Acceleration of Thrombomodulin Gene Transcription by Retinoic
Acid
RETINOIC ACID RECEPTORS AND Sp1 REGULATE THE PROMOTER ACTIVITY
THROUGH INTERACTIONS WITH TWO DIFFERENT SEQUENCES IN THE 5'-FLANKING
REGION OF HUMAN GENE*
Shuichi
Horie
,
Hidemi
Ishii§,
Fumiko
Matsumoto,
Masao
Kusano,
Keiichiro
Kizaki,
Juzo
Matsuda, and
Mutsuyoshi
Kazama
From the Department of Clinical Biochemistry, Faculty of
Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui,
Kanagawa 199-0195 and the § Department of Public Health,
Showa College of Pharmaceutical Sciences, Higashi Tamagawagakuen,
Machida, Tokyo 194-8543, Japan
Received for publication, June 7, 2000, and in revised form, October 13, 2000
 |
ABSTRACT |
The interactions between retinoic acid-
(RA)-dependent transcriptional regulatory sequences of the
5'-untranslated region of the thrombomodulin gene and nuclear
RA-responsive proteins were studied using human pancreas BxPC-3 cells.
Deletion mutants of pTM-CAT plasmid revealed the presence of distal and
proximal RA-responsive regions containing direct repeat with 4 spaces
(DR4) and three of four Sp1 sites, respectively. Cotransfection of a pTM-CAT plasmid with expression plasmids of RA receptors (RAR
, RAR
, and RAR
) augmented the promoter activity under the condition of lower retinoid X receptor-
(RXR
) expression, whereas the activity was greatly diminished when RXR
was highly expressed. An
electrophoretic mobility shift assay with cDNA containing the DR4
indicated that heterodimers of RAR and RXR
interacted with the DR4
site, although the interaction gradually disappeared with the increase
in the ratio of RXR
/RAR. On the other hand, Sp1 protein interacted
especially with the tandem Sp1 site corresponding to the first and
second Sp1 sequences of the four Sp1 sites in the proximal
RA-responsive region. The binding of Sp1 to Sp1 sites was independent
of RAR-RXR heterodimer but increased with the increase in Sp1
concentration in the presence of unknown factor(s) of reticulocyte
lysate. Upon treatment of the cells with RA, time-dependent increases in the ratio of RAR
to RXR
and the phosphorylated form
of Sp1 were observed. We concluded that two genomic DNA regions, the
DR4 site (
1531 to
1516) and the first and second Sp1-binding sites
(
145 to
121), were involved in the RA-dependent
augmentation of thrombomodulin gene expression through increased
interactions of the two regions with heterodimer of RAR-RXR
and
nuclear Sp1, respectively.
 |
INTRODUCTION |
Thrombomodulin (TM)1 is
an essential cofactor for activation of protein C by thrombin on
vascular endothelial cells (1-3). TM expression of human endothelial
cells is decreased by tumor necrosis factor-
(4-6), interleukin-1
(6, 7), endotoxin (8), and phorbol ester (5, 6) and oxidized LDL (9, 10), but we have found that it is increased by
all-trans-retinoic acid (t-RA) (11, 12) and/or cAMP (11, 13)
in human umbilical vein endothelial (HUVE) cells, through the
acceleration of transcriptional activity. Several studies on the
regulatory region of human TM gene have been reported (14-17), and
Dittman et al. (17) showed the existence of an RA response
element (RARE) in the 5'-flanking region of the gene. The direct
effects of retinol and its derivatives (retinoids), such as t-RA and
9-cis-RA (9C-RA), on the expressions of many genes are
apparently mediated by nuclear receptor proteins that are members of
the steroid and thyroid hormone receptor (TR) superfamily of
transcriptional regulators (18, 19). Nuclear retinoid receptor dimers,
of which retinoid X receptor (RXR) is a mandatory constituent, are
required for effective activation of the t-RA and/or 9C-RA response
pathways (20-25). However, the direct repeats of RARE separated by 4 base sequences (DR4) at
1531 to
1516 from the transcription start
site of TM may not be a candidate for nuclear retinoid receptors,
because the DR4 sequence has been shown to be specific for a
heterodimer of RXR and TR (25). It is also known that dimers of RA
receptors such as homodimer of RXR
and heterodimers of RARs and RXR
interact with repeated RAREs spaced by 1 base (DR1) and 5 bases (DR5), respectively (26-28). Accordingly, it is unclear whether or not the
RARE of the TM gene in human cells is functionally responsive to RARs
or RXR, although Dittman et al. (17) recognized ligand dependence of RARE in the TM gene using Chinese hamster ovary cells.
On the other hand, it is well known that the transcription factor Sp1
binds to GC boxes (GGGCGG or CCCGCC) and activates transcription of a
subset of genes that contain those boxes in animal cells (29, 30). Four
Sp1-binding sites are located just upstream of the TATA box in the 5'
region of the TM gene (14-16). Darrow et al. (31) indicated
that RA-induced expression of the tissue plasminogen activator gene
during F9 teratocarcinoma cell differentiation might involve Sp1. The
binding abilities and functional significance of Sp1 for the promoters
of various genes, such as murine ornithine decarboxylase (32), tissue
factor (33), interleukin-6 (34), and prolactin receptor (35) genes,
have been reported. However, there is no direct evidence concerning
interactions between Sp1, RA receptor proteins, and the four Sp1
sequences in the TM gene, especially in the case of HUVE cells treated
with retinoids.
The results of transactivation and electrophoretic mobility shift assay
(EMSA) indicate that up-regulation of TM gene expression by retinoid is
associated with increases in the ratio of RARs to RXRs and in the
amount of Sp1, followed by enhancement of the interactions between
heterodimers of RARs-RXR
and the DR4 sequence and between Sp1 and
the consensus binding sites of the TM gene. Furthermore, our results
suggest that retinoid itself does not function directly as the
accelerator of these bindings and that the bindings of Sp1 to the Sp1
sites is dependent on the phosphorylation of the protein.
 |
EXPERIMENTAL PROCEDURES |
Materials--
pMAM-neo and pMAM-neo-CAT plasmids were purchased
from Toyobo Biochemicals, Osaka, Japan, and pGEM-4Z and
pSV-
-galactosidase control plasmids and TransFast were from
Promega. Expression plasmids pCMX-hRAR
, pCMX-hRAR
, pCMX-hRAR
,
and pCMX-hRXR
were kindly donated by Prof. R. M. Evans, Howard
Hughes Medical Institute, the Salk Institute. BxPC-3 cells, a human
pancreas primary adenocarcinoma cell line, were obtained from Dainippon
Pharmaceutical Co., Osaka, Japan. Recombinant human Sp1 and AP2, and
affinity-purified rabbit polyclonal antibodies against RARs, RXR
,
and Sp1 were purchased from Santa Cruz. [
-32P]dCTP,
the random primer extension labeling system and Renaissance Western
blot Chemiluminescence Reagent were obtained from PerkinElmer Life
Sciences. ISOGEN, t-RA, 9C-RA, Ch55, T7 RNA polymerase, and RNase
inhibitor were from Wako Pure Chemical, Osaka, Japan. Rabbit reticulocyte lysate system, [
-32P]ATP,
[
-32P]UTP, and nitrocellulose paper were obtained from
Amersham Pharmacia Biotech. Biotinylated anti-rabbit IgG and
horseradish peroxidase-conjugated streptavidin were obtained from
Vector Laboratories.
Construction of Plasmids--
The 5'-flanking DNA fragment of
the TM gene was obtained by PCR from a genomic DNA template that was
prepared from HUVE cells. The primers were designed based on the
sequences reported by Shirai et al. (14) and Dittman
et al. (17). The PCR products of 689- and 1319-bp fragments
were ligated at the XhoI site to prepare a 1702-bp TM
fragment (from
1730 to
29 with respect to the untranslated site,
which corresponds to
1562 to +140 with respect to the transcription starting site). The DNA fragments were subcloned into the pGEM-4Z vector, and the sequences were determined by the 373S DNA sequencer (Applied Biosystems Inc.). Expression plasmids, designated as pTM549-CAT and pTM1562-CAT, were constructed by inserting the TM
cDNA (689 or 1702 bp) at the SalI site of a promoterless
pMAM-CAT vector, which was prepared by the ligation of the CAT
expression fragment derived from pMAM-neo-CAT vector and the neo
gene-deleted pMAM-neo vector after deletion of the Rous sarcoma
virus-long terminal repeat sequences. The TM gene was systematically
digested with exonuclease III and mung bean nuclease after treatments
with NheI and SphI. Deletion end sites were
confirmed by DNA sequencing with the 5'-DNA primers derived from pMAM plasmid.
Preparation of Mutant Plasmids and Purification of Plasmid
DNA--
The deletion mutants of pTM-CAT plasmid were prepared by
ligation of PCR products over the mutation sites. pTM346-CAT plasmid was digested with MluI and XhoI, and then the
appropriate ligation fragment was introduced. To introduce a mutation
at the DR4 site of the gene, pTM1562-CAT plasmid was digested with
PstI and ligated after Klenow fragment treatment (the
plasmid was designated as pTM1562-CAT d(
1524/
1521)). Amplified
plasmid DNAs in JM109 cells were purified by CsCl density gradient
centrifugation in a Hitachi RP120VT rotor.
Cell Culture and Transfection--
BxPC-3 cells were cultured
into 60-mm diameter noncoated dishes with RPMI 1640 medium containing
10% fetal calf serum (FCS), and the medium was replaced with MCDB
medium without FCS at ~50% density. Plasmid DNAs treated with a
cationic liposomes, TransFast, were prepared in polystyrene tubes and
added to the cell cultures. They were allowed to stand for 2 h in
a CO2 incubator, and then the culture medium was added to
the cells. Plasmid DNA used amounted to 10 and 5 µg/dish for pTM-CATs
and pSV-
-galactosidase, respectively, and various amounts for
pCMX-RARs. 24 h after the addition of culture medium, t-RA (10 µM in 0.1% dimethyl sulfoxide
(Me2SO), final concentration) or 9C-RA (10 µM in 0.1% Me2SO, final concentration) was
added to the DNA-transfected cells for 24 h. The cells were recovered and sonicated, and the resulting supernatants were used for
determinations of CAT and
-galactosidase activities. HUVE cells were
cultured in Dulbecco's modified Eagle's medium containing 20% FCS
without endothelial cell growth supplement and heparin, and secondary
cultures on collagen-coated dishes were used for experiments (11,
36).
CAT and
-Galactosidase Assays and Measurement of TM Antigen
Level--
For measurement of CAT activity,
1-deoxy[dichloroacetyl-1-14C]chloramphenicol
was used as the substrate, and the 3'-acetylated form of
chloramphenicol was detected by autoradiography following thin layer
chromatography on a Silica Gel 60 plate using a solvent of
chloroform/methanol (19:1, v/v).
-Galactosidase activity was measured according to Promega's protocol, and the activity was used as
an internal control to normalize the transfection efficiency of
individual pTM-CAT plasmids. Total TM antigen in HUVE and BxPC-3 cells
was measured after solubilization of cells with 50 mM
Tris-HCl, pH 7.5, containing 0.15 M NaCl, 0.5% (w/v)
Triton X-100, and 1 mM benzamidine hydrochloride for 30 min
at 4 °C (12).
Northern Blot and Nuclear Run-on Analysis--
Total RNA was
isolated from HUVE and BxPC-3 cells (100-mm diameter dishes) with an
ISOGEN kit according to the recommended protocol. Northern blot
hybridization was performed as described previously (11). For RARs and
RXR
, RNAs were absorbed on a filter using a slot blotter after the
confirmation of each specific transcript on 1% denatured agarose gel
electrophoresis. A 2057-bp TM cDNA fragment (corresponding
188 to
+1869, where +1 is the first translated base, subcloned into pUC 119 at
HindIII and BamHI sites), which was cloned from a
placenta cDNA library, was used as a probe. Hybridization probes
for RAR
, RAR
, RAR
, and RXR
were obtained by treatment of
pCMX plasmids with appropriate endonucleases, whereas Sp1 (DNASIS
accession number J03133) probe was prepared by PCR using specific
primer and cDNA of HUVE cells as a template. The identification of
probes was performed by means of endonuclease treatments. The lengths
of the probes used were 823 (RAR
), 882 (RAR
), 804 (RAR
), 1074 (RXR
), and 732 bp (Sp1). Probes were labeled with
[
-32P]dCTP by using the random primer extension
labeling system. Nuclear run-on study was performed using
[
-32P]dUTP-labeled RNA of nuclear fraction of cells
treated or not treated with 10 µM t-RA for 6 h. The
labeled RNA probe was purified by DNase I and proteinase K treatments,
then absorbed on a nitrocellulose filter, and further purified with
trichloroacetic acid washing and additional DNase I treatment. The
probe eluted by SDS treatment was recovered by ethanol precipitation.
Linearized pCMX and pCMX-TM plasmids, of which the latter was
constructed by insertion of the 2.6-kb TM cDNA fragment into
HindIII and BamHI sites of pCMX, were absorbed on
a Hybond-N filter using a slot-blotter and fixed by a transilluminator.
The filter was incubated with the labeled RNA probe for 36 h at
42 °C in the presence of 40% formamide and 10% dextran sulfate.
The intensities of the signals developed by exposure of filters on
x-ray films were determined using a Fuji BAS 1500 imaging analyzer
(Fuji Photo Film Co., Tokyo, Japan) and expressed relative to the
signal of the control band.
Preparations of Nuclear Extracts and in Vitro Translation
Receptors--
BxPC-3 cells transfected with pCMX-hRARs and/or
pCMX-hRXR
were cultured for 24 h in the presence or absence of
t-RA (or 9C-RA). The cells were recovered with a cell scraper and
centrifuged at 2,000 rpm for 5 min. The resulting pellet was
homogenized in a Potter-Elvehjem type homogenizer and further
centrifuged at 2,000 rpm for 5 min to obtain the nuclear fraction. This
fraction was suspended in a buffer containing 50 mM Tris-Cl
(pH 8.3), 20% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride and stored at
80 °C until used as a nuclear extract. RAR
,
RAR
, and RXR
were also prepared in a rabbit reticulocyte lysate
translation system using linearized pCMX-hRARs and pCMX-hRXR
plasmids as templates for RNA synthesis with T7 RNA polymerase
according to the manufacturer's instruction. The amounts of translated
proteins were estimated by Western blotting after SDS-polyacrylamide
gel electrophoresis (SDS-PAGE).
EMSA and Supershift Assay--
Double-stranded oligonucleotides
(25-mer each) containing DR4 or Sp1 sites were end-labeled with
[
-32P]ATP by T4 polynucleotide kinase. The
upper strand of oligonucleotides with consensus sequences
underlined were 5'-CGTTTGGTCACTGCAGGTCAGTCCA-3' for DR4;
5'-CTGTGTTGCACGTGCAAGCTCCGTA-3' for scramble DR4;
5'-CCTGTCGGCCCCGCCCGAGAACCTC-3' for the third Sp1 site;
5'-ATCCCATGCGCGAGGGCGGGCGCAA-3' for the second Sp1 site;
5'-GCGCAAGGGCGGCCAGAGAACCCAG-3' for the first Sp1
site; and 5'-GCGAGGGCGGGCGCAAGGGCGGCCA-3'
for the tandem Sp1 sites containing second and first Sp1 sites.
The reaction mixtures for the EMSA contained 0.5 ng of labeled probe
(1 × 105 cpm/ng), 1 µg of poly(dI-dC), and 1 µl
of nuclear retinoid receptors or 1 unit of Sp1 or AP2 in a final volume
of 6 µl of 20 mM Hepes-KOH, pH 7.8, 50 mM
KCl, 5 mM MgCl2, 1 mM
ZnCl2, 0.5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM spermidine, 0.008%
Nonidet P-40, and 15% glycerol. As sources of nuclear retinoid
receptors, nuclear extracts of BxPC-3 cells transfected with pCMX-hRARs
and/or pCMX-hRXR
plasmids or in vitro translation
products were used. As a reference, the preparation obtained by using
the same procedure without any plasmid was employed. Competition
experiments were performed with a 100-fold excess of unlabeled probe.
The mixture was incubated for 30 min at 25 °C and subjected to 4%
PAGE using a buffer consisting of 10 mM Tris-HCl, pH 7.9, 1.5 mM EDTA, and 5 mM sodium acetate. For the
supershift assay, 0.5 µg of the respective antibody was further
added, and the mixture was incubated for an additional 15 min at
25 °C. After electrophoresis, the gels were dried on filter paper
and subjected to autoradiography.
Western Blot Analysis--
Nuclear extracts were subjected to
SDS-PAGE according to the method described previously (37). After
SDS-PAGE, protein was transferred to a nitrocellulose membrane and
incubated with polyclonal antibodies of RARs, RXR
, or Sp1.
Detections of RA receptors and Sp1 were carried out using biotinylated
anti-rabbit IgG and horseradish peroxidase-conjugated streptavidin, and
the resulting light emission developed by Renaissance Western blot
Chemiluminescence Reagent was captured on Kodak X-Omat autoradiography film.
 |
RESULTS |
RA-dependent Augmentation of TM Expression and
Acceleration of the Transcriptional Rate--
In the present study, we
used human pancreas BxPC-3 cells instead of HUVE cells, for the
following reasons. 1) BxPC-3 cells show less damage than HUVE cells
after treatment with cationic liposomes, one of the mildest
transfection methods (38); 2) their characteristics of
t-RA-dependent TM expression are similar to those of HUVE
cells (see below); and 3) they can be easily maintained under
homogeneous conditions. Fig. 1 shows the
changes in antigen and mRNA levels of TM in BxPC-3 cells after
treatment with t-RA. Total TM antigen in BxPC-3 cells was 14.1 ± 1.1 ng/1 × 105 cells (HUVE cells contained 17.3 ± 1.4 ng/1 × 105 cells) and increased to 2.8 times
the control on treatment with 10 µM t-RA for 24 h
(HUVE cells showed an increase to 2.5 times the control on similar
treatment) (Fig. 1A). The TM mRNA level of BxPC-3 cells
was increased by t-RA treatment and reached 3.7 times the control at 10 µM t-RA for 24 h (Fig. 1B). The time
courses of the t-RA-dependent increase in TM antigen and in
mRNA levels of BxPC-3 cells were also similar to those of HUVE
cells (data not shown). These dose- and time-dependent
increases in TM antigen and mRNA levels of BxPC-3 cells after t-RA
treatment were in accordance with the result in HUVE cells reported
previously (11). To assess the effect of t-RA on TM gene transcription
rate, a nuclear run-on study was performed on nuclei prepared from both
HUVE and BxPC-3 cells treated or not treated with 10 µM
t-RA for 6 h (Fig. 1C). Treatment with t-RA increased
the TM transcription rate to 310 and 325% of the respective control
level for HUVE and BxPC-3 cells (Fig. 1C). Furthermore,
TNF-
-dependent down-regulation of the TM antigen level
in BxPC-3 cells was counteracted by synchronized treatment with t-RA
(data not shown), as seen in HUVE cells (12).

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Fig. 1.
t-RA increases antigen and mRNA levels of
TM in BxPC-3 cells, a human pancreas carcinoma cell line. When
BxPC-3 cells cultured with RPMI 1640 medium containing 10% FCS became
confluent, t-RA was added to the culture medium at various
concentrations for 24 h and TM antigen (A) and TM
mRNA (B) levels of the cells were measured as
described under "Experimental Procedures." C, nuclear
run-on assays was performed on nuclei obtained from HUVE and
BxPC-3 cells treated with 10 µM t-RA or 0.1%
Me2SO for 6 h as described under "Experimental
Procedures."
|
|
RA-dependent Regulatory Sequence of TM Gene--
We
examined the promoter activity of TM by transient expression assay
using constructs of pTM-CAT plasmids. Although we could not clearly
identify the transcription initiation site for human TM mRNA by S1
nuclease protection analysis, a faint signal at
168 base from the ATG
codon of the translation site was consistent with one of the two sites
reported by Yu et al. (15), and thus the first untranscribed
base
1 was defined as the
169 base from the ATG codon in this
paper. Fig. 2 shows the sequence-specific promoter activity of TM as determined by measuring the CAT activity of
BxPC-3 cells transfected with pTM-CAT plasmids, which have different TM
5'-flanking sequences. Compared with the CAT activity of BxPC-3 cells
transfected with pTM1562-CAT, the activities of cells transfected with
the deletion plasmids decreased gradually when the deletions of TM
5'-flanking sequences were extended to
549. Although the CAT activity
of cells transfected with pTM549-CAT was half that in the case of
pTM1562-CAT, plasmids with more extensive deletions, such as pTM479-CAT
and pTM404-CAT, showed some recovery in CAT activity. When pTM213-CAT
was transfected, the CAT activity was markedly increased as compared
with that of pTM244-CAT, and a further deleted plasmid (pTM119-CAT)
decreased the activity to a level less than that of pTM244-CAT plasmid.
The results suggest that negative- and positive-acting elements are
present in the sequences around
244 to
214 and
213 to
120,
respectively, of the TM gene. The effect of t-RA on the promoter
activity of TM was measured (Fig. 3). The
ratio of increase of CAT activity by t-RA was different for each
plasmid used. The 4.1-fold increase of the activity by t-RA treatment
in the case of pTM1562-CAT decreased to ~2.3-fold when pTM-CAT
plasmids containing the 5'-site from
1491 to
213 of the TM sequence
were transfected, and further deletion of the 5'-sequence abolished the
t-RA-dependent increase in the activity. These results
indicate the existence of two independent RA-responsive regions (one
localized at
1562 to
1492 bp and the other localized at
213 to
120 bp of the transcription site) in the TM promoter sequence.

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Fig. 2.
Transcriptional activity of TM gene
determined by transient CAT expression assay of BxPC-3 cells.
BxPC-3 cells were transfected with pTM1562-CAT or various 5'-deletion
mutants as well as pSV- -galactosidase as described under
"Experimental Procedures." The culture medium was changed after
2 h of cationic liposome transfection, and 48 h thereafter,
activities of CAT and -galactosidase in the cell extracts were
determined. The CAT activities normalized with respect to
-galactosidase activities were expressed as a percentage of that
with pTM1562-CAT. Results are the average values of three independent
experiments. 5'-Sites of the TM promoter sequence in pTM-CAT plasmid,
in the order from left to right, are 1562,
1491, 1272, 960, 549, 479, 404, 346, 290, 244, 213,
119, 47, and +63. Representative RARE sites and four Sp1 sites in
addition to the TATA box are shown at the bottom.
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Fig. 3.
t-RA-dependent increase in
transcriptional activity of the TM gene determined by transient CAT
expression assay of BxPC-3 cells. The transfection procedure was
the same as that in Fig. 2. At 24 h after the medium change, 10 µM t-RA or its vehicle (0.1% Me2SO) was
added to the culture medium, and the cells were incubated for an
additional 24 h. The CAT activities normalized with respect to
-galactosidase activities were expressed as a percentage of that of
BxPC-3 cells treated with 0.1% Me2SO after transfection of
pTM1562-CAT plasmid. Open and closed bars show
relative CAT activities of the transformed cells treated with 0.1%
Me2SO or t-RA, respectively, and values in
parentheses on the right indicate the ratio of
the activity in each transformant. Open bars are the same
data as the values in Fig. 2. Results are the average values of three
independent experiments.
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|
To identify the RA-responsive sequences, mutations were introduced at
DR4 of RARE in the pTM1562-CAT plasmid and at two consensus Sp1 sites
in the pTM346-CAT plasmid (Fig. 4). As
shown in Fig. 4A, mutation at DR4 reduced the
t-RA-dependent increase in CAT activity of pTM1562-CAT
without extensive reduction of the normal CAT activity, and the
increase ratio became 2.4-fold, which is comparable to that in the case
of pTM1491-CAT (Fig. 3). Each deletion at the third or second Sp1 motif
in the TM promoter sequence of pTM346-CAT produced different responses
(Fig. 4B). pTM346-CAT d(
208/
201) showed a slight
decrease in the CAT activity compared with that of the wild-type
plasmid and exhibited a mild response to t-RA treatment, whereas
pTM346-CAT d(
142/
135) decreased the CAT activity, irrespective of
t-RA treatment. The reduction with the plasmid deleted at the second
Sp1 motif of the TM gene sequence reveals the significance of this
region in the primary and t-RA-dependent enhancement of the
transcriptional activity. The third Sp1 motif might contribute to
cooperative interactions of other transcription factors with Sp1
protein. On the other hand, a plasmid deleted from
221 to
214
(pTM346-CAT d(
221/
214)) enhanced the CAT activity of the
transfectant to 165% of that of pTM346-CAT, and further enhancement of
the activity was observed after treatment with t-RA. As expected from
the result in Fig. 2, this deleted region might act negatively, in
concert with an unknown factor, on the TM promoter activity.

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Fig. 4.
Deletions of DR4 and Sp1 sites in TM gene
reduce the RA-dependent increase in promoter activity of
the gene. A, pTM1562-CAT and its deletion mutant at the
DR4 site located at bases 1531 to 1516 and pTM1562-CAT
d( 1524/ 1521) were transfected into BxPC-3 cells, and the cells were
treated with 10 µM t-RA or the vehicle (0.1%
Me2SO) as described under "Experimental Procedures."
The CAT activities normalized with respect to -galactosidase
activities were expressed as a percentage of the activity of BxPC-3
cells treated with the vehicle alone after transfection of pTM1562-CAT
plasmid. Results are average values with standard deviations of three
independent experiments. The sequence on the left of
A is that from 1531 to 1516 of the human TM gene. Four
bases between the direct repeats of RARE are underlined.
B, pTM346-CAT, the third Sp1 site mutant, pTM346-CAT
d( 208/ 201), or the second Sp1 site mutant, pTM346-CAT
d( 142/ 135), and pTM346-CAT d( 221/ 214) were transfected into
BxPC-3 cells, and the cells were treated with 10 µM t-RA
or the vehicle (0.1% Me2SO) as described under
"Experimental Procedures." The CAT activities normalized with
respect to -galactosidase activities were expressed as a percentage
of that of BxPC-3 cells treated with the vehicle alone after
transfection with pTM346-CAT. Results are average values with S.D. of
three independent experiments. The sequences on the left in
B are the bases from 223 to 200 and 144 to 133 of
the human TM gene. The Sp1 sites are boxed. Numbers 3, 2, 1 or T in circles, and
diamond mean third Sp1 site, second Sp1 site, first
Sp1 site or TATA box, and deleted site, respectively. Open
column, 0.1% Me2SO-treated; closed column,
t-RA-treated.
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Expressions of RA Receptors Modulate Promoter Activity of
TM--
To examine the effect of changes in concentration of RA
receptors on the promoter activity of TM in BxPC-3 cells, expression plasmids of RA receptors were cotransfected with pTM1562-CAT plasmid. Cotransfection of the pTM-CAT plasmid and each of pCMX-hRAR
, pCMX-hRAR
, and pCMX-hRAR
plasmid into BxPC-3 cells resulted in a
significant increase in CAT expression in the absence of retinoid,
especially when more than 0.1 µg of pCMX-hRARs was cotransfected (Fig. 5). However, enhancement of the
promoter activity of the cells by t-RA or 9C-RA treatment did not
increase further after the cotransfection of each RAR expression
plasmid. As shown in Fig. 5D, cotransfection of pCMX-hRXR
and pTM1562-CAT plasmids into the cells resulted in little increase
in the promoter activity in the absence of retinoid treatment,
and a remarkable decrease in the activity was seen in the cells
transfected with more than 1 µg of pCMX-RXR
, despite treatment of
the cells with t-RA or 9C-RA. The results suggest that the
retinoid-dependent increase in TM promoter activity
involves at least two different regulations, that is retinoid
receptors-dependent and -independent pathways. RAR-RXR
heterodimers activate transcription in response to t-RA or 9C-RA by
binding to target gene response elements consisting of DR5 or DR2 (26,
39, 40), whereas RXR-RXR homodimers activate transcription in response
to 9C-RA by binding to DR1 (28, 40-42). Since the levels of activation
of TM promoter activity were the same in the cells treated with t-RA
and 9C-RA (Fig. 5), it appears that the homodimer of RXR
(RXR
-RXR
) does not function as an activator of the promoter
activity. Furthermore, suppression of the promoter activity by
RXR
-RXR
homodimer may also be excluded, because RXR
did not
bind to the DR4 element of the TM gene (see below).

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Fig. 5.
Transcriptional activation by
RAR , RAR ,
RAR , and RXR in the
presence or absence of t-RA or 9C-RA. BxPC-3 cells cultured in
60-mm dishes were cotransfected with pTM1562-CAT (10 µg) and various
amounts of expression plasmid for RARs or RXR in addition to
pSV- -galactosidase (5 µg) as described under "Experimental
Procedures." At 24 h after the medium change, 0.1%
Me2SO, 10 µM t-RA, or 10 µM
9C-RA was added to the culture medium, and the cells were further
incubated for 24 h. The CAT activities normalized with respect to
the -galactosidase activities were expressed as a percentage
of that of BxPC-3 cells neither transfected with expression
plasmid of RA receptor nor treated with t-RA or 9C-RA. Receptors
expressed are RAR (A), RAR (B), RAR
(C), and RXR (D). Open column,
0.1% Me2SO-treated; shaded column,
t-RA-treated; closed column, 9C-RA-treated.
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Effect of Concentration of Retinoid Receptor on Promoter Activity
of TM--
In the present study, RXR
was used as the representative
of RXRs, because both HUVE and BxPC-3 cells express its mRNA and protein (see the data in Figs. 10 and 11), and because it has been reported that RXR isoforms such as RXR
, RXR
, and RXR
behave similarly in heterodimeric complex formation with RARs or TR (23, 43-45). It is also reported that 9C-RA has the highest affinity for
RXR
among RXR isoforms (44). The promoter activity of pTM1562-CAT plasmid in the case of simultaneous expressions of RAR
or RAR
and
RXR
in the cells was examined (Fig.
6). The RAR
- and
RAR
-dependent increases in promoter activities were
further enhanced by cotransfection of pCMX-RXR
(0.1 µg) (Fig. 6,
C and D). However, cotransfection with
pCMX-RAR
(or pCMX-RAR
) and a higher dose of pCMX-RXR
(1 µg)
drastically reduced the promoter activity, even when 0.1 µg of
pCMX-RARs was used (Fig. 6, E and F). These
results demonstrate that TM promoter activity is controlled by the
expressions of RARs and RXR
and that the extent of the expression of
the latter is critical for gene expression of TM.

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Fig. 6.
RXR modulates
RAR - and RAR -mediated
transactivation of pTM-CAT. BxPC-3 cells cultured in 60-mm dishes
were cotransfected with pTM1562-CAT (10 µg), pSV- -galactosidase (5 µg), and various amounts of expression plasmid for RXR in addition
to that for RAR (0-5 µg) or RAR (0-5 µg). Cultures and
treatment of cells with 0.1% Me2SO were as described in
the legend to Fig. 5. The CAT activities normalized with respect to the
-galactosidase activities were expressed as a percentage of that of
BxPC-3 cells not transfected with expression plasmid for RA receptor.
Amounts of expression plasmid for RXR used were none (A
and B), 0.1 µg (C and D), and 1 µg
(E and F).
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Sequence-specific Acceleration of TM Promoter Activity by RAR-RXR
Expressions--
To confirm that RA receptors modulate TM gene
expression through the RARE element, we determined the promoter
activity of BxPC-3 cells transfected with DR4-deleted pTM1562-CAT
plasmid (pTM1562-CAT d(
1524/
1521)) or pTM346-CAT plasmid instead of pTM1562-CAT plasmid (Fig. 7). The
following observations were made. First, the promoter activity of the
cells transfected with pTM1562-CAT plasmid was increased by the
coexpression of RARs and RXR
in the absence of retinoid treatment,
as already shown in Figs. 5 and 6, whereas the promoter activity of
those transfected with pTM1562-CAT d(
1524/
1521) or pTM346-CAT was
not increased under the same conditions. Second, the promoter
activities of cells transfected with these pTM-CAT plasmids were
increased by treatment with t-RA or 9C-RA under conditions where
retinoid receptors were not expressed, but the increase ratio after
retinoid treatment was the highest when pTM1562-CAT plasmid was used.
Third, the 9C-RA-dependent increase in the promoter
activity of pTM1562-CAT in the cells, which expressed RARs and a small
amount of RXR
, was not greater than the t-RA-dependent
increase. These results suggest that 1) the interaction between
heterodimer of RAR-RXR
and the DR4 element in TM gene participates
in part in the retinoid-dependent increase in the promoter
activity of TM; 2) ligand-independent dimerization of RARs-RXR
occurs, although we cannot exclude the possibility that a small but
sufficient amount of t-RA exists in the cell in the absence of retinoid
treatment; and 3) formation of RAR-RXR rather than RXR-RXR is
significant when RXR
is not highly expressed.

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Fig. 7.
Expressions of RAR
and RXR or RAR
and RXR fail to transactivate
DR4-deleted pTM-CAT and pTM346-CAT plasmids in the absence of t-RA and
9C-RA. BxPC-3 cells cultured in 60-mm dishes were cotransfected
with a pTM-CAT plasmid (10 µg), pSV- -galactosidase (5 µg), and
0.1 µg of expression plasmid for RAR , RAR , and/or RXR .
Cultures and treatment of cells were as described in the legend to Fig.
5. The CAT activities normalized with respect to the -galactosidase
activities were expressed as a percentage of that of BxPC-3 cells
neither transfected with expression plasmid for RA receptor nor treated
with t-RA or 9C-RA. The reporter plasmid used was pTM1562-CAT
(A), pTM1562-CAT d( 1524/ 1521) (B), and
pTM346-CAT (C). Open column, 0.1%
Me2SO-treated; shaded column, t-RA-treated;
closed column, 9C-RA-treated.
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Interaction of RAR-RXR Heterodimer with DR4 Sequence of TM
Gene--
To test the idea, that RAR-RXR
heterodimer binds to the
DR4 site of the TM gene, interaction between RARs, RXR
, and a DNA probe containing the DR4 sequence of the TM gene was examined by EMSA
(Fig. 8). When a nuclear fraction of
BxPC-3 cells transfected with each pCMX-hRAR plasmid was incubated with
the probe, a retarded band was observed, regardless of the isoform,
although that of cells transfected with RXR
expression plasmid
showed only a weak band (Fig. 8A). By using a nuclear
fraction prepared from cells cotransfected with one RAR isoform and
RXR
expression plasmids, a more distinct band with the same mobility
was observed. The same band on the EMSA was also obtained when the
cDNA probe was incubated with a sample, which was prepared by
mixing the nuclear fraction expressing RAR
(or RAR
) with that
expressing RXR
(Fig. 8B, lane 3). The binding specificity
of these receptors to the radiolabeled probe corresponding to the DR4
sequence of TM gene was confirmed by competition with an excess amount
of the unlabeled cDNA probe. Moreover, these retarded bands were
further shifted in the presence of RAR
and RXR
antibodies
(lanes 7 and 9), and therefore it was
confirmed that RAR
-RXR
heterodimer bound to the consensus
sequence of the DR4 element in the TM gene. To exclude contamination
with endogenous receptors, RA receptors synthesized with a cell-free
reticulocyte lysate system were also subjected to EMSA using the same
DNA probe (Fig. 8C). Since the RAR
- or RAR
-dependent retarded band was observed in the absence
of endogenous RXR
in the cell-free system, it is possible that the
homodimer of each RAR could bind to DR4 of the TM sequence.
Furthermore, it became apparent that homodimer (or monomer) of RXR
did not bind to the DR4 site (lane 3). Specific binding of
the heterodimer binding to this DR4 sequence was further confirmed
using a scramble probe of the DR4 sequence as a competitor (lane
8).

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Fig. 8.
Binding of retinoid receptors to the DR4
element in the TM gene. EMSA was carried out using radiolabeled
oligonucleotide containing the DR4 element in the TM gene, and the
probe was incubated with nuclear extracts of BxPC-3 cells transfected
with pCMX-hRARs and/or pCMX-hRXR plasmids (A and
B) or in vitro translation products of the
respective mRNAs (C). After incubation for 30 min as
described under "Experimental Procedures," the incubation mixture
was subjected PAGE under a low salt condition. After electrophoresis,
the gels were dried on filter paper and subjected to autoradiography.
A, lanes 1 and 2, RAR expressed;
lanes 3 and 4, RAR expressed; lanes
5 and 6, RAR expressed; lanes 7 and
8, RXR expressed; lanes 9 and 10,
RAR and RXR coexpressed; lanes 11 and 12,
RAR and RXR coexpressed; lanes 13 and 14,
RAR and RXR coexpressed; lanes 15 and 16,
2-fold amounts of samples (RXR expressed) in lanes 7 and
8; even-numbered lanes, in the presence of
100-fold excess of DR4 competitor. B, lanes 1 and
2, RAR expressed; lanes 3 and
4, mixture of samples that expressed RAR and RXR
separately; lanes 5-10, RAR and RXR coexpressed;
lanes 7 and 9, in the presence of RAR
antibody; lanes 8 and 9, in the
presence of RXR antibody; lanes 2, 4, 6, and
10, in the presence of 100-fold excess of DR4 competitor.
C, lane 1, RAR ; lane 2, RAR ;
lane 3, RXR ; lanes 4 and 5, mixture
of RAR and RXR ; lanes 6-8, mixture of RAR and
RXR ; lanes 5 and 7, in the presence of
100-fold excess of DR4 competitor; lane 8, in the presence
of 100-fold excess of the scrambled DR4 probe.
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Effect of Ratio of RAR/RXR on Binding of RAR-RXR to DR4
Sequence--
Since an RXR
concentration-dependent
decrease in the promoter activity was observed in Fig. 6, the effect of
the ratio of RAR
to RXR
on interaction of the heterodimer with
the DR4 sequence was investigated (Fig.
9). Nuclear samples of BxPC cells
cotransfected with RAR
and RXR
at various ratios were prepared,
and the binding ability was examined using DR4 probe. The intensity of
the band corresponding to the retardate complex with RAR
-RXR
heterodimer initially increased with the increase in the expression of
RXR
, but a further increase in the RXR
expression apparently
decreased the intensity and no retardate band was observed at 5-fold
excess of RXR
over RAR
(Fig. 9B). When RAR
and
RXR
, prepared in a reticulocyte lysate system instead of by
cotransfection of cells with both plasmids, were mixed in the reaction
mixture at various ratios, virtually the same results were obtained
(Fig. 9B), indicating rapid formation of RAR
-RXR
dimer. These results suggest that the ratio of RARs to RXR
is
critical for not only formation of RAR-RXR heterodimer but also binding
of the heterodimer to the DR4 sequence. Since the maximum binding was
observed when a higher ratio of RXR
compared with RAR
was applied
(Fig. 8B), the formation of RXR
-RXR
heterodimer and/or
the presence of RXR
monomer may be possible under the cell-free
condition.

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Fig. 9.
Effect of ratio of RXR to RAR on the
heterodimer binding to the DR4 element. EMSA was carried out using
radiolabeled oligonucleotide containing the DR4 element in the TM gene,
and the probes were incubated with nuclear extracts of BxPC-3 cells
(A) or in vitro translation products of RAR
and RXR mRNAs (B). Experimental conditions were the
same as described in the legend to Fig. 8. A, nuclear
fractions prepared from BxPC-3 cells that were cotransfected with
pCMX-hRAR and pCMX-hRXR plasmid at different ratios as shown at
the bottom. Lane 7, in the presence of 100-fold excess of
DR4 competitor. B, samples were mixed with different ratios
of RAR and RXR as shown at the bottom. Lane
8, in the presence of 100-fold excess of DR4 competitor.
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Effect of t-RA or 9C-RA on mRNA and Protein Levels of Retinoid
Receptors--
Changes in mRNA levels of retinoid receptors in
BxPC-3 cells after treatment with t-RA or 9C-RA were measured (Fig.
10). The mRNA level of TM increased
in a time-dependent manner, which was compatible with the
results in Fig. 1. BxPC-3 cells contained mRNA of all four RA
receptors, and RAR
mRNA was markedly augmented after the
treatment with t-RA. The level of RAR
mRNA also increased within
6 h after t-RA treatment, whereas RAR
mRNA increased only slightly up to 24 h after the treatment. On the other hand, the RXR
mRNA level decreased with increase in the time after t-RA treatment. Changes in the mRNA levels of these four RA receptors in
HUVE cells after treatment with t-RA were also examined, and quite
similar results to those in BxPC-3 cells were obtained (data not
shown). The presence of RAR
and RAR
mRNAs in normal HUVE cells has already been reported by Fesus et al. (46), and
Kooistra et al. (47) observed the expression of transcripts
of three RAR and two RXR subtypes in normal HUVE cells. They also found induction of RAR
and RXR
mRNA after exposure of the cells to t-RA. Although there was definitive difference in changes in RXR
level after t-RA treatment between their results and ours, similar patterns of increase in RAR
mRNA level and decrease in RXR
mRNA level in BxPC-3 cells were observed when 9C-RA instead of t-RA was used (Fig. 10, C and D). Western blot
analysis showed that these changes in mRNA levels of RAR
and
RXR
reflected those of the protein levels in the nuclear fraction of
the cells after treatment with t-RA (Fig.
11). Thus, treatment with t-RA
gradually increased and decreased the amounts of RAR
and RXR
in
the nuclear fraction of BxPC-3 cells, respectively.

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Fig. 10.
Changes in mRNA levels of TM in BxPC-3
cells treated with t-RA. BxPC-3 cells cultured in 100-mm dishes
were treated with 10 µM t-RA for 0, 3, 6, 12, and 24 h (A and B) or treated with 10 µM
9C-RA for 0 and 24 h (C and D), and total
RNAs were prepared. mRNA levels were determined qualitatively by
performing slot blot hybridization with the respective
32P-labeled probes, and then two transcripts for RAR
( 3.0 and 3.6 kb) and RAR ( 3.5 and faint 2.8 kb) were
combined signals, respectively, in this result. The transcript for
RAR ( 3.3 kb) or RXR ( 5.0 kb) was single. A and
C, autoradiograms. B and D, the
mRNA level without incubation was defined as 1, and the relative
mRNA levels were plotted against incubation time after quantitative
analysis of the bands with a BAS 1500 imaging analyzer. Values were
normalized with respect to the levels of actin mRNA.
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Fig. 11.
Changes in antigen levels of
RAR and RXR in BxPC-3
cells treated with t-RA. BxPC-3 cells cultured in 100-mm dishes
were treated with 10 µM t-RA for 0, 6, and 24 h, and
nuclear extracts were prepared as described under "Experimental
Procedures." After SDS-PAGE, protein was transferred to a
nitrocellulose membrane and incubated with polyclonal antibodies to
RAR and RXR . A, four isoforms of RAR corresponding
56 to 47 kDa (48), as shown by an arrow, were recognized,
and a low molecular band (arrowhead) seems to be a
degradation product of RAR . B, RXR corresponding 53 kDa (49) was recognized. Detection was carried out using antibodies to
RAR and RXR followed by biotinylated anti-rabbit IgG and
horseradish peroxidase-conjugated streptavidin, and the light emission
developed was captured on an autoradiography film. Open
arrowheads in A and B indicated nonspecific
bands.
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Interaction of Nuclear Sp1 Protein with Sp1-dependent
Sequence in TM Gene--
Next we focused on Sp1 as a candidate factor
to interact with the GC box of the TM gene, and EMSA was performed
after incubation of 32P-labeled TM cDNA probes
containing these Sp1 sites with Sp1 or AP2 (the latter is also a
transcription factor that binds to GC-rich sequences (50) and has been
shown to play a role in some retinoid-affected morphogenetic processes
(51)). The promoter activity of pTM346-CAT was not influenced by
coexpression of RARs and/or RXR
(Fig. 7), but recently Suzuki
et al. (52) showed that RAR-RXR interacts physically with
Sp1 and that the complex of RAR-RXR-Sp1 binds to the GC box of the
urokinase promoter sequence. Therefore, the probe was incubated with
recombinant Sp1 in the presence or absence of both RAR and RXR prepared
in a reticulocyte lysate system, followed by electrophoresis. Fig.
12A shows that Sp1 could
form a retarded complex with labeled probes containing an Sp1-binding site in the presence of RAR
and RXR
. The intensity of the
retarded bands of cDNA probes containing the three Sp1-binding
sites were in the following order: second > third
first
Sp1 sites (lanes 2, 5, and 8, respectively).
However, these Sp1 sites were not target sequences for AP2 (data not
shown). As shown in Fig. 4, the significance of the second Sp1-binding
site among the these sites in the TM promoter region was evaluated by
measuring the promoter activity of a TM-CAT plasmid in which this
second Sp1 site was deleted. The shifted bands with slower and faster
migrations were Sp1-dependent and -independent complexes,
respectively, since the lower band with fast migration appeared in the
absence of Sp1 in the reaction mixture (lanes 1, 4,
and 7). The most intense shifted band was seen with a
cDNA probe containing tandem Sp1 sites with 6 spaces
corresponding to the second and first Sp1-binding sites of the TM gene
(lane 11).

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Fig. 12.
Sp1 interacts with the Sp1 sites of the TM
promoter gene. EMSA was carried out using
32P-end-labeled oligonucleotide (25 bp) with the consensus
sequence of the Sp1 site or a tandem Sp1 site containing the second and
the first Sp1 sites in the TM gene in the absence of t-RA. A labeled
DNA probe and recombinant Sp1 was incubated for 30 min and subjected to
PAGE as described under "Experimental Procedures." The
closed and open arrowheads indicate the
Sp1-dependent and -independent DNA-protein complexes,
respectively. A, lanes 1-3, probe ( 217 to 193)
containing the third Sp1 site; lanes 4-6, probe ( 154 to
130) containing the second Sp1 site; lanes 7-9, probe
( 135 to 111) containing the first Sp1 site; lanes
10-12, probe ( 145 to 121) containing the tandem Sp1 site
corresponding to the second and the first Sp1 sites. Probes were
incubated with recombinant Sp1 (lanes 2, 3, 5, 6, 8, 9, 11, and 12) in the presence of in vitro translation
products from RAR and RXR mRNAs by a reticulocyte lysate
system. As a competitor, 100-fold unlabeled probe was added to the
incubation mixture (lanes 3, 6, 9, and 12).
B, probe containing a tandem Sp1 site incorporating the
second and the first Sp1 sites of the TM promoter sequence was
incubated with recombinant Sp1 (lanes 1-16, 18, 21, and 22) in the presence of in vitro
translation products of RAR (lanes 1, 2, 7, and
8), RAR (lanes 3 and 4, and
9-18), and RXR (lanes 5-18) or the
reticulocyte lysate solution (RL) as a reference for these
retinoid receptors (lanes 19-22). Anti-Sp1 IgG (lanes
11, 15, and 16), anti-RAR IgG (lanes 12, 14 and 15) and anti-RXR IgG (lanes 13, 14 and 16) were added at 15 min before electrophoresis. As a
competitor, 100-fold unlabeled probe was added to the incubation
mixture (lanes 2, 4, 6, 8, 10, 20, and 22).
C, probe containing the tandem Sp1 site was incubated with
various concentrations of recombinant Sp1 (lanes 4-8 and
10-17) or AP2 (lanes 18 and 19) in
the presence of the reticulocyte lysate solution (RL)
(lanes 2-8 (1 µl) and lanes 9-19 (0.05 µl)). As a competitor, 100-fold unlabeled probe was added to the
incubation mixture (lanes 2, 15, 17, and 19).
Anti-Sp1 IgG was added in lane 16.
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|
The effect of RA receptors on the Sp1 binding to this tandem Sp1 site
was further investigated by EMSA (Fig. 12B), because a
curious result was obtained as described below. The
Sp1-dependent retarded band with slow migration was
detected on EMSA when this probe was incubated with Sp1 and one
retinoid receptor, such as RAR
, RAR
, or RXR
(lanes 1, 3 and 5), and all of the corresponding bands
disappeared upon addition of excess competitor (lanes 2, 4, and 6). However, the coexistence of RARs and RXR
did not
increase the intensity of the band in the presence of Sp1 (lanes
7 and 9), and the bands were not supershifted by the
addition of the respective retinoid receptor antibody (lanes
12-14). The supershifted band was restricted to the case of Sp1
antibody (lanes 11, 15, and 16). Surprisingly, as
shown in lane 21, the Sp1-dependent retarded
band was detected in the absence of RARs and RXR
but in the presence
of reticulocyte lysate solution as the control for all the RA
receptors. These results suggest that interaction between the Sp1 and
Sp1 sites of the TM promoter sequence is dependent on the presence of
unknown factor(s) in the reticulocyte lysate solution and that the
interaction is independent of formation of the multicomplex of
Sp1-RAR-RXR
. Therefore, we concluded that homodimer and heterodimer
of retinoid receptors do not participate directly or indirectly in the
interaction between Sp1 and the tandem Sp1 sites of TM gene. This is
consistent with the finding in Fig. 7 that expressions of RARs and/or
RXR
did not induce transactivation activity of pTM346-CAT, which
contains all four Sp1 sites of the TM gene. Nevertheless, it is true
that Sp1 binds specifically to these Sp1 probes, because all the
retarded bands were eliminated by an excess of each unlabeled probe.
Further increase in the intensity of the retarded band on EMSA was
directly proportional to the concentration of Sp1 in the presence of a small amount of reticulocyte lysate solution (Fig. 12C,
lanes 3-8 and 9-14). In addition, the retarded
band with slower migration was supershifted in the presence of the
specific antibody of Sp1 (lane 16), and it was also
confirmed that AP2 was unable to form a retarded complex with the same
cDNA probe (lanes 18). Binding of Sp1 to Sp1 sites of
the TM sequence was also independent of the presence of t-RA in EMSA
(data not shown).
RA-dependent Changes in mRNA and Protein Levels of
Sp1--
The time course of changes in mRNA level of Sp1 after
treatment of BxPC-3 and HUVE cells with t-RA was examined (Fig.
13). The Sp1 mRNA was expressed not
only in BxPC-3 cells but also in HUVE cells. Sp1 mRNA increased
rapidly after treatment of BxPC-3 cells with t-RA, and the maximum
increase was ~2.5-fold from the control level. The increase in the
ratio and the time course of change of each mRNA level in HUVE
cells after treatment with t-RA were almost the same as those in BxPC-3
cells. The time course of changes in Sp1 content after treatment of
BxPC-3 cells with t-RA was examined (Fig. 13C). The
appearance of a 105-kDa band, a phosphorylated form of Sp1 (53), was
observed, in addition to a rapid increase in the 95-kDa Sp1, the
dephosphorylated form (53). Upon alkaline phosphatase treatment of a
sample, disappearance of the 105-kDa band followed by increase in the
95-kDa band was observed, indicating the existence of the two forms in
the cells. These results suggest that treatment of human cells with
t-RA modulates the TM gene promoter activity in part through increasing Sp1 content and phosphorylation in the nuclear fraction.

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Fig. 13.
t-RA up-regulates mRNA and protein
levels of Sp1 in BxPC-3 and HUVE cells. A and
B, BxPC-3 and HUVE cells cultured in 100-mm diameter dishes
were treated with 10 µM t-RA for 0, 3, 6, and 24 h.
Total RNA was prepared, and Northern blot hybridization was performed
using Sp1 and -actin probes after 1% denatured agarose gel
electrophoresis. Two transcripts for Sp1 ( 8.0 and 5.5 kb) were
detected. C, nuclear fraction of BxPC-3 cells treated with
t-RA until 24 h was prepared, and SDS-PAGE was performed. Proteins
transferred to a nitrocellulose membrane were incubated with polyclonal
antibody of Sp1 as described under "Experimental Procedures." Two
bands corresponding to 95 and 105 kDa are the dephosphorylated and
phosphorylated forms, respectively, of Sp1 (53).
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|
 |
DISCUSSION |
In this paper we have shown that t-RA-dependent
enhancement of human TM gene expression is regulated by two sites in
the 5'-flanking region. The t-RA-responsive distal region of the TM
gene contains an RARE composed of TGGTCANNNNAGGTCA. Our transient
expression studies revealed that the TM promoter could be
transactivated by the human RARs, especially by RAR
and RAR
. On
the basis of transient expression assay using F9 embryonal carcinoma
cells, Weiler-Guettler et al. (54) suggested that the TM
promoter could be activated by RAR
. By the use of an RAR
antagonist, on the other hand, Shibakura et al. (55)
obtained results indicating a major role of RAR
in TM up-regulation
by retinoids in leukemia and HUVE cells. Treatment with t-RA (or 9C-RA)
in the present study, however, caused a much greater increase in the
RAR
level compared with that of RAR
, and no difference was
observed between RAR
- and RAR
-dependent
transactivation of TM promoter activity. Furthermore, the binding
ability of RAR
and RAR
with the DR4 sequence of the TM gene in
the presence or absence of RXR
was the same when equivalent amounts
of both RAR receptors were used. Therefore, the increase in the total
amount of all the RARs might be a dominant factor for the up-regulation
of transcriptional activity of TM, rather than the changes in the
concentrations of individual RAR subtypes. Coexpression of RXR
with
RAR
or RAR
showed a different mode of promoter activity compared
with that in the case of expression of a single RAR receptor. When RXR
was highly expressed in BxPC-3 cells, the increase in the transactivation activity by RAR
or RAR
was eliminated, whereas it
was augmented at a lower expression level of the RXR
receptor. Furthermore, the binding of RAR
-RXR
to RARE of the TM gene was closely related to the proportion of RAR
/RXR
, and the levels of
RARs increased in both BxPC-3 and HUVE cells after t-RA or 9C-RA
treatment, whereas that of RXR
decreased under the same treatment.
It seems very probable that t-RA and 9C-RA contributed to the
acceleration of TM promoter activity through both an increase in RARs
and a decrease in RXR
, followed by appropriate formation of
RARs-RXR
heterodimers that can bind to the DR4 site of the TM gene.
In this connection, we have examined the changes in the levels of RARs
and RXR
of HUVE cells after treatment with a synthetic RARs-selective retinoid, Ch55. The results supported the above finding
that an increase in TM promoter activity is closely related to the
increasing ratio of RARs/RXR
.
To access the action of t-RA on the DR4 site-dependent
enhancement of promoter activity of TM, a ligand-dependent
change in the binding of RAR
-RXR
to the DR4 site of the TM gene
was investigated. In our EMSA experiment, essentially no increase in
the intensity of the retarded band of the DR4 probe was observed in the
presence of t-RA and/or 9C-RA in the reaction mixture, although a small increase in the intensity was observed at higher concentrations (10
6 and 10
5
M) (data not shown). Therefore, it is plausible that t-RA
and 9C-RA are not stimulators of the interaction between the
heterodimer and DR4 of the TM gene, in a similar manner to the Sp1 and
Sp1 sites of the TM gene (see below), although we cannot exclude the possibility that both retinoids act as accelerators of the heterodimer formation of RAR-RXR, regardless of the presence or absence of the
template DNA.
On the other hand, the protein that interacts with the proximal
sequence for transcriptional up-regulation appeared to be Sp1. Among
the four Sp1 sites in the 5' region from the TATA box of the gene, the
major role of the second Sp1 site, rather than the other sites, was
found. Although it has been shown that one Sp1 site is sufficient for
the Sp1-dependent augmentation of the promoter activities
of cytochrome P450IA1 (56) and
2(I) collagen (57), there was a
significant cooperative role of the second Sp1 site with the other
adjacent Sp1 sites of the TM gene, especially the first Sp1 site. It is
probable that multiple or adjacent Sp1 sites in the TM gene are
essential for the maximal activation of the promoter, as reported for
other genes (32, 58-61). It is known that the binding and subsequent
transactivation activities of Sp1 can be modulated by
post-translational modification, i.e. glycosylation (62, 63)
and phosphorylation (53, 64-66). After t-RA treatment, the
phosphorylated form of Sp1 (105 kDa) was increased (Fig. 13), and the
binding activity of Sp1 to the consensus Sp1 site of the TM gene was
lost when reticulocyte lysate in the reaction mixture was previously
heat-inactivated (data not shown). These results suggest that the
phosphorylation of Sp1 is important for the interaction between Sp1 and
these Sp1 sites of the TM sequence. In our preliminary experiment, the
binding of Sp1 to the Sp1 sites of the TM gene was increased or
decreased after Sp1 was phosphorylated or dephosphorylated,
respectively. Further studies on the functional regulation of TM
promoter activity by such modifications of the Sp1 molecule in HUVE
cells are in progress in our laboratory. In addition, the effects of
Sp1-related factors, which recognize GC boxes, on the promoter activity
should be determined, because some papers indicated that Sp1-mediated
promoter activation was accelerated and attenuated by Sp1-related
family members (58, 67, 69).
It could be assumed that interaction between Sp1 and RARs-RXR
does
not serve as a cooperative process for Sp1 site-dependent promoter activity of TM. However, we cannot exclude the possible formation of a complex between Sp1 and RAR-RXR, which binds to sites
other than Sp1 sequences of the TM gene. Synergistic action of Sp1 with
glucocorticoid receptor (70) or estrogen receptor (71, 72) has been
reported. Krey et al. (61) demonstrated that Sp1 was not
able to interact synergistically with peroxisome proliferator-activated
receptor-RXR, although these three nuclear proteins could bind
simultaneously to the acyl-CoA oxidase gene. However, in our
experiment, an increase in the TM transcription activity by 9C-RA was
not further elevated by the synchronous treatment of HUVE cells with
9C-RA and TR activator (T3) or activators of PPAR
subtypes such as Wy-14,643 and BRL49653 (data not shown). RXR can form
heterodimers with many partners, including orphan receptors (for
reviews, see Refs. 73-75), and thus it has been accepted that RXR is a
key regulator of various cellular events in
receptor-dependent signaling. Since the total amount of all the RXR subtypes was larger than that of all the RAR subtypes (68), the
transcriptional activity of TM might be affected by changes in the
amounts of various nuclear proteins that could form heterodimers with
RXRs under various conditions.
 |
FOOTNOTES |
*
This work was supported in part by a Grant-in-aid from the
Ministry of Education, Science, and Culture of Japan 06672202 and by a
grant from the Japan Foundation of Cardiovascular Research.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 Clinical
Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Tsukui, Kanagawa 199-0195, Japan. Tel.: 81-426-85-3757; Fax: 81-426-85-2577; E-mail: shorie-v@pharm.
teikyo-u.ac.jp.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M004942200
 |
ABBREVIATIONS |
The abbreviations used are:
TM, thrombomodulin;
t-RA, all-trans-retinoic acid;
9C-RA, 9-cis-retinoic acid;
CAT, chloramphenicol acetyltransferase;
RAR, retinoic acid receptor;
RXR, retinoid X receptor;
RARE, retinoic
acid response element;
TR, thyroid hormone receptor;
DR4, direct
repeats of RARE containing 4 base;
HUVE, human umbilical vein
endothelial;
FCS, fetal calf serum;
Me2SO, dimethyl
sulfoxide;
EMSA, electrophoretic mobility shift assay;
PAGE, polyacrylamide gel electrophoresis;
bp, base pair;
kb, kilobase pair;
PCR, polymerase chain reaction.
 |
REFERENCES |
1.
|
Esmon, C. T.,
and Owen, W. G.
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
2249-2252[Abstract]
|
2.
|
Esmon, C. T.
(1989)
J. Biol. Chem.
264,
4743-4746[Free Full Text]
|
3.
|
Dittman, W. A.,
and Majerus, P. W.
(1990)
Blood
75,
329-336[Medline]
[Order article via Infotrieve]
|
4.
|
Nawroth, P. P.,
and Stern, D. M.
(1986)
J. Exp. Med.
163,
740-745[Abstract]
|
5.
|
Scarpati, E. M.,
and Sadler, J. E.
(1989)
J. Biol. Chem.
264,
20705-20713[Abstract/Free Full Text]
|
6.
|
Archipoff, G.,
Beretz, A.,
Freyssinet, J. M.,
Klein-Soyer, C.,
Brisson, C.,
and Cazenave, J. P.
(1991)
Biochem. J.
273,
679-684[Medline]
[Order article via Infotrieve]
|
7.
|
Nawroth, P. P.,
Handley, D. A.,
Esmon, C. T.,
and Stern, D. M.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
3460-3464[Abstract]
|
8.
|
Moore, K. L.,
Andreoli, S. P.,
Esmon, N. L.,
Esmon, C. T.,
and Bang, N. U.
(1987)
J. Clin. Invest.
79,
124-130[Medline]
[Order article via Infotrieve]
|
9.
|
Weis, J. R.,
Pitas, R. E.,
Wilson, B. D.,
and Rogers, G. M.
(1991)
FASEB J.
5,
2459-2465[Abstract/Free Full Text]
|
10.
|
Ishii, H.,
Kizaki, K.,
Horie, S.,
and Kazama, M.
(1996)
J. Biol. Chem.
271,
8458-8465[Abstract/Free Full Text]
|
11.
|
Horie, S.,
Kizaki, K.,
Ishii, H.,
and Kazama, M.
(1992)
Biochem. J.
281,
149-154[Medline]
[Order article via Infotrieve]
|
12.
|
Ishii, H.,
Horie, S.,
Kizaki, K.,
and Kazama, M.
(1992)
Blood
80,
2556-2562[Abstract]
|
13.
|
Ishii, H.,
Kizaki, K.,
Uchiyama, H.,
Horie, S.,
and Kazama, M.
(1990)
Thromb. Res.
59,
841-850[Medline]
[Order article via Infotrieve]
|
14.
|
Shirai, T.,
Shiojiri, S.,
Ito, H.,
Yamamoto, S.,
Kusumoto, H.,
Deyashiki, Y.,
Maruyama, I.,
and Suzuki, K.
(1988)
J. Biochem. (Tokyo)
103,
281-285[Abstract]
|
15.
|
Yu, K.,
Morioka, H.,
Fritza, L. M. A.,
Beeler, D. L.,
Jackman, R. W.,
and Rosenberg, R. D.
(1992)
J. Biol. Chem.
267,
23237-23247[Abstract/Free Full Text]
|
16.
|
Tazawa, R.,
Hirosawa, S.,
Suzuki, K.,
Hirokawa, K.,
and Aoki, N.
(1993)
J. Biochem (Tokyo)
113,
600-606[Abstract]
|
17.
|
Dittman, W. A.,
Nelson, S. T.,
Greer, P. K.,
Horton, E. T.,
Palomba, M. L.,
and McCachren, S. S.
(1994)
J. Biol. Chem.
269,
16925-16932[Abstract/Free Full Text]
|
18.
|
Petkovich, M.,
Brand, N. J.,
Krust, A.,
and Chambon, P.
(1987)
Nature
330,
444-450[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Giguere, V.,
Ong, E. S.,
Segui, P.,
and Evans, R. M.
(1987)
Nature
330,
624-629[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Yu, V. C.,
Delsert, C.,
Andersen, B.,
Holloway, J. M.,
Devary, O. V.,
Naar, A. M.,
Kim, S. Y.,
Boutin, J.-M.,
Glass, C. K.,
and Rosenfeld, M. G.
(1991)
Cell
67,
1251-1266[Medline]
[Order article via Infotrieve]
|
21.
|
Zhang, X.-K.,
Hoffmann, B.,
Tran, P. B-V.,
Graupner, G.,
and Pfahl, M.
(1992)
Nature
355,
441-446[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Kliewer, S. A.,
Umesono, K.,
Mangelsdorf, D. J.,
and Evans, R. M.
(1992)
Nature
355,
446-449[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Leid, M.,
Kastner, P.,
Lyons, R.,
Nakshatri, H.,
Saunders, M.,
Zacharewski, T.,
Chen, J.-Y.,
Staub, A.,
Garnier, J.-M.,
Mader, S.,
and Chambon, P.
(1992)
Cell
68,
377-395[Medline]
[Order article via Infotrieve]
|
24.
|
Bugge, T. H.,
Pohl, J.,
Lonnoy, O.,
and Stunnenberg, H. G.
(1992)
EMBO J.
11,
1409-1418[Abstract]
|
25.
|
Marks, M. S.,
Hallenbeck, P. L.,
Nagata, T.,
Segars, J. H.,
Appella, E.,
Nikodem, V. M.,
and Ozato, K.
(1992)
EMBO J.
11,
1419-1435[Abstract]
|
26.
|
Umesono, K.,
Murakami, K. K.,
Thompson, C. C.,
and Evans, R. M.
(1991)
Cell
65,
1255-1266[Medline]
[Order article via Infotrieve]
|
27.
|
Naar, A. M.,
Boutin, J. M.,
Lipkin, S. M., Yu, V. C.,
Holloway, J. M.,
Glass, C. K.,
and Rosenfeld, M. G.
(1991)
Cell
65,
1267-1279[Medline]
[Order article via Infotrieve]
|
28.
|
Mangelsdorf, D. J.,
Umesono, K.,
Kliewer, S. A.,
Borgmeyer, U.,
Ong, E. S.,
and Evans, R. M.
(1991)
Cell
66,
555-561[Medline]
[Order article via Infotrieve]
|
29.
|
Dynan, W. S.,
and Tjian, R.
(1983)
Cell
35,
79-87[Medline]
[Order article via Infotrieve]
|
30.
|
Kadonaga, J. T.,
Carner, K. R.,
Masiarz, F. R.,
and Tjian, R.
(1987)
Cell
51,
1079-1090[Medline]
[Order article via Infotrieve]
|
31.
|
Darrow, A. L.,
Rickles, R. J.,
Pecorino, L. T.,
and Strickland, S.
(1990)
Mol. Cell. Biol.
10,
5883-5893[Medline]
[Order article via Infotrieve]
|
32.
|
Kumar, A. P.,
Mar, P. K.,
Zhao, B.,
Montgomery, R. L.,
Kang, D.-C.,
and Butler, A. P.
(1995)
J. Biol. Chem.
270,
4341-4348[Abstract/Free Full Text]
|
33.
|
Cui, M.-Z.,
Parry, G. C. N.,
Oeth, P. O.,
Larson, H.,
Smith, M.,
Huang, R.-P.,
Adamson, E. D.,
and Mackman, N.
(1996)
J. Biol. Chem.
271,
2731-2739[Abstract/Free Full Text]
|
34.
|
Kang, S.-H.,
Brown, D. A.,
Kitajima, I.,
Xu, X.,
Heidenreich, O.,
Gryaznov, S.,
and Nerenberg, M.
(1996)
J. Biol. Chem.
271,
7330-7335[Abstract/Free Full Text]
|
35.
|
Hu, Z.-Z.,
Zhuang, L.,
Meng, J.,
and Dufau, M. L.
(1998)
J. Biol. Chem.
273,
26225-26235[Abstract/Free Full Text]
|
36.
|
Horie, S.,
Ishii, H.,
Hara, H.,
and Kazama, M.
(1994)
Biochem. J.
301,
683-691[Medline]
[Order article via Infotrieve]
|
37.
|
Hamada, H.,
Ishii, H.,
Sakyo, K.,
Horie, S.,
Nishiki, K.,
and Kazama, M.
(1995)
Blood
86,
225-233[Abstract/Free Full Text]
|
38.
|
Gao, X.,
and Huang, L.
(1991)
Biochem. Biophys. Res. Commun.
179,
280-285[Medline]
[Order article via Infotrieve]
|
39.
|
Rusten, L. S.,
Dybedal, I.,
Blomhoff, R.,
Smeland, E. B.,
and Jacobsen, S. E. W.
(1996)
Blood
87,
1728-1736[Abstract/Free Full Text]
|
40.
|
Zhang, X.-K.,
Lehmann, J.,
Hoffmann, B.,
Dawson, M. I.,
Cameron, J.,
Graupner, G.,
Hermann, T.,
Tran, P.,
and Pfahl, M.
(1992)
Nature
358,
587-591[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Lehmann, J. M.,
Jong, L.,
Fanjul, A.,
Cameron, J. F.,
Ping, L. X.,
Haefner, P.,
Dawson, M. I.,
and Pfahl, M.
(1992)
Science
258,
1944-1946[Medline]
[Order article via Infotrieve]
|
42.
|
Kliewer, S. A.,
Umesono, K.,
Heyman, R. A.,
Mangelsdorf, D. J.,
Dyck, J. A.,
and Evans, R. M.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
1448-1452[Abstract]
|
43.
|
Nagpal, S.,
Friant, S.,
Nakshatri, H.,
and Chambon, P.
(1993)
EMBO J.
12,
2349-2360[Abstract]
|
44.
|
Mangelsdorf, D. J.,
Borgmeyer, U.,
Heyman, R. A.,
Zhou, J. Y.,
Ong, E. S.,
Oro, A. E.,
Kakizuka, A.,
and Evans, R. M.
(1992)
Genes Dev.
6,
329-344[Abstract]
|
45.
|
Fawell, S. E.,
Lees, J. A.,
White, R.,
and Parker, M. G.
(1990)
Cell
60,
953-963[Medline]
[Order article via Infotrieve]
|
46.
|
Fesus, L.,
Nagy, L.,
Basilion, J. P.,
and Davies, J. A.
(1991)
Biochem. Biophys. Res. Commun.
179,
32-38[Medline]
[Order article via Infotrieve]
|
47.
|
Kooistra, T.,
Lansink, M.,
Arts, J.,
Sitter, T.,
and Toet, K.
(1995)
Eur. J. Biochem.
232,
425-432[Abstract]
|
48.
|
Rochette, E. C.,
Gaub, M. P.,
Lutz, Y.,
Ali, S.,
Scheuer, I.,
and Chambon, P.
(1992)
Mol. Endocrinol.
6,
2197-2209[Abstract]
|
49.
|
Duprez, E.,
Lillehaug, J. R.,
Gaub, M. P.,
and Lanotte, M.
(1996)
Oncogene
12,
2443-2450[Medline]
[Order article via Infotrieve]
|
50.
|
Mitchell, P. J.,
Wang, C.,
and Tjian, R.
(1987)
Cell
50,
847-861[Medline]
[Order article via Infotrieve]
|
51.
|
Mitchell, P. J.,
Timmons, P. M.,
Hebert, J. M.,
Rigby, P. W. J.,
and Tjian, R.
(1991)
Genes Dev.
5,
105-119[Abstract]
|
52.
|
Suzuki, Y.,
Shimada, J.,
Shudo, K.,
Matsumura, M.,
Crippa, M. P.,
and Kojima, S.
(1999)
Blood
93,
4264-4276[Abstract/Free Full Text]
|
53.
|
Jackson, S. P.,
MacDonald, J. J.,
Lees-Miller, S.,
and Tjian, R.
(1990)
Cell
63,
155-165[Medline]
[Order article via Infotrieve]
|
54.
|
Weiler-Guettler, H., Yu, K.,
Soff, G.,
Gudas, L. J.,
and Rosenberg, R. D.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
2155-2159[Abstract]
|
55.
|
Shibakura, M.,
Koyama, T.,
Saito, T.,
Shudo, K.,
Miyasaka, N.,
Kamiyama, R.,
and Hirosawa, S.
(1997)
Blood
90,
1545-1551[Abstract/Free Full Text]
|
56.
|
Imataka, H.,
Sogawa, K.,
Yasumoto, K.,
Kikuchi, Y.,
Sasano, K.,
Kobayashi, A.,
Hayami, M.,
and Fujii-Kuriyama, Y.
(1992)
EMBO J.
11,
3663-3671[Abstract]
|
57.
|
Tamaki, T.,
Ohnishi, K.,
Hartl, E.,
LeRoy, C.,
and Trojanowska, M.
(1995)
J. Biol. Chem.
270,
4299-4304[Abstract/Free Full Text]
|
58.
|
Pascal, E.,
and Tjian, R.
(1990)
Genes Dev.
5,
1646-1656[Abstract]
|
59.
|
Hagen, G.,
Dennig, J.,
Preiß, A.,
Beato, M.,
and Suske, G.
(1995)
J. Biol. Chem.
270,
24989-24994[Abstract/Free Full Text]
|
60.
|
Li, R.,
Hodny, Z.,
Luciakova, K.,
Barath, P.,
and Nelson, B. D.
(1996)
J. Biol. Chem.
271,
18925-18930[Abstract/Free Full Text]
|
61.
|
Krey, G.,
Mahfoudi, A.,
and Wahli, W.
(1995)
Mol. Endocrinol.
9,
219-231[Abstract]
|
62.
|
Jackson, S. P.,
and Tjian, R.
(1988)
Cell
55,
125-133[Medline]
[Order article via Infotrieve]
|
63.
|
Su, K.,
Roos, M. D.,
Yang, X.,
Han, I.,
Paterson, A. J.,
and Kudlow, J. E.
(1999)
J. Biol. Chem.
274,
15194-15202[Abstract/Free Full Text]
|
64.
|
Leggett, R. W.,
Armstrong, S. A.,
Barry, D.,
and Mueller, C. R.
(1995)
J. Biol. Chem.
270,
25879-25884[Abstract/Free Full Text]
|
65.
|
Zutter, M. M.,
Ryan, E. E.,
and Painter, A. D.
(1997)
Blood
90,
678-689[Abstract/Free Full Text]
|
66.
|
Rohlff, C.,
Ahmad, S.,
Borellini, F.,
Lei, J.,
and Glazer, R. I.
(1997)
J. Biol. Chem.
272,
21137-21141[Abstract/Free Full Text]
|
67.
|
Hagen, G.,
Müller, S.,
Beato, M.,
and Suske, G.
(1994)
EMBO J.
13,
3843-3851[Abstract]
|
68.
|
Ulven, S. M.,
Natarajan, V.,
Holven, K. B.,
Lovdal, T.,
Berg, T.,
and Blomhoff, R.
(1998)
Eur. J. Cell Biol.
77,
111-116[Medline]
[Order article via Infotrieve]
|
69.
|
Hata, Y.,
Duh, E.,
Zhang, K.,
Robinson, G. S.,
and Aiello, L. P.
(1998)
J. Biol. Chem.
273,
19294-19303[Abstract/Free Full Text]
|
70.
|
Strahle, U.,
Schmid, W.,
and Schutz, G.
(1988)
EMBO J.
7,
3389-3395[Abstract]
|
71.
|
Schule, R.,
Muller, M.,
Kaltschmidt, C.,
and Renkawitz, R.
(1988)
Science
242,
1418-1420[Medline]
[Order article via Infotrieve]
|
72.
|
Duan, R.,
Porter, W.,
and Safe, S.
(1998)
Endocrinology
139,
1981-1990[Abstract/Free Full Text]
|
73.
|
Giguere, V.
(1994)
Endocr. Rev.
15,
61-77[Medline]
[Order article via Infotrieve]
|
74.
|
Mangelsdorf, D. J.,
and Evans, R. M.
(1995)
Cell
83,
841-850[Medline]
[Order article via Infotrieve]
|
75.
|
Gronemeyer, H.,
and Laudet, V.
(1995)
Protein Profile
2,
1173-1308[Medline]
[Order article via Infotrieve]
|
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