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From the Department of Oral Microbiology, Meikai University School
of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan
ABSTRACT We now report that transforming growth factor
INTRODUCTION Bone remodeling is regulated by a network of local and systemic
cytokines and hormones (1, 2, 3, 4). Of them, TGF- RA is an important regulatory hormone for the proliferation and
differentiation of a variety of human and mouse cells (13, 14, 15, 16). RA
exerts its biological effects transcriptionally through the function of
two distinct classes of receptors, RAR (RAR MATERIALS AND METHODS Human platelet-derived TGF- Anti-mouse
RAR The osteoblastic cell line and its culture were as follows. Cells of
clonal osteoblastic MC3T3-E1 cell line (provided kindly by Dr. H. Kodama, Ohu University, Koriyama, Japan) were cultured to the
subconfluent state in Each plasmid containing the
mouse RAR or RXR Total cellular RNA was extracted
by the guanidine isothiocyanate procedure (24). The RNA was subjected
to 1% agarose electrophoresis and blotted onto a nylon membrane (MSI
Magnagraph, Westboro, MA). The membranes were baked, prehybridized, and
then hybridized to each cDNA probe labeled with
[ Nuclei were prepared
essentially as described by Dignam et al. (25), and a
"run-on" assay was performed according to the method of Groudine
et al. (26) as described previously (27, 28). In brief, the
cells were or were not treated with TGF- Subconfluent
monolayers in 15-cm diameter dishes were or were not treated with
TGF- Binding reactions were performed
for 20 min on ice with 5 µg of sample protein in 20 µl of binding
buffer (for AP-1 binding to TRE: 2 mM HEPES, pH 7.9, 8 mM NaCl, 0.2 mM EDTA, 12% (v/v) glycerol, 5 mM DTT, 0.5 mM PMSF; for DR5 binding: 2 mM HEPES, pH 7.9, 100 mM KCl, 12% (v/v)
glycerol, 1 mM DTT, 0.5 mM PMSF) and 1 µg of
poly(dI-dC) containing 20,000 cpm of each 32P-labeled
oligonucleotide in a final volume of 20 µl. Poly(dI-dC) and nuclear
extracts were first incubated at 4 °C for 10 min before addition of
the labeled oligodeoxynucleotide. Double-stranded oligonucleotides for
5 In some experiments, after a further incubation for 5 min on ice,
antibodies specific for RAR and RXR were added, and the reaction
mixture was maintained on ice for 15 min before being loaded onto
polyacrylamide gels. 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 Antisense fos
(5 H-7 was obtained from Seikagaku Kougyo,
Tokyo, Japan. Curcumin was from Nakarai Tesque, Inc., Kyoto, Japan.
RESULTS We first examined the possible regulatory effect
of TGF-
We next examined the time course of gene expression of RAR We furthermore examined whether TGF-
Since
TGF-
As shown in our previous study (30),
TGF-
Since these observations indicated a role for AP-1 in the expression of
these genes in the cells, we looked, using antisense c-fos
and c-jun oligonucleotides, to see whether either one of the
c-fos and c-jun protooncogenes or both are
necessary for these expressions. The cells were pretreated for 3 h
with each antisense and/or sense oligonucleotide (2.5 µM)
and thereafter treated with TGF-
Furthermore, to verify the AP-1 induction in the cells treated with
TGF-
As demonstrated by several studies (32, 33, 34), the members of
nonsteroid nuclear receptor families can recognize the common consensus
half-site sequence AGGTCA in the form of a direct repeat. And RAR-RXR
heterodimers bind efficiently to the DR5 sequence that encodes two
copies of -AGGTCA- in directed repeat separated by 5 nucleotides and
promote transcriptional activation in a ligand-dependent manner. Thus it is very important to demonstrate whether TGF-
To confirm the specificity of RAR As recent studies (35, 36, 37, 38, 39)
have demonstrated that the TGF-
DISCUSSION TGF- We had an interest in determining which among the subclasses of RAR and
RXR genes in MC3T3-E1 cells are regulated negatively and which are
regulated positively by TGF- Recently, the TGF- It was also of interest to us to understand which transcriptional
factor is involved in TGF- For the nonsteroid members of the receptor superfamily, the hormone
response elements consist of a minimal core consensus sequence,
-AGGTCA-, that can be configured into a variety of structured motifs.
Several studies (32, 33, 34) have shown that heterodimers of RAR-RXR bind
to DR5, which encodes two copies of AGGTCA in directed repeat separated
by 5 nucleotides. Since the binding of the heterodimers to DR5
activates transcription from DNA containing DR5, it was very important
to examine by the gel mobility shift assay the binding activity of
nuclear proteins to DR5 in TGF- In conclusion, this study suggests a novel pathway of TGF- FOOTNOTES Acknowledgment We thank Dr. P. Chambon for generous gifts of
RAR and RXR cDNAs, and also for antibodies to RAR and RXR.
REFERENCES
Volume 271, Number 49,
Issue of December 6, 1996
pp. 31602-31606
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
of the Expression of Retinoic Acid and Retinoid X Receptor
Genes in Osteoblastic Cells Is Mediated through AP-1*
,
1 (TGF-1), a potent regulatory cytokine of bone remodeling, is a
powerful stimulator for gene expression of retinoic acid receptors
(RARs) and retinoid X receptors (RXRs) in osteoblastic MC3T3-E1 cells. TGF-1 transcriptionally stimulated the expression of RAR
, RAR
, and RXR genes, but did not do so for RAR, RXR, and RXR
genes. We also observed that AP-1, a transcriptional factor, plays an important role in the signal pathway for expression of RAR, RAR, and RXR genes stimulated by TGF-1 because stimulation of the expression of these genes in the cytokine-treated cells was markedly inhibited by a mixture of antisense c-fos and
c-jun. A gel mobility shift assay demonstrated that
TGF-1 is able to increase, in a dose-dependent manner,
the binding of nuclear proteins to direct repeat 5, a consensus
sequence with high affinity for RAR-RXR heterodimers. The mobility
shift assay, using specific antibody for each receptor, showed that
direct repeat 5-binding proteins may be RAR and RXR isoforms. The
stimulated binding to direct repeat 5 was inhibited strongly by H-7, an
inhibitor of serine/threonine kinase, and by curcumin, an inhibitor of
AP-1. The present study suggests a novel pathway for TGF-1 action in
osteoblastic cells via stimulation of RAR-RXR transcriptional activity
in a ligand-dependent fashion.
1 is produced by osteoblastic cells
in bone tissues, and this factor accumulates abundantly in bone matrix
tissues. Thus, this cytokine may play a central role in the cytokine
network of bone remodeling. In fact, many studies (5, 6, 7, 8, 9, 10, 11, 12) have
demonstrated a functional role for this cytokine in the regulation of
the proliferation and differentiation of osteoblastic and osteoclastic
cells.
, RAR, and RAR) and
RXR (RXR, RXR, and RXR), that bind to their respective target
DNA sequences. Also, more recent studies (17, 18, 19, 20, 21) have demonstrated
that RXR, as an auxiliary protein, forms heterodimers with vitamin
D3 and thyroid hormone and may even affect signaling of the
steroid hormone receptor family. Therefore, RXRs play an important role
in the regulation of signal transduction of RA and of vitamin
D3 and thyroid hormone. In the present study, we examined
whether TGF-1 regulates gene expression of RARs and RXRs in
osteoblastic MC3T3-E1 cells. We show here that TGF-1 regulates
positively, at the transcriptional level, RAR, RAR, and RXR
genes. These results also suggest that TGF-1 may effect positive
regulation of RA in osteoblasts via the stimulation of its nuclear
receptors.
1, purified to
homogeneity (>98%, determined by SDS-polyacrylamide gel
electrophoresis), was purchased from King Brewer (Kakogawa, Japan).
-MEM was obtained from Flow Laboratories (McLean, VA) and fetal calf
serum from Hyclone (Logan, UT). [-32P]dCTP and
megaprimed DNA labeling system were from Amersham Japan (Tokyo,
Japan), and [-32P]UTP and oligonucleotide
5
-end-labeling system were from DuPont NEN. [-32P]ATP
was purchased from Amersham Japan.
and RAR and RXR Antisera
, RAR, and RXR antisera was kindly supplied by Dr. P. Chambon (Institut de Chimie Biologique Facultte de Medecine,
France). The characteristics and specificity of these antisera were
described previously (22).
-MEM containing 10% fetal calf serum at
37 °C and 5% CO2 in air, washed, and then incubated
overnight in serum-free -MEM. They were then washed further and
treated with test samples at various concentrations.
, , or cDNA sequences was provided
by Dr. P. Chambon. Plasmid containing -actin cDNA was obtained
from Oncor (Gaithersburg, MA). The methods used for plasmid
preparation were described earlier (23).
-32P]dCTP by use of a megaprimed DNA labeling system.
After hybridization, the membranes were washed, dried, and exposed to
x-ray film (Eastman Kodak Co., Rochester, NY) at 
70 °C. -Actin
was used as an internal standard for quantification of total mRNA
in each lane of the gel.
1. After 2.5 h, the
cells were scraped from the dishes and suspended in pellet lysis buffer
(10 mM Tris, pH 7.4, 3 mM MgCl2, 10 mM NaCl, and 0.5% Nonidet P-40). The nuclei were separated
from the cytosol by centrifugation. Transcription initiated in intact
cells was allowed to proceed for 30 min at 30 °C in the presence of [-32P]UTP, and the RNA was isolated and hybridized to
slot-blotted cDNA probes (5 µg/slot). Blots were hybridized for
72 h and autoradiographed for 3 days. The -actin gene was
utilized as an internal standard.
1 as indicated in the figure legends, and their nuclei were
isolated as described above. Next, the nuclei were treated with buffer
A (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT) and
further treated by stirring for 60 min at 4 °C in buffer B (for
AP-1: 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.42 M NaCl,
25% glycerol, 0.5 mM DTT, 0.5 mM PMSF; for
DR5: 10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.6 M KCl, 0.5 mM DTT). Nuclear extracts were obtained by centrifugation for 60 min at 25,000 × g and demineralized through a
Sephadex G-25 column equilibrated with buffer C (5 mM
HEPES, pH 7.9, 0.02 M KCl, 0.04 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF). Protein concentration was
measured by the method of Bradford (29).
-AGCTTCAGGTCACCAGGAGGTCAGAGAGCT-3 of the DR5 binding site with high
affinity for RAR-RXR heterodimers and 30-mer double-stranded
oligonucleotides containing the consensus sequence TGACTCA (Oncogene
Science, Inc.) of the AP-1 binding site, i.e. TRE, were
end-labeled with [-32P]ATP by the oligonucleotide
5-end-labeling method.
70 °C.
-TGC-GTT-GAA-GCC-CGA-GAA-3) and jun
(5-CGT-TTC-CAT-CTT-TGC-AGT-3) unsubstituted oligodeoxynucleotides, and their corresponding sense oligonucleotides were synthesized and
purified as described previously (27). These nucleotide sequences were
complementary to the first 18 nucleotides following the AUG sequence of
fos and jun mRNAs.
1 Stimulates Expression of RAR and RAR Genes in
MC3T3-E1 Cells
1 on gene expression of , , and subclasses of RAR
in MC3T3-E1 cells. As shown in Fig. 1A, the
cytokine at 0.05 ng/ml stimulated gene expression of both RAR and
RAR. The stimulated expression of both genes was
dose-dependent. On the other hand, we observed that the
RAR gene was not expressed constitutively in the cells and that the
gene expression was not induced by the cytokine (data not shown).
Fig. 1.
TGF-1 stimulates gene expression of RAR
and RAR in MC3T3-E1 cells. A, the cells were or were not
treated with TGF-1, and then total RNA was prepared at 3 h
after the initiation of the treatment. Northern blot analysis was
performed with RAR, RAR, and -actin cDNAs used as probes.
B, the cells were or were not treated with TGF-1 at 1 ng/ml, and then total RNA was prepared at selected times after the
initiation of the treatment. Northern blot analysis was performed with
RAR(
), RAR (
), and -actin cDNAs used as probes.
Quantification of both receptor gene expressions was done by
densitometry and is expressed as a percentage of the maximum. An
identical experiment independently performed gave similar
results.
[View Larger Version of this Image (25K GIF file)]
and
RAR stimulated by TGF-1. Fig. 1B shows that the
cytokine clearly stimulated expression of both genes, maximally at
3 h after initiation of the cytokine treatment. Although the
stimulated expression of the RAR gene was short lived, the RAR
gene expression continued to increase in a treatment
time-dependent manner. These results show that TGF-1
positively regulates expression of the RAR and RAR genes of
MC3T3-E1 cells.
1 Also Stimulates Expression of RXR Gene in MC3T3-E1
Cells
1 also regulates
gene expression of RXRs, the other class of RAR, in MC3T3-E1 cells. As
shown in Fig. 2A, although the cytokine at
0.5 ng/ml stimulated RXR gene expression, alteration of RXR gene
expression was not observed with the doses tested. We also found that
the RXR gene was not expressed at all in the cells (data not shown).
Fig. 2B shows the time course of the expression of the
RXR gene stimulated by TGF-1. Peak expression of the RXR gene
was observed at 1 h after initiation of the cytokine treatment,
and the expression decreased quickly thereafter, reaching base line by
3 h. These results suggest that TGF-1 also stimulates
expression of the RXR gene but not that of the RXR in MC3T3-E1
cells.
Fig. 2.
TGF-1 stimulates gene expression of RXR
in MC3T3-E1 cells. A, the cells were treated or not with
TGF-1, and then total RNA was prepared at 1 h after the
initiation of the treatment. Northern blot analysis was performed with
RXR, RXR, and -actin cDNAs used as probes. B,
the cells were or were not treated with TGF-1 at 1 ng/ml, and then
total RNA was prepared at selected times after the initiation of the
treatment. Northern blot analysis was performed with RXR and
-actin cDNAs used as probes. Quantification of RXR gene
expression was done by densitometry and is expressed as a percentage of
the maximum. An identical experiment independently performed gave
similar results.
[View Larger Version of this Image (26K GIF file)]
1 Stimulates the Expression of RAR, RAR, and RXR
Genes at the Level of Transcription in MC3T3-E1 Cells
1 stimulated expression of and subclasses of the RAR
gene and the subclass of the RXR gene in MC3T3-E1 cells, we next
explored the possibility, by using the run-on assay, that the factor
stimulates the rate of transcription of these genes in the
cytokine-treated cells. As shown in Fig. 3, A
and B, TGF-1 clearly increased the transcriptional rate
of these genes. These results strongly suggest that TGF-1 is a
potent stimulator for transcription of RAR, RAR, and RXR genes
in MC3T3-E1 cells.
Fig. 3.
TGF-1 stimulates transcription of RAR,
RAR, and RXR genes in MC3T3-E1 cells. The cells were or were
not treated for 2.5 or 0.5 h with TGF-1 (1 ng/ml) for RAR,
RAR, or RXR genes, respectively, and then their nuclei were
incubated for 30 min in the presence of [-32P]UTP,
after which the RNA was isolated. Transcriptional activity assay
(run-on assay) was performed with RAR (A), RAR
(A), and RXR (B) cDNAs and -actin
cDNA. pBR322, the vector plasmid, was used as a negative control.
An identical experiment independently performed gave similar
results.
[View Larger Version of this Image (29K GIF file)]
1-stimulated Expression of RAR, RAR, and RXR Genes
Is Mediated via AP-1
1 strongly induced expression of the c-fos and
c-jun genes, which encode the two components of the
transcriptional factor AP-1, in MC3T3-E1 cells. Huang et al.
(31) demonstrated that curcumin, a potent inhibitor of tumor promotion,
inhibits gene expression of c-jun induced by TPA and
suppresses the TPA-induced TRE binding activity of c-jun/AP-1 protein but does not affect TPA-induced
c-fos gene expression. Therefore, we examined, using
curcumin, the probable involvement of AP-1 in RAR, RAR, and
RXR gene expressions in the cells. Fig. 4,
A and B show that curcumin strongly inhibited the
stimulated expression of RAR, RAR, and RXR genes in the cytokine-treated cells.
Fig. 4.
Inhibitory effect of curcumin on
TGF-1-stimulated expression of RAR, RAR, and RXR genes.
A, the cells were or were not treated with curcumin in the
presence or absence of TGF-1 (1 ng/ml), and then total RNA was
prepared at 3 h after the initiation of the treatment. Northern
blot analysis was performed with RAR, RAR, and -actin
cDNAs used as probes. B, the cells were or were not
treated with curcumin in the presence or absence of TGF-1 (1 ng/ml),
and then total RNA was prepared at 1 h after the initiation of the
treatment. Northern blot analysis was performed with RXR and
-actin cDNAs used as probes. An identical experiment
independently performed gave similar results.
[View Larger Version of this Image (24K GIF file)]
1 (1 ng/ml). The gene expressions
were then analyzed by the Northern blot assay. As shown in Fig.
5, A-D, simultaneous addition of
c-fos and c-jun antisense oligonucleotides to the
cells inhibited TGF-1-stimulated RAR, RAR, and RXR gene
expressions. On the other hand, neither sense oligonucleotide inhibited
the expression of these genes.
Fig. 5.
TGF-1-stimulated expression of RAR,
RAR, and RXR genes is mediated by AP-1. The cells were or
were not treated with antisense or sense c-fos and
c-jun oligonucleotides, all at 2.5 µM, for
3 h and then incubated in the presence or absence of TGF-1 (1 ng/ml). Total RNA was prepared at 3 h for RAR and RAR genes
or at 1 h for the RXR gene after the initiation of the cytokine
treatment. Northern blot analysis was performed with RAR
(A), RAR (A), RXR (C), and
-actin cDNAs used as probes. Quantification of RAR
(closed column) and RAR (open column) in
(B) and RXR gene expressions in (D) was done
by densitometry and is expressed as percent inhibition of
maximum.
[View Larger Version of this Image (41K GIF file)]
1, we employed the gel mobility shift assay to look for its
presence in the cytokine-treated cells. The cytokine stimulated the
binding of nuclear proteins to the TRE sequence in a
dose-dependent manner. The stimulated appearance of the
nuclear proteins was eliminated by the use of unlabeled competitor
(Fig. 6). These results imply that AP-1 plays a
functional role as a transcriptional factor in RAR, RAR, and
RXR gene expressions in TGF-1-treated MC3T3-E1 cells.
Fig. 6.
TGF-1 stimulates binding of AP-1 to TRE in
MC3T3-E1 cells. The cells were treated or not with TGF-1. After
3 h, the nuclear proteins were prepared. Gel mobility shift assay
was performed with 32P-labeled oligonucleotide containing
the TRE sequence in the presence of the nuclear proteins. Unlabeled
oligonucleotide containing the AP-1 sequence was used as a competitor.
An identical experiment independently performed gave similar results.
The arrow indicates position of DNA-protein complexes.
[View Larger Version of this Image (33K GIF file)]
1 Stimulates Binding of Nuclear Proteins to DR5 in MC3T3-E1
Cells
1 stimulates binding of nuclear proteins to the DR5 in MC3T3-E1 cells. We
prepared nuclear proteins from TGF-1-treated and -untreated cells
and examined by the gel mobility shift assay to determine whether the
cytokine stimulates the binding of nuclear proteins to DR5 in the
cells. As shown in Fig. 7A, TGF-1
increased the binding of protein (presumably RAR-RXR heterodimers)
in vitro in a dose-dependent manner. The
TGF-1-stimulated binding was eliminated when an unlabeled DR5
oligonucleotide was used as a competitor. These results suggest the
possibility that the cytokine may stimulate RAR-RXR binding to DR5 in
the MC3T3-E1 cells.
Fig. 7.
TGF-1 stimulates binding of nuclear
proteins to DR5 in MC3T3-E1 cells. A, the cells were treated
or not with TGF-1. After 6 h, the nuclear proteins were
prepared. Gel mobility shift assay was performed with
32P-labeled oligonucleotide containing the DR5 sequence in
the presence of the nuclear proteins. Unlabeled oligonucleotide
containing the DR5 sequence was used as a competitor. An identical
experiment independently performed gave similar results. The
arrow indicates position of DNA-protein complexes.
B, the nuclear extracts prepared as described in panel
A were or were not treated with specific antibody against RAR,
RAR, or RXR. The gel mobility shift assay was performed with
32P-labeled oligonucleotides containing the DR5 sequence in
the presence of their nuclear proteins. Lower arrow
indicates the position of the DNA-protein complexes. Upper
arrow indicates the shifted complex formed in the presence of each
antibody to RAR, RAR, or RXR.
[View Larger Version of this Image (24K GIF file)]
, , and RXR stimulation in
the cytokine-treated cells, we also performed the gel mobility shift
assay using antibodies to each receptor. As shown in Fig. 7B, the DNA and protein complexes were shifted to a position
indicating slower migration when the extracts were treated with
specific antibody against each receptor. However, such a shift in
protein complexes was not detected when normal rabbit serum was used. Thus, these observations strongly suggest that these DR5-binding proteins are RAR, RAR, and RXR.
1-stimulated Binding of Nuclear Proteins to DR5 in MC3T3-E1
Cells Is Inhibited by H-7 and Curcumin
receptor has an inner domain
expressing serine/threonine kinase activity, a serine/threonine kinase
may be involved in signal transduction of the cytokine. In fact, we observed that H-7 (10 µM), a potent inhibitor of
serine/threonine kinases, especially of protein kinase C, also
inhibited expression of RAR, RAR, and RXR genes in the cells
(Fig. 8). Therefore, to understand the signal pathway of
TGF-1-stimulated expression of these genes, we examined the effect
of H-7 and curcumin on the binding of nuclear proteins to DR5 in
TGF-1-treated MC3T3-E1 cells. Fig. 9 shows that
either H-7 or curcumin, at 10 µM, markedly inhibited the
binding of nuclear proteins to DR5. These results indicate that
TGF-1 activates H-7-sensitive protein kinase and stimulates the
binding of nuclear proteins to DR5 via induction of AP-1.
Fig. 8.
Inhibitory effect of H-7 on
TGF-1-stimulated expression of RAR, RAR, and RXR genes.
A, the cells were or were not treated with H-7 (10 µM) in the presence or absence of TGF-1 (1 ng/ml), and
then total RNA was prepared at 3 h after the initiation of the
treatment. Northern blot analysis was performed with RAR, RAR,
and -actin cDNAs used as probes. B, the cells were or
were not treated with H-7 (10 µM) in the presence or
absence of TGF-1 (1 ng/ml), and then total RNA was prepared at
1 h after the initiation of the treatment. Northern blot analysis
was performed with RXR and -actin cDNAs used as probes. An
identical experiment independently performed gave similar
results.
[View Larger Version of this Image (28K GIF file)]
Fig. 9.
TGF-1-stimulated binding of nuclear
proteins from MC3T3-E1 cells to DR5 was inhibited by H-7 and
curcumin. The cells were or were not treated with H-7 or curcumin
(both at 10 µM) in the presence or absence of TGF-1 at
1 ng/ml. After 6 h, the nuclear proteins were prepared. Gel
mobility shift assay was performed with 32P-labeled
oligonucleotide containing the DR5 sequence in the presence of the
nuclear proteins. An identical experiment independently performed gave
similar results. The arrow indicates position of DNA-protein
complexes formed.
[View Larger Version of this Image (31K GIF file)]
is a multifunctional growth factor that often has opposite
effects on cellular responses. This cytokine regulates bone remodeling
by acting like a "coupling factor." It is well known that RA is
also a potent regulator of osteoblasts and osteoclasts (28, 40, 41, 42).
Biological actions of this hormone are mediated by its nuclear
receptors, which act as transcriptional factors. It is thus of much
interest to determine whether TGF-1 regulates expression of the RAR
and RXR genes in osteoblastic cells. In the present study, we sought to
demonstrate the regulatory effect of the cytokine on RAR and RXR gene
expressions and the signal pathway for their receptor expressions. We
showed here that TGF-1 transcriptionally stimulates the expression
of RAR, RAR, and RXR genes in osteoblastic MC3T3-E1 cells via
AP-1 and increases the binding of nuclear proteins to DR5, whose
sequence has a high affinity for RAR-RXR heterodimers.
1 because an interesting study (43) has
shown that RAR plays an important role with RA in murine bone
formation and also because RXRs are auxiliary proteins that
heterodimerize with RA, vitamin D3, and thyroid hormone
receptors, all of which are potent regulators of bone remodeling.
TGF-1 stimulated RAR, RAR, and RXR gene expressions in the
cells. However, RAR and RXR genes were not induced by the
cytokine. On the other hand, a constitutive RXR gene expression of
high intensity was observed although the gene expression was not
altered by the cytokine. These observations suggest that RXR may
play a functional role as the predominant receptor in heterodimer formation with RA, vitamin D3, and thyroid hormone
receptors in response to TGF-1 stimulation.
-type I receptor has been identified as a
serine/threonine kinase receptor that is related to the type II
receptor family (35, 36, 37, 38, 39). Thus, these type I and II heterometric
complexes play an important role in the cytokine signaling. We actually
showed that TGF-1-stimulated expressions of RAR, RAR, and
RXR genes in MC3T3-E1 cells were inhibited by H-7, a potent
inhibitor of serine/threonine kinases. Therefore, these results suggest
involvement of an H-7-sensitive protein kinase, probably a
serine/threonine kinase receptor, in the cytokine transducing pathway
for these gene expressions. This possibility is also supported by our
observation that TGF-1-stimulated binding of nuclear proteins to DR5
was inhibited markedly by H-7 treatment.
1-induced expression of RAR and RXR genes
in MC3T3-E1 cells. Thus, we examined by using actinomycin D and the
run-on assay whether the cytokine stimulates the expression of RAR,
RAR, and RXR genes at the transcriptional level. Actinomycin D
inhibited these gene expressions in TGF-1-treated cells (data not
shown), and the run-on assay showed stimulation of the transcriptional rate of these genes in the cytokine-treated cells. Since it has been
shown in MC3T3-E1 cells that TGF-1 is able to strongly induce the
expression of c-jun and c-fos genes, which encode
AP-1, it was reasonable to examine the involvement of AP-1 in the
cytokine-stimulated expression of each RA receptor. We observed that
TGF-1-stimulated expression of RAR, RAR, and RXR genes was
inhibited by antisense c-fos and c-jun
oligonucleotides. And an inhibitor of AP-1, curcumin, markedly
inhibited these genes stimulated by TGF-1. These observations suggest the involvement of AP-1 in the cytokine stimulation for expression of these genes. In fact, our gel mobility shift assay actually showed that the cytokine increased, in a
dose-dependent manner, the AP-1 binding to TRE.
1-treated cells. In doing so, we
observed that the cytokine increased, in a dose-dependent
manner, the binding of nuclear proteins to DR5. We also demonstrated by
the gel mobility shift assay, using antibody specific for each
receptor, the specificity of RAR, RAR, and RXR stimulation in
the cytokine-treated cells. These results suggest the possiblity that
TGF-1 may stimulate the RAR-RXR transcriptional activity in a
ligand-dependent manner through induction of their mRNA
expression. Furthermore, we observed that curcumin strongly inhibited
the stimulated binding of nuclear proteins to DR5 in TGF-1-treated
MC3T3-E1 cells. Thus, in consideration of all of these observations, we
suggest here that TGF-1 induces AP-1 by activation of H-7-sensitive
protein kinase and stimulates RAR, RAR, and RXR gene
expressions via AP-1 in MC3T3-E1 cells.
1 action
for proliferation and differentiation of osteoblastic cells via
stimulation of RAR-RXR transcriptional activity in a ligand-dependent fashion.
Should be considered as equal first author.
§
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:
492-85-1035.
1
The abbreviations used are: TGF-,
transforming growth factor ; RA, retinoic acid; MEM, minimum
essential medium; PMSF, phenylmethylsulfonyl fluoride; RAR, RA
receptor; RXR, retinoid X receptor; DR5, direct repeat 5; TRE,
12-O-tetradecanoylphorbol-13-acetate-responsive element;
DTT, dithiothreitol.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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  S. Tejerina, A. De Pauw, S. Vankoningsloo, A. Houbion, P. Renard, F. De Longueville, M. Raes, and T. Arnould Mild mitochondrial uncoupling induces 3T3-L1 adipocyte de-differentiation by a PPAR{gamma}-independent mechanism, whereas TNF{alpha}-induced de-differentiation is PPAR{gamma} dependent J. Cell Sci., January 1, 2009; 122(1): 145 - 155. [Abstract] [Full Text] [PDF]
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X. Tan, T. Weng, J. Zhang, J. Wang, W. Li, H. Wan, Y. Lan, X. Cheng, N. Hou, H. Liu, et al. Smad4 is required for maintaining normal murine postnatal bone homeostasis J. Cell Sci., July 1, 2007; 120(13): 2162 - 2170. [Abstract] [Full Text] [PDF]
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 S. Taschner, C. Koesters, B. Platzer, A. Jorgl, W. Ellmeier, T. Benesch, and H. Strobl Down-regulation of RXR{alpha} expression is essential for neutrophil development from granulocyte/monocyte progenitors Blood, February 1, 2007; 109(3): 971 - 979. [Abstract] [Full Text] [PDF]
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S. Vankoningsloo, A. De Pauw, A. Houbion, S. Tejerina, C. Demazy, F. de Longueville, V. Bertholet, P. Renard, J. Remacle, P. Holvoet, et al. CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes J. Cell Sci., April 1, 2006; 119(7): 1266 - 1282. [Abstract] [Full Text] [PDF]
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 S. Vankoningsloo, M. Piens, C. Lecocq, A. Gilson, A. De Pauw, P. Renard, C. Demazy, A. Houbion, M. Raes, and T. Arnould Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells: role of fatty acid {beta}-oxidation and glucose J. Lipid Res., June 1, 2005; 46(6): 1133 - 1149. [Abstract] [Full Text] [PDF]
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 S. W. Kubalak, D. R. Hutson, K. K. Scott, and R. A. Shannon Elevated transforming growth factor {beta}2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor {alpha} knockout embryos Development, January 2, 2002; 129(3): 733 - 746. [Abstract] [Full Text] [PDF]
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 E. Junn, K. N. Lee, H. R. Ju, S. H. Han, J. Y. Im, H. S. Kang, T. H. Lee, Y. S. Bae, K. S. Ha, Z. W. Lee, et al. Requirement of Hydrogen Peroxide Generation in TGF-{beta}1 Signal Transduction in Human Lung Fibroblast Cells: Involvement of Hydrogen Peroxide and Ca2+ in TGF-{beta}1-Induced IL-6 Expression J. Immunol., August 15, 2000; 165(4): 2190 - 2197. [Abstract] [Full Text] [PDF]
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 T. Shimizu and K. Takeda Induction of Retinoic Acid Receptor-{{alpha}} by Granulocyte Macrophage Colony-stimulating Factor in Human Myeloid Leukemia Cell Lines Cancer Res., August 1, 2000; 60(16): 4544 - 4549. [Abstract] [Full Text]
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 A. Takeshita, K. Imai, and S. Hanazawa CpG Motifs in Porphyromonas gingivalis DNA Stimulate Interleukin-6 Expression in Human Gingival Fibroblasts Infect. Immun., September 1, 1999; 67(9): 4340 - 4345. [Abstract] [Full Text] [PDF]
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 T. Arnould, L. Sellin, T. Benzing, L. Tsiokas, H. T. Cohen, E. Kim, and G. Walz Cellular Activation Triggered by the Autosomal Dominant Polycystic Kidney Disease Gene Product PKD2 Mol. Cell. Biol., May 1, 1999; 19(5): 3423 - 3434. [Abstract] [Full Text] [PDF]
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 O. Eickelberg, A. Pansky, R. Mussmann, M. Bihl, M. Tamm, P. Hildebrand, A. P. Perruchoud, and M. Roth Transforming Growth Factor-beta 1 Induces Interleukin-6 Expression via Activating Protein-1 Consisting of JunD Homodimers in Primary Human Lung Fibroblasts J. Biol. Chem., April 30, 1999; 274(18): 12933 - 12938. [Abstract] [Full Text] [PDF]
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K. Matsushita, R. Motani, T. Sakuta, S. Nagaoka, T. Matsuyama, K. Abeyama, I. Maruyama, H. Takada, and M. Torii Lipopolysaccharide Enhances the Production of Vascular Endothelial Growth Factor by Human Pulp Cells in Culture Infect. Immun., April 1, 1999; 67(4): 1633 - 1639. [Abstract] [Full Text] [PDF]
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C. A. Riddick, K. J. Serio, C. R. Hodulik, W. L. Ring, M. S. Regan, and T. D. Bigby TGF-{beta} Increases Leukotriene C4 Synthase Expression in the Monocyte-Like Cell Line, THP-1 J. Immunol., January 15, 1999; 162(2): 1101 - 1107. [Abstract] [Full Text] [PDF]
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 Y.-G. Chen, A. Hata, R. S. Lo, D. Wotton, Y. Shi, N. Pavletich, and J. Massagué Determinants of specificity in TGF-beta signal transduction Genes & Dev., July 15, 1998; 12(14): 2144 - 2152. [Abstract] [Full Text]
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T. Virolle, M.-N. Monthouel, Z. Djabari, J.-P. Ortonne, G. Meneguzzi, and D. Aberdam Three Activator Protein-1-binding Sites Bound by the Fra-2·JunD Complex Cooperate for the Regulation of Murine Laminin alpha 3A (lama3A) Promoter Activity by Transforming Growth Factor-beta J. Biol. Chem., July 10, 1998; 273(28): 17318 - 17325. [Abstract] [Full Text] [PDF]
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A. Takeshita, K. Imai, S. Kato, S. Kitano, and S. Hanazawa 1alpha ,25-Dehydroxyvitamin D3 Synergism toward Transforming Growth Factor-beta 1-induced AP-1 Transcriptional Activity in Mouse Osteoblastic Cells via Its Nuclear Receptor J. Biol. Chem., June 12, 1998; 273(24): 14738 - 14744. [Abstract] [Full Text] [PDF]
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J. A. Uria, M. G. Jimenez, M. Balbin, J. M. P. Freije, and C. Lopez-Otin Differential Effects of Transforming Growth Factor-beta on the Expression of Collagenase-1 and Collagenase-3 in Human Fibroblasts J. Biol. Chem., April 17, 1998; 273(16): 9769 - 9777. [Abstract] [Full Text] [PDF]
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W. Tang, L. Yang, Y.-C. Yang, S. X. Leng, and J. A. Elias Transforming Growth Factor-beta Stimulates Interleukin-11 Transcription via Complex Activating Protein-1-dependent Pathways J. Biol. Chem., March 6, 1998; 273(10): 5506 - 5513. [Abstract] [Full Text] [PDF]
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W. Wang, G. Zhou, M. C.-T. Hu, Z. Yao, and T.-H. Tan Activation of the Hematopoietic Progenitor Kinase-1 (HPK1)-dependent, Stress-activated c-Jun N-terminal Kinase (JNK) Pathway by Transforming Growth Factor beta (TGF-beta )-activated Kinase (TAK1), a Kinase Mediator of TGF beta Signal Transduction J. Biol. Chem., September 5, 1997; 272(36): 22771 - 22775. [Abstract] [Full Text] [PDF]
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A. Takeshita, A. Watanabe, Y. Takada, and S. Hanazawa Selective Stimulation by Ceramide of the Expression of the alpha Isoform of Retinoic Acid and Retinoid X Receptors in Osteoblastic Cells. A ROLE OF SPHINGOSINE 1-PHOSPHATE-MEDIATED AP-1 IN THE LIGAND-DEPENDENT TRANSCRIPTIONAL ACTIVITY OF THESE RECEPTORS J. Biol. Chem., October 6, 2000; 275(41): 32220 - 32226. [Abstract] [Full Text] [PDF]
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J. Qing, Y. Zhang, and R. Derynck Structural and Functional Characterization of the Transforming Growth Factor-beta -induced Smad3/c-Jun Transcriptional Cooperativity J. Biol. Chem., December 1, 2000; 275(49): 38802 - 38812. [Abstract] [Full Text] [PDF]
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S. Dennler, C. Prunier, N. Ferrand, J.-M. Gauthier, and A. Atfi c-Jun Inhibits Transforming Growth Factor beta -mediated Transcription by Repressing Smad3 Transcriptional Activity J. Biol. Chem., September 8, 2000; 275(37): 28858 - 28865. [Abstract] [Full Text] [PDF]
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