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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Characterization of the Target...
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Characterization of the Target...
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go 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-{kappa}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.

 
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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Characterization of the Target...
 MATERIALS AND METHODS
 REFERENCES
 
Enhanced Transactivation of GC Box-Containing Gene Promoters by RAR{alpha}/RXR{alpha}
Cellular levels of RAR{alpha} 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{alpha} 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. 2AGo, lane and column 2). In contrast, the mRNA and protein levels of Sp1 were not significantly altered by atRA (Fig. 2Go, B and C). The enhancement of UK, TGase, and TGFß RII was suppressed by CHX (Fig. 2AGo, 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 {alpha}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 15–30 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 2–4, 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).

 
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{alpha}/RXR{alpha} (Fig. 3Go). As depicted by columns 1–4, 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{alpha}/RXR{alpha} enhanced these activities 6- to 8-fold (column 4). Introduction of either RAR{alpha} or RXR{alpha} 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{alpha}/RXR{alpha} (panel C), suggesting an existence of RARE/RXRE-independent pathway. The result in Fig. 2Go 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{alpha}/RXR{alpha} (columns 2 and 3), whereas enhanced transactivation activities in RAR{alpha}/RXR{alpha}-transfected cells were RA-independent (columns 5 and 6). We predict that this is because saturating concentrations of RAR{alpha} and RXR{alpha} are expressed in the transfected cells, such that RA does not further up-regulate RAR{alpha}/RXR{alpha}, 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{alpha}/RXR{alpha} 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{alpha}-pSG5 and RXR{alpha}-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 1–6, Without MTM; columns 7–12, with MTM. Columns 1 and 7, Reporter alone; columns 2 and 8, + atRA; columns 3 and 9, + 9cRA; columns 4 and 10, + RAR{alpha}/RXR{alpha}; columns 5 and 11, + RAR{alpha}/RXR{alpha} + atRA; columns 6 and 12, + RAR{alpha}/RXR{alpha} + 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.

 
The involvement of Sp1 in RAR{alpha}/RXR{alpha}-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{alpha}/RXR{alpha} (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{alpha}/RXR{alpha}. 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{alpha}/RXR{alpha} might act on the GC-rich region of each promoter. Indeed, we found that the reactivity to RAR{alpha}/RXR{alpha} was preserved in the GC-rich region of each promoter (Fig. 4Go), suggesting that this region is sufficient to confer responsiveness to RA via RAR{alpha}/RXR{alpha}. Sp1 is the major protein that binds to the GC box motifs (19, 20). However, the transactivation activity detected in Fig. 4Go 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{alpha}/RXR{alpha}. Transactivation of the promoters dealt in Fig. 4Go was uniformly enhanced by overexpressed Sp1, and introduction of a combination of low amounts of Sp1 and RAR{alpha}/RXR{alpha} 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{alpha}/RXR{alpha} alone, cotransfection of these resulted in a 7-fold increase in reporter activity. This suggested a functional interaction between RAR{alpha}/RXR{alpha} and Sp1.



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Figure 4. Conservation of Effect of RAR{alpha}/RXR{alpha} in GC-Rich Region

BAEC cultures were cotransfected with a combination of either 500 ng of pSG5 or 250 ng each of RAR{alpha}-pSG5 and RXR{alpha}-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{alpha}/RXR{alpha}. 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.

 
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{alpha}/RXR{alpha} 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. 3AGo) and GC3-Luc (Fig. 3BGo), respectively. Figure 5BGo shows the results of gel shift assays using various GC box motifs as probes, whose sequences are presented in Fig. 5AGo, and whose locations are indicated by solid triangles in Fig. 1Go. Incubation of Sp1 with RAR{alpha}-glutathione S-transferase (GST)/RXR{alpha}-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{alpha}-GST/RXR{alpha}-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{alpha}-GST or RXR{alpha}-GST alone (data not shown), but not with 100 ng each of GST or BSA (lanes 4 and 5 in each set, respectively).



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Figure 5. Potentiation by RAR{alpha}/RXR{alpha} of Binding of Sp1 to GC Box Motifs Within Several RA-Inducible Genes

A, Sequences of various gene GC box oligonucleotides used as probes. Canonical and atypical GC box core motif sequences are indicated by solid and dotted arrows, respectively. B, After 15 ng of Sp1 alone (lane 1), 50 ng each of RAR{alpha}-GST/RXR{alpha}-GST alone (lane 2), a combination of 15 ng of Sp1 plus 50 ng each of RAR{alpha}-GST/RXR{alpha}-GST (lane 3), a combination of 15 ng of Sp1 plus 100 ng of GST (lane 4), or a combination of 15 ng of Sp1 plus 100 ng of BSA (lane 5) was preincubated, the reaction mixture was incubated with 32P-labeled-various gene GC box oligonucleotides. Protein-DNA complexes were separated through a 4% polyacrylamide gel electrophoresis and visualized on an image analyzer. Representative results from three independent experiments with similar results are shown.

 
Figure 6Go is the result of Coomassie Brilliant Blue (CBB) staining and Western blot analyses of RAR{alpha}-GST/RXR{alpha}-GST and Sp1 used in the gel shift assays. As seen in panel A, RAR{alpha}- or RXR{alpha}-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{alpha}-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{alpha}-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{alpha}-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{alpha} and RXR{alpha}, 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{alpha}

A, CBB staining of RAR{alpha}-GST (lane 1) and RXR{alpha}-GST (lane 3) and their Western blot analyses using specific antibody to RAR{alpha} (lane 2) or RXR{alpha} (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{alpha}-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{alpha}-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 1–3, CBB staining; lanes 4–12, Western blotting; lanes 4–10, 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{alpha}-GST; lanes 3, 6, and 12, Sp1+RAR{alpha}-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.

 
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 7Go shows an example obtained using nuclear extracts from RA-treated (panel A) and RAR{alpha}/RXR{alpha}-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{alpha}/RXR{alpha} 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{alpha}-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{alpha}/RXR{alpha} (B)

BAECs grown in 15-cm dishes were treated with 5 µM atRA or transfected with 18 µg of either vacant pSG5 or RAR{alpha}-pSG5/RXR{alpha}-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 2–8, atRA-treated cells (A) or RAR{alpha}-pSG5/RXR{alpha}-pSG5-transfected cells (B). Lanes 3–8, 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{alpha} IgG. The concentration of each antibody was 1 µg/ml. Representative results from three independent experiments with similar results are shown.

 
Consistent with the results in a previous report (17), we could not detect the RAR-Sp1/GC box complex on the gel. Anti-RAR{alpha} antibody did not supershift the bands (lane 8). In addition, neither anti-RARß antibody nor anti-RAR{gamma} 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. 6BGo). 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{alpha}/RXR{alpha} 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.


    Characterization of the Target GC Box Motifs
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Characterization of the Target...
 MATERIALS AND METHODS
 REFERENCES
 
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. 8AGo. 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{alpha}/RXR{alpha}’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{alpha}/RXR{alpha} completely (column 18), as was observed in the presence of MTM (Fig. 3Go). 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 Sp1’s 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{alpha}-GST/RXR{alpha}-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 1–3), in contrast to the canonical GC box (II)/atypical GC box (III)-containing oligonucleotide (lane 4) and oligonucleotide containing the GC box (I) (lanes 7–9).



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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{alpha} and RXR{alpha} 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 3–18, 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{alpha} and RXR{alpha}. 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{alpha}/RXR{alpha} 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{alpha}-GST/RXR{alpha}-GST, and Sp1 in the presence of RAR{alpha}-GST/RXR{alpha}-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. 9AGo. Lanes 1–3, UK GC box (IV)-containing oligonucleotide; lanes 4–6, UK GC boxes (III) and (II)-containing oligonucleotide; lanes 7–9, UK GC box (I)-containing oligonucleotide. Lanes 1, 4, and 7, Sp1 alone; lanes 2, 5, and 8, RAR{alpha}-GST/RXR{alpha}-GST alone; lanes 3, 6, and 9, Sp1 plus RAR{alpha}-GST/RXR{alpha}-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{alpha} 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 9AGo summarizes the results of gel shift assays shown in Fig. 8CGo 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 10–15), 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 16–20) 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 16–20 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. 9BGo). In general, mutant GC boxes containing five tandem purine or pyrimidine bases at nucleotide numbers 16–20 showed no or very weak binding to Sp1 even in the presence of RAR{alpha}-GST/RXR{alpha}-GST (lanes 1–6), 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 5–9) did not abrogate DNA binding (lanes 7–12) 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. 8CGo) are indicated by O, {Delta}, or X at the right of each sequence. O, Strong binding; {Delta}, 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{alpha}-GST/RXR{alpha}-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. 10AGo). 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{alpha}-GST/RXR{alpha}-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{alpha}-GST/RXR{alpha}-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.

 
The role of the 3'-flanking region of core GC box sequence has been addressed in a number of studies (See Fig. 10BGo 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{alpha} and RARß, in a ligand-dependent manner (17). RARs then physically interact with Sp1 with the help of RXRs, especially RXR{alpha} (17), and strengthen Sp1’s 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-{kappa}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Characterization of the Target...
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-GST and pRXR{alpha}-GST that express fusion proteins between either RAR{alpha} or RXR{alpha} 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 {alpha}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. 1Go. 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{alpha} or RXR{alpha} 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 [{gamma}-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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