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Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 160-167
Anticoagulant Effects of 1 ,25-Dihydroxyvitamin D3 on
Human Myelogenous Leukemia Cells and Monocytes
By
Takatoshi Koyama,
Misako Shibakura,
Mai Ohsawa,
Ryuichi Kamiyama, and
Shinsaku Hirosawa
From the School of Allied Health Sciences and the First Department of
Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
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ABSTRACT |
The hormonally active form of vitamin D is 1 ,25-dihydroxyvitamin
D3 [1,25(OH)2D3], which is a
principal regulator of calcium homeostasis. It also
affects hormone secretion, cell differentiation, and proliferation by a
mode of action that involves stereospecific interaction with an
intracellular vitamin D receptor (VDR). We recently found that
retinoids, which are vitamin A derivatives, exert anticoagulant effects
by upregulating thrombomodulin (TM) and downregulating tissue factor
(TF) expression in acute promyelocytic leukemia cells and monoblastic
leukemia cells. Both the VDR and retinoid receptors belong to the same
family of receptors. A heterodimer consisting of the retinoid X
receptor and the VDR binds to vitamin D responsive elements on genes
regulated by vitamin D. To determine whether
1,25(OH)2D3 would exhibit anticoagulant effects
similar to retinoids, we measured the antigen level, activity, and mRNA level of TM and TF in human leukemic cells, vascular endothelial cells,
and monocytes treated with 1,25(OH)2D3. We
found that 1,25(OH)2D3 upregulates antigen
expression, activity, and mRNA levels of TM and downregulates antigen
expression, activity, and mRNA levels of TF in human monocytic leukemia
cells, some acute myelogenous leukemia cells, and monocytes, but not in
umbilical vein endothelial cells. Transient transfection studies with
reporter plasmids in monocytic leukemia cells and mobility gel-shift
assay showed interaction with 1,25(OH)2D3 and
functional retinoic acid responsive elements present in the
5 -flanking region of the TM gene. However, auxiliary factors or
other elements in the TM gene may contribute to VDR specificity and
transactivation of the gene in specific target cells. These findings
indicate that 1,25(OH)2D3 resembles the retinoids in its control of the transcription of the TM and TF genes in
human monocytic cells. Analogs of 1,25(OH)2D3
with anticoagulant activity may serve as adjunctive antithrombotic
agents in monocytic leukemia and atherosclerotic disease.
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INTRODUCTION |
VITAMIN D METABOLITES are involved in
many cellular processes, including calcium homeostasis, the immune
response, cell differentiation, and the regulation of gene
transcription.1,2 The hormonally active form of vitamin D,
1,25-dihydroxyvitamin D3
[1,25(OH)2D3], may generate biological
responses via both the regulation of gene transcription and nongenomic
pathways.1,2
The biological effects of 1,25(OH)2D3 are
mediated mainly by intranuclear receptors, specifically, by the vitamin
D receptors (VDRs), which belong to the same family as the steroid and
retinoid receptors. However, receptor-independent effects have also
been reported.1 VDRs are ligand-activated transcriptional
factors that interact with their cognate responsive elements, called
vitamin D responsive elements (VDREs), on vitamin D-regulated genes
either as homodimers or as heterodimers with retinoid X receptors
(RXRs). RXR/VDR heterodimers bind to VDREs with a much higher affinity than VDR homodimers and are thought to be a physiologically relevant form of the transcriptionally active complex. It is proposed that binding specificity can be conferred by nucleotide spacing between two
half-sites of a VDRE. Spacer regions of 3, 4, and 5 nucleotides confer
recognition for VDR, thyroid hormone (T3) receptor
(T3R) and retinoic acid receptor (RAR).3
We recently found that several vitamin A derivatives, or retinoids,
exert anticoagulant effects by upregulating the expression of
thrombomodulin (TM) and downregulating that of tissue factor (TF) in
acute promyelocytic leukemia (APL) and monoblastic leukemia cells.4-6 The anticoagulant action of retinoic acids (RAs)
has been implicated in patients with APL.4 An RA-responsive
element, RARE, was identified in the 5-flanking region of the TM
gene (-1537 TGGTCA- CTGC- AGGTCA -1522).7 The spacer region
of this element consists of 4 nucleotides, which implicates the
recognition of T3. However, in preliminary experiments,
T3 has no effect on TM expression. To determine the effect
of 1,25(OH)2D3 on the expression of TM and TF,
we measured the antigen level, activity, and mRNA level of each TM and
TF in human leukemic cells, monocytes, and umbilical vein endothelial
cells (HUVECs) treated with 1,25(OH)2D3. Because the TM gene does not contain a consensus sequence of VDRE, which consists of two direct repeats separated by three nucleotides that would confer binding specificity of the VDR, we further evaluated whether 1,25(OH)2D3 combines with and
transactivates RARE in the TM gene.
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MATERIALS AND METHODS |
Reagents.
Chemicals were purchased from the following suppliers. Thyroid hormone
3,5,3-triiodothyronine (T3),
1,25(OH)2D3, and human thrombin (1,400 NIH U/mg
protein) were obtained from Sigma (St Louis, MO). Recombinant human
soluble TM, which possesses the entire extracellular TM domain, was a
gift from Asahi Chemical Industry Co (Shizuoka, Japan). Human
placenta-derived TF (Thromborel S) was from Behringwerke AG (Marburg,
Germany). The chromogenic substrate S2266 was from Chromogenix
(Stockholm, Sweden). Human protein C (inactivated and activated) was
generously provided by the Chemo-Sero-Therapeutic Research Institute
(Kumamoto, Japan). Recombinant human hirudin was from American
Diagnostica Inc (Greenwich, CT). Monoclonal mouse anti-human TM
IgG KA-4 and polyclonal rabbit anti-human TM antibody were from Teijin,
Ltd (Tokyo, Japan). Monoclonal mouse anti-human TF IgG ADI 4509 was
from American Diagnostica Inc (Greenwich, CT). Polyclonal rabbit
anti-mouse RAR and polyclonal anti-human VDR antibodies were from
BIOMOL Research Laboratories, Inc (Plymouth Meeting, PA) and Affinity
BioReagents, Inc (Golden, CO), respectively. All other chemicals were
reagent-grade products and were purchased from Wako Pure Chemicals
(Osaka, Japan), unless otherwise indicated.
Peripheral blood (PB) cells.
PB was drawn from 7 patients with acute myelogenous leukemia (AML), 1 patient with biphenotypic leukemia (Bi-1), and 2 patients with acute
lymphocytic leukemia (ALL) (ALL-1, 2). PB samples were obtained at
diagnosis, before the administration of chemotherapy, and the
mononuclear fraction was isolated as already described.5 Blood samples were selected for study only when they contained more
than 85% blasts. Leukemic cell subtypes were classified according to
French-American-British (FAB) classification. Whole blood was collected
from healthy donors and anticoagulated with heparin (10 U/mL blood).
Mature monocytes were isolated as previously described.8
Cell culture.
The following human leukemic and vascular endothelial cell lines were
used in this study. A monoblastic leukemia cell line, U937, and an
erythroleukemia cell line, K562, were provided by the Japanese Cancer
Research Bank (Tokyo, Japan). Acute myelomonocytic leukemia (AML M4)
cell lines, OCI-AML2 and OCI-AML3,9 and a B-lymphocytic
leukemia cell line, TMD2,4 were generously provided by Dr
S. Tohda (Tokyo Medical and Dental University, Tokyo, Japan). An APL
cell line, NB4, was kindly provided by Dr M. Lanotte (Hôpital Saint Louis, Paris, France).10 An acute megakaryoblastic
leukemia (AML M7) cell line, UT7,4 was a gift from Dr N. Komatsu (Jichi Medical School, Tochigi, Japan). Human vascular
endothelial cell line ECV30411 was kindly provided by Dr K. Takahashi (Tokyo Medical College, Ibaraki, Japan). Cell lines, leukemia
cells from patients, monocytes, and HUVECs were cultured as already
described.5,6,8,11 The 1,25(OH)2D3
was dissolved in absolute ethanol and further diluted into growth media
to the desired concentration. The final concentration of ethanol
in the culture media was less than 0.1%. Cells exhibited no
damage at this concentration. The culture media without
1,25(OH)2D3 or T3, used for the
control cells, contained the same concentration of ethanol as the
culture media used for the treated cells. All of the procedures
involving 1,25(OH)2D3 were performed under
subdued light.
Measurement of TM and TF antigens.
Leukemic cells were incubated with 1,25(OH)2D3
for 24 hours and then washed three times with phosphate-buffered saline
(PBS). Cells were counted and their concentration was adjusted to
107 cells/mL. Cell lysates were prepared as described
previously.4 Total TM and TF antigens in cell lysates were
measured by enzyme-linked immunosorbent assay (ELISA) using an EIA TM
kit Teijin (Teijin Inc, Tokyo, Japan) and IMUBIND Tissue Factor ELISA
Kit (American Diagnostica Inc, Greenwich, CT) according to the
manufacturer's instructions.
Cell-surface TM cofactor activity.
Cell-surface TM cofactor activity was measured as previously
described.6,12,13 In this assay, exogenous protein C (0.16 µmol/L) was activated by intact cells in the presence of thrombin (3.3 NIH U/mL) and Ca2+ (1.3 mmol/L). Cleavage of the small
molecular weight substrate S-2266 by activated protein C was measured
with a spectrophotometer. The results were expressed as
 OD405nm/minute or as the percentage of the initial
velocity of activated protein C formation (with 100% considered as the
rate of formation of activated protein C by cell-surface TM under basal
conditions). No activation of protein C was observed in control cells
in the absence of thrombin or protein C.
Cell-surface TF cofactor activity: Analysis of procoagulant activity
in clotting assays.
Leukemic cell suspensions were adjusted to 1 × 107/mL
in PBS. Normal plasma-based one-stage recalcification clotting time was determined as described previously.4 Our previous study
showed that the procoagulant activity on the surface of U937 cells
increases with the expression of TF,4 thus prolongation of
recalcification time is mainly caused by the downregulation of TF
expression by 1,25(OH)2D3. TF cofactor activity
was quantitated by reference to standard curves (log-log plot)
constructed using human placenta TF. TF activity with a 50-second
recalcification time was defined as 1 U/mL.
Immunoblotting analysis.
Western blot analysis of TM and TF in cell lysates was performed
essentially as previously described.6 Horseradish
peroxidase-conjugated KA-4 anti-TM monoclonal antibody (MoAb) and
anti-TF MoAb ADI 4509 were used for the detection of TM and TF,
respectively. Equal amounts of lysates from 5 × 106
cells were applied to each lane. Rabbit anti-human VDR antibody and
horseradish peroxidase-conjugated goat antibody to rabbit IgG was used
for detection of VDR. Equal amounts of lysates from 1 × 106 cells were applied to each lane.
Quantitative reverse transcription-polymerase chain reaction
(RT-PCR).
Levels of TM and TF mRNA in U937 cells were determined by quantitative
RT-PCR assays using the SuperScript Preamplification system (Life
Technologies, Inc, Gaithersburg, MD) as previously described.6 For mRNA quality control, glyceralde
hyde-3-phosphate dehydrogenase (GAPDH) was amplified.
Northern blotting.
The levels of TM mRNA in U937 cells and monocytes were also measured by
Northern blot analysis as reported previously.5  -actin
was used as an mRNA quality control.
Constructs and transfection.
The control plasmid pUC18tkLUC contains a thymidine kinase promoter and
Photinus pyralis luciferase (LUC) gene in pUC18 vector. OCI-AML3 cells were transfected with the plasmid, PTM-2RAREtkLUC, that
contained two tandem copies of the RARE from the 5-flanking region of the human TM gene upstream of the thymidine kinase
promoter.7 The ability of
1,25(OH)2D3 or all-trans RA (ATRA) to
induce LUC production was determined using PicaGene Dual Sea Pansy
(Wako Pure Chemicals, Osaka, Japan). OCI-AML3 cells, grown in RPMI 1640 (Nissui Pharmaceutical Co, LTD, Tokyo, Japan) with 10% fetal calf serum (FCS), were washed twice in PBS, and the concentration of cells
was adjusted to 2 × 107 cells/mL with PBS. A 250 µL
aliquot of cells was transfected with 10 µg of the plasmids and 1 µg of a control reporter vector, pRL-tk, which encoded
Renilla (Sea Pansy) LUC. The pRL-tk reporter vector was used
for normalization of transfection efficiency. Electroporation was
performed at 960 µF and 350 V using a Bio-Rad Genepulser (Bio-Rad,
Hercules, CA). The mixtures were immediately diluted into RPMI 1640 with 10% FCS in the presence or absence of
1,25(OH)2D3 or ATRA. After incubation, cells
were lysed according to the manufacturer's instructions. ECV304 cells
were transfected with the plasmids using LipofectAMINE Reagent (Life
Technologies, Inc, Gaithersburg, MD). LUC assays were sequentially
analyzed in a Lumat (Berthold, Wildbad, Germany).
Gel mobility shift assay.
The electrophoretic gel-shift assay was performed as described
previously14 using a digoxigenin (DIG)-labeled
double-stranded 22-bp oligonucleotide probe containing RARE from
the 5-flanking region of the human TM gene. A
DIG-labeled TM RARE probe was incubated with nuclear extracts
from OCI-AML3 cells that had been incubated with or without
1,25(OH)2D3 for 24 hours. Gel-shift reaction, 0.25% polyacrylamide gel electrophoresis, contact blotting, and chemiluminescent detection were performed as indicated by the manufacturer using DIG Gel Shift Kit (Boehringer Mannheim, Mannheim, Germany). In competition experiments, increasing doses of unlabeled competitors were added to the reaction mixture. Polyclonal antibody against VDR or RAR was also included in the reaction mixture.
Statistical analysis.
The significance of differences between data of groups was determined
with a two-group t-test. A P value of >.05 was
considered to represent a statistically nonsignificant change.
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RESULTS |
Effects of 1,25(OH)2 D3 or T3 on TM
and TF antigen levels in leukemic cell lines and PB cells.
TM and TF antigen levels in untreated PB leukemic cells freshly
isolated from patients with leukemia and untreated leukemic cell lines
were between 0 and 101 ng TM protein, and 0 and 6.63 ng TF protein per
107 cells. After incubation with
1,25(OH)2D3, total TM antigen levels were
increased in several AML cells including biphenotypic leukemia cells
freshly isolated from patients, and AML cell lines, OCI-AML 2 and 3, and U937 (Fig 1A). Levels of TM antigen
were increased more than 10-fold in two monocytic leukemia cell lines,
U937 and OCI-AML2, as well as in cells from a patient with acute
monocytic leukemia (M5b-1). A monocytic leukemia cell line, OCI-AML3,
exhibited a high baseline expression of TM that was further increased
by treatment with 1,25(OH)2D3. Although the
increase of the TM expression by 1,25(OH)2D3
was found in several types of AML and biphenotypic leukemia cells, high
induction of the TM levels were found in a type of monocytic leukemia.
In contrast, incubation with 1,25(OH)2D3 resulted in a decrease in levels of TF antigen in four of seven AML
cells freshly isolated from patients M2-1, M2-3, M4-1, and M5b-1; one
of two ALL cells, ALL-2; and AML cell lines OCI-AML3, U937, and NB4
(Fig 1B). TM expression was not detected in the presence
or in the absence of 1,25(OH)2D3 in three
lymphocytic cells, ALL-1, ALL-2 or TMD2 cells. Levels of TM and TF
antigen were not affected by incubating any of the leukemic cell lines with 0.1 to 10 µg/dL T3 (data not shown). Since the
remarkable change in levels of TM and TF antigen was observed in the
U937 cell line, we next examined the dose dependency of the effects of
1,25(OH)2D3 on levels of TM and TF antigen in
these cells.
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| Fig 1.
Effects of 1,25(OH)2D3 on total
TM and TF antigen in several leukemic cell lines and PB cells freshly
isolated from patients with leukemia. Leukemic cells were incubated
with 1,25(OH)2D3 (0.1 µmol/L) for 24 hours.
Total TM (A) and TF (B) antigen levels are shown on ordinates. Data
represent means of duplicate measurements.
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Effects of 1,25(OH)2D3 on total TM and TF
antigen in U937 cells.
U937 cells were incubated for 24 hours with various concentrations of
1,25(OH)2D3. Levels
of TM antigen were increased dose dependently with up to 0.1 µmol/L
1,25(OH)2D3 (Fig 2A). In contrast, 1,25(OH)2D3 treatment led to a decrease in
levels of TF antigen at concentrations of
1,25(OH)2D3 as low as 1 nmol/L (Fig 2B). The
change in the levels of TM and TF antigen after treatment of the U937
cells with 1,25(OH)2D3 resembled those
previously reported in NB4 cells treated with ATRA or 9-cis
RA.4,5
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| Fig 2.
Effects of 1,25(OH)2D3 on total
TM and TF antigen in U937 cells. Dose-dependent effects of
1,25(OH)2D3 on TM (A) and TF (B) antigen
expression in U937 cells. U937 cells were incubated with 1,25(OH)2D3 (0.1 nmol/L to 5 µmol/L) for 24 hours. These assays were repeated independently three times and the
results are expressed as the mean ± SD (standard deviation).
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Cell-surface TM and TF activities.
U937 cells or monocytes from PB treated for 24 hours with
1,25(OH)2D3 showed an increase in cell-surface
TM activity. In contrast, treatment of HUVECs with
1,25(OH)2D3 had no effect on the cell-surface activity of TM (Fig 3A). Levels of TM
cofactor activity were also unaffected by treating the HUVECs with
T3 (data not shown). Treatment of U937 cells with
1,25(OH)2D3 decreased cell-surface TF activity (Fig 3B). The decrease of the cell-surface TF activity after 7 days'
incubation with 1,25(OH)2D3 was more dramatic
than that after 1 day of incubation.
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| Fig 3.
Changes in TM cofactor activity for protein C activation
and TF cofactor activity on U937 cells, monocytes, or HUVECs surfaces after exposure to 1,25(OH)2D3. Cells were
exposed to 1,25(OH)2D3 (0.1 µmol/L) for 24 hours. Cell-surface TM activity was determined for suspended cells as
described in Materials and Methods. Basal  OD405nm/min
levels were 0.063 ± 0.002/min/5 × 106 U937 cells (61 ± 2 ng activated protein C/106 cells), 0.062 ± 0.005/min/106 monocytes (300 ± 24 ng activated protein
C/106 cells), and 0.251 ± 0.027/min/106
HUVECs (1,305 ± 235 ng activated protein C/106 cells).
Cell-surface TF activity was determined by normal plasma-based one-stage recalcification clotting time and was quantitated by reference to standard curves constructed using human placenta TF as
described in Materials and Methods. These assays were repeated independently three times and the results are expressed as the mean ± SD. The difference between TM cofactor activity on the surface of
1,25(OH)2D3-treated U937 cells or monocytes and
that of untreated cells is statistically significant (P < .05). The difference between TF cofactor activity on the surface of
U937 cells which had been 1,25(OH)2D3-treated
for 7 days and that of untreated cells is also statistically
significant (P < .05).
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Western blot analysis for TM, TF, and VDR.
After treatment with 1,25(OH)2D3, the
nonreduced form of TM was identified as a prominent band at
approximately 75 kD in U937 and OCI-AML3 cells using the MoAb KA-4
(Fig 4A). This size resembled that of
placental and HUVEC-derived TM. Recombinant soluble TM exhibited a band
at 65 kD. In contrast, a marked reduction in the strength of the TF
band at 45 kD in U937 cells was observed after treatment with
1,25(OH)2D3 (Fig 4B). A VDR of 48 kD
was expressed in all leukemic cell lines studied (data not shown). The
expression of VDR was higher in the K562 cells than in the U937 cells.
The VDR protein levels appeared to be unaffected by stimulation of
1,25(OH)2D3. The levels of VDR expression were not correlated with the anticoagulant activities of
1,25(OH)2D3 in the leukemic cell lines.
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| Fig 4.
Western blot analysis of TM and TF in leukemic cell lines
treated with 1,25(OH)2D3. Cell lysates were
subjected to immunoblotting analysis using a monoclonal anti-TM
antibody (A), and a monoclonal anti-TF antibody (B). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed
under nonreducing conditions. All cell lines were incubated either with
(+) or without ( ) 0.1 µmol/L 1,25(OH)2D3
for 24 hours. Molecular-weight markers are shown along the left margin.
Soluble recombinant TM (A, lane 5) and placenta TF (B, lane 3) were
used as controls.
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RT-PCR and Northern blot analyses of TM mRNA in U937 cells and
monocytes.
An increased expression of specific mRNA for TM was detected when U937
cells were treated with 0.1 µmol/L
1,25(OH)2D3 for 5 hours
(Fig 5A and C). A similar
induction of TM mRNA was found in monocytes obtained from the PB of a
normal subject (Fig 5C).
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| Fig 5.
RT-PCR and Northern blot analyses of TM and TF mRNA in
U937 cells and monocytes treated with
1,25(OH)2D3. Total RNA was extracted from
cultured U937 cells and monocytes after exposure to 0.1 µmol/L of
1,25(OH)2D3 (lane 2) for 5 hours. RT-PCR
analysis of TM (A) or TF (B) mRNA and Northern blot analysis of TM mRNA
(C) were performed as described in Materials and Methods. Base-pair
markers are shown to the left. GAPDH and -actin were used as quality controls of mRNA.
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RT-PCR analysis of TF mRNA in U937 cells.
Treatment with 1,25(OH)2D3 (0.1 µmol/L) for 5 hours markedly reduced the expression of TF mRNA in U937 cells (Fig
5B).
Transfections.
Because U937 cells had a low transfection efficiency, we used the acute
myelomonocytic cell line, OCI-AML3, in the transfection studies. We
chose these cells because they showed more monocytic character than did
the OCI-AML2 cell line. Suspended OCI-AML3 cells that were transfected
with a plasmid containing the RARE of the human TM gene resulted in a
dose-dependent induction of LUC activity when treated with various
concentrations of ATRA. When treated with
1,25(OH)2D3, the transfection with the
constructs containing the RARE resulted in modest induction of LUC
activity in OCI-AML3 cells (Fig 6A). The
same plasmids were also transfected into a HUVEC line, adherent ECV304
cells, by using LipofectAMINE. ATRA induced LUC activity up to twofold
in these cells, whereas 1,25(OH)2D3 did not
increase the LUC activity (Fig 6B).
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| Fig 6.
1,25(OH)2D3 and ATRA
responsiveness of the TM RARE in OCI-AML3 cells and ECV304. OCI-AML3
and ECV304 cells were transfected with pTM-2RAREtkLUC containing two
copies of the RARE from the TM gene, thymidine kinase promotor and LUC
gene, and internal control plasmids. After 48 hours, ATRA ( ) or
1,25(OH)2D3 ( ) were added and the cells were
incubated for an additional 24 hours. Cells were procured, and cell
extracts were assayed for LUC activity. Experiments were repeated
independently four times. Values are means ± SD. *Significantly
different from untreated controls (P < .05).
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Gel mobility shift assay.
Gel-shift assay was used to test if VDR binds specifically to TM RARE.
Two shifted bands were observed when a TM RARE probe was
incubated with OCI-AML3 nuclear extracts
(Fig 7, lanes 2 and 3). Sixteen- and
69-fold molar excess of an unlabeled TM-RARE oligonucleotide
efficiently competed only for the upper band formation (Fig
7, lanes 4 and 5), which suggests that the lower band may be
nonspecific. Anti-VDR (Fig 7, lanes 6 through 8) and anti-RAR (Fig 7, lanes 9 through 11) antibodies supershifted the upper band, while an irrelevant antibody did not affect the band (data not
shown).
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| Fig 7.
Gel mobility shift assay with a DIG-labeled
oligonucleotide probe containing TM RARE. Gel-shift assay was performed
as described in Materials and Methods. Lane 1 corresponds to the
DIG-labeled TM RARE probe incubated in the absence of nuclear extracts.
One microgram of nuclear extracts of OCI-AML3 cells was incubated with
the DIG-labeled probe in the absence (lanes 2, 3, and 6 through 11) or
in the presence of 16- and 69-fold molar excess amount of unlabeled TM
RARE (lanes 4 and 5). Increasing amounts of anti-VDR (lanes 6 through
8) or anti-RAR (lanes 9 through 11) antibody was included in the
reaction mixture. The position of the specific complex is indicated by
the closed arrow and the supershifted band is indicated by the open
arrow.
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Influence of 1,25(OH)2D3 on TM upregulation
by ATRA.
We have also examined if 1,25(OH)2D3 influences
TM upregulation by ATRA and vice versa with various concentrations of
both reagents, as indicated in Fig 8. When
both 1,25(OH)2D3 and ATRA were included as
stimulants for U937 cells, moderate synergism was observed in TM
upregulation (Fig 8).
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| Fig 8.
The influence of 1,25(OH)2D3 on
TM upregulation by ATRA. The various combinations of
1,25(OH)2D3 and ATRA were incubated with U937
cells for 24 hours. TM upregulation was expressed as increase of TM
antigen levels in the cell lysate. Experiments were repeated independently three times. Values are means ± SD.
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| |
DISCUSSION |
The present study showed that the expression of TM was upregulated by
1,25(OH)2D3 in several myelogenous leukemia
cells and mature monocytes from the PB but not in HUVECs. We have shown that the induction of TM by 1,25(OH)2D3 was
most marked in monocytic leukemia cells and mature monocytes, and
mediated by the TM RARE. A downregulation of TF was observed in the
most of the monocytic leukemia cells.
1,25(OH)2D3 exerted its anticoagulant effects in the monocytic leukemia cells by upregulating TM and downregulating TF. In contrast, the induction of TM and the reduction of TF by ATRA
were marked in APL and monocytic leukemia cells.5
An upregulated expression of TM by 1,25(OH)2D3
has been reported in osteoblasts.15 Functional TM
expression was also observed in human blood monocytes and in the human
synovial tissue lining macrophages.16 When the AML M2 cell
line, HL-60, was differentiated into a monocytic lineage, the induction
of TM was also observed.17 As previously
reported,4 the induction of TM and the reduction of TF by
ATRA create a favorable environment for the improvement of the
disseminated intravascular coagulation syndrome (DIC) in patients with
APL. Because there is a relatively high incidence of DIC complications
in patients with acute monocytic leukemia, the induction of TM and the
reduction of TF in monocytic cells may improve DIC in these patients.
The activities of 1,25(OH)2D3 are
mediated by the specific nuclear receptor VDR, which is widely
distributed in mammalian tissues including intestine, bone, and
kidney.1,2 The receptor has been found also in such blood
cells as stimulated lymphocytes, promyelocytes, and precursors of
monocytes.2 The expression levels of VDR in different cell
types may affect the function of 1,25(OH)2D3.
However, VDR levels in the cell lines, U937 and NB4, responsive to
1,25(OH)2D3, resembled those of unresponsive cell line K562. The treatment with 1,25(OH)2D3
did not increase the VDR expression in all of those cell lines. We
suggest that the presence of coactivator(s) or repressor(s) is also
important for the function of 1,25(OH)2D3 in
leukemia cells. The activities of 1,25(OH)2D3
are mediated principally via the RXR/VDR heterodimers, not the VDR/VDR
homodimers.1 Coactivator(s) and repressor(s) are involved
in the function of the RXR/RAR heterodimer.18 A selective
cascade of interactions between receptor dimers, cofactors, silencing
mediators for VDR, and members of the transcriptional machinery, such
as TATA-binding proteins and TATA-associated factors, may be required
for the induction of TM. These interactions may result from a specific
change in conformation within the RXR/VDR heterodimer which binds to
its ligand. In addition, multifunctional regulators such as
YY119 interfere with interactions of the VDR with VDRE and
transcription factor IIB, thereby regulating the enhancement of TM gene
transcription by 1,25(OH)2D3.
Retinoids induce the transcriptional activation of TM expression via a
specific ligand-RAR binding site, RARE, in the 5-flanking region
of the TM gene.7 The TM gene does not contain a typical VDRE, which is a direct repeat sequence with a spacer of 3 bp. Therefore, a reporter gene that contained two copies of the RARE of the
TM gene was transfected into OCI-AML3 and ECV304 cells to determine
whether the RARE with a spacer of 4 bp mediates the activity of
1,25(OH)2D3. Treatment with
1,25(OH)2D3 increased the activity of LUC in
OCI-AML3 cells, but not in ECV304 cells. The responsiveness of the RARE
to 1,25(OH)2D3 indicates that strict adherence
to a spacer region of 3 bp for VDR is questioned. In fact, VDR and RAR
recognize a common response element in the osteocalcin gene.20 To strengthen the data of transfection, we have
also performed a gel-shift assay using antibodies to VDR and RAR. The assay has shown that VDR in addition to RAR binds to the TM RARE
with similar mobility (Fig 7) because VDR and RAR have similar
molecular weights, 48 kD and 45 kD, respectively. We have used
anti-RAR antibody as well as anti-VDR antibody to observe the
supershift of the complex band, because we have already found that
retinoids mediate TM upregulation via RAR subtype.6
While RXR-RAR heterodimers bind to the RARE sequence even in the
absence of retinoids,18 RXR-VDR heterodimers may also bind
to the TM RARE without stimulation of
1,25(OH)2D3, as shown in Fig 7, lane 2. These
results adequately support our hypothesis that both
1,25(OH)2D3 and ATRA mediate upregulation of TM
via the similar response element. Although TM was induced by both
1,25(OH)2D3 and ATRA through an interaction
with the RARE of the TM gene, there may be different degrees of
binding of 1,25(OH)2D3 and ATRA to the TM RARE.
In addition, other additional factor(s), corepressors, and coactivators may be involved in TM induction. A recent report showed that the biological activities of potent vitamin D3 analogs did not
correspond to the ability of these cells to bind to VDR and to
transactivate a VDRE.21 Therefore, the anticoagulant
activities of 1,25(OH)2D3 may not correlate to
its ability to interact with the RARE of the TM gene, which is VDRE.
Further studies are necessary to elucidate mechanism of TM upregulation
mediated via 1,25(OH)2D3.
TF is constitutively expressed by several types of cells present
outside the vessels and induced in monocytes and endothelial cells
within the vasculature by cytokines such as tumor necrosis factor and
interleukin-1 (IL-1). The levels of TF in the monocytic leukemia cells
were decreased by 1,25(OH)2D3. Treatment of
these cells with 1,25(OH)2D3 may repress the
expression of TF through the binding of activated VDRs to the
transcriptional activation factor AP-1 (Jun/Fos) in a mechanism similar
to that observed in retinoids.22 In addition, sterical
competition23 for the binding sites of AP-1 on the TF gene
may occur due to the formation of complexes between RXR/VDR
heterodimer, auxiliary factor(s), and their binding sites on the TF
gene. The RXR-VDR heterodimer blocks NFATp (a T-cell-specific
transcription factor)/AP-1 complex formation and then stably associates
with the NFAT-1 element in the IL-2 gene, thereby
1,25(OH)2D3 transcriptionally represses the
IL-2 gene.24 The mode of direct or indirect, genomic or nongenomic control of TF expression by
1,25(OH)2D3 has yet to be determined. TF
expression initiates thrombotic episodes associated with various
diseases, including atherosclerosis, septic shock, and malignancy. ATRA
is an effective downregulator of TF in monocytes25 and of
TF and cancer procoagulant in APL cells.4-6,26 The
administration of retinoids in the clinical setting is expected to
result in the differentiation of APL cells. In addition, retinoids may
be useful in a new form of anticoagulant therapy which changes the procoagulant character of cells, including malignant cells, diseased endothelial cells, and diseased monocytes, by regulating TM and TF
expression at the level of transcription. Similar regulation of TM and
TF in monocytic leukemia cells by 1,25(OH)2D3
may be effective for the control of DIC in the patients with monocytic leukemia. Furthermore, the differentiation effect of monocytic leukemia
cells by 1,25(OH)2D3 has also been
reported.1
It is suggested that hyperactive monocytes with a high expression of TF
and a low expression of TM favor the development of atherosclerosis.27 Platelets may also play a primary role
in the pathogenesis of atherosclerosis where platelet activation is a
pivotal risk factor for the development of thrombotic episodes. Antiplatelet action is induced when 1,25(OH)2D3
and its analog 22-oxa-1,25(OH)2D3 stimulate the
production of prostacyclin by vascular tissues.28 In
addition to the treatment of DIC and cell differentiation therapy in
patients with monocytic leukemia, anticoagulant and antiplatelet
effects of 1,25(OH)2D3 may be useful for
preventing and treating atherosclerotic thrombotic disease.
Physiological concentration of 1,25(OH)2D3 in
serum is less than 0.1 nmol/L. Pharmacological concentration of
1,25(OH)2D3 in serum is around 0.2 nmol/L.29 It has been shown that side effects related to
hypercalcemia may occur when concentrations of
1,25(OH)2D3 in serum exceed 0.2 nmol/L.2,29 Therefore, it may not be possible to administer
either 1,25(OH)2D3 or 1(OH)D3 at
doses sufficient to produce an anticoagulant effect without inducing
hypercalcemia. One resolution may be to synthesize analogs that are
especially effective in inducing cell differentiation, anti-osteoporosis, and anticoagulation without inducing side effects related to hypercalcemia. Both 1,25(OH)2D3 and
its analogs are promising differentiation-inducing agents and are
important modulators of cellular proliferation for a number of
malignant cell types.30 1,25(OH)2D3
is capable of inducing differentiation of human myeloid leukemia cells
from both patients and cell lines.31 A 20-epi-vitamin D3 analog induces differentiation and potentially inhibits
clonal growth of human breast cancer cell lines.32 Although
studies in vivo suggest that 1,25(OH)2D3 can
prolong the survival of mice injected with myeloid leukemia cells, oral
administration of 1,25(OH)2D3 to preleukemic
patients did not have an enduring therapeutic effect.33 The
reason may also be that the concentration of
1,25(OH)2D3 in the serum of patients is too low
to affect differentiation and growth of leukemia cells. The growing
information on VDRs and the signaling pathways of
1,25(OH)2D3 activity will ultimately permit the
synthesis of 1,25(OH)2D3 analogs for treating
medical conditions, including those requiring anticoagulant therapy.
Another strategy may be to identify agents that are synergistic with
1,25(OH)2D3 so that lower serum concentrations
of 1,25(OH)2D3 can be used in the therapy. As
shown in Fig 8, there appeared to be a modest synergism between
1,25(OH)2D3 and ATRA in TM upregulation. It reminds us of similar synergism between 20-epi-vitamin D3
analog and 9-cis RA on inhibition of clonal growth and
induction of apoptosis in NB4 cells.30 The mechanism is
still unclear but the investigators have suggested that the combination
may cause the activation of RXR/VDR, RXR/RAR, and VDR/RAR heterodimers,
as well as VDR/VDR, RAR/RAR, and RXR/RXR homodimers.30
Our present study has linked vitamin D3 and retinoid
signaling pathway in the regulation of TM and TF, and outlined the
profundity of nature's anticoagulant system.
| |
FOOTNOTES |
Submitted October 14, 1997;
accepted March 4, 1998.
Supported in part by a research grant from the Ministry of Education,
Science, Sports and Culture, and a grant for intractable diseases from
the Ministry of Health and Welfare of Japan.
Presented in part at the XVIth Congress of International Society on
Thrombosis and Haemostasis, Florence, Italy, June 8, 1997.
Address reprint requests to Takatoshi Koyama, MD, The First Department
of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo, 113-8519 Japan; e-mail:
int1koya.mtec{at}med.tmd.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Keiko Yamamoto and Prof Sachiko Yamada, Institute
for Medical and Dental Engineering of Tokyo Medical and Dental
University for helpful discussions.
| |
REFERENCES |
1.
Christakos S,
Raval-Pandya M,
Wernyj RP,
Yang W:
Genomic mechanisms involved in the pleiotropic action of 1, 25-dihydroxyvitamin D3.
Biochem J
316:361,
1996[Medline]
[Order article via Infotrieve]
2.
DeLuca HF,
Krisinger J,
Darwish H:
The vitamin D system: 1990.
Kidney Int
38:S2,
1990
3.
Umesono K,
Murakami KK,
Thompson CC,
Evans RM:
Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors.
Cell
65:1255,
1991[Medline]
[Order article via Infotrieve]
4.
Koyama T,
Hirosawa S,
Kawamata N,
Tohda S,
Aoki N:
All-trans retinoic acid (ATRA) upregulates thrombomodulin and downregulates tissue factor expression in acute promyelocytic leukemia cells: Distinct expression of thrombomodulin and tissue factor in human leukemic cells.
Blood
84:3001,
1994[Abstract/Free Full Text]
5.
Saito T,
Koyama T,
Nagata K,
Kamiyama R,
Hirosawa S:
Anticoagulant effects of retinoic acids on leukemia cells.
Blood
87:657,
1996[Abstract/Free Full Text]
6.
Shibakura M,
Koyama T,
Saito T,
Miyasaka N,
Kamiyama R,
Hirosawa S:
Anticoagulant effects of synthetic retinoids mediated via different receptors on human leukemia and umbilical vein endothelial cells.
Blood
90:1545,
1997[Abstract/Free Full Text]
7.
Dittman WA,
Nelson SC,
Greer PK,
Horton ET,
Palomba ML,
McCachren SS:
Characterization of thrombomodulin expression in response to retinoic acid and identification of a retinoic acid response element in the human thrombomodulin gene.
J Biol Chem
269:16925,
1994[Abstract/Free Full Text]
8.
Napoleone E,
Di Santo A,
Lorenzet R:
Monocytes upregulate endothelial cell expression of tissue factor: A role for cell-cell contact and cross-talk.
Blood
89:541,
1997[Abstract/Free Full Text]
9.
Tohda S,
Shudo K,
Minden MD:
The effects of retinoic acid analogues on the blast cells of acute myeloblastic leukemia in culture.
Int J Oncol
4:1311,
1994
10.
Lanotte M,
Martin-Thouvenin V,
Najiman S,
Balerini P,
Valensi F,
Berger R:
NB-4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3).
Blood
77:1080,
1991[Abstract]
11.
Takahashi K,
Sawasaki Y,
Hata J,
Mukai K,
Goto T:
Spontaneous transformation and immortalization of human endothelial cells.
In Vitro Cell Dev Biol
25:265,
1990
12.
Koyama T,
Parkinson JF,
Aoki N,
Bang NU,
Müller-Berghaus G,
Preissner KT:
Relationship between post-translational glycosylation and anticoagulant function of secretable, recombinant mutants of human thrombomodulin.
Br J Haematol
78:515,
1991[Medline]
[Order article via Infotrieve]
13.
Hirokawa K,
Aoki N:
Up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro.
J Biochem
108:839,
1990[Abstract]
14.
Yamamoto K,
Quelle FW,
Thierfelder WE,
Kreider BL,
Gilbert DJ,
Jenkins NA,
Copeland NG,
Silvennoinen O,
Ihle JN:
Stat 4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.
Mol Cell Biol
14:4342,
1994[Abstract]
15.
Maillard C,
Berruyer M,
Serre CM,
Amiral J,
Dechavanne M,
Delmas PD:
Thrombomodulin is synthesized by osteoblasts, stimulated by 1,25-(OH)2D3 and activates protein C at their cell membrane.
Endocrinology
133:668,
1993[Abstract]
16.
McCachren SS,
Diggs J,
Weinberg JB,
Dittman WA:
Thrombomodulin expression by human blood monocytes and by human synovial tissue lining macrophages.
Blood
78:3128,
1991[Abstract]
17.
Kizaki K,
Naito S,
Horie S,
Ishii H,
Kazama M:
Different thrombomodulin induction in monocytic, macrophagic and neutrophilic cells differentiated from HL-60 cells.
Biochem Biophys Res Commun
193:175,
1993[Medline]
[Order article via Infotrieve]
18.
Chen JD,
Evans RM:
A transcriptional co-repressor that interacts with nuclear hormone receptors.
Nature
377:454,
1995[Medline]
[Order article via Infotrieve]
19.
Guo B,
Aslam F,
van Wijnen AJ,
Roberts SGE,
Frenkel B,
Green MR,
DeLuca H,
Lian JB,
Stein GS,
Stein JL:
YY1 regulates vitamin D receptor/retinoid X receptor mediated transactivation of the vitamin D responsive osteocalcin gene.
Proc Natl Acad Sci USA
94:121,
1997[Abstract/Free Full Text]
20.
Schule R,
Umesono K,
Mangelsdorf DJ,
Bolado J,
Pike JW,
Evans RM:
Jun-Fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene.
Cell
61:495,
1990
21.
Imai Y,
Pike JW,
Koeffler HP:
Potent vitamin D3 analogs: Their abilities to enhance transactivation and to bind to the vitamin D3 responsive element.
Leuk Res
19:147,
1995[Medline]
[Order article via Infotrieve]
22.
Schule R,
Rangarajan P,
Yang N,
Kliewer S,
Ransone LJ,
Bolado J,
Verma IM,
Evans RM:
Retinoic acid is a negative regulator of AP-1-responsive genes.
Proc Natl Acad Sci USA
88:6092,
1991[Abstract]
23.
Jääskeläinen T,
Pirskanen A,
Ryhänen S,
Palvimo JJ,
DeLuca HF,
Mäenpää PH:
Functional interference between AP-1 and the vitamin D receptor on osteocalcin gene expression in human osteosarcoma cells.
Eur J Biochem
224:11,
1994[Abstract]
24.
Alroy I,
Towers TL,
Freedman LP:
Transcriptional repression of the interleukin-2 gene by vitamin D3: Direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor.
Mol Cell Biol
15:5789,
1995[Abstract]
25.
Barstad RM,
Hamers MJAG,
Stephens RW,
Sakariassen S:
Retinoic acid reduces induction of monocyte tissue factor and tissue factor/factor VIIa-dependent arterial thrombus formation.
Blood
86:212,
1995[Abstract/Free Full Text]
26.
Falanga A,
Iacoviello L,
Evangelista V,
Belotti D,
Consonni R,
D'Orazio A,
Robba L,
Donati MB,
Barbui T:
Loss of blast cell procoagulant activity and improvement of hemostatic variables in patients with acute promyelocytic leukemia administered all-trans-retinoic acid.
Blood
86:1072,
1995[Abstract/Free Full Text]
27.
Osterud B:
A global view on the role of monocytes and platelets in atherogenesis.
Thromb Res
85:1,
1997[Medline]
[Order article via Infotrieve]
28.
Inoue M,
Wakasugi M,
Wakao R,
Gan N,
Tawata M,
Nishii Y,
Onaya T:
A synthetic analogue of vitamin D3, 22-oxa-1,25-dihydroxy-vitamin D3, stimulates the production of prostacyclin by vascular tissues.
Life Sci
51:1105,
1992[Medline]
[Order article via Infotrieve]
29.
Seino Y,
Tanaka H,
Yamaoka K,
Yabuuchi H:
Circulating 1,25-dihydroxyvitamin D3 levels after a single dose of 1,25-dihydroxyvitamin D3 or 1 -hydroxyvitamin D3 in normal men.
Bone Miner
2:479,
1987[Medline]
[Order article via Infotrieve]
30.
Elstner E,
Linker-Israeli M,
Le J,
Umiel T,
Michl P,
Said JW,
Binderup L,
Reed JC,
Koeffler HP:
Synergistic decrease of clonal proliferation, induction of differentiation, and apoptosis of acute promyelocytic leukemia cells after combined treatment with novel 20-epi vitamin D3 analogs and 9-cis retinoic acid.
J Clin Invest
99:349,
1997[Abstract/Free Full Text]
31.
Munker R,
Norman A,
Koeffler HP:
Vitamin D compounds. Effect on clonal proliferation and differentiation of human myeloid cells.
J Clin Invest
78:424,
1986[Medline]
[Order article via Infotrieve]
32.
Elstner E,
Linker-Israeli M,
Le J,
Said J,
Umiel T,
de Vos S,
Shintaku IP,
Heber D,
Binderup L,
Uskokovic M,
Koeffler HP:
20-epi-vitamin D3 analogues: A novel class of potent inhibitors of proliferation and inducers of differentiation of human breast cancer cell lines.
Cancer Res
55:2822,
1995[Abstract]
33.
Koeffler HP,
Hirji K,
Itri L and the Southern California Leukemia Group:
1,25-dihydroxyvitamin D3: In vivo and in vitro effects on human preleukemic and leukemic cells.
Cancer Treat Rep
69:1399,
1985[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract