Antagonistic Action of Novel 1
,25-Dihydroxyvitamin
D3-26,23-lactone Analogs on Differentiation of Human
Leukemia Cells (HL-60) Induced by 1
,25-Dihydroxyvitamin
D3*
Daishiro
Miura
,
Kenji
Manabe§,
Keiichi
Ozono¶,
Mariko
Saito¶,
Qingzhi
Gao§,
Anthony W.
Norman
**, and
Seiichi
Ishizuka§
From the
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
Department of
Biochemistry and Division of Biomedical Sciences, University of
California, Riverside, California 92521
 |
ABSTRACT |
We examined the effects of two novel
1
,25-dihydroxyvitamin D3-26,23-lactone
(1
,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 1
,25-dihydroxyvitamin D3
(1
,25(OH)2D3) and its analogues. We found
that the 1
,25-lactone analogues
(23S)-25-dehydro-1
-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and
(23R)-25-dehydro-1
-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S,25R)-1
,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 1
,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 1
,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 1
,25-lactone analogues, TEI-9647 and
TEI-9648, are specific antagonists of
1
,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 |
It is widely accepted that the fundamental biological activities
of the hormonal form of vitamin D3,
1
,25-dihydroxyvitamin D3
(1
,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).
1
,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
1
,25(OH)2D3 that can modulate or antagonize
these interactions. However, to date the only known antagonist of
1
,25(OH)2D3 is the analogue
1
,25(OH)2D3, which blocks rapid nongenomic
responses but is without effect on the classical nuclear VDR (15).
(23S,25R)-1
,25-dihydroxyvitamin
D3-26,23-lactone
((23S, 25R)-1
,25(OH)2
D3-26,23-lactone) was found by Ishizuka et al.
(16-19) as a major metabolite of
1
,25(OH)2D3 both in vivo and
in vitro. They reported that the naturally occurring
(23S,
25R)-1
,25(OH)2D3-26,23-lactone has unique biological features in comparison with
1
,25(OH)2D3. First of all, the VDR binding
affinity of the naturally occurring 1
,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
1
,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), 1
,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
1
,25(OH)2D3 through a VDR/DRE-mediated
pathway (29); as such this is a useful system to study genomic actions
of 1
,25(OH)2D3 and related analogues.
We have recently synthesized various analogues of 1
,25-lactone to
investigate which functionality of the 1
,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 1
,25-lactone analogues
((23S)-25-dehydro-1
(OH)D3-26,23-lactone) (TEI-9647) and
((23S)-25-dehydro-1
(OH)D3-26,23-lactone)
(TEI-9648). These analogues were found to block both
1
,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.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
25-Hydroxyvitamin D3
(25(OH)D3), 1
,25(OH)2D3,
1
,25-dihydroxyvitamin D3
(1
,25(OH)2D3),
1
,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
1
,25(OH)2D3-26,23-lactone and its analogues
are shown in Fig. 1. The
20-epi-22-oxa-24a,26a,27a-trihomo-1
,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]1
,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]1
,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
1
,25(OH)2D3 and 1
,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]1
,25(OH)2D3
(specific activity, 179 Ci/mmol, 15,000 dpm, 15.7 pg) and various
amounts of 1
,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 1
,25-lactone analogues was performed as described previously (33).
Cytohistochemical Assay--
Cell morphology, NBT-reducing
activity and monocytic cell-specific esterase (
-naphthylbutyrate
(
-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.
1
,25(OH)2D3 and 1
,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.
-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
-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
-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
-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
-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
1
,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
-galactosidase activity.
Metabolism of [1-3H]1
,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]1
,25(OH)2D3 (specific
activity 16.2 Ci/mmol, 1.62 µCi), and 10
7 M
1
,25-lactone analogues, and then cultured for the indicated time.
[1-3H]1
,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 1
,25(OH)2D3,
24-oxo-1
,25(OH)2D3,
24,25,26,27-tetranor-1
,25(OH)2D3, (23S,25R)-1
,25(OH)2D3-26,23-lactone
and 1
,24R,25(OH)3D3 eluted at
16.3, 19.3, 23.8, 29.0, and 29.3 min, respectively.
 |
RESULTS |
Fig. 1 indicates the structures of
the naturally occurring 1
,25-lactone and its three analogues.
TEI-9616 is a 25-dehydroxylated version of the naturally occurring
(23S,25R)-1
,25(OH)2D3-26,23-lactone. TEI-9647 and TEI-9648 are both 25-dehydrated lactones of the
(23S,25R)- and
(23R,25R)-1
,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
1 ,25(OH)2D3-26,23-lactone
analogues. The
(23S,25R)-1 ,25(OH)2D3-26,23-lactone
is a naturally occurring metabolite derived from
1 ,25(OH)2D3 (55).
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|
The receptor binding affinities of 1
,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
1
,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)-1
,25(OH)2D3-26,23-lactone. In contrast, the binding affinities of TEI-9616 and the naturally occurring
(23S,25R)-1
,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 1
,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
1 ,25(OH)2D3-26,23-lactone
analogues. A, HL-60 cell VDR binding assay with
1 ,25(OH)2D3 and
1 ,25(OH)2D3-26,23-lactone analogues in
comparison with
[3H]1 ,25(OH)2D3. B,
chick intestinal VDR binding assay with
1 ,25(OH)2D3 and
1 ,25(OH)2D3-26,23-lactone analogues in
comparison with
[3H]1 ,25 (OH)2D3.
C, competitive binding assay of DBP in FCS for
1 ,25(OH)2D3 and
1 ,25(OH)2D3-26,23-lactone analogues in
comparison with [3H]25(OH)D3. Analogs are
25(OH)D3 ( ), 1 ,25(OH)2D3
( ), TEI-9647 ( ), TEI-9648 ( ), TEI-9616 ( ) and
(23S,25R)-1 ,25(OH)2D3-26,23-lactone
( ). Points are means of triplicate determinations. All values
are within 5% of mean.
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Table I
Biological characteristics of
1 ,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 1 ,25(OH)2D3, which was set to 100%.
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(23S,25R)-1
,25(OH)2D3-26,23-lactone
bound to the plasma DBP 6.2 times stronger than
1
,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 1
,25(OH)2D3
(Table I).
Our preliminary data indicated that the natural
(23S,25R)-1
,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 1
,25-lactone analogues might be more potent in HL-60 cell
differentiation than the natural
(23S,25R)-1
,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)-1
,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
1
,25(OH)2D3 dose dependently induced
differentiation of HL-60 cells; a concentration of 10
8
M of 1
,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 1
,25(OH)2D3. Although
undifferentiated HL-60 cells showed promyelocytic features, cells
differentiated by 1
,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
1
,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
-NB esterase activity, are known to be up-regulated by
1
,25(OH)2D3. Therefore, we examined the
effect of TEI-9647 and TEI-9648 to mediate the up-regulation of
differentiation markers induced by
1
,25(OH)2D3. TEI-9647 or TEI-9648 alone
could not induce activation of NBT-reducing activity or
-NB esterase
activity. In contrast, they both markedly suppressed the up-regulation
induced by 1
,25(OH)2D3 (Fig. 3, B
and C).

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Fig. 3.
Effects of TEI-9647 and TEI-9648 on
1 ,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 1 ,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, -NB esterase
activity.
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Next we examined separately the inhibitory effects of TEI-9647 and
TEI-9648 on 1
,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 1
,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)-1
,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
1 ,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 1 ,25(OH)2D3 for 96 h,
and NBT-reducing activity was examined. Rectangles and
bars show mean ± S.D. of triplicates,
respectively.
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A particularly potent analogue of
1
,25(OH)2D3 is KH-1060, which has been shown
to have a 5,000-10,000-fold more potent cell differentiating activity
than 1
,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.
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Fig. 6 shows the consequences of TEI-9647
and TEI-9648 on the changes of cell surface marker expression. In HL-60
cells, 1
,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 1
,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
1 ,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
1 ,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
1
,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 1
,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 1
,25(OH)2D3 regulated gene expression. The gene expression of p21WAF1,CIP1 was
clearly up-regulated by 10
8 M of
1
,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
1
,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
1 ,25(OH)2D3 for the indicated time. Total
RNA was extracted, and reverse transcription PCR of
p21WAF1,CIP1 or -actin was done as described under
"Experimental Procedures."
|
|
Fig. 8 reports the antagonistic action of
TEI-9647 on 1
,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,
1
,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
1
,25(OH)2D3 at 10
8
M.

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|
Fig. 8.
Antagonistic action of TEI-9647 on
1 ,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 -galactosidase
(beta-gal). Then the cells were treated for
48 h with 1 ,25(OH)2D3 or TEI-9647 alone
or in combination, and the luciferase activity of the lysate was
determined and adjusted for the -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 1
,25(OH)2D3 in
the cell culture system over 24 h (Fig. 7) to 48 h. (Fig. 8).
Thus, the apparent inhibition of the
1
,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]1
,25(OH)2D3 induced by
the 1
,25-lactone analogues in HL-60 cells. When TEI-9647, TEI-9648,
TEI-9616, or
(23S,25R)-1
,25(OH)2D3-lactone were separately added to the
[1-3H]1
,25(OH)2D3, there was a
consistent reduction in the rate of catabolism of the
[1-3H]1
,25(OH)2D3. These
results suggest that the antagonistic actions of TEI-9647 and TEI-9648
occur despite blocking of the metabolism of
1
,25(OH)2D3.

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Fig. 9.
Evaluation of the catabolism of
[3H]1 ,25(OH)2D3-1 ,25(OH)2D3
induced by
1 ,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]1 ,25(OH)2D3-1 ,25(OH)2D3
10 8 M and the indicated
1 ,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." ,
[1-3H]1 ,25(OH)2D3 only in
cell-free culture; ,
[1-3H]1 ,25(OH)2D3 in HL-60
cells; , [1-3H]1 ,25(OH)2D3 + TEI-9647 10 7 M; ,
[1-3H]1 ,25(OH)2D3 + TEI-9648
10 7 M; ,
[1-3H]1 ,25(OH)2D3 + TEI-9616
10 7 M; ,
[1-3H]1 ,25(OH)2D3 + (23S,25R)-1 ,25(OH)2D3-lactone
10 7 M.
|
|
 |
DISCUSSION |
In the process of characterizing the relative importance of the
26,23-lactone ring present on the naturally occurring metabolite (23S,25R)-1
,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)-1
,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
1
,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
1
,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 1
,25(OH)2D3
action (Fig. 8). Secondly, TEI-9647 antagonized gene expression of
p21WAF1,CIP1 regulated by
1
,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 1
,25(OH)2D3.
It is well known that 1
,25(OH)2D3 is
initially deactivated and metabolized to
1
,24R,25-trihydroxyvitamin D3 (1
,
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-1
,23-dihydroxyvitamin D3
(24,25,26,27-tetranor-1
,23(OH)2D3) in HL-60
cells (39-41). Moreover, 1
,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
1
,25(OH)2D3 might be a consequence of
enhanced catabolism of 1
,25(OH)2D3, we have
investigated the effect of the 1
,25-lactone analogues on the
metabolism of 10
8 M
[3H]1
,25(OH)2D3 in HL-60 cells
(Fig. 9). In a control experiment in the absence of antagonist,
1
,25-(OH)2D3 was metabolized to 1
,24R,25(OH)3D3,
24-oxo-1
,25-dihydroxyvitamin D3
(24-oxo-1
,25(OH)2D3), and
24,25,26,27-tetranor-1
,23(OH)2D3 at 8 h
after incubation with HL-60 cells (data not presented), and the
concentration of the 1
, 25(OH)2D3 was
markedly decreased by 24 h. The amounts of the
1
,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]1
,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)-1
,25(OH)2D3-26,23-lactone did not inhibit the metabolism of
1
,25(OH)2D3. Collectively the results
suggest that the antagonistic actions of TEI-9647 and TEI-9648 clearly
occur despite blocking the metabolism of
1
,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 1
,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
1
,25(OH)2D3 acts as an antagonist of vitamin
D3-induced nongenomic action. In these reports,
1
,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 1
,25(OH)2D3 (13).
In contrast, 1
,25(OH)2D3 did not antagonize
HL-60 cell differentiation induced by
1
,25(OH)2D3, which is thought to be a
genomic action of vitamin D3 (data not presented and Ref.
15). Considering these data, 1
,25(OH)2D3 would not be an antagonist of VDR/DRE-mediated genomic action of
1
,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 1
,25(OH)2D3,
from which we conclude that they are antagonists of
1
,25(OH)2D3-induced genomic action. It is
not yet clear whether the lactones may function as antagonists of 1
,25(OH)2D3-mediated rapid nongenomic actions.
The biological significance of the natural
(23S,25R)-1
,25(OH)2D3-26,23-lactone
is not yet fully understood, though it is a major metabolite of
1
,25(OH)2D3 under physiological conditions (50). The metabolic pathways leading to 1
,25-lactone production from
1
,25(OH)2D3 are well investigated (19),
whereas the further metabolism of 1
,25-lactone is not entirely
known. It has been previously reported that the 25-dehydration reaction
of 1
,25(OH)2D3 can occur in vivo,
resulting in the production of both 24-dehydro-1
-hydroxyvitamin D3 and 25-dehydro-1
-hydroxyvitamin D3 (51).
In the case of the naturally occurring 1
,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
1
,25(OH)2D3 in vivo. We are now trying to identify further metabolites of the natural 1
,25-lactone in vivo and determining whether they include TEI-9647.
In conclusion, our data strongly suggest that the novel 1
,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 1
,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:
1
,25(OH)2D3, 1
,25-dihydroxyvitamin
D3;
1
,25-lactone, 1
,25-dihydroxyvitamin
D3-26,23-lactone;
25(OH)D3-24-hydroxylase, 25-hydroxyvitamin D3-24-hydroxylase;
25(OH)D3, 25-hydroxyvitamin D3;
(23S,25R)-1
,25(OH)2D3-26,23-lactone, (23S,25R)-1
,25-dihydroxyvitamin
D3-26,23-lactone;
1
,24R, 25(OH)3D3,
1
,24R,25-trihydroxyvitamin D3;
24,25,26,27-tetranor-1
,23(OH)2D3, 24,25,26,27-tetranor-1
,23-dihydroxyvitamin D3;
(23S)-1
(OH)D3-26,23-lactone (TEI-9616), (23S)-1
-hydroxyvitamin D3-26,23-lactone;
(23S)-25-dehydro-1
(OH)D3-26,23-lactone
(TEI-9647), (23S)-25-dehydro-1
-hydroxyvitamin
D3-26,23-lactone;
(23R)-25dehydro-1
(OH)D3-26,23-lactone
(TEI-9648), (23R)-25-dehydro-1
-hydroxyvitamin
D3-26,23-lactone;
KH-1060, 20-epi-22-oxa-24a,26a-, 27a-trihomo-1
,25(OH)2D3;
VDR, vitamin D nuclear receptor;
DRE, vitamin D-responsive element;
DBP, vitamin D-binding protein;
NBT, nitro blue tetrazolium;
-NB,
-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.
 |
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