpha ,25-(OH)2D3 was metabolized to 1alpha ,24R,25(OH)3D3, 24-oxo-1alpha ,25-dihydroxyvitamin D3 (24-oxo-1alpha ,25(OH)2D3), and 24,25,26,27-tetranor-1alpha ,23(OH)2D3 at 8 h after incubation with HL-60 cells (data not presented), and the concentration of the 1alpha , 25(OH)2D3 was markedly decreased by 24 h. The amounts of the 1alpha ,25(OH)2D3 metabolites reached maximum levels 48-72 h after the cultivation. When 10-7 M TEI-9647 was added to the above culture system, it significantly inhibited the metabolism of [3H]1alpha ,25(OH)2D3. The same is also true for TEI-9648, but the inhibitory action of TEI-9647 was stronger than that of TEI-9648. On the other hand, TEI-9616 and (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone did not inhibit the metabolism of 1alpha ,25(OH)2D3. Collectively the results suggest that the antagonistic actions of TEI-9647 and TEI-9648 clearly occur despite blocking the metabolism of 1alpha ,25(OH)2D3.

Many reports have described that the activation mechanism of steroid nuclear receptor families' function involves complex formation with partner proteins after ligand/receptor binding (44-46). For example, VDR-retinoid X receptor complex formation is thought to be essential for initiating 1alpha ,25(OH)2D3 responses (47). A possible consequence of TEI-9647 and TEI-9648 antagonistic action may be to prevent heterodimer complex formation or the recruitment by the VDR receptor co-activator proteins like NCoA-62 (48) or steroid co-activator-1 (49). At present, we are carrying out further studies concerning the mode of action of TEI-9647 and TEI-9648.

Norman et al. (13, 15) reported that 1beta ,25(OH)2D3 acts as an antagonist of vitamin D3-induced nongenomic action. In these reports, 1beta ,25(OH)2D3 suppressed up-regulation of calcium transport in intestinal epithelium (transcaltachia) (15) and stimulation of whole cell chloride currents in osteoblastic ROS 17/2.8 cells briefly exposed to 1alpha ,25(OH)2D3 (13). In contrast, 1beta ,25(OH)2D3 did not antagonize HL-60 cell differentiation induced by 1alpha ,25(OH)2D3, which is thought to be a genomic action of vitamin D3 (data not presented and Ref. 15). Considering these data, 1beta ,25(OH)2D3 would not be an antagonist of VDR/DRE-mediated genomic action of 1alpha ,25(OH)2D3, but of nongenomic actions. The action spectrum of our novel antagonists, TEI-9647 and TEI-9648, is quite different from that of 1beta ,25(OH)2D3, from which we conclude that they are antagonists of 1alpha ,25(OH)2D3-induced genomic action. It is not yet clear whether the lactones may function as antagonists of 1alpha ,25(OH)2D3-mediated rapid nongenomic actions.

The biological significance of the natural (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone is not yet fully understood, though it is a major metabolite of 1alpha ,25(OH)2D3 under physiological conditions (50). The metabolic pathways leading to 1alpha ,25-lactone production from 1alpha ,25(OH)2D3 are well investigated (19), whereas the further metabolism of 1alpha ,25-lactone is not entirely known. It has been previously reported that the 25-dehydration reaction of 1alpha ,25(OH)2D3 can occur in vivo, resulting in the production of both 24-dehydro-1alpha -hydroxyvitamin D3 and 25-dehydro-1alpha -hydroxyvitamin D3 (51). In the case of the naturally occurring 1alpha ,25-lactone, a similar 25-dehydration reaction may possibly take place resulting in the production of TEI-9647. If our hypothesis is true, TEI-9647 should be present under physiological conditions and could possibly act as a negative regulator of hormonal action of 1alpha ,25(OH)2D3 in vivo. We are now trying to identify further metabolites of the natural 1alpha ,25-lactone in vivo and determining whether they include TEI-9647.

In conclusion, our data strongly suggest that the novel 1alpha ,25-lactone analogues, TEI-9647 and TEI-9648, may be antagonists of VDR/DRE-mediated genomic actions. They are the first antagonists that possess such properties, and they may be useful compounds for basic research on vitamin D3 action. Also, it is clear that the 26,23-lactone ring functionality can change the biological properties of 1alpha ,25(OH)2D3 in an unexpected fashion.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 909-787-4777; Fax: 909-787-4784; E-mail: norman{at}ucrac1.ucr.edu.

    ABBREVIATIONS

The abbreviations used are: 1alpha ,25(OH)2D3, 1alpha ,25-dihydroxyvitamin D3; 1alpha ,25-lactone, 1alpha ,25-dihydroxyvitamin D3-26,23-lactone; 25(OH)D3-24-hydroxylase, 25-hydroxyvitamin D3-24-hydroxylase; 25(OH)D3, 25-hydroxyvitamin D3; (23S,25R)-1alpha ,25(OH)2D3-26,23-lactone, (23S,25R)-1alpha ,25-dihydroxyvitamin D3-26,23-lactone; 1alpha ,24R, 25(OH)3D3, 1alpha ,24R,25-trihydroxyvitamin D3; 24,25,26,27-tetranor-1alpha ,23(OH)2D3, 24,25,26,27-tetranor-1alpha ,23-dihydroxyvitamin D3; (23S)-1alpha (OH)D3-26,23-lactone (TEI-9616), (23S)-1alpha -hydroxyvitamin D3-26,23-lactone; (23S)-25-dehydro-1alpha (OH)D3-26,23-lactone (TEI-9647), (23S)-25-dehydro-1alpha -hydroxyvitamin D3-26,23-lactone; (23R)-25dehydro-1alpha (OH)D3-26,23-lactone (TEI-9648), (23R)-25-dehydro-1alpha -hydroxyvitamin D3-26,23-lactone; KH-1060, 20-epi-22-oxa-24a,26a-, 27a-trihomo-1alpha ,25(OH)2D3; VDR, vitamin D nuclear receptor; DRE, vitamin D-responsive element; DBP, vitamin D-binding protein; NBT, nitro blue tetrazolium; alpha -NB, alpha -naphthylbutyrate; TPA, 12-O-tetradecanoylphorbol-13-acetate; ATRA, all-trans retinoic acid; 9-cis-RA, 9-cis-retinoic acid; PCR, polymerase chain reaction; FACS, fluorescent-activated cell sorter; FBS, fetal bovine serum; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Nemere, I., and Norman, A. W. (1991) Handbook of Physiology, American Physiological Society, Bethesda, MD
  2. Haussler, M. R. (1986) Annu. Rev. Nutr. 6, 527-562[CrossRef][Medline] [Order article via Infotrieve]
  3. Bouillon, R., Okamura, W. H., and Norman, A. W. (1995) Endocr. Rev. 16, 200-257[Medline] [Order article via Infotrieve]
  4. Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S., and Suda, T. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4990-4994[Abstract]
  5. Lemire, J. M., Archer, D. C., Beck, L., and Spiegelberg, H. L. (1995) J. Nutr. 125, 1704-1708
  6. Norman, A. W., Frankel, B. J., Heldt, A. M., and Grodsky, G. M. (1980) Science 209, 823-825[Medline] [Order article via Infotrieve]
  7. Cade, C., and Norman, A. W. (1987) Endocrinology 120, 1490-1497[Abstract]
  8. Neveu, I., Naveihan, C., Menna, D., Wion, D., Brachet, P., and Garabedian, M. (1994) J. Neurosci. Res. 38, 214-220[Medline] [Order article via Infotrieve]
  9. Wion, D., MacGrogan, D., Neveu, I., Jehan, F., Houglatte, R., and Brachet, P. (1991) J. Neurosci. Res. 28, 110-114[Medline] [Order article via Infotrieve]
  10. Pike, J. W. (1997) in The Vitamin D Receptor and Its Gene (Feldman, D., Glorieux, F. H., and Pike, J. W., eds), pp. 105-125, Academic Press, San Diego
  11. Haussler, M. R., Whitfield, G. K., Haussler, C. A., Hsieh, J. C., Thompson, P. D., Selznick, S. H., Dominquez, C. E., and Jurutka, P. A. (1998) J. Bone Miner. Res. 13, 325-349[Medline] [Order article via Infotrieve]
  12. Norman, A. W., Okamura, W. H., Hammond, M. W., Bishop, J. E., Dormanen, M. C., Bouillon, R., van Baelen, H., Ridal, A. L., Daane, E., Khoury, R., and Farach-Carson, M. C. (1997) Mol. Endocrinol. 11, 1518-1531[Abstract/Free Full Text]
  13. Zanello, L. P., and Norman, A. W. (1997) J. Biol. Chem. 272, 22617-22622[Abstract/Free Full Text]
  14. Song, X., Bishop, J. E., Okamura, W. H., and Norman, A. W. (1997) Endocrinology 139, 457-465[Abstract/Free Full Text]
  15. Norman, A. W., Bouillon, R., Farach-Carson, M. C., Bishop, J. E., Zhou, L. X., Nemere, I., Zhao, J., Muralidharan, K. R., and Okamura, W. H. (1993) J. Biol. Chem. 268, 20022-20030[Abstract/Free Full Text]
  16. Ishizuka, S., Yamaguchi, H., Yamada, S., Nakayama, K., and Takayama, H. (1981) FEBS Lett. 134, 207-211[CrossRef][Medline] [Order article via Infotrieve]
  17. Ishizuka, S., Ishimoto, S., and Norman, A. W. (1984) J. Steroid Biochem. 20, 611-615[CrossRef][Medline] [Order article via Infotrieve]
  18. Ishizuka, S., Ishimoto, S., and Norman, A. W. (1984) Biochemistry 23, 1473-1478[Medline] [Order article via Infotrieve]
  19. Ishizuka, S., and Norman, A. W. (1987) J. Biol. Chem. 262, 7165-7170[Abstract/Free Full Text]
  20. Ishizuka, S., Oshida, J., Tsuruta, H., and Norman, A. W. (1985) Arch. Biochem. Biophys. 242, 82-89[Medline] [Order article via Infotrieve]
  21. Kiyoki, M., Kurihara, N., Ishizuka, S., Ishii, S., Hakeda, Y., Kumegawa, M., and Norman, A. W. (1985) Biochem. Biophys. Res. Commun. 127, 693-698[Medline] [Order article via Infotrieve]
  22. Ishizuka, S., Kiyoki, M., Kurihara, N., Hakeda, Y., Ikeda, K., Kumegawa, M., and Norman, A. W. (1988) Mol. Cell. Endocrinol. 55, 77-86[CrossRef][Medline] [Order article via Infotrieve]
  23. Ishizuka, S., Kurihara, N., Hakeda, S., Maeda, N., Ikeda, K., Kumegawa, M., and Norman, A. W. (1988) Endocrinology 123, 781-786[Abstract]
  24. Ishizuka, S., Sumitani, K., Hiura, K., Kawata, T., Okawa, M., Hakeda, Y., and Kumegawa, M. (1990) Endocrinology 127, 695-701[Abstract]
  25. Ishizuka, S., Mimura, H., Hayashi, T., Oshida, J., Ishizeki, K., Takigawa, M., and Norman, A. W. (1997) in Vitamin D: Chemistry, Biology and Clinical Applications of Steroid Hormone (Norman, A. W., Bouillon, R., and Thomasset, M., eds), pp. 683-684, University of California, Riverside, CA
  26. Collins, S. J. (1987) Blood 70, 1233-1244[Abstract]
  27. Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2936-2940[Abstract]
  28. Rovera, G., Santoli, D., and Damsky, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2779-2783[Abstract]
  29. Lee, Y., Inaba, M., DeLuca, H. F., and Mellon, W. S. (1989) J. Biol. Chem. 264, 13701-13705[Abstract/Free Full Text]
  30. Manabe, K., Ishizuka, S., Tabe, M., Tanaka, H., Gao, Q., Furuya, M., Tomimori, K., Sakuma, Y., and Hazato, A. (1997) in Vitamin D: Chemistry, Biology and Clinical Applications of Steroid Hormone (Norman, A. W., Bouillon, R., and Thomasset, M., eds), pp. 79-80, University of California, Riverside, CA
  31. Eisman, J. A., Hamstra, A. J., Kream, B. E., and DeLuca, F. H. (1976) Arch. Biochem. Biophys. 176, 235-243[Medline] [Order article via Infotrieve]
  32. Inaba, M., and DeLuca, H. F. (1989) Biochim. Biophys. Acta 1010, 20-27[Medline] [Order article via Infotrieve]
  33. Honda, A., Nakashima, N., Mori, Y., Katsumata, T., and Ishizuka, S. (1992) J. Steroid Biochem. Mol. Biol. 41, 109-112[CrossRef][Medline] [Order article via Infotrieve]
  34. Collins, S. J., Ruscetti, F. W., Gallagher, R. E., and Gallo, R. C. (1979) J. Exp. Med. 149, 969-974[Abstract]
  35. Ohyama, Y., Ozono, K., Uchida, M., Yoshimura, M., Shinki, T., Suda, T., and Yamamoto, O. (1996) J. Biol. Chem. 271, 30381-30385[Abstract/Free Full Text]
  36. Yoshida, M., Ishizuka, S., and Hoshi, A. (1984) J. Pharm. Dyn. 7, 962-968[Medline] [Order article via Infotrieve]
  37. Zhou, J. Y., Norman, A. W., Lubbert, M., Collins, E. D., Uskokovic, M. R., and Koeffler, H. P. (1989) Blood 74, 82-93[Abstract]
  38. Binderup, L., Latini, S., Binderup, E., Bretting, C., Calverley, M., and Hansen, K. (1991) Biochem. Pharmacol. 42, 1569-1575[CrossRef][Medline] [Order article via Infotrieve]
  39. Honda, A., Nakashima, N., Shida, Y., Mori, Y., Nagata, A., and Ishizuka, S. (1993) Biochem. J. 295, 509-516[Medline] [Order article via Infotrieve]
  40. Reddy, G. S., Ishizuka, S., Tserng, K. Y., and Spagnuolo, P. J. (1987) J. Bone Miner. Res. 2, 36 (suppl.) (Abstr. 78)
  41. Inaba, M., Burgos-Trinidad, M., and DeLuca, H. F. (1991) Arch. Biochem. Biophys. 284, 257-263[Medline] [Order article via Infotrieve]
  42. Ohyama, Y., Ozono, K., Uchida, M., Shinki, T., Kato, S., Suda, T., Yamamoto, O., Noshiro, M., and Kato, Y. (1994) J. Biol. Chem. 269, 10545-10550[Abstract/Free Full Text]
  43. Zierold, C., Hisham, M. D., and DeLuca, H. F. (1995) J. Biol. Chem. 270, 1675-1678[Abstract/Free Full Text]
  44. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83, 835-839[Medline] [Order article via Infotrieve]
  45. Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841-850[Medline] [Order article via Infotrieve]
  46. Beato, M., Herrlich, P., and Schutz, G. (1995) Cell 83, 851-857[Medline] [Order article via Infotrieve]
  47. Liu, Y-Y., Collins, E. D., Norman, A. W., and Peleg, S. (1997) J. Biol. Chem. 272, 3336-3345[Abstract/Free Full Text]
  48. Baudino, T. A., Kraicheley, D. M., Jefcoat, S. C., Jr., Winchester, S. K., Partridge, N. C., and MacDonald, P. N. (1998) J. Biol. Chem. 273, 16434-16441[Abstract/Free Full Text]
  49. Gill, R. K., Atkins, L. M., Hollis, B. W., and Bell, N. H. (1998) Mol. Endocrinol. 12, 57-65[Abstract/Free Full Text]
  50. Ishizuka, S., Ohba, T., and Norman, A. W. (1988) in Vitamin D: Molecular, Cellular and Clinical Endocrinology (Norman, A. W., Schaefer, K., and Grigoleit, H. G., eds), pp. 143-144, Walter de Gruyter & Co., Berlin
  51. Onisko, B. L., Esvelt, P., Schnoes, H. K., and DeLuca, H. F. (1980) Biochemistry 19, 4124-4130[Medline] [Order article via Infotrieve]


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