1␣,25-Dihydroxyvitamin D3 and Its Potent Synthetic Analogs
Downregulate Tissue Factor and Upregulate
Thrombomodulin Expression in Monocytic Cells,
Counteracting the Effects of Tumor Necrosis Factor
and Oxidized LDL
Mai Ohsawa, MS; Takatoshi Koyama, MD; Keiko Yamamoto, PhD; Shinsaku Hirosawa, MD;
Sachiko Kamei, MD; Ryuichi Kamiyama, MD
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Background—We have recently found that a hormonally active form of vitamin D, 1␣,25-dihydroxyvitamin D3
[1,25(OH)2D3], exerts anticoagulant effects by upregulating the expression of an anticoagulant glycoprotein, thrombomodulin (TM), and downregulating the expression of a critical coagulation factor, tissue factor (TF), in monocytic cells
including human peripheral monocytes. In this study, we investigated the counteracting effects of 1,25(OH)2D3 and its
potent analogs on TF induction and TM downregulation by tumor necrosis factor and oxidized LDL in monocytic cells
and the modulatory effects of potent analogs on TF and TM expression.
Methods and Results—Effects of 1,25(OH)2D3 and its potent synthetic analogs (22R)-22-methyl-20-epi-1,25(OH)2D3
(KY3) and 22-oxacalcitriol on TF and TM antigen levels, cell surface activities, and mRNA levels in monocytic cells
were examined. 1,25(OH)2D3 and its potent analogs showed anticoagulant effects in monocytic cells by downregulating
TF and upregulating TM expression, counteracting the effects of tumor necrosis factor and oxidized LDL. KY3 was
most potent in its regulatory effect on TF and TM expression.
Conclusions—Because KY3 has the highest affinity for vitamin D receptor, our findings suggest that TF and TM
regulation by 1,25(OH)2D3 analogs is also mediated by vitamin D receptor. The 1,25(OH)2D3 analogs KY3 and
22-oxacalcitriol may have the potential to serve as an agent for preventing and treating atherosclerotic and other
cytokine-mediated thrombotic diseases and as a tool for studying the molecular mechanisms of TF and TM regulation.
(Circulation. 2000;102:2867-2872.)
Key Words: anticoagulants 䡲 coagulation 䡲 thrombosis
V
itamin D metabolites influence the expression of various
genes whose products are involved in calcium homeostasis, cell differentiation, and regulation of the immune
response.1 Expression of these genes is mediated by the
nuclear vitamin D receptor (VDR), which belongs to the same
family as the steroid and retinoid receptors. Although the
molecular mechanisms of gene regulation are still under
investigation, it is clear that binding of vitamin D to the VDR
initiates a sequence of events resulting in the activation or
repression of transcription. Thus, the VDR–vitamin D complex is most directly relevant to the ultimate biological effect.
We have recently found that a hormonally active form of
vitamin D, 1␣,25-dihydroxyvitamin D3 [1,25(OH)2D3],2 as
well as vitamin A derivatives, retinoic acids,3,4 exerts
anticoagulant effects by upregulating the expression of an
anticoagulant glycoprotein, thrombomodulin (TM), and
downregulating the expression of a critical coagulation
factor, tissue factor (TF), in monocytic cells including
human peripheral monocytes. Because 1,25(OH)2D3 derivatives are expected to be adjunctive antithrombotic agents,
its analogs are promising as useful therapeutic agents
without adverse effects.
22-Oxa-1,25(OH)2 D 3 (22-oxacalcitriol, maxacalcitol;
OCT) (Figure 1) is a so-called “noncalcemic” vitamin D
analog with accentuated differentiation-inducing/antiproliferative properties and reduced ability to cause hypercalcemia.5 Its affinity to vitamin D– binding protein is 500 times
lower than that of 1,25(OH)2D3, which means that OCT can
achieve higher cellular levels for a much shorter duration.1
(22R)-22-Methyl-20-epi-1,25(OH)2D3 (KY3) (Figure 1)
was recently synthesized and found to have the highest VDR
binding affinity so far known.6 KY3 has ⬇20 times higher
Received April 18, 2000; revision received June 28, 2000; accepted June 30, 2000.
From the School of Allied Health Sciences (M.O., T.K., S.K., R.K.), Institute for Biomaterials and Bioengineering (K.Y.), and First Department of
Internal Medicine (S.H.), Tokyo Medical and Dental University, Tokyo, Japan.
Correspondence to Takatoshi Koyama, MD, School of Allied Health Sciences, Faculty of Medicine, Tokyo Medical and Dental University 1-5-45
Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail [email protected]
© 2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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December 5, 2000
previously described.4 The percentage of monocytes was between
15% and 25% before adhesion. The isolated cells were cultured as
U937 cells.
Measurement of Levels of TF and TM Antigens
Figure 1. Structure of 1,25(OH)2D3 and its potent synthetic
analogs.
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
affinity for VDR than 1,25(OH)2D3 and does not bind to the
transport protein vitamin D– binding protein.6
In cultured endothelial cells, expression of TF procoagulant activity and/or mRNA is induced and expression of TM
anticoagulant activity and/or mRNA is suppressed by tumor
necrosis factor (TNF), lipopolysaccharides (LPS), and oxidized LDL (ox-LDL).7–9 Similar results have been found in
monocytic cells, in which expression of TF procoagulant
activity and/or mRNA is stimulated by LPS and ox-LDL.7,10
On the other hand, all-trans retinoic acid (ATRA) downregulates the induction of TF by TNF or LPS in vascular
endothelial cells11 or monocytes.12
In this study, we investigated the counteracting effects of
1,25(OH)2D3 and its potent analogs on TF induction and TM
downregulation by TNF and ox-LDL in monocytic cells and
the modulatory effects of potent analogs on TF and TM
expression.
Methods
Reagents
Human thrombin (1400 NIH U/mg protein) and ATRA were
purchased from Sigma. Human placenta– derived TF (Thromborel S)
was from Behringwerke AG. The chromogenic substrate S2266 was
from Chromogenix. Human protein C (inactivated and activated)
was generously provided by the Chemo-Sero-Therapeutic Research
Institute. Recombinant human hirudin was from American Diagnostica Inc. KY3 was synthesized as described previously.6 OCT was
kindly provided by Chugai Pharmaceutical Co Ltd.
All other chemicals were reagent-grade products and were purchased from Wako Pure Chemicals unless otherwise indicated.
Cell Culture
Because anticoagulant effects have been detected in human peripheral monocytes as well as in monocytic leukemia cell lines,2 we
mainly used U937 cells in this study as a model of monocytic cells.
Monoblastic leukemia cell lines U937 and THP-1 were provided by
Health Science Research Resources Bank, Osaka, Japan. U937 and
THP-1 cells were cultured for the indicated periods in RPMI medium
supplemented with 10% FCS, sodium carbonate, glutamine, penicillin, and streptomycin with vitamin D derivatives at the indicated
concentrations. Vitamin D derivatives were dissolved in absolute
ethanol and then added to the growth media at the desired final
concentration. The final concentration of ethanol in the culture media
was ⬍0.1%. At this concentration, the cells exhibited no signs of
damage. The culture media without vitamin D derivatives, 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 vitamin D derivatives were performed under subdued
light.
Whole blood was collected from healthy donors and treated with
heparin (10 U/mL blood) to prevent coagulation. The mononuclear
cell fraction was freshly isolated from 4 different healthy donors as
Leukemic cells were incubated with vitamin D derivatives for 24
hours and then washed with PBS 3 times. Cell numbers were
determined and adjusted. To prepare cell lysates, the cells were then
treated with 0.5% Triton X-100 in PBS for 30 minutes at 4°C.
Cellular debris was removed by centrifugation at 12 000g for 20
minutes. The cell lysates were stored at ⫺80°C until assay. Total
levels of TF and TM antigens in cell lysates were measured by
ELISA, with the IMUBIND Tissue Factor ELISA Kit, American
Diagnostica Inc, and the EIA TM kit Teijin, Teijin Inc, according to
the manufacturer’s instructions.
Cell Surface TF Cofactor Activity: Analysis of
Procoagulant Activity in Clotting Assays
Suspensions of U937, THP-1, and freshly isolated mononuclear cells
were prepared in PBS. The U937 cell suspension was adjusted to
1⫻107 cells/mL in PBS. A portion of the cell suspension (106 cells)
was added to 0.1 mL of pooled human normal plasma. After
incubation at 37°C for 3 minutes, 0.1 mL of 25 mmol/L calcium
chloride was added and the plasma recalcification time was determined with a CA-100 semiautomatic coagulator (Sysmex). Our
previous study showed that the procoagulant activity associated with
the surface of U937 cells determined as described above was
attributable to the occurrence of TF expression2 and thus the
prolongation of the recalcification time is mainly due to downregulation of TF expression by vitamin D3 derivatives. TF cofactor
activity was quantitatively measured by reference to standard curves
(log-log plot) constructed with human placental TF, and the amount
of TF activity that yielded a 50-second recalcification time was
defined as 1 U/mL.
Measurement of Cell Surface TM
Cofactor Activity
Cell surface TM cofactor activity was measured as previously
described.2 In this assay, exogenous protein C (0.16 ␮mol/L) was
activated by intact cells in the presence of thrombin (0.83 NIHU/mL,
6.5 nmol/L) and Ca2⫹ (1.3 mmol/L). After completely inactivating
the thrombin activity by treatment with hirudin, cleavage of the small
molecular weight substrate S2266 by activated protein C was
measured with a spectrophotometer. The results were expressed as
the changes in optical density at 405 nm per minute or as the
percentage of the initial velocity of activated protein C formation
(with 100% taken to be the rate in the case of cell surface TM under
basal conditions). Controls with cells in the absence of thrombin and
protein C were treated similarly, and no activation of protein C was
observed.
Quantitative Reverse Transcription Polymerase
Chain Reaction and Densitometric Analysis
TF and TM mRNA levels in U937 cells were measured by performing quantitative reverse transcription polymerase chain reaction
(RT-PCR) assays with the Super-Script Preamplification System,
Life Technologies, Inc, as described previously.2 Relative signal
intensity was determined on Scion Image Software and standardized
by use of the relative density of glyceraldehydes-3-dehydrogenase
(GAPDH) signal. TM mRNA half-life was calculated with actinomycin D. Briefly, U937 cells were exposed to 0.1 ␮mol/L
1,25(OH)2D3 for 1 hour; 10 ␮g/mL actinomycin D was then added,
and total RNA was extracted at 0, 2.5, and 5 hours later. RT-PCR and
the determination of signal intensity were performed as well.
Results
Effects of 1,25(OH)2D3 and Its Potent Synthetic
Analogs on Total Levels of TF and TM Antigen in
U937 Cells
U937 cells were incubated for 24 hours with various concentrations of 1,25(OH)2D3 and its synthetic analogs, as indicated
Ohsawa et al
Antithrombogenic Effects of 1,25(OH)2D3 Analogs
2869
Figure 2. Effects of 1,25(OH)2D3 and its
potent synthetic analogs KY3 and OCT
on total TF and TM antigen expression
in U937 cells. U937 cells were incubated with indicated concentrations of
1,25(OH)2D3, KY3, and OCT for 24
hours. TF (A) and TM (B) antigen levels
were measured by ELISA. These assays
were repeated independently 3 times;
results are expressed as mean⫾SD.
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in Figure 2. Both KY3 and OCT decreased the total level of
TF antigen in a dose-dependent manner (up to 1 nmol/L)
(Figure 2A) while increasing the total level of TM antigen
dose-dependently (up to 0.1 ␮mol/L) (Figure 2B). The
changes in the levels of TF and TM antigen resembled those
observed in the case of 1,25(OH)2D3. KY3 was more efficient
in regulation of the levels of TF and TM antigen at a lower
concentration than 1,25(OH)2D3 or OCT.
RT-PCR Analysis of TF and TM mRNA Levels in
U937 Cells
To examine the changes in TF and TM mRNA levels, U937
cells were treated with 0.1 nmol/L 1,25(OH)2D3, KY3, or
OCT. The pharmacological concentration of 1,25(OH)2D3 is
0.1 nmol/L in serum. Both KY3 and OCT markedly decreased the expression of TF mRNA and increased the
expression of TM mRNA, as observed in the case of
1,25(OH)2D3 (Figure 3). These effects were in parallel with
the changes in TF and TM antigen levels. TM mRNA
half-life was not different in the absence and presence of
1,25(OH)2D3 (3.0⫾0.42 and 2.75⫾0.75 hours, n⫽3), which
indicates that TM gene is regulated at the level of
transcription.
Counteracting Effects of 1,25(OH)2D3 and Its
Potent Analogs on Upregulation of TF and
Downregulation of TM by TNF or Ox-LDL in
Monocytic Cells
We next examined whether 1 nmol/L 1,25(OH)2D3 or its
analogs could suppress the induction of TF activity in U937
cells treated with 1 nmol/L TNF or 50 ␮g/mL ox-LDL
(Figure 4A) and the decrease in TM activity caused by TNF
(Figure 4B). TF activity on the surface of U937 cells was
upregulated by both ox-LDL and TNF (Figure 4A, lanes 5
and 9), whereas TM activity was downregulated by TNF
(Figure 4B, lane 9). Induction of TF activity was more
Figure 3. RT-PCR analysis of TF
and TM mRNA levels in U937 cells
treated with 1,25(OH)2D3 and its
potent synthetic analogs KY3 and
OCT. Total RNA was extracted
from cultured U937 cells after
exposure to 0.1 nmol/L
1,25(OH)2D3, KY3, or OCT for 5
hours. RT-PCR analysis of TF (A)
and TM (B) mRNA levels was performed; relative signal intensity
was determined as described in
Methods. Basepair markers are
shown on left. GAPDH mRNA was
used as a mRNA quantity control
indicator.
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December 5, 2000
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Figure 4. Effects of 1,25(OH)2D3 and its
potent synthetic analogs in suppressing
upregulation of TF activity on surface
of U937 cells treated with ox-LDL or
TNF and downregulation of TM activity
caused by TNF. U937 (A, B) or freshly
isolated mononuclear cells (C, D) were
treated with 50 ␮g/mL ox-LDL or 1
nmol/L TNF for 24 hours with or without preincubation with 1 nmol/L
1,25(OH)2D3 (D3), KY3, or OCT for 1
hour. 1,25(OH)2D3 and its analogs were
added before incubation with TNF or
ox-LDL and were present throughout
culture. Cell-surface TF (A, C) and TM
(B, D) activity levels were measured as
described in Methods. In mononuclear
cells (MNCs) isolated from 4 different
donors, TF activity induced by TNF
was defined as 100%. These assays
were repeated independently 4 times;
results are expressed as mean⫾SD.
Differences in both activities comparing
levels in absence and presence of each
vitamin D3 analog were statistically significant (P⬍0.01 by t test).
marked when the cells were treated with TNF. Ox-LDL
mildly upregulated TM activity as reported in the case of
THP-1 cells, another monocytic cell line (Figure 4B, lane 5).
Preincubation with 1,25(OH)2D3, KY3, or OCT for 1 hour
counteracted the upregulation of TF activity by ox-LDL
(Figure 4A, lanes 6 to 8) and TNF (Figure 4A, lanes 10 to 12)
and the downregulation of TM activity by TNF (Figure 4B,
lane 10 to 12). TF activity induced by TNF was suppressed to
11% by KY3. Although somewhat weaker, similar effects
were observed when U937 cells were treated with
1,25(OH)2D3 1 hour after the start of incubation with TNF or
ox-LDL (data not shown). Similar results were obtained in
experiments using THP-1 cells (data not shown) or peripheral
mononuclear cells (Figure 4C and D) rather than U937 cells.
KY3 suppressed TF activity induced by TNF in mononuclear
cells weaker than in U937 cells.
Levels of TF and TM mRNA in U937 cells treated with
ox-LDL, TNF, and/or 1,25(OH)2D3 were measured by the
RT-PCR method (Figure 5). The increase in TF mRNA levels
induced by ox-LDL (Figure 5A, lane 2) or TNF (Figure 5B,
lane 5) as determined after 5 hours of incubation was
antagonized by 1,25(OH)2D3 (Figure 5A, lane 3 and Figure
5B, lane 6) in cells preincubated with 1,25(OH)2D3 for 1 hour.
TM mRNA levels were markedly increased when the cells
were preincubated with 1,25(OH)2D3 (Figure 5C, lane 3) as
compared with incubation with ox-LDL alone (Figure 5C,
lane 2). The decrease in TM mRNA levels induced by TNF as
determined after 5 hours of incubation (Figure 5D, lane 5)
was markedly counteracted by 1,25(OH)2D3 (Figure 5D, lane
6) in cells preincubated with 1,25(OH)2D3 for 1 hour.
Discussion
In this study, we have shown that 1,25(OH)2D3 and its potent
analogs, KY3 and OCT, exert anticoagulant effects in monocytic cells by downregulating TF expression and upregulating
TM expression, counteracting the effects of TNF and oxLDL. KY3 was most potent in its regulatory effect on TF and
TM expression. This is compatible with the findings described in our recent report indicating that KY3 shows
stronger activity than 1,25(OH)2D3 in VDR binding, stimulation of transcription of the osteopontin gene, promotion of the
differentiation of HL60 cells, and inhibition of the proliferation of MCF-7 cells and keratinocytes, because KY3 has the
highest affinity for VDR.13 Our findings support the view that
TF downregulation and TM upregulation by 1,25(OH)2D3 and
its analogs are mediated by VDR.
Ohsawa et al
Antithrombogenic Effects of 1,25(OH)2D3 Analogs
2871
Figure 5. Effects of 1,25(OH)2D3 in
counteracting changes in TF and TM
mRNA levels induced by ox-LDL or
TNF. U937 cells were treated with 50
␮g/mL ox-LDL or 1 nmol/L TNF for 5
hours with or without preincubation
with 0.1 ␮mol/L 1,25(OH)2D3 for 1
hour. RT-PCR analysis of TF (A, B)
and TM (C, D) mRNA levels and determination of relative signal intensity
were performed as described in Methods. Lanes 1 and 4 represent
untreated control.
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Monocyte TF activity is induced by a variety of agents,
such as LPS in bacterial infection, cytokines TNF and
interleukin-1, antigen-antibody complexes and complement
fragments in inflammatory diseases, and ox-LDL in atherosclerotic diseases. Furthermore, LPS and TNF downregulate
TM expression in monocytes. In patients with infections or
inflammatory diseases, upregulation of TF and downregulation of TM expression in endothelial cells and monocytes/
macrophages are major mechanisms involved in triggering
thrombosis.
Monocytes/macrophages in atheromatous plaques display
TF activity, and it is well established that these cells have
thrombogenic activity.14 Monocytes in patients with coronary
ischemic syndrome bear TF antigen.15 Ox-LDL has been
shown to be present in human atherosclerotic lesions, and it
enhances the progression of these lesions. Ox-LDL induces
macrophage foam cell generation, smooth muscle cell proliferation, platelet adhesion, and aggregation as well as triggering thrombosis by inducing TF expression.
In this context, upregulation of TM and downregulation of
TF expression in monocytic cells by 1,25(OH)2D3 analogs
may be an intriguing novel strategy to prevent thrombosis in
inflammatory and atherosclerotic diseases.
Enhancement of TM mRNA by 1,25(OH)2D3 analogs is not
due to higher stability of mRNA but due to an increase in the
transcription of the TM gene. The more potent effect of KY3
on TM mRNA expression may be due to its higher affinity to
VDR. 1,25(OH)2D3 analogs may mediate upregulation of TM
expression at least in part by the retinoic acid-responsive
element (RARE) of the TM gene.2 The TM RARE is in a
manner a vitamin D–responsive element, which suggests a
cross-talk between the cellular RAR and VDR pathways.
Whereas ATRA upregulates TM expression in both monocytic and vascular endothelial cells, 1,25(OH)2D3 does not in
the latter.2 Such a cell-specific response in terms of TM
regulation may be associated with cell- or tissue-specific
expression and regulation of VDR. On the other hand, the
mechanism of TF gene repression is not yet clear. The TF
promoter contains 2 activator protein-1 (AP-1)-binding sites
and a nuclear factor-␬B (NF-␬B) site, which serve as binding
sites for c-Fos/c-Jun and c-Rel/p65 heterodimers, respectively. Functional interactions between these transcription
factors are required for maximal induction of TF gene
transcription by TNF in vascular endothelial cells.7 Similar
interactions are necessary for induction of TF gene transcription by LPS in monocytic cells such as THP-1 cells.7 It has
been reported that 1,25(OH)2D3 inhibits interleukin-12 production by activated THP-1 cells through downregulation of
NF-␬B activation.16 On the other hand, ATRA selectively
inhibits LPS induction of TF gene expression in THP-1 cells
by a mechanism that does not involve repression of AP-1 and
NF-␬B-mediated transcription.17 Effects of 1,25(OH)2D3 analogs on AP-1 and NF-␬B activation are now under
investigation.
In contrast with downregulation of TM expression in
endothelial cells,9 ox-LDL induces mild upregulation of TM
expression in human monocytic leukemia cell line, U937 and
THP-1 cells,18 by a mechanism that is not yet known.
It has been shown also that increased expression of TF in
human malignant cells such as melanoma cells promotes
metastasis by enhancing angiogenesis or by other mechanisms.19 On the other hand, diminished expression of TM
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December 5, 2000
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appears to play an important role in the metastatic process of
esophageal20 and hepatic cancers.21 Upregulation of TM and
downregulation of TF expression by 1,25(OH)2D3 or ATRA
have also been observed in several cancer cell lines (our
unpublished observations). Thus, 1,25(OH)2D3 analogs may
be novel agents capable of preventing cancer cell metastasis
through induction of TM expression and suppression of TF
expression.
In summary, 1,25(OH)2D3 analogs, with a potent capacity
to suppress TF expression and to induce TM expression, are
expected to serve as adjunctive antithrombotic agents in
treatment of inflammatory and atherosclerotic diseases.
1,25(OH)2D3 analogs such as OCT, already in use in clinical
trials as agents for treatment of secondary hyperparathyroidism and psoriasis, and KY3 should be examined further in in
vivo models of thrombotic disease. OCT and KY3 are
expected to avoid an adverse effect of hypercalcemia. Although KY3 is twice as hypercalcemic in vivo than
1,25(OH)2D3, its binding affinity to VDR or biological
activities are far stronger (20-fold or ⬎100-fold) than
1,25(OH)2D3.13 The 1,25(OH)2D3 analogs examined in this
study may have the potential to serve as an agent for
preventing and treating atherosclerotic and other cytokinemediated thrombotic diseases and as a tool for studying the
molecular mechanisms of TF and TM regulation.
Acknowledgments
This work was supported by research grants from the Atsuko Ouchi
Memorial Fund, Tokyo Medical and Dental University, from the
Research Foundation for Clinical Pharmacology, and from the
Ministry of Education, Science, Sports, and Culture.
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1α,25-Dihydroxyvitamin D3 and Its Potent Synthetic Analogs Downregulate Tissue Factor
and Upregulate Thrombomodulin Expression in Monocytic Cells, Counteracting the
Effects of Tumor Necrosis Factor and Oxidized LDL
Mai Ohsawa, Takatoshi Koyama, Keiko Yamamoto, Shinsaku Hirosawa, Sachiko Kamei and
Ryuichi Kamiyama
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Circulation. 2000;102:2867-2872
doi: 10.1161/01.CIR.102.23.2867
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