Transactivation via RAR/RXR-Sp1 Interaction: Characterization of Binding Between Sp1 and GC Box Motif
Jun Shimada,
Yasuhiro Suzuki,
Seong-Jin Kim,
Pi-Chao Wang,
Masatoshi Matsumura and
Soichi Kojima
Laboratory of Molecular Cell Sciences, Tsukuba Institute, RIKEN,
Koyadai, Tsukuba, Ibaraki 305-0074, Japan (J.S., Y.S., S.K.); Institute
of Applied Biochemistry, University of Tsukuba, Tennoudai, Tsukuba,
Ibaraki 305-0006, Japan (J.S., Y.S., P.-C.W., M.M.); and Laboratory of
Cell Regulation and Carcinogenesis, Division of Basic Science, National
Cancer Institute, National Institute of Health, Bethesda, Maryland
20892 (S.-J.K.)
Address all correspondence and requests for reprints to: Soichi Kojima, Ph.D., Laboratory of Molecular Cell Sciences, Tsukuba Institute, RIKEN, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. E-mail:
kojima{at}rtc.riken.go.jp
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ABSTRACT
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Modulation of Sp1 activity by nuclear receptors is a novel
mechanism by which fat-soluble hormones regulate gene expression. We
previously established that upon autoinduction of RARs by RA, RARs/RXRs
physically interact with Sp1, potentiate Sp1 binding to the GC box
motifs, and thus enhance transactivation of the urokinase promoter,
which lacks a canonical RAR-responsive element/RXR-responsive
element. Here, we examined whether a similar mechanism might
participate in transcriptional regulation of other key RA-inducible
genes in endothelial cells and characterized binding between Sp1 and GC
box motifs. Northern blot analyses showed that in addition to
urokinase, after induction of RARs, RA up-regulates GC-rich
region-dependent mRNA expression of transglutaminase, TGFß1, and
types I and II TGFß receptors. RA failed to alter the expression of
Sp1 at both mRNA and protein levels. Reporter and gel shift assays and
Western blot analyses suggested that either RA-treatment or
RAR/RXR-overexpression enhances transactivation of these genes through
a GC-rich region and strengthens the affinity of Sp1 to GC box motifs,
accompanying a potential conformational change of Sp1 as reflected in
its increased immunogenicity. Detailed analyses of the GC box motifs
within the urokinase and other promoters indicate that interaction
between RAR/RXR and Sp1 does not occur in the presence of nonfunctional
GC box motifs containing five tandem purine or pyrimidine bases at the
3'-flanking region of hexanucleotide core sequence. These findings
provide insight into the molecular mechanisms underlying
RARE/RXRE-independent transactivation of RA-inducible gene
promoters.
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INTRODUCTION
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RETINOL (VITAMIN A) and its
derivatives (retinoids) exert profound effects on the regulation of
cell growth and differentiation, mainly through two families of nuclear
receptors, the RARs and the RXRs (1, 2). These receptors
belong to a family of nuclear hormone receptors (NHRs) and are
ligand-dependent transcription factors that bind to
cis-acting DNA sequences, called RAR-responsive elements
(RAREs) and RXR-responsive elements (RXREs), located in the promoter
region of their target genes (1, 2). Expression of RARs
increases in an autocrine manner after stimulation with RA because
these receptors contain a typical RARE sequence (1, 2, 3).
RARs bind to the RARE in response to both all-trans-RA
(atRA) and 9-cis-RA (9cRA), whereas RXRs bind and activate
transcription in response to only 9cRA. RARs/RXRs, upon binding to
ligands, promote transcription through interaction with coactivators
such as steroid receptor coactivator-1, GR-interacting protein
1, p300/cAMP-response element binding protein (CREB)-binding
protein (CBP) and p300/CBP co-integrator associate protein after
dissociation from corepressors such as nuclear receptor corepressor and
silencing mediator of retinoid and thyroid hormone receptor
(4, 5, 6). However, not all RA-inducible genes contain
RARE/RXRE sequence(s) within their promoter. For example, the urokinase
(UK) gene has no canonical RARE/RXRE sequence within its promoter
(7).
Recently, NHRs have been shown to modulate activity of the ubiquitous
transcription factor Sp1, leading to Sp1-mediated responses to NHR
signaling in the absence of direct NHR interactions with the target
genes (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). We reported a physical interaction between
RAR/RXR and Sp1 as the second step in a molecular pathway by which RA
induces the UK gene in vascular endothelial cells (17). RA
first induces the expression of RARs through RARE (1, 2, 3).
RARs then physically interact with Sp1 with the help of RXR and
potentiate binding of Sp1 to GC box motifs within the UK promoter,
leading to Sp1-mediated induction of UK gene expression. RARs and RXRs
have an equivalent ability to modulate Sp1 activity, and in
vivo they appear to act additively (17). Whereas the
first step is ligand-dependent, the subsequent steps appear to be
ligand-independent, at least for the transactivation of naked DNA
transfected into the cells (17). Similar interactions with
Sp1 were originally found for other NHRs, especially ER
(8, 9, 10, 11, 12).
Sp1 binds to canonical GGGCGG or its atypical hexanucleotide sequence,
called "GC box" motif, of several cellular and viral genes and
activates transcription of these genes by RNA polymerase II (19, 20). The canonical GC box motif has higher affinity to Sp1 than
atypical GC box motifs containing one or two substitutions in the
hexanucleotide sequence (21). It is of interest to know
whether any GC box motifs in any genes can serve as targets for
RAR/RXR-Sp1 interaction and are responsible for the induction by RA.
Potentiation of Sp1 binding is observed also for a consensus GC box,
implying that the mechanism might be universal (17). RA
induces various genes in vascular endothelial cells
(22, 23, 24, 25, 26, 27). For example, in bovine aortic endothelial cells
(BAECs), in addition to UK, RA induces transglutaminase (TGase)
(23), TGFß1 (24), and types I and II TGFß
receptors (TGFß RI and RII) (26). Namely, RA enhances
fibrinolytic levels through rapid stimulation of the expression of UK
and related genes, resulting in induction of active TGFß, which
subsequently mediates some of the actions of RA in BAECs (24, 26, 28). Some of these gene promoters have a canonical RARE/RXRE
(27, 29, 30), but others do not. On the other hand, an
important role of Sp1 and GC box motif(s) (see Fig. 1
for location in each gene promoter) in
constitutive and inducible transcription of these genes has been
addressed (31, 32, 33, 34).

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Figure 1. Binding Sites for Nuclear Factors Within the
Promoter Regions of the UK, GC3, TGase, TGFß1, TGFß RI, and TGFß
RII Genes
Locations for canonical GC boxes, AP-1 binding sites, AP-2 binding
sites, TATA boxes, a CAAT box, NF-1 binding sites, a NF- B binding
site, and a CREB binding site, are presented as ovals with
different colors as indicated. In addition, atypical GC boxes
used as probes in gel shift assays are depicted as light blue
ovals. The underlined section indicates the
region used for promoter assays. Closed triangles
represent the GC box motifs used in gel shift assays, the results of
which are presented in this paper. Cross-bars represent
nonfunctional GC boxes.
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In the current study, we have examined whether a similar
mechanism of RAR/RXR-Sp1 interaction might underlie transcriptional
regulation by RA of the genes listed above in BAECs and have
characterized the GC box motifs involved. We investigated whether
induction of these genes by RA is dependent on both RAR and Sp1 and
whether either RA treatment or RAR/RXR overexpression can transactivate
promoters of these genes through GC box motifs. We have also explored
the potentiation of Sp1 binding to GC box motifs and characterized the
functional and nonfunctional GC box motifs required for this effect.
Here, we report that upon autoinduction of RARs by RA, RAR/RXR
interacts with Sp1. The interaction appears to change the conformation
of Sp1 and induces gene expression, at least in part, by potentiating
Sp1 binding to the functional GC box motifs that have the 3'-flanking
region consisting of the mixture of purine and pyrimidine bases.
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RESULTS AND DISCUSSION
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Enhanced Transactivation of GC Box-Containing Gene Promoters by
RAR
/RXR
Cellular levels of RAR
and RARß increase after exposure of
BAECs to RA, followed by augmentation of UK gene expression caused by
physical interaction between RARs and Sp1. This in turn potentiates Sp1
binding to the GC box motifs in the UK promoter and thus enhances
Sp1-mediated transactivation (17). Potentiation of Sp1
binding was observed also for the consensus GC box (17),
and a combination of previous studies indicates that mRNA levels of
tissue TGase, TGFß1, and TGFß RI and RII also increase after
autoinduction of RAR
and RARß (17, 23, 24, 26).
Except for the TGase promoter (30), other gene promoters
do not have a canonical RARE/RXRE sequence. These observations prompted
us to examine whether induction of these additional RA-responsive genes
might be also dependent upon the physical interaction between RARs and
Sp1. We first performed Northern blot analyses in BAECs after treatment
with atRA in the absence and presence of a protein synthesis inhibitor,
cycloheximide (CHX), which inhibits synthesis of RAR proteins
(17), and a GC box inhibitor, mithramycin (MTM), which
specifically blocks interaction between Sp1 and GC box motif by
obscuring GC-rich sequences (17, 35, 36). AtRA enhanced
mRNA levels of UK as well as TGase, TGFß1, TGFß RI, and TGFß RII
(Fig. 2A
, lane and column 2). In
contrast, the mRNA and protein levels of Sp1 were not significantly
altered by atRA (Fig. 2
, B and C). The enhancement of UK, TGase, and
TGFß RII was suppressed by CHX (Fig. 2A
, lane 3). For TGFß1 and the
TGFß RI, superinduction was provoked by CHX, and atRA did not further
increase their mRNA levels. On the other hand, MTM suppressed both the
basal expression as well as the enhancement by atRA (lane
4). These results inferred a potential involvement of both newly
synthesized RARs and preexisting Sp1 in transcriptional regulation of
target genes by RA.

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Figure 2. Effect of RA on mRNA Levels of UK, TGase, TGFß1,
TGFß RI, TGFß RII, and mRNA and Protein Levels of Sp1 in BAECs
A, Cell lysates were prepared from confluent BAEC cultures after
incubation for 24 h in MEM-BSA with or without 5
µM atRA, in the absence or presence of 40
µM CHX or 12.5 nM MTM. Total RNA was isolated
from each cell lysate, and approximately 1530 µg of each RNA were
subjected to Northern blot analyses for mRNA levels of each gene. The
ethidium bromide-labeled 28S RNA is shown as an internal standard.
Representative bands from five independent experiments are shown.
Relative intensity of each band was quantified densitometrically and
expressed as a percent of control for each gene. Lanes and columns 1,
RA-untreated control cells; lanes and columns 24, RA-treated cells.
Lanes and columns 3, +CHX; lanes and columns
4, +MTM. Each value represents the mean ±
SD (n = 5). An asterisk and dagger
indicate a significant difference (P < 0.05)
compared with control (column 1) and RA-treated (column 2) cells,
respectively. B and C, Cell lysates were prepared from confluent BAEC
cultures untreated or treated with 5 µM atRA for 24
h, and total RNA and nuclear extracts were obtained. Changes in mRNA
levels of Sp1 were assessed by Northern blotting, quantified, and
plotted as panel B, and changes in protein levels of Sp1 as well as
cdc2 kinase (internal) in nuclear extracts bearing 20 µg of proteins
were assessed by Western blotting with rabbit antibodies, quantified,
and plotted as panel C. Lanes and columns 1, RA-untreated control
cells; lanes and columns 2, RA-treated cells. Each value
represents the mean ± SD (n = 4).
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We next performed reporter assays in BAECs transfected with various
gene promoter-luciferase constructs after treatment with atRA or 9cRA
and/or cotransfection of RAR
/RXR
(Fig. 3
). As depicted by columns
14, in addition to the pUK-Luc (panel A) and
GC3-Luc (panel B), either atRA (column 2) or 9cRA (column 3)
enhanced the luciferase activities of the reporter fused with promoter
region of TGase (panel C), TGFß1 (panel D), TGFß RI (panel E), and
TGFß RII (panel F) about 3- to 4- fold, and introduction of
RAR
/RXR
enhanced these activities 6- to 8-fold (column
4). Introduction of either RAR
or RXR
alone enhanced the
activities 2- to 3-fold, and similar effects were observed for other
subtypes of RARs/RXRs (data not shown). These results suggest that
augmentation of RAR expression either by RA treatment or by
transfection of exogenous cDNA enhances the transactivation activities;
namely an involvement of RARs in RA induction of these genes. The
transactivation activity of 1.6-kb TGase promoter, which lacks a
canonical RARE/RXRE sequence at -1.7 kb (30), was
enhanced 3-fold by treatment with RAs and 7-fold by cotransfection with
RAR
/RXR
(panel C), suggesting an existence of
RARE/RXRE-independent pathway. The result in Fig. 2
suggested that
this pathway would be predominant, because MTM almost completely
blocked RA enhancement of TGase mRNA. Transactivation activities of
every reporter examined were enhanced in a RA-dependent manner in the
cells untransfected with RAR
/RXR
(columns 2 and 3), whereas
enhanced transactivation activities in RAR
/RXR
-transfected cells
were RA-independent (columns 5 and 6). We predict that this is
because saturating concentrations of RAR
and RXR
are
expressed in the transfected cells, such that RA does not further
up-regulate RAR
/RXR
, a mechanism different from the usual
ligand-dependent transcriptional regulation via RARE.

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Figure 3. Enhanced Transactivation of GC Box-Containing
Promoters by RA Treatment and RAR /RXR Overexpression
BAECs were cotransfected with a combination of either each of six
different reporter constructs (500 ng/dish) plus pSG5
(500 ng/dish) or RAR -pSG5 and
RXR -pSG5 (250 ng each/dish), along with
pRL-CMV (Renilla luciferase, 100
ng/dish). Half of the dishes were treated with 12.5 nM MTM
for 24 h. The next day after transfection, the cells were or were
not treated with 5 µM atRA or 9cRA for 24 h.
Luciferase activity of each cell was measured, and changes in firefly
luciferase activity were calculated and plotted after normalization to
Renilla luciferase activity of MTM-untreated cells.
Panel A, pUK-Luc; panel B, GC3-Luc; panel
C, pTGase-Luc; panel D, pTGF-ß1-Luc;
panel E, pTGF-ß RI-Luc; panel F, pTGF-ß
RII-Luc. Columns 16, Without MTM; columns
712, with MTM. Columns 1 and 7, Reporter alone;
columns 2 and 8, + atRA; columns 3 and 9, + 9cRA; columns 4 and 10, +
RAR /RXR ; columns 5 and 11, + RAR /RXR + atRA; columns 6 and
12, + RAR /RXR + 9cRA. Each value represents the mean ±
SD (n = 3). An asterisk indicates a
significant difference (P < 0.05) compared with
control (column 1 in each set). Representative results from three
independent experiments with similar results are shown.
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The involvement of Sp1 in RAR
/RXR
-mediated transactivation
of these promoters was suggested by testing the effect of MTM. In the
presence of MTM, all the reporter constructs showed lower basal
activities (column 7) and a significantly attenuated response to RA
(columns 8 and 9), RAR
/RXR
(column 10), or their combination
(columns 11 and 12). This suggested that the effects of RA might be
mediated through GC box motifs, i.e. that Sp1 and its
related proteins accounted for the action of RA and RAR
/RXR
.
However, it was also possible that this might be simply because Sp1-
and/or its related protein-dependent basal transcription was inhibited
by MTM and thus the RA signal would act on a region other than the GC
box motifs. Therefore, we examined whether RAR
/RXR
might act on
the GC-rich region of each promoter. Indeed, we found that the
reactivity to RAR
/RXR
was preserved in the GC-rich region of each
promoter (Fig. 4
), suggesting that this
region is sufficient to confer responsiveness to RA via RAR
/RXR
.
Sp1 is the major protein that binds to the GC box motifs (19, 20). However, the transactivation activity detected in Fig. 4
does not necessarily represent only Sp1, as increasing members of
proteins that share homologous C-terminal zinc finger DNA-binding
domain have been suggested to act on the GC box motifs
(37, 38, 39). Therefore, we overexpressed Sp1 to confirm that
Sp1 could act on this region via functional interaction with
RAR
/RXR
. Transactivation of the promoters dealt in Fig. 4
was
uniformly enhanced by overexpressed Sp1, and introduction of a
combination of low amounts of Sp1 and RAR
/RXR
functioned
cooperatively. For example, when transactivation activity of the TGFß
RI promoter was enhanced about 2-fold by transfection of either 250
ng/dish Sp1 or 100 ng each/dish RAR
/RXR
alone, cotransfection of
these resulted in a 7-fold increase in reporter activity. This
suggested a functional interaction between RAR
/RXR
and Sp1.

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Figure 4. Conservation of Effect of RAR /RXR in GC-Rich
Region
BAEC cultures were cotransfected with a combination of either 500 ng of
pSG5 or 250 ng each of RAR -pSG5 and
RXR -pSG5 plus 500 ng of each of reporter constructs,
along with 100 ng of pRL-CMV. Cell lysates were
prepared, and luciferase activity normalized to Renilla
luciferase activity was calculated. Data are expressed as relative
luciferase activity compared with the activity of each
promoter-luciferase cotransfected with pSG5. The
numbers in parentheses to the right of each bar indicate
fold-induction calculated for each reporter. Columns 1, 3, 5, 7, 9, 11,
13, 15, 17, and 19, reporter + pSG5; columns 2, 4, 6, 8,
10, 12, 14, 16, 18, and 20, reporter + RAR /RXR . Columns 1 and 2,
pUK-Luc; columns 3 and 4, pUK GC-Luc;
columns 5 and 6, pTGase-Luc; columns 7 and 8,
pTGase GC-Luc; columns 9 and 10,
pTGF-ß1-Luc; columns 11 and 12, pTGF-ß1
GC-Luc; columns 13 and 14, pTGF-ß RI-Luc;
columns 15 and 16, pTGF-ß RI GC-Luc; columns 17 and
18, pTGF-ß RII-Luc; columns 19 and 20, pTGF-ß
RII GC-Luc. Each value represents the mean ±
SD (n = 3). An asterisk indicates a
significant difference (P < 0.05) compared with
samples from pSG5-transfected cells for each reporter.
Representative results from three independent experiments with similar
results are shown.
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Collectively, the above results suggest that RA induction of UK, TGase,
TGFß1, and its signaling receptors is mediated by both newly
synthesized RARs and preexisting Sp1 through GC box motifs within their
promoters.
Physical Interaction Between RAR
/RXR
and Sp1 and Potentiation
of Sp1 Binding to GC Box Motifs
As we described previously (17), in BAEC nuclear
extracts RARs/RXRs physically interact with Sp1 in a specific manner.
This interaction may result in a potentiation of Sp1 binding to the GC
box motifs both in the UK promoter and in consensus sequence, leading
to enhanced transactivation of the pUK-Luc (Fig. 3A
) and
GC3-Luc (Fig. 3B
), respectively. Figure 5B
shows the results of gel shift assays
using various GC box motifs as probes, whose sequences are presented in
Fig. 5A
, and whose locations are indicated by solid
triangles in Fig. 1
. Incubation of Sp1 with RAR
-glutathione
S-transferase (GST)/RXR
-GST enhanced the binding of Sp1
to the GC box motifs within the UK promoter as well as a GC box motif
within consensus sequence and the TGase, the TGFß1, the TGFß RI,
and the TGFß RII promoters (lane 3 in each set).
RAR
-GST/RXR
-GST alone did not bind to these GC box motifs (lane 2
in each set). A similar but weaker potentiation was obtained by
incubating Sp1 with 50 ng each of RAR
-GST or RXR
-GST alone (data
not shown), but not with 100 ng each of GST or BSA (lanes 4 and 5 in
each set, respectively).
Figure 6
is the result of Coomassie
Brilliant Blue (CBB) staining and Western blot analyses of
RAR
-GST/RXR
-GST and Sp1 used in the gel shift assays. As seen in
panel A, RAR
- or RXR
-GST preparation showed 80-kDa major bands in
both CBB staining (lanes 1 and 3) and Western blotting (lanes 2 and 4).
In Western blotting, 60-kDa minor bands were also detected (lanes 2 and
4), which are likely to be a degradation product. On the other hand,
the Sp1 preparation showed three bands in CBB staining, of which the
middle one was predominant (panel B, lane 1). These bands appear to be
a dimer of Sp1 (190 kDa), monomeric Sp1 (95 kDa), and a degradation
product (92 kDa), as has been reported previously (34, 40). Incubation of Sp1 with RAR
-GST did not alter the
staining pattern (lane 3). These results suggested the purity of the
preparations. Western blotting using monoclonal anti-Sp1 antibody
detected only monomeric Sp1 (lane 4), whose immunogenicity was
significantly enhanced upon incubation with RAR
-GST (lane 6)
compared with GST or BSA (lanes 8 and 10). We first doubted an
involvement of proteases possibly contaminating in preparations as a
reason for this increased immunogenicity. However, we could not
obtain data supporting this possibility. The effect was not dependent
on either temperature or incubation time and was not prevented by
inclusion of protease inhibitors (data not shown). Furthermore, the
increased immunogenicity was no longer detectable with the rabbit
polyclonal antibody (lanes 11 and 12). Therefore, we speculate that the
current data may represent a potential conformational change of Sp1. A
similar result was obtained with RXR
-GST (data not shown). Together
with the previous result that RAR/RXR-Sp1 forms a complex detectable by
immunoprecipitation/Western blot and GST-pull down (17),
these results implied that a conformational change would be induced in
Sp1 upon association with RAR
and RXR
, and this may link to
increased affinity of Sp1 to the GC box motifs.

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Figure 6. Enhancement of Immunogenicity of Sp1 After
Interaction with RAR
A, CBB staining of RAR -GST (lane 1) and RXR -GST (lane 3) and
their Western blot analyses using specific antibody to RAR (lane 2)
or RXR (lane 4) after SDS-PAGE with 9% resolving gels under
reducing conditions. The amount of protein loaded was 1 µg each for
CBB staining and 400 ng each for Western blotting. B, CBB staining of
Sp1, RAR -GST, and their mixture, as well as Western blot analyses of
these samples using specific antibody to Sp1 after SDS-PAGE with 9%
resolving gels under reducing conditions. The amounts of Sp1 and
RAR -GST used were 600 and 800 ng, respectively, for CBB staining and
30 and 100 ng, respectively, for Western blotting. For Western
blotting, control experiments with 100 ng each of GST or BSA were
performed. Lanes 13, CBB staining; lanes 412, Western blotting;
lanes 410, detected with monoclonal anti-Sp1 antibody (Mo Ab); lanes
11 and 12, detected by rabbit polyclonal anti-Sp1 antibody (Poly Ab).
Lanes 1, 4, and 11, Sp1; lanes 2 and 5, RAR -GST; lanes 3, 6, and 12,
Sp1+RAR -GST; lane 7, GST; lane 8, Sp1+GST; lane 9, BSA; lane 10,
Sp1+BSA. For panels A and B, representative results from three
independent experiments with similar results are shown.
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We next confirmed that endogenous Sp1 binds to GC box motifs and that
either RA treatment or RAR/RXR overexpression results in an enhancement
of Sp1 binding. Figure 7
shows an example
obtained using nuclear extracts from RA-treated (panel A) and
RAR
/RXR
-transfected (panel B) BAECs, respectively, with respect
to the binding to the consensus GC box. Three specific bands were
detected using nuclear extracts derived from untreated or untransfected
control cells (lane 1). Both RA treatment and RAR
/RXR
transfection uniformly increased the amounts of these bands (lane 2),
which disappeared in the presence of a 100-fold excess of unlabeled
probe, ensuring the specificity of these bands (lane 3). A similar
potentiating effect was obtained for transfection with other subtypes
of RARs/RXRs (data not shown). The uppermost band and lower two bands
were supershifted with anti-Sp1 (lane 5) and anti-Sp3 (lane 7)
antibodies, respectively, whereas anti-Sp2 antibody did not have any
effects (lane 6). For unknown reasons, the ratio between Sp1 and Sp3
and their supershift pattern varied, depending upon the experimental
conditions. Both stimulatory and inhibitory roles of Sp3 have been
reported, depending upon the target genes and cell types (11, 37, 41). For example, ER
-Sp3 interaction resulted in decreased
expression of the vascular endothelial growth factor (11).
We are now examining a role of Sp3 in our proposed regulatory pathway.
Similar results were obtained with GC box motifs derived from other
gene promoters (data not shown). Enhancement of Sp1 (or Sp3) binding
has been evoked by interaction of Sp1 (or Sp3) with other NHRs
(8, 9, 10, 11, 12, 13, 14, 15, 16). Furthermore, NHR-enhanced Sp1 (or Sp3)-DNA
binding is not an isolated phenomenon and has been observed for many
other DNA-bound proteins where binding is enhanced by other proteins
(8, 42, 43, 44).

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Figure 7. Potentiation of the DNA Binding Activity of
Endogenous Sp1 After Either Treatment with atRA (A) or Transfection
with RAR /RXR (B)
BAECs grown in 15-cm dishes were treated with 5 µM atRA
or transfected with 18 µg of either vacant pSG5 or
RAR -pSG5/RXR -pSG5. After 48 h nuclear
extracts were prepared, and subjected to gel shift assays using the
consensus GC box as a probe. Lane 1, untreated cells (A) or
pSG5-transfected cells (B); lanes 28, atRA-treated
cells (A) or RAR -pSG5/RXR -pSG5-transfected cells
(B). Lanes 38, control experiments. Lane 3, + 100-fold unlabeled
probe (cold); lane 4, + nonimmune antibody (NI IgG); lane 5, + anti-Sp1
IgG; lane 6, + anti-Sp2 IgG; lane 7, + anti-Sp3 IgG; lane 8, +
anti-RAR IgG. The concentration of each antibody was 1 µg/ml.
Representative results from three independent experiments with similar
results are shown.
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Consistent with the results in a previous report (17), we
could not detect the RAR-Sp1/GC box complex on the gel. Anti-RAR
antibody did not supershift the bands (lane 8). In addition, neither
anti-RARß antibody nor anti-RAR
antibody supershifted the bands
(data not shown). These suggest that the increased band does not
contain RARs. The RAR-Sp1 complex could not be also detected in
both CBB staining and Western blotting (Fig. 6B
). A similar phenomenon
has been reported for physical interaction between ER and Sp1 (9, 11). On the other hand, Husmann et al.
(18) have recently reported the formation of RAR-Sp1-GC
box motifs detectable in supershift experiments. Furthermore, not only
the intensity but also mobility of Sp1 band was increased by RAR in
their gel shift assays (18). Currently, we have no obvious
explanation for these differences, except the differences in the
experimental conditions, i.e. the kind of antibody,
incubation time, and reaction temperature. As discussed above, we
predict that in our current experimental condition the formation of an
RAR-Sp1 complex might be unstable, so that RAR will eventually
dissociate, as has been proposed for physical interaction between p300
and Sp1 (42). Also, the complex might be disrupted during
electrophoresis (17).
Collectively, the above results suggest that RAR
/RXR
enhances the
transactivation of some GC box-containing gene promoters through
physical interaction with Sp1, which may lead to a conformation change
and potentiate Sp1 binding to GC box motifs.
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Characterization of the Target GC Box Motifs
|
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Identification of Functional and Nonfunctional GC Box Motifs.
It remained unclear whether all GC box motifs could serve equally well
as targets for transcriptional regulation via RAR/RXR-Sp1 interaction.
We addressed this issue using the UK GC box motifs, which contain three
contiguous canonical GC boxes and one atypical GC box. First, we
performed reporter assays using wild-type and mutated UK GC box
promoter-luciferase constructs, the GC box motif sequences of which are
presented in Fig. 8A
.
As illustrated in panel B, mutation in the 5'-site canonical
GC box (IV) did not affect either basal or stimulated transactivation
(columns 3 and 4, respectively), whereas mutation in either the middle
canonical GC box (II) or 3'-site canonical GC box (I) resulted in lower
basal activities (columns 5, 7, 9, 11, 13, and 15), but did not affect
RAR
/RXR
s potentiating effect (columns 6, 8, 10, 12, 14, and
16). Mutation in the GC box (II) reduced the potentiation significantly
(columns 5, 9, 13, and 15). Additional mutation in the atypical GC box
(III) eliminated most of the basal activity (column 17) and eliminated
the response to RAR
/RXR
completely (column 18), as was observed
in the presence of MTM (Fig. 3
). We observed an exactly parallel change
in binding activity of Sp1 to the wild type and various mutated UK GC
box motifs by gel shift assays (data not shown), confirming a direct
correlation between Sp1 binding and its transactivating activity. This
suggests that modulation of Sp1 activity by RAR/RXR may be due to
potentiation of Sp1s binding affinity to target GC box motifs and
therefore depend upon whether Sp1 can interact directly with the GC box
motifs. Panel C shows the result of gel shift assays using
oligonucleotides containing one of three canonical UK GC boxes, in the
absence and presence of RAR
-GST/RXR
-GST. In keeping with the
results obtained with mutated GC box motifs, the Sp1-GC box complex was
barely detected with oligonucleotide containing the canonical GC box
(IV) (lanes 13), in contrast to the canonical GC box (II)/atypical GC
box (III)-containing oligonucleotide (lane 4) and oligonucleotide
containing the GC box (I) (lanes 79).

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Figure 8. Identification of Functional and
Nonfunctional GC Box Motifs within the UK Promoter
A, Sequences of the UK promoter GC box as well as its mutants used in
the experiments. Three canonical GC boxes (solid lines)
are represented by uppercase W (wild) or T (mutation to
T) and an atypical GC box (a dotted line) is represented
by lowercase W or T. B, BAECs were cotransfected with a
mixture of RAR and RXR expressing vectors,
pRL-CMV, and UK GC box promoter construct (WwWW) or its
mutant promoter constructs in which one of three canonical GC boxes
(TwWW, WwTW, WwWT), two of three canonical GC boxes (WwTT, TwWT, TwTW),
all three canonical GC boxes (TwTT), or all three canonical GC boxes
plus an atypical GC box (TtTT) were mutated. Luciferase activity
normalized Renilla luciferase activity was calculated.
Data are expressed after subtracting the basal activity level obtained
for the cells transfected with vacant pGL3 vector.
Columns 1 and 2, Wild-type UK GC box; columns 318,
mutant UK GC boxes as indicated. Columns 1, 3, 5, 7, 9, 11, 13, 15, and
17, reporter alone; columns 2, 4, 6, 8, 10, 12, 14, 16 and 18, reporter
plus RAR and RXR . Each value represents the mean ±
SD (n = 3). An asterisk indicates a
significant difference (P < 0.05) obtained by a
comparison to samples without RAR /RXR transfection for each
reporter. A dagger indicates a significant difference
(P < 0.05) in basal activity between each reporter
and wild-type pUK-GC Luc (column 1). C, The binding of
Sp1, RAR -GST/RXR -GST, and Sp1 in the presence of
RAR -GST/RXR -GST to oligonucleotides containing one of three
canonical UK GC boxes was tested by gel shift assays. Five base pairs
of both 5'- and 3'-site flanking sequences of the core GC box sequence
were substituted with those of each UK canonical GC box. Sequences of
these oligonucleotides are presented in Fig. 9A . Lanes 13, UK GC box
(IV)-containing oligonucleotide; lanes 46, UK GC boxes (III) and
(II)-containing oligonucleotide; lanes 79, UK GC box (I)-containing
oligonucleotide. Lanes 1, 4, and 7, Sp1 alone; lanes 2, 5, and 8,
RAR -GST/RXR -GST alone; lanes 3, 6, and 9, Sp1 plus
RAR -GST/RXR -GST. For panels B and C, representative results from
three independent experiments with similar results are shown.
|
|
Collectively, these results suggest that there are both functional and
nonfunctional GC box motifs; canonical GC boxes (I) and (II) have
medium and high affinities to Sp1, respectively, and therefore serve as
functional targets motifs, whereas a canonical GC box (IV) has very low
affinity to Sp1 and therefore does not serve as a target. Similar
results have been reported in studies exploring interactions between
ER
and Sp1 (45, 46, 47).
Influence of the Flanking Sequences of the Hexanucleotide Core
GC Box Motif.
We analyzed the sequence difference(s) between functional and
nonfunctional GC box motifs. Figure 9A
summarizes the results of gel shift assays shown in Fig. 8C
along with
sequences of each oligonucleotide used as probes as well as consensus
GC box. All oligonucleotides share the same hexanucleotide GGGCGG core
sequence (nucleotide numbers 1015), suggesting that in addition to
the core sequence, its flanking sequences may be important for binding
to Sp1. Therefore, utilizing many mutant oligonucleotides we carefully
analyzed roles of the 5'- and 3'-flanking sequences and found that the
3'-flanking region (nucleotide numbers 1620) was important. The
sequence of this region within UK GC box (IV) consists of only five
purine bases and that within UK GC box (I) consists of four pyrimidine
bases following G at nucleotide number 16. In contrast, in both the
consensus GC box and the GC box (III/II), no more than three tandem
purine or pyrimidine bases exist in this region, implying that
existence of continuing five purine bases at nucleotide numbers 1620
might be a cause for the reduced affinity of the nonfunctional GC box
motifs. This hypothesis was tested by gel shift assays using mutant
consensus GC box oligonucleotides (Fig. 9B
). In general, mutant GC
boxes containing five tandem purine or pyrimidine bases at nucleotide
numbers 1620 showed no or very weak binding to Sp1 even in the
presence of RAR
-GST/RXR
-GST (lanes 16), although
some enhanced binding was observed with AGAGA and CCCCC mutants (lanes
3 and 4, respectively). The introduction of such sequences
into the immediate 5'-flanking region (nucleotide numbers 59) did not
abrogate DNA binding (lanes 712) except substitution with CCCCC
(lane 10).

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Figure 9. Effect on DNA Binding of Five Tandem
Purine/Pyrimidine Bases at Flanking Region of the Core GC Box Sequence
A, Sequence of consensus GC box and oligonucleotides containing one of
three UK canonical GC boxes. The results of the binding as assessed by
gel shift assays (Fig. 8C ) are indicated by O, , or X at the
right of each sequence. O, Strong binding; , weak
binding; X, almost no binding. B, Five base pairs of either 3'- or
5'-site flanking sequence in addition to the hexanucleotide core GC box
sequence in the consensus GC box oligonucleotide were substituted with
five adenine nucleotides, five guanine nucleotides, or their mixture,
or five cytosine nucleotides, five thymine nucleotides, or their
mixture, and the binding of Sp1 to these oligonucleotides in the
presence of RAR -GST/RXR -GST was assessed by gel shift assays as
before. Representative results from four independent experiments with
similar results are shown.
|
|
Furthermore, two GC boxes within the TGFß1 promoter, a 5'-site GC box
within the TGFß RI promoter and a 3'-site GC box within the TGFß
RII promoter, were also nonfunctional (Fig. 10A
). The 3'-flanking region of these
nonfunctional GC box motifs consists only of pyrimidine bases (CTCCCC
and CCCCC for TGFß1 promoter GC box motifs at -216 and -118,
respectively) or purine bases (AGGGGG for TGFß RI promoter GC box
motif at -957; AGAGAGG for TGFß RII promoter GC box motif at -22)
(32, 33, 48). These results suggest that the presence of
five tandem purine or pyrimidine bases at 3'-flanking region may
interfere with interaction between Sp1 and GC box motifs, although the
extent of interference varies depending upon the sequences. Namely, the
presence of mixed purine and pyrimidine bases within the immediate
3'-flanking region of the core sequence may favor the GC box serving as
the target for Sp1 and therefore as the target for RAR/RXR. This
feature affects the interaction between Sp1 and GC box motif but does
not affect the interaction between RAR/RXR and Sp1.

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Figure 10. Examples of Nonfunctional GC Box Motifs
A, No or weaker binding of Sp1 to putative nonfunctional GC box motifs
within the promoter region of TGFß1, TGFß RI, and TGFß RII genes.
The binding of Sp1 to oligonucleotides containing various putative
nonfunctional GC box motifs was compared by gel shift assays between
with and without RAR -GST/RXR -GST. Seven base pairs of 3'-site
flanking sequences of the consensus core GC box sequence were
substituted with those of TGFß1 (-216), TGFß1 (-118), TGFß RI
(-957), or TGFß RII (-22) GC box and used as probes. Lanes 1 and 2,
Consensus GC box; lanes 3 and 4, TGFß1 GC box (-216)-containing
oligonucleotide; lanes 5 and 6, TGFß1 GC box (-118)-containing
oligonucleotide; lanes 7 and 8, TGFß RI GC box (-957)-containing
oligonucleotide; lanes 9 and 10, TGFß RII GC box (-22)-containing
oligonucleotide. Lanes 1, 3, 5, 7, and 9, Sp1 alone; lanes 2, 4, 6, 8,
and 10, Sp1 plus RAR -GST/RXR -GST. Representative results from
three independent experiments with similar results are shown. B,
Sequences of the GC box motifs in the MDR1, E1B, and IGFBP-2 genes.
Putative functional and nonfunctional GC box motifs are indicated by
enclosing core sequences with shaded and open
boxes, respectively. Sequences of continuous purines or
pyrimidines at the 3'-flanking region of each core GC box sequence are
double underlined.
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|
The role of the 3'-flanking region of core GC box sequence has been
addressed in a number of studies (See Fig. 10B
for the sequences). For
example, 1) Sp1 activates the MDR1 promoter through binding to a
GC-rich region that has mixed purine and pyrimidine bases at the
3'-site of the core sequence, GGGCGT, leading to transactivation of
this promoter, whereas Sp1 cannot bind to an inhibitory GC-rich region
within the same gene promoter, consisting of a GGGCCG core motif
followed by continuous purine bases (GGAGCAG)
at the 3'-flanking site (49); 2) Sp1 binds with high
affinity to an E1B promoter GC box motif (-47
-42, GGGCGG) that has
mixed purine and pyrimidine bases at its 3'-flanking site, and
introduction of mutations into this region to make five continuous
pyrimidine bases (GGCCCTC) reduces the binding of Sp1 to
this GC box motif (50); 3) Moreover, among four tandem GC
box motifs identified in the IGFBP-2 promoter, the GC box 4 containing
a mixture of purine/pyrimidine bases at the 3'-site flanking region
exhibits a 10- to 20-fold higher affinity to both endogenous and
exogenous Sp1 than the other three GC boxes containing approximately
five to eight continuous purine or pyrimidine bases at this region
(51). We are now examining whether these genes bearing
functional GC box motifs can be transcriptionally regulated by RA in
BAECs via RAR/RXR-Sp1 interaction.
Collectively, we demonstrated here that transactivation via RAR/RXR-Sp1
interaction is dependent on the 3'-flanking sequence of the target GC
box motifs.
In summary, we described a potential regulatory pathway through
which RA induces the expression of several genes independently of
RARE/RXRE in BAECs via GC box motifs. RA first induces the expression
of RARs, especially RAR
and RARß, in a ligand-dependent manner
(17). RARs then physically interact with Sp1 with the help
of RXRs, especially RXR
(17), and strengthen Sp1s
affinity to the functional GC box motifs, leading to enhanced
transactivation of several target gene promoters. Through this
mechanism, RA will induce UK, TGase, TGFß1, and its receptors,
namely, the production of (latent) TGFß and its activators (UK and
TGase) to generate active ligand (22, 23, 24). In addition,
this mechanism accounts for increased expression of TGFß receptors,
which increases responsiveness to the ligand (26), leading
to TGFß-mediated regulation of endothelial cell functions (24, 26, 28).
We suggest that the functional GC box motif can serve as a target for
RARs/RXRs, but not that functional GC box motifs are the only sites
responsible for all the response to RA. Several important issues remain
unresolved: 1) It will be important to elucidate whether other nuclear
factors, including corepressors and coactivators, modulate the
interaction between RAR/RXR and Sp1 and subsequent transcriptional
regulation, as has been demonstrated for interactions between other
NHRs and Sp1 (12, 16); 2) We anticipate that
polypurine/polypyrimidine in the 3'-flanking region may disrupt Sp1
binding to the core GC box sequence through a steric hindrance, which
may be a result of either alteration in the structure of DNA strands
because purine-pyrimidine step forms two stable conformations
(52), or formation of triple-stranded complexes
(53); 3) There remains a possibility that the modest
modifications such as phosphorylation or acetylation may alter the
conformation of Sp1. NMR analysis is required to directly confirm a
conformational change in Sp1 by RAR/RXR as well as to determine the
effect of polypurine/polypyrimidine; 4) Because functional repression
of gene expression through interaction between RAR and AP-1, nuclear
factor (NF)-IL-6, or Myb has been reported (54, 55, 56), one
may ask whether the RAR/RXR-Sp1 interaction affects other transcription
factors shown to be important for basal as well as induced
transcription from the genes studied here, such as Egr-1, NF-
B,
CREB, and AP-1 (57, 58, 59, 60, 61); 5) The relevance of this
observation to the transcriptional activation observed in
vivo has yet to be established. We have been mapping the
interaction site(s) in both RAR and Sp1 molecules as the first step
toward answering these questions. Preliminary results suggest that DNA
binding domains within both molecules are important for their
interaction (Suzuki, Y., and S. Kojima, unpublished observation) as has
been reported in other systems (10, 18). Ongoing studies
will further identify the molecular mechanisms by which RA induces the
expression of other RARE-less genes and should provide evidence of a
more generalized role of Sp1 in retinoid responsiveness.
 |
MATERIALS AND METHODS
|
---|
Materials
AtRA, 9cRA, and CHX were purchased from Sigma (St.
Louis, MO). MTM was purchased from Calbiochem (La Jolla,
CA). The concentration of MTM used in the experiments was not toxic to
the cells, and the specificity of the inhibitor has been ensured in
previous reports (17, 35, 36). Construction of the
pRAR
-GST and pRXR
-GST
that express fusion proteins between either RAR
or RXR
and GST,
and purification of these fusion proteins from Escherichia
coli BL21 were performed as described previously
(17). The human Sp1 expression vector,
Sp1-pCIneo, was also as described (17). Human
Sp1 was obtained from Promega Corp. (Madison, WI).
Antibodies used for Western blotting and supershift experiments were
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)
except rabbit polyclonal anti-Sp1 antibody was from Sigma.
Second antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Isolation of RNA and Northern Blot Analysis
BAECs were isolated and maintained in
MEM containing 10%
newborn calf serum. Isolation of RNA and Northern blot analyses were
performed as described previously (17). Membranes were
hybridized with cDNA probes for either bovine UK (22),
bovine TGase (23), murine TGFß1 (24), human
TGFß RI and RII (26), and human Sp1 (a
NotI/EcoRI fragment of Sp1-pCIneo).
The conditions for hybridization and washings were as described
(17). Autoradiography was performed using a Fujix BAS 2500
Bio-imaging analyzer (Fuji Photo Film Co., Ltd., Tokyo,
Japan). Each band was scanned, and the signal intensity was normalized
to that obtained with ethidium bromide-labeled 28S RNA as internal,
because MTM treatment affects mRNA levels of glyceraldehyde-3-phosphate
dehydrogenase.
Construction of Reporter Genes
Reporter plasmids encoding chimeric promoter fused to
luciferase were prepared as described (7, 17, 31, 32, 33). The
schematic structures of these promoters are illustrated in Fig. 1
. The
pUK-Luc and pUK GC-Luc were constructed as
described previously (7, 17). The GC3-Luc,
which contains three sequential repeats of consensus GC boxes and
TATA box
(5'-CCCGGGGCGGGGCGAGCTGGGGCGGGGCGAGCTCGGGGCGGGGCCTGATATACGG-3')
before the luciferase cDNA, was generated by inserting synthesized
oligo DNA into the KpnI/BglII site of the
pGL3 vector (Promega Corp.). Other luciferase
fusion constructs include promoters of human tissue TGase
(-1665
+72) or its GC-rich region (-122
+72) (generous gifts from
Dr. P. J. A. Davies, University of Texas Medical School; Ref.
31), human TGFß1 (-1362
+819) or its GC-rich region
(-453
+11; Ref. 31), human TGFß RI (-1422
-65) or its GC-rich
region (-425
-65; Ref. 33), and human TGFß RII
(-1,670
+36) or its GC-rich region (-219
+36; Ref. 33). UK
promoter mutants consisting of wild-type or various mutated GC box(es)
and TATA box (corresponding to -68
-20) before luciferase reporter
were generated by inserting synthesized oligonucleotides into the
MluI/BglII site of the pGL3
vector.
Transient Transfection and Luciferase Assay
Transient transfections using Lipofectamine Plus reagent
(Life Technologies, Inc., Gaithersburg, MD) and luciferase
assays using the Dual-Luciferase Reporter Assay System (Promega Corp.) were performed as described previously
(17).
Western Blotting
Western blotting was performed as described previously
(39) after SDS-PAGE with 9% resolving gels under reducing
conditions using a combination of rabbit anti-RAR
or RXR
polyclonal antibody (final 1:200 dilution), mouse monoclonal
(Santa Cruz Biotechnology, Inc.), or rabbit polyclonal
(Sigma) anti-Sp1 antibody (both final 1:50 dilution), or
rabbit anti-cdc2 kinase antibody (final 1:500 dilution), and goat
antirabbit IgG or antimouse IgG antibody conjugated with peroxidase
(final 1:1,500 dilution). The signals were detected with an
Amersham Pharmacia Biotech-ECL Plus system (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Preparation of Nuclear Extracts and Gel Shift Assay
Nuclear extracts were prepared in 200 µl of BL buffer (10
mM potassium phosphate-10 mM Tris-HCl, pH 8.0,
containing 0.4 M LiCl and 0.1% NP-40) as described
previously (17). The protein concentrations were
determined by BCA assays (Pierce Chemical Co., Rockford,
IL). Oligonucleotides containing various GC box motifs and their
mutants were synthesized, double-stranded, and end-labeled with
[
-32P] ATP by T4 polynucleotide kinase using
the kit from Takara Biomedicals (Tokyo, Japan). Gel shift assays were
performed as described previously (17).
Statistics
Significance was determined by the two-tailed t
test.
 |
ACKNOWLEDGMENTS
|
---|
We thank J. T. Kadonaga, P. Chambon, and P. J. A.
Davies for constructs, S. L. Friedman for critical reading of the
manuscript, and C. Iijima, M. Tokunami, M. Kobayashi, M. Yoshizawa, and
K. Akita for technical assistance.
 |
FOOTNOTES
|
---|
This work was supported, in part, by a Grant for Multibioprobe Research
Program from RIKEN and The Special Coordination Funds of the
Ministry of Education, Culture, Sports, Science and Technology.
Abbreviations: atRA, All-trans-RA; BAEC, bovine
aorta endothelial cell; CBB, Coomassie Brilliant Blue; CHX,
cycloheximide; 9cRA, 9-cis-RA; GST,
glutathione-S-transferase; MTM, mithramycin; NF, nuclear
factor; NHR, nuclear hormone receptor; RARE, RAR-responsive element;
RXRE, RXR-responsive element; TGase, transglutaminase; UK,
urokinase.
Received for publication August 21, 2000.
Accepted for publication June 8, 2001.
 |
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