From the Department of Oral Microbiology, Meikai University School
of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan and the
Institute of Molecular and Cellular Biosciences,
University of Tokyo, Bunkyo-Ku, Tokyo 113, Japan
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ABSTRACT |
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The present study demonstrates
1,25-dehydroxyvitamin D3
(1
-25-(OH)2D3) synergism toward
transforming growth factor (TGF)-
1-induced activation protein-1
(AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear
receptor of the vitamin. 1
-25-(OH)2D3
synergistically stimulated TGF-
1-induced expression of the
c-jun gene in the cells but not that of the
c-fos gene. We actually showed by a gel mobility shift
assay 1
-25-(OH)2D3 synergism of
TGF-
1-induced AP-1 binding to the
12-(O-tetradecanoylphorbol-13-acetate response element
(TRE). 1
-25-(OH)2D3 markedly stimulated the
transient activity of TGF-
1-induced AP-1 in the cells transfected
with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-
1-induced CAT activity was observed in
the cells cotransfected with an expression vector encoding vitamin
D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we
observed 1
-25-(OH)2D3 synergism of
TGF-
1-induced expression of the c-jun gene in the cells
transfected with the VDR expression vector and also found that the
synergistic action was clearly blocked by VDR antisense oligonucleotide
pretreatment. The present study strongly suggests a novel positive
regulation by 1
-25-(OH)2D3 of
TGF-
1-induced AP-1 activity in osteoblasts via "genomic
action."
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INTRODUCTION |
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TGF-11 is locally
produced by osteoblasts and accumulates abundantly in bone matrix
tissue (1-3). This local cytokine plays an important role as a
"coupling factor" in bone remodeling (4-7). We (8, 9) previously
demonstrated that TGF-
1 is a potent activator of AP-1, a
transcriptional factor that is a heterodimer of FOS and JUN proteins,
in mouse osteoblastic MC3T3-E1 cells. AP-1 activates the transcription
of target genes by binding to specific promoter elements called TRE. In
fact, several studies (10-16) have suggested that AP-1 is a regulatory
factor in bone metabolism. Therefore, it is of interest to investigate
the mechanism by which AP-1 regulates the metabolism of
TGF-
1-treated bone cells, which regulation may occur in an autocrine
and/or paracrine fashion.
1-25-(OH)2D3 also is an important systemic
hormone in bone metabolism and regulates transcriptionally the
expression of several genes involved in the differentiation of
osteoblastic cells (17-23). Many studies (20-26) have shown that the
hormone binds to its receptor, VDR, in the cell nucleus and acts via
binding of this complex to the VDR response element. This
VDR-dependent action is referred to as the "genomic
action" of the hormone. On the other hand, other recent studies
(27-37) have suggested that the hormone is also able to induce several
biological activities via protein kinase C, ceramide signaling
pathways, and via an increase in the intracellular calcium
concentration, which are called "nongenomic action."
Several investigators (21-24) have demonstrated negative regulation
between AP-1 and VDR of the transcriptional activity of osteocalcin and
collagen genes, both of which are involved in bone formation. However,
positive regulation by 1-25-(OH)2D3 of AP-1
transcriptional activity in osteoblastic cells has not been
demonstrated in detail. Therefore, it is of interest to explore the
possibility of 1
-25-(OH)2D3 positive
regulation of AP-1 transcriptional activity via "genomic" or
"nongenomic" action.
In this regard, we investigated in the present study the regulation by
1-25-(OH)2D3 of TGF-
1-induced AP-1
transcriptional activity in osteoblastic MC3T3-E1 cells. As a result,
we demonstrated the presence of 1
-25-(OH)2D3
synergism toward TGF-
1-induced AP-1 transcriptional activity via
genomic action. This demonstration suggests the presence of a novel
positive regulation by 1
-25-(OH)2D3 of AP-1
transcriptional activity in osteoblastic cells via the VDR-dependent pathway (genomic action).
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MATERIALS AND METHODS |
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Reagents--
Human recombinant TGF-1 was purified to
homogeneity (>98.9%, determined by SDS-polyacrylamide gel
electrophoresis analysis: King Brewer, Kakogawa, Japan).
1
-25-(OH)2D3,
24,25-(OH)2D3, and 22-oxa-1,25-dihydroxyvitamin
D3 (OCT) were kindly provided by Chugai Pharmaceutical Co.,
Ltd. (Tokyo, Japan).
-MEM was obtained from Flow Laboratories
(McLean, VA). FCS was from HyClone (Logan, UT).
5'-[
-32P]dCTP megaprime DNA labeling system and
[
-32P]ATP were purchased from Amersham Pharmacia
Biotech (Tokyo, Japan). 5'-[
-32P]UTP and T4
polynucleotide kinase were from NEN Life Science Products.
Cell Culture--
Clonal osteoblastic MC3T3-E1 cells derived
from C57BL/6 mouse calvaria were cultured in -MEM supplemented with
10% FCS in plastic dishes at 37 °C and 5% CO2 in air
and subcultured every 3 days as described previously (8, 38). The cells
(3 × 105 cells) were cultured at 37 °C under an
atmosphere of 5% CO2 in air in
-MEM supplemented with
10% FCS in 90-mm plastic dishes until nearly confluent. Then the cells
were washed, incubated for 24 h in serum-free
-MEM, and
subsequently treated for the desired periods in serum-free
-MEM
with or without test samples at various concentrations.
Northern Blot Analysis--
Total cellular RNA was extracted by
the guanidine isothiocyanate procedure (39). As described previously
(9, 38), the RNA was subjected to 1% agarose electrophoresis and
blotted onto a nylon membrane (MSI Magnagraph, Westboro, MA), and the
membranes were subsequently baked, prehybridized, and then hybridized
with mouse c-fos cDNA (Oncor, Gaithersburg, MD), mouse
c-jun cDNA (ATCC, Rockville, MD), and human VDR cDNA
(ATCC) probes labeled with 5'-[32P]dCTP by use of the
megaprime DNA labeling system. After hybridization, the membranes were
washed, dried, and exposed to x-ray film (Eastman Kodak Co.) at
70 °C.
-Actin was used as an internal standard for
quantification of total RNA in each lane of the gel.
Nuclear Transcriptional (Run-on) Assay--
This assay was
performed according to the method of Groudine et al. (40) as
described previously (8, 41). Nuclei were prepared essentially as
described by Diagnam et al. (42). In brief, cells (5 × 107 cells) were cultured at 37 °C under an atmosphere of
5% CO2 in air in -MEM supplemented with 10% FCS in
15-cm plastic dishes until nearly confluent. Then the cells were washed
and incubated for 24 h in serum-free
-MEM. In addition, the
cells were next treated or not for 24 h with
1
-25-(OH)2D3 and then for 40 min with
TGF-
1, scraped into phosphate-buffered saline, and centrifuged. Subsequently, the cell pellet was suspended in a lysis buffer (10 mM Tris (pH 7.4), 3 mM MgCl2, 10 mM NaCl, 0.5% Nonidet P-40) after which the nuclei were
separated from the cytosol by centrifugation at 3,000 × g for 15 min. Transcription initiated in intact cells was
allowed to proceed for 30 min at 30 °C in the presence of 5'-[
-32P]UTP, and the RNA was then isolated and
hybridized to slot-blotted cDNA probes (5 µg/slot). Blots were
hybridized for 72 h and autoradiographed for 3 days.
-Actin
gene was utilized as an internal standard.
Preparation of Nuclear Extracts-- Confluent monolayers in 15-cm diameter dishes were treated with test samples as indicated in the figure legends, and then their nuclei were isolated as described above, and extracts of them were prepared as described previously (8, 9). Protein concentration was measured by the method of Bradford (43).
Gel Mobility Shift Assay--
This assay was carried out
as described previously (8, 9). Binding reactions were performed for 20 min on ice with 5 µg of nuclear protein of 20 µl of binding buffer
(2 mM HEPES (pH 7.9), 8 mM NaCl, 0.2 mM EDTA, 12% (v/v) glycerol, 5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg of poly(dI-dC)) containing 20,000 cpm of 32P-labeled
oligonucleotide in the absence or presence of nonlabeled oligonucleotide. Poly(dI-dC) and nuclear extract were first incubated at 4 °C for 10 min before addition of the labeled oligonucleotide. 30-Mer double-stranded oligonucleotides containing the -TGACTCA- sequence (Oncogene Science, Inc., Manhasset, NY) of the AP-1 binding site were end-labeled by the oligonucleotide 5'-end-labeling
[-32P]ATP method. Reaction mixtures for the binding
were incubated for 15 min at room temperature after addition of the
labeled oligonucleotide. Unlabeled double-stranded oligonucleotide was
used as the competitor. DNA-protein complexes were electrophoresed on
native 6% polyacrylamide gels in 0.25× TBE buffer (22 mM
Tris, 22 mM boric acid, and 0.5 mM EDTA (pH
8.0)). Gels were vacuumed, dried, and exposed to Kodak x-ray film at
70 °C.
Plasmid Construction for Transient Expression Assay-- The plasmid pTRE-TK-CAT was constructed by inserting a synthetic oligonucleotide containing the -TGACTCA- motif with HindIII-XbaI sites into the corresponding sites of pTK-CAT, which contains the herpes simplex virus-thymidine kinase promoter enhancer region located upstream of the CAT gene. The pGL-TK used also contains the herpes simplex virus-thymidine kinase promoter enhancer region, but it is located upstream of the luciferase gene, and this plasmid was made from a pGL3-Enhancer Vector (Promega, Madison, WI). pSG-VDR is a rat VDR expression vector.
Transient Expression Assay--
This assay was performed
according to the method of Felgner et al. (44, 45). The
cells (1 × 106 cells) were inoculated in 5-cm
diameter dishes and incubated for 12 h in 10% FCS containing
-MEM. Then the cells were washed 3 times with Opti-MEM (Life
Technologies, Inc.), transfected with a total of 7 µg of DNA by use
of 10 µg of LipofectAMINE (Life Technologies, Inc.), and incubated
for 6 h in serum-free Opti-MEM. The cells were transfected with 2 µg of reporter plasmid, and the expression vector for the nuclear
receptor (4 µg of each expression vector) was used. The assay was
performed in the presence of 1 µg of pGL-TK, a luciferase expression
plasmid, used as an internal control to normalize for variations in
transfection efficiency. Bluescribe M13+ (Stratagene, La
Jolla, CA) was used as a carrier to adjust the total amount of DNA. The
cells were washed three times after the transfection, and
1
-25-(OH)2D3 at 10
8
M or its analogs at 10
8 M in
serum-free
-MEM was then added. After a 24-h incubation, the cells
were treated for 6 h with TGF-
1. The cellular extracts were
prepared by use of Reporter Lysis Buffer (Promega) and subjected to the
CAT assay after normalizing luciferase activity. CAT activity was
determined by autoradiography of thin layer chromatography (TLC) plates
following completion of the CAT reaction using the appropriate
concentration of
D-threo-[dichloroacetyl-1-14C]chloramphenicol
(Amersham Pharmacia Biotech, Japan) as described previously (46,
47).
Preparation of VDR Antisense or Sense Oligonucleotide-- VDR antisense (5'-GCT GGC TGC CAT TGC CTC-3') phosphorothioate oligodeoxynucleotide was synthesized and purified as described previously (8, 9, 41). These nucleotide sequences were complementary to the first 18 bases following the AUG sequence of mouse VDR mRNA. Also, the corresponding sense oligonucleotide was prepared and used as a control.
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RESULTS |
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We (8, 9) previously demonstrated by the gel mobility shift assay
that TGF-1 induces the binding of AP-1 to TRE in osteoblastic
MC3T3-E1 cells. Since AP-1 typically appeared when the cells were
treated for 3 h with TGF-
1 at 1 ng/ml, in this study we
investigated the regulatory action of
1
-25-(OH)2D3 on TGF-
1-induced AP-1
activity in the cells under these experimental conditions.
1-25-(OH)2D3 Synergistically Stimulates
Expression of the TGF-
1-induced c-jun Gene in MC3T3-E1
Cells--
First, we examined the effect of
1
-25-(OH)2D3 on TGF-
1-induced expression
of the c-jun and c-fos genes in the cells. The cells were treated or not with TGF-
1 at 1 ng/ml before the vitamin D3 was added to the cell cultures. As shown in Fig.
1A,
1
-25-(OH)2D3 stimulated synergistically
TGF-
1-induced expression of the c-jun gene in the cells.
The synergistic action was observed at a physiological concentration
(10
10 M) of the hormone. However, such action
was not observed in the expression of the cytokine-induced
c-fos gene in the cells. Also, the synergistic effect of
1
-25-(OH)2D3 on the expression of the c-jun gene in the cells was observed even when the hormone
pretreatment time was only 1 h before the cells were incubated
with TGF-
1 and tended to be pretreatment time-dependent
(Fig. 1B).
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1-25-(OH)2D3 Synergistic Effect on
TGF-
1-induced Expression of the c-jun Gene in MC3T3-E1 Cells Occurs
at the Transcriptional Level--
Next we examined, using the run-on
assay, whether or not the 1
-25-(OH)2D3
synergistic action toward TGF-
1-induced expression of the
c-jun gene in the cells operates at the transcriptional level. The cells were pretreated or not for 24 h with
1
-25-(OH)2D3 at 10
8
M and then were treated or not for 1 h with TGF-
1
at 1 ng/ml. Thereafter, the run-on assay was performed using nuclei
isolated from the cells. Fig. 2,
A and B, shows that
1
-25-(OH)2D3 clearly stimulated the
transcriptional activity of the TGF-
1-induced c-jun gene.
These results indicate the synergistic action of
1
-25-(OH)2D3 at the transcriptional level
for TGF-
1-induced expression of c-jun gene in the
cells.
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Synergistic Effect of 1-25-(OH)2D3 on
AP-1 Binding to TRE in TGF-
1-treated MC3T3-E1 Cells--
AP-1 is a
heterodimer of FOS and JUN proteins and binds to the TRE consensus
sequence (48). Synergistic stimulation by
1
-25-(OH)2D3 of TGF-
1-induced expression
of the c-jun gene in the cells suggested to us that the
hormone may remarkably increase AP-1 binding to TRE in the
cytokine-treated cells. Therefore, we examined this point by the gel
mobility shift assay. As we expected, and as shown in Fig.
3,
1
-25-(OH)2D3 caused a synergistic increase
in AP-1 binding to TRE in the TGF-
1-treated cells in a
dose-dependent manner. The stimulated binding was
completely inhibited by the unlabeled oligonucleotide containing TRE
(data not shown).
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1-25-(OH)2D3 Stimulates TGF-
1-induced
AP-1 Transcriptional Activity in MC3T3-E1 Cells--
In addition, we
explored whether 1
-25-(OH)2D3 is able,
moreover, to stimulate TGF-
1-induced AP-1 transcriptional activity in the cells. We investigated this possibility by employing the transient CAT assay on cells transfected with plasmids containing with
TRE-TK-CAT reporter gene (pTRE-TK-CAT). As shown in Fig. 4, A and B,
1
-25-(OH)2D3 pretreatment clearly stimulated
TGF-
1-induced AP-1 transcriptional activity in the cells. No CAT
activity was observed in the cells transfected with the TK-CAT reporter
plasmid (pTK-CAT).
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Effect of 1-25-(OH)2D3 Analogs on
TGF-
1-induced AP-1 Transcriptional Activity in MC3T3-E1
Cells--
Several recent studies (27-37) have suggested the presence
of VDR-dependent (genomic) and -independent (non-genomic)
pathways in 1
-25-(OH)2D3 signal
transduction. Therefore, it was of interest to determine whether the
synergistic action of 1
-25-(OH)2D3 toward AP-1 transcriptional activity in TGF-
1-treated cells was
VDR-dependent or -independent. In this regard, some studies
(34, 49) have shown that OCT, a 1
-25-(OH)2D3
derivative, has a high affinity for VDR and that its biological
activity is generated via the VDR-dependent (genomic)
pathway. In contrast, 24,25-(OH)2D3 is an
analog that has very low affinity for VDR (29, 49). Therefore, using
these two analogs, we examined their effect on TGF-
1-induced expression of the c-jun gene in the cells. And also we
explored whether the synergistic action of
1
-25-(OH)2D3 toward the cytokine-induced AP-1 transcriptional activity in the cells was
VDR-dependent or -independent.
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Effect of VDR Expression on 1-25-(OH)2D3
Synergy in c-jun Gene Expression in TGF-
1-treated MC3T3-E1
Cells--
To ensure that 1
-25-(OH)2D3
synergy toward TGF-
1-induced AP-1 transcriptional activity in the
cells is mediated through VDR, it is significant to understand the
precise role of VDR in the hormone synergy toward c-jun gene
expression in the cells. Therefore, we explored using a VDR expression
vector (pSG-VDR) the functional role of endogenous VDR in this synergy.
The cells were transfected with pSG-VDR or control vector pSG-5 and
then pretreated or not with 1
-25-(OH)2D3.
Subsequently, the cells were treated or not with TGF-
1, and
c-jun gene expression was analyzed by the Northern blot
assay. As shown in Fig. 6, A
and B, 1
-25-(OH)2D3 markedly
stimulated TGF-
1-induced expression of the c-jun gene in
pSG-VDR-transfected cells when the gene expression was compared with
that of the control and pSG-5 vector-transfected cells. These results
confirm the functional role of endogenous VDR in
1
-25-(OH)2D3 synergy in TGF-
1-induced
expression of the c-jun gene in the cells.
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Synergistic Stimulation by 1-25-(OH)2D3
of TGF-
1-induced AP-1 Transcriptional Activity in MC3T3-E1 Cells Is
Mediated via VDR--
1
-25-(OH)2D3 synergy
in TGF-
1-induced expression of the c-jun gene in the
cells transfected with the VDR expression vector suggested to us the
possibility that 1
-25-(OH)2D3 stimulates synergistically TGF-
1-induced AP-1-transcriptional activity in the
cells via VDR. Therefore, we explored this possibility by using a
cotransfection system with VDR expression vector (pSG-VDR) and
pTRE-TK-CAT. Fig. 7, A and
B, shows that TGF-
1-induced AP-1 transcriptional activity
in pSG-VDR-transfected cells was approximately 1.5 times that in the
cells transfected with pSG-5. In a CAT assay with the TK-CAT reporter
plasmid (pTK-CAT), no effect of pSG-VDR or pSG-5 was observed (data not
shown). These results indicate strongly that
1
-25-(OH)2D3 stimulation of TGF-
1-induced
AP-1 transcriptional activity in the cells occurs via VDR.
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Involvement of Endogenous VDR in
1-25-(OH)2D3 Synergy toward TGF-
1-induced
Expression of the c-jun Gene and AP-1 Transcriptional Activity in
MC3T3-E1 Cells--
As described above, since our data suggested
involvement of an endogenous VDR in TGF-
1-induced expression of the
c-jun gene and in AP-1 transcriptional activity in the
cells, in addition, we used mouse VDR antisense oligonucleotide to
investigate the functional role of VDR in this synergy. Fig.
8A shows that VDR antisense,
but not sense, oligonucleotide treatment clearly inhibited the
synergistic action of 1
-25-(OH)2D3 in the
cytokine-induced expression of the c-jun gene in the
cells.
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1-25-(OH)2D3 Stimulates at the
Transcriptional Level the AP-1-dependent Expression of the
Osteopontin Gene in TGF-
1-treated MC3T3-E1 Cells--
It is of
interest to determine whether the
1
-25-(OH)2D3 synergism toward
TGF-
1-induced AP-1 transcriptional activity actually operates
physiologically in osteoblastic cells. It is well known that
osteopontin, an important matrix protein, is a marker of osteoblastic
cell differentiation (50-53). Although the data are not shown, we
observed that TGF-
1-stimulated expression of the osteopontin gene in
MC3T3-E1 cells is mediated via AP-1. And, in fact, it has been
demonstrated that a TRE sequence is located on the promoter region of
the murine osteopontin gene (52, 53). Therefore, finally, we
investigated 1
-25-(OH)2D3 synergism toward TGF-
1-stimulated expression of the osteopontin gene in the cells. As
we expected, 1
-25-(OH)2D3 pretreatment at
10
8 M stimulated expression of the
osteopontin gene in the cytokine-treated cells (Fig.
9A), and a run-on assay (Fig.
9B) showed that 1
-25-(OH)2D3 operates at the transcriptional level in the synergistic action. Also,
such synergism was observed with OCT pretreatment (Fig. 9C).
These observations allow us to propose that
1
-25-(OH)2D3 actually stimulates
AP-1-mediated differntiation of TGF-
1-treated osteoblastic
cells.
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DISCUSSION |
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TGF-1 and 1
-25-(OH)2D3 are important
local and systemic regulatory factors in bone remodeling (4-7,
54-56). For osteoblastic cells, both are potent factors in growth and
differentiation (7-9, 20-25). We previously demonstrated that
TGF-
1 strongly induces AP-1 in mouse osteoblastic MC3T3-E1 cells via
the activation of serine/threonine kinase (8, 9). Several studies
(21-23) have documented that AP-1 regulates the differentiation of
osteoblastic cells by interacting with VDR, which functions as a
transcriptional factor. In general,
1
-25-(OH)2D3 may exhibit multiple effects on
gene expression in osteoblastic cells via its
receptor-dependent mechanism or genomic action. On the
other hand, interestingly, recent studies (20-26) have suggested that
the hormone exerts several effects on cells by a receptor-independent
mechanism, i.e. by nongenomic action. In this regard, it is
of interest to investigate whether
1
-25-(OH)2D3 negatively or positively
regulates TGF-
1-induced AP-1 transcriptional activity in mouse
MC3T3-E1 cells. And our interest also was to define whether the
1
-25-(OH)2D3 action on the AP-1 activity
would be genomic or nongenomic (27-37). Of these two mechanisms, the
present study demonstrates that the synergistic action of
1
-25-(OH)2D3 toward TGF-
1-induced AP-1
transcriptional activity in the cells occurs via the genomic activity
(the receptor-dependent mechanism).
Several studies (10-16, 21-23) have well documented that AP-1 in
osteoblasts is an important transcriptional factor in bone formation
and resorption. We observed here that TGF-1-induced expression of
the c-jun gene in MC3T3-E1 cells was synergistically stimulated by 1
-25-(OH)2D3 pretreatment,
although such synergistic effect was not observed for c-fos
gene expression. The synergistic action of
1
-25-(OH)2D3 toward c-jun gene
expression was dose- and pretreatment-dependent. Our run-on
assay indicated that the 1
-25-(OH)2D3
synergistic action in the c-jun gene expression in
TGF-
1-treated cells resulted from stimulation of its transcription. Since these observations suggested the synergistic increase by the
hormone of AP-1 binding to TRE in the cells, we explored this point by
the gel mobility shift assay. This assay clearly demonstrated that the
cytokine-induced AP-1 binding to TRE in the cells was synergistically
increased by 1
-25-(OH)2D3 pretreatment. Of
more interest was whether the hormone would be actually able to
stimulate synergistically AP-1 transcriptional activity. In fact, the
TRE-TK-CAT assay showed that 1
-25-(OH)2D3
clearly stimulated the AP-1 transcriptional activity in
TGF-
1-treated cells. In this regard, Sassone-Corsi and co-workers
(48, 57) showed that AP-1 transcriptional activity in mouse embryonal
carcinoma F9 cells, which express constitutively c-jun and
c-fos genes at a low level, is induced by transfecting the
cells with a c-jun autonomous expression vector. We (8, 58)
observed that curcumin, a potent inhibitor of c-jun gene expression, completely inhibited the synergistic effect of
1
-25-(OH)2D3 on TGF-
1-induced AP-1
transcriptional activity in MC3T3-E1 cells (data not shown). These
observations of vitamin D3 support the notion that the
stimulated expression of the c-jun gene provided an
important clue in the mechanism of
1
-25-(OH)2D3 synergism toward
TGF-
1-induced AP-1 transcriptional activity in the cells.
It is well documented that multiple biological actions of
1-25-(OH)2D3 in osteoblastic cells are
mediated via interaction of its receptor complex with specific DNA
sequences (20-22, 24, 25). On the other hand, recent studies
(27-37) have demonstrated that 1
-25-(OH)2D3
expresses several biological actions via ceramide and protein kinase C
signaling pathways and also via intracellular calcium signals. Thus, it
was of interest to demonstrate whether 1
-25-(OH)2D3 synergy in the stimulation of
AP-1 transcriptional activity of TGF-
1-treated MC3T3-E1 cells is
genomic action-dependent or -independent. Therefore, we
explored this point by using 1) 1
-25-(OH)2D3
analogs having high and low affinities to VDR, 2) transfection assay
with VDR expression and TRE-TK-CAT vectors, and 3) VDR antisense
oligonucleotide to eliminate production of endogenous VDR. We observed
that OCT synergy in the expression of the c-jun gene in
TGF-
1-treated MC3T3-E1 cells was the same as that of
1
-25-(OH)2D3. Since OCT is a ligand having
high affinity for VDR (33, 37), these observations suggested to us that 1
-25-(OH)2D3 synergy in the stimulation of
AP-1 transcriptional activity of TGF-
1-treated cells may be genomic
in nature. We proved this suggestion by using a cotransfection assay
with VDR expression and TRE-TK-CAT vectors.
1
-25-(OH)2D3 or OCT markedly stimulated
TGF-
1-induced AP-1 transcriptional activity in the cells when they
were cotransfected with VDR expression and TRE-TK-CAT vectors. In
addition, VDR antisense oligonucleotide pretreatment inhibited
approximately 40% the synergistic action of
1
-25-(OH)2D3 toward TGF-
1-induced AP-1
transcriptional activity in the cells. These results together with the
above demonstrate that 1
-25-(OH)2D3 synergism toward TGF-
1-induced AP-1 transcriptional activity in
MC3T3-E1 cells is mediated via a VDR-dependent mechanism
(genomic action).
Several studies (59-62) have shown that the combination of
1-25-(OH)2D3 and TGF-
1 functionally
regulates bone formation. Our previous study (9) showed that
TGF-
1-induced expression of RAR-
, RAR-
, and retinoic X
receptor-
is mediated via the cytokine-induced AP-1 signaling
pathway in MC3T3-E1 cells. Most recently, we observed that the
cytokine-induced expression of RAR-
, -
, and retinoic X
receptor-
genes were remarkably stimulated by pretreatment with
1
-25-(OH)2D3.2
In addition, as we also showed in this study, the hormone stimulated synergistically TGF-
1-induced expression of the osteopontin gene at
the transcriptional level in the cells. Since osteopontin is one of the
extracellular non-collagenous matrix proteins and also is a marker of
differentiation of osteoblastic cells (20, 50-53), these observations
are significant with respect to bone metabolism.
In further experiments, our interest will be to define the mechanism of
1-25-(OH)2D3 synergism operating in
TGF-
1-induced AP-1 transcriptional activity in mouse osteoblastic
cells. We are examining two possibilities as follows: one is that the
hormone stimulates c-jun kinase activity, and the other one
is stimulation by the hormone of TGF-
1 receptor-associated Smads in
a VDR-dependent manner.
In conclusion, our present study demonstrates
1-25-(OH)2D3 synergism toward
TGF-
1-induced AP-1 transcriptional activity in mouse osteoblastic
MC3T3-E1 cells via VDR action (genomic action). This mode of action
proposes a positive regulation by
1
-25-(OH)2D3 and TGF-
1 of AP-1-mediated
expression of several genes in osteoblastic cells.
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ACKNOWLEDGEMENT |
---|
We thank Chugai Pharmaceutical Co., Ltd., for
providing 1-25-(OH)2D3 and its analogs.
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FOOTNOTES |
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* 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: Dept. of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan. Tel.: 492-85-5511; Fax: 492-87-6657.
1
The abbreviations used are: TGF-1,
transforming growth factor-
1;
1
-25-(OH)2D3, 1
,25-dehydroxyvitamin
D3; 24,25-(OH)2D3, 24, 25-dihydroxyvitamin D3; OCT, 22-oxa-1,25-dihydroxyvitamin D3; VDR, vitamin D3 receptor; AP-1, activation
protein-1;
-MEM,
-minimum modified essential medium; FCS, fetal
calf serum; TRE, 12-tetra-decanoyl phorbol-13-acetate-responsive
element; CAT, chloramphenicol acetyltransferase; RAR, retinoic acid
receptor.
2 A. Takeshita, K. Imai, S. Kato, S. Kitano, and S. Hanazawa, unpublished data.
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REFERENCES |
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