Acceleration of Thrombomodulin Gene Transcription by Retinoic Acid

RETINOIC ACID RECEPTORS AND Sp1 REGULATE THE PROMOTER ACTIVITY THROUGH INTERACTIONS WITH TWO DIFFERENT SEQUENCES IN THE 5'-FLANKING REGION OF HUMAN GENE*

Shuichi HorieDagger, Hidemi Ishii§, Fumiko Matsumoto, Masao Kusano, Keiichiro Kizaki, Juzo Matsuda, and Mutsuyoshi Kazama

From the Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui, Kanagawa 199-0195 and the § Department of Public Health, Showa College of Pharmaceutical Sciences, Higashi Tamagawagakuen, Machida, Tokyo 194-8543, Japan

Received for publication, June 7, 2000, and in revised form, October 13, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The interactions between retinoic acid- (RA)-dependent transcriptional regulatory sequences of the 5'-untranslated region of the thrombomodulin gene and nuclear RA-responsive proteins were studied using human pancreas BxPC-3 cells. Deletion mutants of pTM-CAT plasmid revealed the presence of distal and proximal RA-responsive regions containing direct repeat with 4 spaces (DR4) and three of four Sp1 sites, respectively. Cotransfection of a pTM-CAT plasmid with expression plasmids of RA receptors (RARalpha , RARbeta , and RARgamma ) augmented the promoter activity under the condition of lower retinoid X receptor-alpha (RXRalpha ) expression, whereas the activity was greatly diminished when RXRalpha was highly expressed. An electrophoretic mobility shift assay with cDNA containing the DR4 indicated that heterodimers of RAR and RXRalpha interacted with the DR4 site, although the interaction gradually disappeared with the increase in the ratio of RXRalpha /RAR. On the other hand, Sp1 protein interacted especially with the tandem Sp1 site corresponding to the first and second Sp1 sequences of the four Sp1 sites in the proximal RA-responsive region. The binding of Sp1 to Sp1 sites was independent of RAR-RXR heterodimer but increased with the increase in Sp1 concentration in the presence of unknown factor(s) of reticulocyte lysate. Upon treatment of the cells with RA, time-dependent increases in the ratio of RARbeta to RXRalpha and the phosphorylated form of Sp1 were observed. We concluded that two genomic DNA regions, the DR4 site (-1531 to -1516) and the first and second Sp1-binding sites (-145 to -121), were involved in the RA-dependent augmentation of thrombomodulin gene expression through increased interactions of the two regions with heterodimer of RAR-RXRalpha and nuclear Sp1, respectively.



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Thrombomodulin (TM)1 is an essential cofactor for activation of protein C by thrombin on vascular endothelial cells (1-3). TM expression of human endothelial cells is decreased by tumor necrosis factor-alpha (4-6), interleukin-1 (6, 7), endotoxin (8), and phorbol ester (5, 6) and oxidized LDL (9, 10), but we have found that it is increased by all-trans-retinoic acid (t-RA) (11, 12) and/or cAMP (11, 13) in human umbilical vein endothelial (HUVE) cells, through the acceleration of transcriptional activity. Several studies on the regulatory region of human TM gene have been reported (14-17), and Dittman et al. (17) showed the existence of an RA response element (RARE) in the 5'-flanking region of the gene. The direct effects of retinol and its derivatives (retinoids), such as t-RA and 9-cis-RA (9C-RA), on the expressions of many genes are apparently mediated by nuclear receptor proteins that are members of the steroid and thyroid hormone receptor (TR) superfamily of transcriptional regulators (18, 19). Nuclear retinoid receptor dimers, of which retinoid X receptor (RXR) is a mandatory constituent, are required for effective activation of the t-RA and/or 9C-RA response pathways (20-25). However, the direct repeats of RARE separated by 4 base sequences (DR4) at -1531 to -1516 from the transcription start site of TM may not be a candidate for nuclear retinoid receptors, because the DR4 sequence has been shown to be specific for a heterodimer of RXR and TR (25). It is also known that dimers of RA receptors such as homodimer of RXRalpha and heterodimers of RARs and RXR interact with repeated RAREs spaced by 1 base (DR1) and 5 bases (DR5), respectively (26-28). Accordingly, it is unclear whether or not the RARE of the TM gene in human cells is functionally responsive to RARs or RXR, although Dittman et al. (17) recognized ligand dependence of RARE in the TM gene using Chinese hamster ovary cells.

On the other hand, it is well known that the transcription factor Sp1 binds to GC boxes (GGGCGG or CCCGCC) and activates transcription of a subset of genes that contain those boxes in animal cells (29, 30). Four Sp1-binding sites are located just upstream of the TATA box in the 5' region of the TM gene (14-16). Darrow et al. (31) indicated that RA-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation might involve Sp1. The binding abilities and functional significance of Sp1 for the promoters of various genes, such as murine ornithine decarboxylase (32), tissue factor (33), interleukin-6 (34), and prolactin receptor (35) genes, have been reported. However, there is no direct evidence concerning interactions between Sp1, RA receptor proteins, and the four Sp1 sequences in the TM gene, especially in the case of HUVE cells treated with retinoids.

The results of transactivation and electrophoretic mobility shift assay (EMSA) indicate that up-regulation of TM gene expression by retinoid is associated with increases in the ratio of RARs to RXRs and in the amount of Sp1, followed by enhancement of the interactions between heterodimers of RARs-RXRalpha and the DR4 sequence and between Sp1 and the consensus binding sites of the TM gene. Furthermore, our results suggest that retinoid itself does not function directly as the accelerator of these bindings and that the bindings of Sp1 to the Sp1 sites is dependent on the phosphorylation of the protein.


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Materials-- pMAM-neo and pMAM-neo-CAT plasmids were purchased from Toyobo Biochemicals, Osaka, Japan, and pGEM-4Z and pSV-beta -galactosidase control plasmids and TransFast were from Promega. Expression plasmids pCMX-hRARalpha , pCMX-hRARbeta , pCMX-hRARgamma , and pCMX-hRXRalpha were kindly donated by Prof. R. M. Evans, Howard Hughes Medical Institute, the Salk Institute. BxPC-3 cells, a human pancreas primary adenocarcinoma cell line, were obtained from Dainippon Pharmaceutical Co., Osaka, Japan. Recombinant human Sp1 and AP2, and affinity-purified rabbit polyclonal antibodies against RARs, RXRalpha , and Sp1 were purchased from Santa Cruz. [alpha -32P]dCTP, the random primer extension labeling system and Renaissance Western blot Chemiluminescence Reagent were obtained from PerkinElmer Life Sciences. ISOGEN, t-RA, 9C-RA, Ch55, T7 RNA polymerase, and RNase inhibitor were from Wako Pure Chemical, Osaka, Japan. Rabbit reticulocyte lysate system, [gamma -32P]ATP, [alpha -32P]UTP, and nitrocellulose paper were obtained from Amersham Pharmacia Biotech. Biotinylated anti-rabbit IgG and horseradish peroxidase-conjugated streptavidin were obtained from Vector Laboratories.

Construction of Plasmids-- The 5'-flanking DNA fragment of the TM gene was obtained by PCR from a genomic DNA template that was prepared from HUVE cells. The primers were designed based on the sequences reported by Shirai et al. (14) and Dittman et al. (17). The PCR products of 689- and 1319-bp fragments were ligated at the XhoI site to prepare a 1702-bp TM fragment (from -1730 to -29 with respect to the untranslated site, which corresponds to -1562 to +140 with respect to the transcription starting site). The DNA fragments were subcloned into the pGEM-4Z vector, and the sequences were determined by the 373S DNA sequencer (Applied Biosystems Inc.). Expression plasmids, designated as pTM549-CAT and pTM1562-CAT, were constructed by inserting the TM cDNA (689 or 1702 bp) at the SalI site of a promoterless pMAM-CAT vector, which was prepared by the ligation of the CAT expression fragment derived from pMAM-neo-CAT vector and the neo gene-deleted pMAM-neo vector after deletion of the Rous sarcoma virus-long terminal repeat sequences. The TM gene was systematically digested with exonuclease III and mung bean nuclease after treatments with NheI and SphI. Deletion end sites were confirmed by DNA sequencing with the 5'-DNA primers derived from pMAM plasmid.

Preparation of Mutant Plasmids and Purification of Plasmid DNA-- The deletion mutants of pTM-CAT plasmid were prepared by ligation of PCR products over the mutation sites. pTM346-CAT plasmid was digested with MluI and XhoI, and then the appropriate ligation fragment was introduced. To introduce a mutation at the DR4 site of the gene, pTM1562-CAT plasmid was digested with PstI and ligated after Klenow fragment treatment (the plasmid was designated as pTM1562-CAT d(-1524/-1521)). Amplified plasmid DNAs in JM109 cells were purified by CsCl density gradient centrifugation in a Hitachi RP120VT rotor.

Cell Culture and Transfection-- BxPC-3 cells were cultured into 60-mm diameter noncoated dishes with RPMI 1640 medium containing 10% fetal calf serum (FCS), and the medium was replaced with MCDB medium without FCS at ~50% density. Plasmid DNAs treated with a cationic liposomes, TransFast, were prepared in polystyrene tubes and added to the cell cultures. They were allowed to stand for 2 h in a CO2 incubator, and then the culture medium was added to the cells. Plasmid DNA used amounted to 10 and 5 µg/dish for pTM-CATs and pSV-beta -galactosidase, respectively, and various amounts for pCMX-RARs. 24 h after the addition of culture medium, t-RA (10 µM in 0.1% dimethyl sulfoxide (Me2SO), final concentration) or 9C-RA (10 µM in 0.1% Me2SO, final concentration) was added to the DNA-transfected cells for 24 h. The cells were recovered and sonicated, and the resulting supernatants were used for determinations of CAT and beta -galactosidase activities. HUVE cells were cultured in Dulbecco's modified Eagle's medium containing 20% FCS without endothelial cell growth supplement and heparin, and secondary cultures on collagen-coated dishes were used for experiments (11, 36).

CAT and beta -Galactosidase Assays and Measurement of TM Antigen Level-- For measurement of CAT activity, 1-deoxy[dichloroacetyl-1-14C]chloramphenicol was used as the substrate, and the 3'-acetylated form of chloramphenicol was detected by autoradiography following thin layer chromatography on a Silica Gel 60 plate using a solvent of chloroform/methanol (19:1, v/v). beta -Galactosidase activity was measured according to Promega's protocol, and the activity was used as an internal control to normalize the transfection efficiency of individual pTM-CAT plasmids. Total TM antigen in HUVE and BxPC-3 cells was measured after solubilization of cells with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl, 0.5% (w/v) Triton X-100, and 1 mM benzamidine hydrochloride for 30 min at 4 °C (12).

Northern Blot and Nuclear Run-on Analysis-- Total RNA was isolated from HUVE and BxPC-3 cells (100-mm diameter dishes) with an ISOGEN kit according to the recommended protocol. Northern blot hybridization was performed as described previously (11). For RARs and RXRalpha , RNAs were absorbed on a filter using a slot blotter after the confirmation of each specific transcript on 1% denatured agarose gel electrophoresis. A 2057-bp TM cDNA fragment (corresponding -188 to +1869, where +1 is the first translated base, subcloned into pUC 119 at HindIII and BamHI sites), which was cloned from a placenta cDNA library, was used as a probe. Hybridization probes for RARalpha , RARbeta , RARgamma , and RXRalpha were obtained by treatment of pCMX plasmids with appropriate endonucleases, whereas Sp1 (DNASIS accession number J03133) probe was prepared by PCR using specific primer and cDNA of HUVE cells as a template. The identification of probes was performed by means of endonuclease treatments. The lengths of the probes used were 823 (RARalpha ), 882 (RARbeta ), 804 (RARgamma ), 1074 (RXRalpha ), and 732 bp (Sp1). Probes were labeled with [alpha -32P]dCTP by using the random primer extension labeling system. Nuclear run-on study was performed using [alpha -32P]dUTP-labeled RNA of nuclear fraction of cells treated or not treated with 10 µM t-RA for 6 h. The labeled RNA probe was purified by DNase I and proteinase K treatments, then absorbed on a nitrocellulose filter, and further purified with trichloroacetic acid washing and additional DNase I treatment. The probe eluted by SDS treatment was recovered by ethanol precipitation. Linearized pCMX and pCMX-TM plasmids, of which the latter was constructed by insertion of the 2.6-kb TM cDNA fragment into HindIII and BamHI sites of pCMX, were absorbed on a Hybond-N filter using a slot-blotter and fixed by a transilluminator. The filter was incubated with the labeled RNA probe for 36 h at 42 °C in the presence of 40% formamide and 10% dextran sulfate. The intensities of the signals developed by exposure of filters on x-ray films were determined using a Fuji BAS 1500 imaging analyzer (Fuji Photo Film Co., Tokyo, Japan) and expressed relative to the signal of the control band.

Preparations of Nuclear Extracts and in Vitro Translation Receptors-- BxPC-3 cells transfected with pCMX-hRARs and/or pCMX-hRXRalpha were cultured for 24 h in the presence or absence of t-RA (or 9C-RA). The cells were recovered with a cell scraper and centrifuged at 2,000 rpm for 5 min. The resulting pellet was homogenized in a Potter-Elvehjem type homogenizer and further centrifuged at 2,000 rpm for 5 min to obtain the nuclear fraction. This fraction was suspended in a buffer containing 50 mM Tris-Cl (pH 8.3), 20% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride and stored at -80 °C until used as a nuclear extract. RARalpha , RARbeta , and RXRalpha were also prepared in a rabbit reticulocyte lysate translation system using linearized pCMX-hRARs and pCMX-hRXRalpha plasmids as templates for RNA synthesis with T7 RNA polymerase according to the manufacturer's instruction. The amounts of translated proteins were estimated by Western blotting after SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

EMSA and Supershift Assay-- Double-stranded oligonucleotides (25-mer each) containing DR4 or Sp1 sites were end-labeled with [gamma -32P]ATP by T4 polynucleotide kinase. The upper strand of oligonucleotides with consensus sequences underlined were 5'-CGTTTGGTCACTGCAGGTCAGTCCA-3' for DR4; 5'-CTGTGTTGCACGTGCAAGCTCCGTA-3' for scramble DR4; 5'-CCTGTCGGCCCCGCCCGAGAACCTC-3' for the third Sp1 site; 5'-ATCCCATGCGCGAGGGCGGGCGCAA-3' for the second Sp1 site; 5'-GCGCAAGGGCGGCCAGAGAACCCAG-3' for the first Sp1 site; and 5'-GCGAGGGCGGGCGCAAGGGCGGCCA-3' for the tandem Sp1 sites containing second and first Sp1 sites. The reaction mixtures for the EMSA contained 0.5 ng of labeled probe (1 × 105 cpm/ng), 1 µg of poly(dI-dC), and 1 µl of nuclear retinoid receptors or 1 unit of Sp1 or AP2 in a final volume of 6 µl of 20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 5 mM MgCl2, 1 mM ZnCl2, 0.5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM spermidine, 0.008% Nonidet P-40, and 15% glycerol. As sources of nuclear retinoid receptors, nuclear extracts of BxPC-3 cells transfected with pCMX-hRARs and/or pCMX-hRXRalpha plasmids or in vitro translation products were used. As a reference, the preparation obtained by using the same procedure without any plasmid was employed. Competition experiments were performed with a 100-fold excess of unlabeled probe. The mixture was incubated for 30 min at 25 °C and subjected to 4% PAGE using a buffer consisting of 10 mM Tris-HCl, pH 7.9, 1.5 mM EDTA, and 5 mM sodium acetate. For the supershift assay, 0.5 µg of the respective antibody was further added, and the mixture was incubated for an additional 15 min at 25 °C. After electrophoresis, the gels were dried on filter paper and subjected to autoradiography.

Western Blot Analysis-- Nuclear extracts were subjected to SDS-PAGE according to the method described previously (37). After SDS-PAGE, protein was transferred to a nitrocellulose membrane and incubated with polyclonal antibodies of RARs, RXRalpha , or Sp1. Detections of RA receptors and Sp1 were carried out using biotinylated anti-rabbit IgG and horseradish peroxidase-conjugated streptavidin, and the resulting light emission developed by Renaissance Western blot Chemiluminescence Reagent was captured on Kodak X-Omat autoradiography film.


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RA-dependent Augmentation of TM Expression and Acceleration of the Transcriptional Rate-- In the present study, we used human pancreas BxPC-3 cells instead of HUVE cells, for the following reasons. 1) BxPC-3 cells show less damage than HUVE cells after treatment with cationic liposomes, one of the mildest transfection methods (38); 2) their characteristics of t-RA-dependent TM expression are similar to those of HUVE cells (see below); and 3) they can be easily maintained under homogeneous conditions. Fig. 1 shows the changes in antigen and mRNA levels of TM in BxPC-3 cells after treatment with t-RA. Total TM antigen in BxPC-3 cells was 14.1 ± 1.1 ng/1 × 105 cells (HUVE cells contained 17.3 ± 1.4 ng/1 × 105 cells) and increased to 2.8 times the control on treatment with 10 µM t-RA for 24 h (HUVE cells showed an increase to 2.5 times the control on similar treatment) (Fig. 1A). The TM mRNA level of BxPC-3 cells was increased by t-RA treatment and reached 3.7 times the control at 10 µM t-RA for 24 h (Fig. 1B). The time courses of the t-RA-dependent increase in TM antigen and in mRNA levels of BxPC-3 cells were also similar to those of HUVE cells (data not shown). These dose- and time-dependent increases in TM antigen and mRNA levels of BxPC-3 cells after t-RA treatment were in accordance with the result in HUVE cells reported previously (11). To assess the effect of t-RA on TM gene transcription rate, a nuclear run-on study was performed on nuclei prepared from both HUVE and BxPC-3 cells treated or not treated with 10 µM t-RA for 6 h (Fig. 1C). Treatment with t-RA increased the TM transcription rate to 310 and 325% of the respective control level for HUVE and BxPC-3 cells (Fig. 1C). Furthermore, TNF-alpha -dependent down-regulation of the TM antigen level in BxPC-3 cells was counteracted by synchronized treatment with t-RA (data not shown), as seen in HUVE cells (12).



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Fig. 1.   t-RA increases antigen and mRNA levels of TM in BxPC-3 cells, a human pancreas carcinoma cell line. When BxPC-3 cells cultured with RPMI 1640 medium containing 10% FCS became confluent, t-RA was added to the culture medium at various concentrations for 24 h and TM antigen (A) and TM mRNA (B) levels of the cells were measured as described under "Experimental Procedures." C, nuclear run-on assays was performed on nuclei obtained from HUVE and BxPC-3 cells treated with 10 µM t-RA or 0.1% Me2SO for 6 h as described under "Experimental Procedures."

RA-dependent Regulatory Sequence of TM Gene-- We examined the promoter activity of TM by transient expression assay using constructs of pTM-CAT plasmids. Although we could not clearly identify the transcription initiation site for human TM mRNA by S1 nuclease protection analysis, a faint signal at -168 base from the ATG codon of the translation site was consistent with one of the two sites reported by Yu et al. (15), and thus the first untranscribed base -1 was defined as the -169 base from the ATG codon in this paper. Fig. 2 shows the sequence-specific promoter activity of TM as determined by measuring the CAT activity of BxPC-3 cells transfected with pTM-CAT plasmids, which have different TM 5'-flanking sequences. Compared with the CAT activity of BxPC-3 cells transfected with pTM1562-CAT, the activities of cells transfected with the deletion plasmids decreased gradually when the deletions of TM 5'-flanking sequences were extended to -549. Although the CAT activity of cells transfected with pTM549-CAT was half that in the case of pTM1562-CAT, plasmids with more extensive deletions, such as pTM479-CAT and pTM404-CAT, showed some recovery in CAT activity. When pTM213-CAT was transfected, the CAT activity was markedly increased as compared with that of pTM244-CAT, and a further deleted plasmid (pTM119-CAT) decreased the activity to a level less than that of pTM244-CAT plasmid. The results suggest that negative- and positive-acting elements are present in the sequences around -244 to -214 and -213 to -120, respectively, of the TM gene. The effect of t-RA on the promoter activity of TM was measured (Fig. 3). The ratio of increase of CAT activity by t-RA was different for each plasmid used. The 4.1-fold increase of the activity by t-RA treatment in the case of pTM1562-CAT decreased to ~2.3-fold when pTM-CAT plasmids containing the 5'-site from -1491 to -213 of the TM sequence were transfected, and further deletion of the 5'-sequence abolished the t-RA-dependent increase in the activity. These results indicate the existence of two independent RA-responsive regions (one localized at -1562 to -1492 bp and the other localized at -213 to -120 bp of the transcription site) in the TM promoter sequence.



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Fig. 2.   Transcriptional activity of TM gene determined by transient CAT expression assay of BxPC-3 cells. BxPC-3 cells were transfected with pTM1562-CAT or various 5'-deletion mutants as well as pSV-beta -galactosidase as described under "Experimental Procedures." The culture medium was changed after 2 h of cationic liposome transfection, and 48 h thereafter, activities of CAT and beta -galactosidase in the cell extracts were determined. The CAT activities normalized with respect to beta -galactosidase activities were expressed as a percentage of that with pTM1562-CAT. Results are the average values of three independent experiments. 5'-Sites of the TM promoter sequence in pTM-CAT plasmid, in the order from left to right, are -1562, -1491, -1272, -960, -549, -479, -404, -346, -290, -244, -213, -119, -47, and +63. Representative RARE sites and four Sp1 sites in addition to the TATA box are shown at the bottom.



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Fig. 3.   t-RA-dependent increase in transcriptional activity of the TM gene determined by transient CAT expression assay of BxPC-3 cells. The transfection procedure was the same as that in Fig. 2. At 24 h after the medium change, 10 µM t-RA or its vehicle (0.1% Me2SO) was added to the culture medium, and the cells were incubated for an additional 24 h. The CAT activities normalized with respect to beta -galactosidase activities were expressed as a percentage of that of BxPC-3 cells treated with 0.1% Me2SO after transfection of pTM1562-CAT plasmid. Open and closed bars show relative CAT activities of the transformed cells treated with 0.1% Me2SO or t-RA, respectively, and values in parentheses on the right indicate the ratio of the activity in each transformant. Open bars are the same data as the values in Fig. 2. Results are the average values of three independent experiments.

To identify the RA-responsive sequences, mutations were introduced at DR4 of RARE in the pTM1562-CAT plasmid and at two consensus Sp1 sites in the pTM346-CAT plasmid (Fig. 4). As shown in Fig. 4A, mutation at DR4 reduced the t-RA-dependent increase in CAT activity of pTM1562-CAT without extensive reduction of the normal CAT activity, and the increase ratio became 2.4-fold, which is comparable to that in the case of pTM1491-CAT (Fig. 3). Each deletion at the third or second Sp1 motif in the TM promoter sequence of pTM346-CAT produced different responses (Fig. 4B). pTM346-CAT d(-208/-201) showed a slight decrease in the CAT activity compared with that of the wild-type plasmid and exhibited a mild response to t-RA treatment, whereas pTM346-CAT d(-142/-135) decreased the CAT activity, irrespective of t-RA treatment. The reduction with the plasmid deleted at the second Sp1 motif of the TM gene sequence reveals the significance of this region in the primary and t-RA-dependent enhancement of the transcriptional activity. The third Sp1 motif might contribute to cooperative interactions of other transcription factors with Sp1 protein. On the other hand, a plasmid deleted from -221 to -214 (pTM346-CAT d(-221/-214)) enhanced the CAT activity of the transfectant to 165% of that of pTM346-CAT, and further enhancement of the activity was observed after treatment with t-RA. As expected from the result in Fig. 2, this deleted region might act negatively, in concert with an unknown factor, on the TM promoter activity.



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Fig. 4.   Deletions of DR4 and Sp1 sites in TM gene reduce the RA-dependent increase in promoter activity of the gene. A, pTM1562-CAT and its deletion mutant at the DR4 site located at bases -1531 to -1516 and pTM1562-CAT d(-1524/-1521) were transfected into BxPC-3 cells, and the cells were treated with 10 µM t-RA or the vehicle (0.1% Me2SO) as described under "Experimental Procedures." The CAT activities normalized with respect to beta -galactosidase activities were expressed as a percentage of the activity of BxPC-3 cells treated with the vehicle alone after transfection of pTM1562-CAT plasmid. Results are average values with standard deviations of three independent experiments. The sequence on the left of A is that from -1531 to -1516 of the human TM gene. Four bases between the direct repeats of RARE are underlined. B, pTM346-CAT, the third Sp1 site mutant, pTM346-CAT d(-208/-201), or the second Sp1 site mutant, pTM346-CAT d(-142/-135), and pTM346-CAT d(-221/-214) were transfected into BxPC-3 cells, and the cells were treated with 10 µM t-RA or the vehicle (0.1% Me2SO) as described under "Experimental Procedures." The CAT activities normalized with respect to beta -galactosidase activities were expressed as a percentage of that of BxPC-3 cells treated with the vehicle alone after transfection with pTM346-CAT. Results are average values with S.D. of three independent experiments. The sequences on the left in B are the bases from -223 to -200 and -144 to -133 of the human TM gene. The Sp1 sites are boxed. Numbers 3, 2, 1 or T in circles, and diamond mean third Sp1 site, second Sp1 site, first Sp1 site or TATA box, and deleted site, respectively. Open column, 0.1% Me2SO-treated; closed column, t-RA-treated.

Expressions of RA Receptors Modulate Promoter Activity of TM-- To examine the effect of changes in concentration of RA receptors on the promoter activity of TM in BxPC-3 cells, expression plasmids of RA receptors were cotransfected with pTM1562-CAT plasmid. Cotransfection of the pTM-CAT plasmid and each of pCMX-hRARalpha , pCMX-hRARbeta , and pCMX-hRARgamma plasmid into BxPC-3 cells resulted in a significant increase in CAT expression in the absence of retinoid, especially when more than 0.1 µg of pCMX-hRARs was cotransfected (Fig. 5). However, enhancement of the promoter activity of the cells by t-RA or 9C-RA treatment did not increase further after the cotransfection of each RAR expression plasmid. As shown in Fig. 5D, cotransfection of pCMX-hRXRalpha and pTM1562-CAT plasmids into the cells resulted in little increase in the promoter activity in the absence of retinoid treatment, and a remarkable decrease in the activity was seen in the cells transfected with more than 1 µg of pCMX-RXRalpha , despite treatment of the cells with t-RA or 9C-RA. The results suggest that the retinoid-dependent increase in TM promoter activity involves at least two different regulations, that is retinoid receptors-dependent and -independent pathways. RAR-RXR heterodimers activate transcription in response to t-RA or 9C-RA by binding to target gene response elements consisting of DR5 or DR2 (26, 39, 40), whereas RXR-RXR homodimers activate transcription in response to 9C-RA by binding to DR1 (28, 40-42). Since the levels of activation of TM promoter activity were the same in the cells treated with t-RA and 9C-RA (Fig. 5), it appears that the homodimer of RXRalpha (RXRalpha -RXRalpha ) does not function as an activator of the promoter activity. Furthermore, suppression of the promoter activity by RXRalpha -RXRalpha homodimer may also be excluded, because RXRalpha did not bind to the DR4 element of the TM gene (see below).



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Fig. 5.   Transcriptional activation by RARalpha , RARbeta , RARgamma , and RXRalpha in the presence or absence of t-RA or 9C-RA. BxPC-3 cells cultured in 60-mm dishes were cotransfected with pTM1562-CAT (10 µg) and various amounts of expression plasmid for RARs or RXRalpha in addition to pSV-beta -galactosidase (5 µg) as described under "Experimental Procedures." At 24 h after the medium change, 0.1% Me2SO, 10 µM t-RA, or 10 µM 9C-RA was added to the culture medium, and the cells were further incubated for 24 h. The CAT activities normalized with respect to the beta -galactosidase activities were expressed as a percentage of that of BxPC-3 cells neither transfected with expression plasmid of RA receptor nor treated with t-RA or 9C-RA. Receptors expressed are RARalpha (A), RARbeta (B), RARgamma (C), and RXRalpha (D). Open column, 0.1% Me2SO-treated; shaded column, t-RA-treated; closed column, 9C-RA-treated.

Effect of Concentration of Retinoid Receptor on Promoter Activity of TM-- In the present study, RXRalpha was used as the representative of RXRs, because both HUVE and BxPC-3 cells express its mRNA and protein (see the data in Figs. 10 and 11), and because it has been reported that RXR isoforms such as RXRalpha , RXRbeta , and RXRgamma behave similarly in heterodimeric complex formation with RARs or TR (23, 43-45). It is also reported that 9C-RA has the highest affinity for RXRalpha among RXR isoforms (44). The promoter activity of pTM1562-CAT plasmid in the case of simultaneous expressions of RARalpha or RARbeta and RXRalpha in the cells was examined (Fig. 6). The RARalpha - and RARbeta -dependent increases in promoter activities were further enhanced by cotransfection of pCMX-RXRalpha (0.1 µg) (Fig. 6, C and D). However, cotransfection with pCMX-RARalpha (or pCMX-RARbeta ) and a higher dose of pCMX-RXRalpha (1 µg) drastically reduced the promoter activity, even when 0.1 µg of pCMX-RARs was used (Fig. 6, E and F). These results demonstrate that TM promoter activity is controlled by the expressions of RARs and RXRalpha and that the extent of the expression of the latter is critical for gene expression of TM.



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Fig. 6.   RXRalpha modulates RARalpha - and RARbeta -mediated transactivation of pTM-CAT. BxPC-3 cells cultured in 60-mm dishes were cotransfected with pTM1562-CAT (10 µg), pSV-beta -galactosidase (5 µg), and various amounts of expression plasmid for RXRalpha in addition to that for RARalpha (0-5 µg) or RARbeta (0-5 µg). Cultures and treatment of cells with 0.1% Me2SO were as described in the legend to Fig. 5. The CAT activities normalized with respect to the beta -galactosidase activities were expressed as a percentage of that of BxPC-3 cells not transfected with expression plasmid for RA receptor. Amounts of expression plasmid for RXRalpha used were none (A and B), 0.1 µg (C and D), and 1 µg (E and F).

Sequence-specific Acceleration of TM Promoter Activity by RAR-RXR Expressions-- To confirm that RA receptors modulate TM gene expression through the RARE element, we determined the promoter activity of BxPC-3 cells transfected with DR4-deleted pTM1562-CAT plasmid (pTM1562-CAT d(-1524/-1521)) or pTM346-CAT plasmid instead of pTM1562-CAT plasmid (Fig. 7). The following observations were made. First, the promoter activity of the cells transfected with pTM1562-CAT plasmid was increased by the coexpression of RARs and RXRalpha in the absence of retinoid treatment, as already shown in Figs. 5 and 6, whereas the promoter activity of those transfected with pTM1562-CAT d(-1524/-1521) or pTM346-CAT was not increased under the same conditions. Second, the promoter activities of cells transfected with these pTM-CAT plasmids were increased by treatment with t-RA or 9C-RA under conditions where retinoid receptors were not expressed, but the increase ratio after retinoid treatment was the highest when pTM1562-CAT plasmid was used. Third, the 9C-RA-dependent increase in the promoter activity of pTM1562-CAT in the cells, which expressed RARs and a small amount of RXRalpha , was not greater than the t-RA-dependent increase. These results suggest that 1) the interaction between heterodimer of RAR-RXRalpha and the DR4 element in TM gene participates in part in the retinoid-dependent increase in the promoter activity of TM; 2) ligand-independent dimerization of RARs-RXRalpha occurs, although we cannot exclude the possibility that a small but sufficient amount of t-RA exists in the cell in the absence of retinoid treatment; and 3) formation of RAR-RXR rather than RXR-RXR is significant when RXRalpha is not highly expressed.



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Fig. 7.   Expressions of RARalpha and RXRalpha or RARbeta and RXRalpha fail to transactivate DR4-deleted pTM-CAT and pTM346-CAT plasmids in the absence of t-RA and 9C-RA. BxPC-3 cells cultured in 60-mm dishes were cotransfected with a pTM-CAT plasmid (10 µg), pSV-beta -galactosidase (5 µg), and 0.1 µg of expression plasmid for RARalpha , RARbeta , and/or RXRalpha . Cultures and treatment of cells were as described in the legend to Fig. 5. The CAT activities normalized with respect to the beta -galactosidase activities were expressed as a percentage of that of BxPC-3 cells neither transfected with expression plasmid for RA receptor nor treated with t-RA or 9C-RA. The reporter plasmid used was pTM1562-CAT (A), pTM1562-CAT d(-1524/-1521) (B), and pTM346-CAT (C). Open column, 0.1% Me2SO-treated; shaded column, t-RA-treated; closed column, 9C-RA-treated.

Interaction of RAR-RXR Heterodimer with DR4 Sequence of TM Gene-- To test the idea, that RAR-RXRalpha heterodimer binds to the DR4 site of the TM gene, interaction between RARs, RXRalpha , and a DNA probe containing the DR4 sequence of the TM gene was examined by EMSA (Fig. 8). When a nuclear fraction of BxPC-3 cells transfected with each pCMX-hRAR plasmid was incubated with the probe, a retarded band was observed, regardless of the isoform, although that of cells transfected with RXRalpha expression plasmid showed only a weak band (Fig. 8A). By using a nuclear fraction prepared from cells cotransfected with one RAR isoform and RXRalpha expression plasmids, a more distinct band with the same mobility was observed. The same band on the EMSA was also obtained when the cDNA probe was incubated with a sample, which was prepared by mixing the nuclear fraction expressing RARalpha (or RARbeta ) with that expressing RXRalpha (Fig. 8B, lane 3). The binding specificity of these receptors to the radiolabeled probe corresponding to the DR4 sequence of TM gene was confirmed by competition with an excess amount of the unlabeled cDNA probe. Moreover, these retarded bands were further shifted in the presence of RARbeta and RXRalpha antibodies (lanes 7 and 9), and therefore it was confirmed that RARbeta -RXRalpha heterodimer bound to the consensus sequence of the DR4 element in the TM gene. To exclude contamination with endogenous receptors, RA receptors synthesized with a cell-free reticulocyte lysate system were also subjected to EMSA using the same DNA probe (Fig. 8C). Since the RARalpha - or RARbeta -dependent retarded band was observed in the absence of endogenous RXRalpha in the cell-free system, it is possible that the homodimer of each RAR could bind to DR4 of the TM sequence. Furthermore, it became apparent that homodimer (or monomer) of RXRalpha did not bind to the DR4 site (lane 3). Specific binding of the heterodimer binding to this DR4 sequence was further confirmed using a scramble probe of the DR4 sequence as a competitor (lane 8).



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Fig. 8.   Binding of retinoid receptors to the DR4 element in the TM gene. EMSA was carried out using radiolabeled oligonucleotide containing the DR4 element in the TM gene, and the probe was incubated with nuclear extracts of BxPC-3 cells transfected with pCMX-hRARs and/or pCMX-hRXRalpha plasmids (A and B) or in vitro translation products of the respective mRNAs (C). After incubation for 30 min as described under "Experimental Procedures," the incubation mixture was subjected PAGE under a low salt condition. After electrophoresis, the gels were dried on filter paper and subjected to autoradiography. A, lanes 1 and 2, RARalpha expressed; lanes 3 and 4, RARbeta expressed; lanes 5 and 6, RARgamma expressed; lanes 7 and 8, RXRalpha expressed; lanes 9 and 10, RARalpha and RXRalpha coexpressed; lanes 11 and 12, RARbeta and RXRalpha coexpressed; lanes 13 and 14, RARgamma and RXRalpha coexpressed; lanes 15 and 16, 2-fold amounts of samples (RXRalpha expressed) in lanes 7 and 8; even-numbered lanes, in the presence of 100-fold excess of DR4 competitor. B, lanes 1 and 2, RARbeta expressed; lanes 3 and 4, mixture of samples that expressed RARbeta and RXRalpha separately; lanes 5-10, RARbeta and RXRalpha coexpressed; lanes 7 and 9, in the presence of RARbeta antibody; lanes 8 and 9, in the presence of RXRalpha antibody; lanes 2, 4, 6, and 10, in the presence of 100-fold excess of DR4 competitor. C, lane 1, RARalpha ; lane 2, RARbeta ; lane 3, RXRalpha ; lanes 4 and 5, mixture of RARalpha and RXRalpha ; lanes 6-8, mixture of RARbeta and RXRalpha ; lanes 5 and 7, in the presence of 100-fold excess of DR4 competitor; lane 8, in the presence of 100-fold excess of the scrambled DR4 probe.

Effect of Ratio of RAR/RXR on Binding of RAR-RXR to DR4 Sequence-- Since an RXRalpha concentration-dependent decrease in the promoter activity was observed in Fig. 6, the effect of the ratio of RARbeta to RXRalpha on interaction of the heterodimer with the DR4 sequence was investigated (Fig. 9). Nuclear samples of BxPC cells cotransfected with RARbeta and RXRalpha at various ratios were prepared, and the binding ability was examined using DR4 probe. The intensity of the band corresponding to the retardate complex with RARbeta -RXRalpha heterodimer initially increased with the increase in the expression of RXRalpha , but a further increase in the RXRalpha expression apparently decreased the intensity and no retardate band was observed at 5-fold excess of RXRalpha over RARbeta (Fig. 9B). When RARbeta and RXRalpha , prepared in a reticulocyte lysate system instead of by cotransfection of cells with both plasmids, were mixed in the reaction mixture at various ratios, virtually the same results were obtained (Fig. 9B), indicating rapid formation of RARbeta -RXRalpha dimer. These results suggest that the ratio of RARs to RXRalpha is critical for not only formation of RAR-RXR heterodimer but also binding of the heterodimer to the DR4 sequence. Since the maximum binding was observed when a higher ratio of RXRalpha compared with RARbeta was applied (Fig. 8B), the formation of RXRalpha -RXRalpha heterodimer and/or the presence of RXRalpha monomer may be possible under the cell-free condition.



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Fig. 9.   Effect of ratio of RXR to RAR on the heterodimer binding to the DR4 element. EMSA was carried out using radiolabeled oligonucleotide containing the DR4 element in the TM gene, and the probes were incubated with nuclear extracts of BxPC-3 cells (A) or in vitro translation products of RARbeta and RXRalpha mRNAs (B). Experimental conditions were the same as described in the legend to Fig. 8. A, nuclear fractions prepared from BxPC-3 cells that were cotransfected with pCMX-hRARbeta and pCMX-hRXRalpha plasmid at different ratios as shown at the bottom. Lane 7, in the presence of 100-fold excess of DR4 competitor. B, samples were mixed with different ratios of RARbeta and RXRalpha as shown at the bottom. Lane 8, in the presence of 100-fold excess of DR4 competitor.

Effect of t-RA or 9C-RA on mRNA and Protein Levels of Retinoid Receptors-- Changes in mRNA levels of retinoid receptors in BxPC-3 cells after treatment with t-RA or 9C-RA were measured (Fig. 10). The mRNA level of TM increased in a time-dependent manner, which was compatible with the results in Fig. 1. BxPC-3 cells contained mRNA of all four RA receptors, and RARbeta mRNA was markedly augmented after the treatment with t-RA. The level of RARalpha mRNA also increased within 6 h after t-RA treatment, whereas RARgamma mRNA increased only slightly up to 24 h after the treatment. On the other hand, the RXRalpha mRNA level decreased with increase in the time after t-RA treatment. Changes in the mRNA levels of these four RA receptors in HUVE cells after treatment with t-RA were also examined, and quite similar results to those in BxPC-3 cells were obtained (data not shown). The presence of RARalpha and RARbeta mRNAs in normal HUVE cells has already been reported by Fesus et al. (46), and Kooistra et al. (47) observed the expression of transcripts of three RAR and two RXR subtypes in normal HUVE cells. They also found induction of RARbeta and RXRalpha mRNA after exposure of the cells to t-RA. Although there was definitive difference in changes in RXRalpha level after t-RA treatment between their results and ours, similar patterns of increase in RARbeta mRNA level and decrease in RXRalpha mRNA level in BxPC-3 cells were observed when 9C-RA instead of t-RA was used (Fig. 10, C and D). Western blot analysis showed that these changes in mRNA levels of RARbeta and RXRalpha reflected those of the protein levels in the nuclear fraction of the cells after treatment with t-RA (Fig. 11). Thus, treatment with t-RA gradually increased and decreased the amounts of RARbeta and RXRalpha in the nuclear fraction of BxPC-3 cells, respectively.



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Fig. 10.   Changes in mRNA levels of TM in BxPC-3 cells treated with t-RA. BxPC-3 cells cultured in 100-mm dishes were treated with 10 µM t-RA for 0, 3, 6, 12, and 24 h (A and B) or treated with 10 µM 9C-RA for 0 and 24 h (C and D), and total RNAs were prepared. mRNA levels were determined qualitatively by performing slot blot hybridization with the respective 32P-labeled probes, and then two transcripts for RARalpha (approx 3.0 and approx 3.6 kb) and RARbeta (approx 3.5 and faint approx 2.8 kb) were combined signals, respectively, in this result. The transcript for RARgamma (approx 3.3 kb) or RXRalpha (approx 5.0 kb) was single. A and C, autoradiograms. B and D, the mRNA level without incubation was defined as 1, and the relative mRNA levels were plotted against incubation time after quantitative analysis of the bands with a BAS 1500 imaging analyzer. Values were normalized with respect to the levels of actin mRNA.



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Fig. 11.   Changes in antigen levels of RARbeta and RXRalpha in BxPC-3 cells treated with t-RA. BxPC-3 cells cultured in 100-mm dishes were treated with 10 µM t-RA for 0, 6, and 24 h, and nuclear extracts were prepared as described under "Experimental Procedures." After SDS-PAGE, protein was transferred to a nitrocellulose membrane and incubated with polyclonal antibodies to RARbeta and RXRalpha . A, four isoforms of RARbeta corresponding 56 to 47 kDa (48), as shown by an arrow, were recognized, and a low molecular band (arrowhead) seems to be a degradation product of RARbeta . B, RXRalpha corresponding 53 kDa (49) was recognized. Detection was carried out using antibodies to RARbeta and RXRalpha followed by biotinylated anti-rabbit IgG and horseradish peroxidase-conjugated streptavidin, and the light emission developed was captured on an autoradiography film. Open arrowheads in A and B indicated nonspecific bands.

Interaction of Nuclear Sp1 Protein with Sp1-dependent Sequence in TM Gene-- Next we focused on Sp1 as a candidate factor to interact with the GC box of the TM gene, and EMSA was performed after incubation of 32P-labeled TM cDNA probes containing these Sp1 sites with Sp1 or AP2 (the latter is also a transcription factor that binds to GC-rich sequences (50) and has been shown to play a role in some retinoid-affected morphogenetic processes (51)). The promoter activity of pTM346-CAT was not influenced by coexpression of RARs and/or RXRalpha (Fig. 7), but recently Suzuki et al. (52) showed that RAR-RXR interacts physically with Sp1 and that the complex of RAR-RXR-Sp1 binds to the GC box of the urokinase promoter sequence. Therefore, the probe was incubated with recombinant Sp1 in the presence or absence of both RAR and RXR prepared in a reticulocyte lysate system, followed by electrophoresis. Fig. 12A shows that Sp1 could form a retarded complex with labeled probes containing an Sp1-binding site in the presence of RARbeta and RXRalpha . The intensity of the retarded bands of cDNA probes containing the three Sp1-binding sites were in the following order: second > third >=  first Sp1 sites (lanes 2, 5, and 8, respectively). However, these Sp1 sites were not target sequences for AP2 (data not shown). As shown in Fig. 4, the significance of the second Sp1-binding site among the these sites in the TM promoter region was evaluated by measuring the promoter activity of a TM-CAT plasmid in which this second Sp1 site was deleted. The shifted bands with slower and faster migrations were Sp1-dependent and -independent complexes, respectively, since the lower band with fast migration appeared in the absence of Sp1 in the reaction mixture (lanes 1, 4, and 7). The most intense shifted band was seen with a cDNA probe containing tandem Sp1 sites with 6 spaces corresponding to the second and first Sp1-binding sites of the TM gene (lane 11).



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Fig. 12.   Sp1 interacts with the Sp1 sites of the TM promoter gene. EMSA was carried out using 32P-end-labeled oligonucleotide (25 bp) with the consensus sequence of the Sp1 site or a tandem Sp1 site containing the second and the first Sp1 sites in the TM gene in the absence of t-RA. A labeled DNA probe and recombinant Sp1 was incubated for 30 min and subjected to PAGE as described under "Experimental Procedures." The closed and open arrowheads indicate the Sp1-dependent and -independent DNA-protein complexes, respectively. A, lanes 1-3, probe (-217 to -193) containing the third Sp1 site; lanes 4-6, probe (-154 to -130) containing the second Sp1 site; lanes 7-9, probe (-135 to -111) containing the first Sp1 site; lanes 10-12, probe (-145 to -121) containing the tandem Sp1 site corresponding to the second and the first Sp1 sites. Probes were incubated with recombinant Sp1 (lanes 2, 3, 5, 6, 8, 9, 11, and 12) in the presence of in vitro translation products from RARbeta and RXRalpha mRNAs by a reticulocyte lysate system. As a competitor, 100-fold unlabeled probe was added to the incubation mixture (lanes 3, 6, 9, and 12). B, probe containing a tandem Sp1 site incorporating the second and the first Sp1 sites of the TM promoter sequence was incubated with recombinant Sp1 (lanes 1-16, 18, 21, and 22) in the presence of in vitro translation products of RARalpha (lanes 1, 2, 7, and 8), RARbeta (lanes 3 and 4, and 9-18), and RXRalpha (lanes 5-18) or the reticulocyte lysate solution (RL) as a reference for these retinoid receptors (lanes 19-22). Anti-Sp1 IgG (lanes 11, 15, and 16), anti-RARbeta IgG (lanes 12, 14 and 15) and anti-RXRalpha IgG (lanes 13, 14 and 16) were added at 15 min before electrophoresis. As a competitor, 100-fold unlabeled probe was added to the incubation mixture (lanes 2, 4, 6, 8, 10, 20, and 22). C, probe containing the tandem Sp1 site was incubated with various concentrations of recombinant Sp1 (lanes 4-8 and 10-17) or AP2 (lanes 18 and 19) in the presence of the reticulocyte lysate solution (RL) (lanes 2-8 (1 µl) and lanes 9-19 (0.05 µl)). As a competitor, 100-fold unlabeled probe was added to the incubation mixture (lanes 2, 15, 17, and 19). Anti-Sp1 IgG was added in lane 16.

The effect of RA receptors on the Sp1 binding to this tandem Sp1 site was further investigated by EMSA (Fig. 12B), because a curious result was obtained as described below. The Sp1-dependent retarded band with slow migration was detected on EMSA when this probe was incubated with Sp1 and one retinoid receptor, such as RARalpha , RARbeta , or RXRalpha (lanes 1, 3 and 5), and all of the corresponding bands disappeared upon addition of excess competitor (lanes 2, 4, and 6). However, the coexistence of RARs and RXRalpha did not increase the intensity of the band in the presence of Sp1 (lanes 7 and 9), and the bands were not supershifted by the addition of the respective retinoid receptor antibody (lanes 12-14). The supershifted band was restricted to the case of Sp1 antibody (lanes 11, 15, and 16). Surprisingly, as shown in lane 21, the Sp1-dependent retarded band was detected in the absence of RARs and RXRalpha but in the presence of reticulocyte lysate solution as the control for all the RA receptors. These results suggest that interaction between the Sp1 and Sp1 sites of the TM promoter sequence is dependent on the presence of unknown factor(s) in the reticulocyte lysate solution and that the interaction is independent of formation of the multicomplex of Sp1-RAR-RXRalpha . Therefore, we concluded that homodimer and heterodimer of retinoid receptors do not participate directly or indirectly in the interaction between Sp1 and the tandem Sp1 sites of TM gene. This is consistent with the finding in Fig. 7 that expressions of RARs and/or RXRalpha did not induce transactivation activity of pTM346-CAT, which contains all four Sp1 sites of the TM gene. Nevertheless, it is true that Sp1 binds specifically to these Sp1 probes, because all the retarded bands were eliminated by an excess of each unlabeled probe. Further increase in the intensity of the retarded band on EMSA was directly proportional to the concentration of Sp1 in the presence of a small amount of reticulocyte lysate solution (Fig. 12C, lanes 3-8 and 9-14). In addition, the retarded band with slower migration was supershifted in the presence of the specific antibody of Sp1 (lane 16), and it was also confirmed that AP2 was unable to form a retarded complex with the same cDNA probe (lanes 18). Binding of Sp1 to Sp1 sites of the TM sequence was also independent of the presence of t-RA in EMSA (data not shown).

RA-dependent Changes in mRNA and Protein Levels of Sp1-- The time course of changes in mRNA level of Sp1 after treatment of BxPC-3 and HUVE cells with t-RA was examined (Fig. 13). The Sp1 mRNA was expressed not only in BxPC-3 cells but also in HUVE cells. Sp1 mRNA increased rapidly after treatment of BxPC-3 cells with t-RA, and the maximum increase was ~2.5-fold from the control level. The increase in the ratio and the time course of change of each mRNA level in HUVE cells after treatment with t-RA were almost the same as those in BxPC-3 cells. The time course of changes in Sp1 content after treatment of BxPC-3 cells with t-RA was examined (Fig. 13C). The appearance of a 105-kDa band, a phosphorylated form of Sp1 (53), was observed, in addition to a rapid increase in the 95-kDa Sp1, the dephosphorylated form (53). Upon alkaline phosphatase treatment of a sample, disappearance of the 105-kDa band followed by increase in the 95-kDa band was observed, indicating the existence of the two forms in the cells. These results suggest that treatment of human cells with t-RA modulates the TM gene promoter activity in part through increasing Sp1 content and phosphorylation in the nuclear fraction.



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Fig. 13.   t-RA up-regulates mRNA and protein levels of Sp1 in BxPC-3 and HUVE cells. A and B, BxPC-3 and HUVE cells cultured in 100-mm diameter dishes were treated with 10 µM t-RA for 0, 3, 6, and 24 h. Total RNA was prepared, and Northern blot hybridization was performed using Sp1 and beta -actin probes after 1% denatured agarose gel electrophoresis. Two transcripts for Sp1 (approx 8.0 and approx 5.5 kb) were detected. C, nuclear fraction of BxPC-3 cells treated with t-RA until 24 h was prepared, and SDS-PAGE was performed. Proteins transferred to a nitrocellulose membrane were incubated with polyclonal antibody of Sp1 as described under "Experimental Procedures." Two bands corresponding to 95 and 105 kDa are the dephosphorylated and phosphorylated forms, respectively, of Sp1 (53).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this paper we have shown that t-RA-dependent enhancement of human TM gene expression is regulated by two sites in the 5'-flanking region. The t-RA-responsive distal region of the TM gene contains an RARE composed of TGGTCANNNNAGGTCA. Our transient expression studies revealed that the TM promoter could be transactivated by the human RARs, especially by RARalpha and RARbeta . On the basis of transient expression assay using F9 embryonal carcinoma cells, Weiler-Guettler et al. (54) suggested that the TM promoter could be activated by RARbeta . By the use of an RARalpha antagonist, on the other hand, Shibakura et al. (55) obtained results indicating a major role of RARalpha in TM up-regulation by retinoids in leukemia and HUVE cells. Treatment with t-RA (or 9C-RA) in the present study, however, caused a much greater increase in the RARbeta level compared with that of RARalpha , and no difference was observed between RARalpha - and RARbeta -dependent transactivation of TM promoter activity. Furthermore, the binding ability of RARalpha and RARbeta with the DR4 sequence of the TM gene in the presence or absence of RXRalpha was the same when equivalent amounts of both RAR receptors were used. Therefore, the increase in the total amount of all the RARs might be a dominant factor for the up-regulation of transcriptional activity of TM, rather than the changes in the concentrations of individual RAR subtypes. Coexpression of RXRalpha with RARalpha or RARbeta showed a different mode of promoter activity compared with that in the case of expression of a single RAR receptor. When RXRalpha was highly expressed in BxPC-3 cells, the increase in the transactivation activity by RARalpha or RARbeta was eliminated, whereas it was augmented at a lower expression level of the RXRalpha receptor. Furthermore, the binding of RARbeta -RXRalpha to RARE of the TM gene was closely related to the proportion of RARbeta /RXRalpha , and the levels of RARs increased in both BxPC-3 and HUVE cells after t-RA or 9C-RA treatment, whereas that of RXRalpha decreased under the same treatment. It seems very probable that t-RA and 9C-RA contributed to the acceleration of TM promoter activity through both an increase in RARs and a decrease in RXRalpha , followed by appropriate formation of RARs-RXRalpha heterodimers that can bind to the DR4 site of the TM gene. In this connection, we have examined the changes in the levels of RARs and RXRalpha of HUVE cells after treatment with a synthetic RARs-selective retinoid, Ch55. The results supported the above finding that an increase in TM promoter activity is closely related to the increasing ratio of RARs/RXRalpha .

To access the action of t-RA on the DR4 site-dependent enhancement of promoter activity of TM, a ligand-dependent change in the binding of RARbeta -RXRalpha to the DR4 site of the TM gene was investigated. In our EMSA experiment, essentially no increase in the intensity of the retarded band of the DR4 probe was observed in the presence of t-RA and/or 9C-RA in the reaction mixture, although a small increase in the intensity was observed at higher concentrations (10-6 and 10-5 M) (data not shown). Therefore, it is plausible that t-RA and 9C-RA are not stimulators of the interaction between the heterodimer and DR4 of the TM gene, in a similar manner to the Sp1 and Sp1 sites of the TM gene (see below), although we cannot exclude the possibility that both retinoids act as accelerators of the heterodimer formation of RAR-RXR, regardless of the presence or absence of the template DNA.

On the other hand, the protein that interacts with the proximal sequence for transcriptional up-regulation appeared to be Sp1. Among the four Sp1 sites in the 5' region from the TATA box of the gene, the major role of the second Sp1 site, rather than the other sites, was found. Although it has been shown that one Sp1 site is sufficient for the Sp1-dependent augmentation of the promoter activities of cytochrome P450IA1 (56) and alpha 2(I) collagen (57), there was a significant cooperative role of the second Sp1 site with the other adjacent Sp1 sites of the TM gene, especially the first Sp1 site. It is probable that multiple or adjacent Sp1 sites in the TM gene are essential for the maximal activation of the promoter, as reported for other genes (32, 58-61). It is known that the binding and subsequent transactivation activities of Sp1 can be modulated by post-translational modification, i.e. glycosylation (62, 63) and phosphorylation (53, 64-66). After t-RA treatment, the phosphorylated form of Sp1 (105 kDa) was increased (Fig. 13), and the binding activity of Sp1 to the consensus Sp1 site of the TM gene was lost when reticulocyte lysate in the reaction mixture was previously heat-inactivated (data not shown). These results suggest that the phosphorylation of Sp1 is important for the interaction between Sp1 and these Sp1 sites of the TM sequence. In our preliminary experiment, the binding of Sp1 to the Sp1 sites of the TM gene was increased or decreased after Sp1 was phosphorylated or dephosphorylated, respectively. Further studies on the functional regulation of TM promoter activity by such modifications of the Sp1 molecule in HUVE cells are in progress in our laboratory. In addition, the effects of Sp1-related factors, which recognize GC boxes, on the promoter activity should be determined, because some papers indicated that Sp1-mediated promoter activation was accelerated and attenuated by Sp1-related family members (58, 67, 69).

It could be assumed that interaction between Sp1 and RARs-RXRalpha does not serve as a cooperative process for Sp1 site-dependent promoter activity of TM. However, we cannot exclude the possible formation of a complex between Sp1 and RAR-RXR, which binds to sites other than Sp1 sequences of the TM gene. Synergistic action of Sp1 with glucocorticoid receptor (70) or estrogen receptor (71, 72) has been reported. Krey et al. (61) demonstrated that Sp1 was not able to interact synergistically with peroxisome proliferator-activated receptor-RXR, although these three nuclear proteins could bind simultaneously to the acyl-CoA oxidase gene. However, in our experiment, an increase in the TM transcription activity by 9C-RA was not further elevated by the synchronous treatment of HUVE cells with 9C-RA and TR activator (T3) or activators of PPAR subtypes such as Wy-14,643 and BRL49653 (data not shown). RXR can form heterodimers with many partners, including orphan receptors (for reviews, see Refs. 73-75), and thus it has been accepted that RXR is a key regulator of various cellular events in receptor-dependent signaling. Since the total amount of all the RXR subtypes was larger than that of all the RAR subtypes (68), the transcriptional activity of TM might be affected by changes in the amounts of various nuclear proteins that could form heterodimers with RXRs under various conditions.


    FOOTNOTES

* This work was supported in part by a Grant-in-aid from the Ministry of Education, Science, and Culture of Japan 06672202 and by a grant from the Japan Foundation of Cardiovascular Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Tsukui, Kanagawa 199-0195, Japan. Tel.: 81-426-85-3757; Fax: 81-426-85-2577; E-mail: shorie-v@pharm. teikyo-u.ac.jp.

Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M004942200


    ABBREVIATIONS

The abbreviations used are: TM, thrombomodulin; t-RA, all-trans-retinoic acid; 9C-RA, 9-cis-retinoic acid; CAT, chloramphenicol acetyltransferase; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; TR, thyroid hormone receptor; DR4, direct repeats of RARE containing 4 base; HUVE, human umbilical vein endothelial; FCS, fetal calf serum; Me2SO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; bp, base pair; kb, kilobase pair; PCR, polymerase chain reaction.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Esmon, C. T., and Owen, W. G. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2249-2252[Abstract]
2. Esmon, C. T. (1989) J. Biol. Chem. 264, 4743-4746[Free Full Text]
3. Dittman, W. A., and Majerus, P. W. (1990) Blood 75, 329-336[Medline] [Order article via Infotrieve]
4. Nawroth, P. P., and Stern, D. M. (1986) J. Exp. Med. 163, 740-745[Abstract]
5. Scarpati, E. M., and Sadler, J. E. (1989) J. Biol. Chem. 264, 20705-20713[Abstract/Free Full Text]
6. Archipoff, G., Beretz, A., Freyssinet, J. M., Klein-Soyer, C., Brisson, C., and Cazenave, J. P. (1991) Biochem. J. 273, 679-684[Medline] [Order article via Infotrieve]
7. Nawroth, P. P., Handley, D. A., Esmon, C. T., and Stern, D. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3460-3464[Abstract]
8. Moore, K. L., Andreoli, S. P., Esmon, N. L., Esmon, C. T., and Bang, N. U. (1987) J. Clin. Invest. 79, 124-130[Medline] [Order article via Infotrieve]
9. Weis, J. R., Pitas, R. E., Wilson, B. D., and Rogers, G. M. (1991) FASEB J. 5, 2459-2465[Abstract/Free Full Text]
10. Ishii, H., Kizaki, K., Horie, S., and Kazama, M. (1996) J. Biol. Chem. 271, 8458-8465[Abstract/Free Full Text]
11. Horie, S., Kizaki, K., Ishii, H., and Kazama, M. (1992) Biochem. J. 281, 149-154[Medline] [Order article via Infotrieve]
12. Ishii, H., Horie, S., Kizaki, K., and Kazama, M. (1992) Blood 80, 2556-2562[Abstract]
13. Ishii, H., Kizaki, K., Uchiyama, H., Horie, S., and Kazama, M. (1990) Thromb. Res. 59, 841-850[Medline] [Order article via Infotrieve]
14. Shirai, T., Shiojiri, S., Ito, H., Yamamoto, S., Kusumoto, H., Deyashiki, Y., Maruyama, I., and Suzuki, K. (1988) J. Biochem. (Tokyo) 103, 281-285[Abstract]
15. Yu, K., Morioka, H., Fritza, L. M. A., Beeler, D. L., Jackman, R. W., and Rosenberg, R. D. (1992) J. Biol. Chem. 267, 23237-23247[Abstract/Free Full Text]
16. Tazawa, R., Hirosawa, S., Suzuki, K., Hirokawa, K., and Aoki, N. (1993) J. Biochem (Tokyo) 113, 600-606[Abstract]
17. Dittman, W. A., Nelson, S. T., Greer, P. K., Horton, E. T., Palomba, M. L., and McCachren, S. S. (1994) J. Biol. Chem. 269, 16925-16932[Abstract/Free Full Text]
18. Petkovich, M., Brand, N. J., Krust, A., and Chambon, P. (1987) Nature 330, 444-450[CrossRef][Medline] [Order article via Infotrieve]
19. Giguere, V., Ong, E. S., Segui, P., and Evans, R. M. (1987) Nature 330, 624-629[CrossRef][Medline] [Order article via Infotrieve]
20. Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, O. V., Naar, A. M., Kim, S. Y., Boutin, J.-M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 67, 1251-1266[Medline] [Order article via Infotrieve]
21. Zhang, X.-K., Hoffmann, B., Tran, P. B-V., Graupner, G., and Pfahl, M. (1992) Nature 355, 441-446[CrossRef][Medline] [Order article via Infotrieve]
22. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992) Nature 355, 446-449[CrossRef][Medline] [Order article via Infotrieve]
23. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J.-Y., Staub, A., Garnier, J.-M., Mader, S., and Chambon, P. (1992) Cell 68, 377-395[Medline] [Order article via Infotrieve]
24. Bugge, T. H., Pohl, J., Lonnoy, O., and Stunnenberg, H. G. (1992) EMBO J. 11, 1409-1418[Abstract]
25. Marks, M. S., Hallenbeck, P. L., Nagata, T., Segars, J. H., Appella, E., Nikodem, V. M., and Ozato, K. (1992) EMBO J. 11, 1419-1435[Abstract]
26. Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) Cell 65, 1255-1266[Medline] [Order article via Infotrieve]
27. Naar, A. M., Boutin, J. M., Lipkin, S. M., Yu, V. C., Holloway, J. M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 65, 1267-1279[Medline] [Order article via Infotrieve]
28. Mangelsdorf, D. J., Umesono, K., Kliewer, S. A., Borgmeyer, U., Ong, E. S., and Evans, R. M. (1991) Cell 66, 555-561[Medline] [Order article via Infotrieve]
29. Dynan, W. S., and Tjian, R. (1983) Cell 35, 79-87[Medline] [Order article via Infotrieve]
30. Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987) Cell 51, 1079-1090[Medline] [Order article via Infotrieve]
31. Darrow, A. L., Rickles, R. J., Pecorino, L. T., and Strickland, S. (1990) Mol. Cell. Biol. 10, 5883-5893[Medline] [Order article via Infotrieve]
32. Kumar, A. P., Mar, P. K., Zhao, B., Montgomery, R. L., Kang, D.-C., and Butler, A. P. (1995) J. Biol. Chem. 270, 4341-4348[Abstract/Free Full Text]
33. Cui, M.-Z., Parry, G. C. N., Oeth, P. O., Larson, H., Smith, M., Huang, R.-P., Adamson, E. D., and Mackman, N. (1996) J. Biol. Chem. 271, 2731-2739[Abstract/Free Full Text]
34. Kang, S.-H., Brown, D. A., Kitajima, I., Xu, X., Heidenreich, O., Gryaznov, S., and Nerenberg, M. (1996) J. Biol. Chem. 271, 7330-7335[Abstract/Free Full Text]
35. Hu, Z.-Z., Zhuang, L., Meng, J., and Dufau, M. L. (1998) J. Biol. Chem. 273, 26225-26235[Abstract/Free Full Text]
36. Horie, S., Ishii, H., Hara, H., and Kazama, M. (1994) Biochem. J. 301, 683-691[Medline] [Order article via Infotrieve]
37. Hamada, H., Ishii, H., Sakyo, K., Horie, S., Nishiki, K., and Kazama, M. (1995) Blood 86, 225-233[Abstract/Free Full Text]
38. Gao, X., and Huang, L. (1991) Biochem. Biophys. Res. Commun. 179, 280-285[Medline] [Order article via Infotrieve]
39. Rusten, L. S., Dybedal, I., Blomhoff, R., Smeland, E. B., and Jacobsen, S. E. W. (1996) Blood 87, 1728-1736[Abstract/Free Full Text]
40. Zhang, X.-K., Lehmann, J., Hoffmann, B., Dawson, M. I., Cameron, J., Graupner, G., Hermann, T., Tran, P., and Pfahl, M. (1992) Nature 358, 587-591[CrossRef][Medline] [Order article via Infotrieve]
41. Lehmann, J. M., Jong, L., Fanjul, A., Cameron, J. F., Ping, L. X., Haefner, P., Dawson, M. I., and Pfahl, M. (1992) Science 258, 1944-1946[Medline] [Order article via Infotrieve]
42. Kliewer, S. A., Umesono, K., Heyman, R. A., Mangelsdorf, D. J., Dyck, J. A., and Evans, R. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1448-1452[Abstract]
43. Nagpal, S., Friant, S., Nakshatri, H., and Chambon, P. (1993) EMBO J. 12, 2349-2360[Abstract]
44. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Zhou, J. Y., Ong, E. S., Oro, A. E., Kakizuka, A., and Evans, R. M. (1992) Genes Dev. 6, 329-344[Abstract]
45. Fawell, S. E., Lees, J. A., White, R., and Parker, M. G. (1990) Cell 60, 953-963[Medline] [Order article via Infotrieve]
46. Fesus, L., Nagy, L., Basilion, J. P., and Davies, J. A. (1991) Biochem. Biophys. Res. Commun. 179, 32-38[Medline] [Order article via Infotrieve]
47. Kooistra, T., Lansink, M., Arts, J., Sitter, T., and Toet, K. (1995) Eur. J. Biochem. 232, 425-432[Abstract]
48. Rochette, E. C., Gaub, M. P., Lutz, Y., Ali, S., Scheuer, I., and Chambon, P. (1992) Mol. Endocrinol. 6, 2197-2209[Abstract]
49. Duprez, E., Lillehaug, J. R., Gaub, M. P., and Lanotte, M. (1996) Oncogene 12, 2443-2450[Medline] [Order article via Infotrieve]
50. Mitchell, P. J., Wang, C., and Tjian, R. (1987) Cell 50, 847-861[Medline] [Order article via Infotrieve]
51. Mitchell, P. J., Timmons, P. M., Hebert, J. M., Rigby, P. W. J., and Tjian, R. (1991) Genes Dev. 5, 105-119[Abstract]
52. Suzuki, Y., Shimada, J., Shudo, K., Matsumura, M., Crippa, M. P., and Kojima, S. (1999) Blood 93, 4264-4276[Abstract/Free Full Text]
53. Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990) Cell 63, 155-165[Medline] [Order article via Infotrieve]
54. Weiler-Guettler, H., Yu, K., Soff, G., Gudas, L. J., and Rosenberg, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2155-2159[Abstract]
55. Shibakura, M., Koyama, T., Saito, T., Shudo, K., Miyasaka, N., Kamiyama, R., and Hirosawa, S. (1997) Blood 90, 1545-1551[Abstract/Free Full Text]
56. Imataka, H., Sogawa, K., Yasumoto, K., Kikuchi, Y., Sasano, K., Kobayashi, A., Hayami, M., and Fujii-Kuriyama, Y. (1992) EMBO J. 11, 3663-3671[Abstract]
57. Tamaki, T., Ohnishi, K., Hartl, E., LeRoy, C., and Trojanowska, M. (1995) J. Biol. Chem. 270, 4299-4304[Abstract/Free Full Text]
58. Pascal, E., and Tjian, R. (1990) Genes Dev. 5, 1646-1656[Abstract]
59. Hagen, G., Dennig, J., Preiß, A., Beato, M., and Suske, G. (1995) J. Biol. Chem. 270, 24989-24994[Abstract/Free Full Text]
60. Li, R., Hodny, Z., Luciakova, K., Barath, P., and Nelson, B. D. (1996) J. Biol. Chem. 271, 18925-18930[Abstract/Free Full Text]
61. Krey, G., Mahfoudi, A., and Wahli, W. (1995) Mol. Endocrinol. 9, 219-231[Abstract]
62. Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133[Medline] [Order article via Infotrieve]
63. Su, K., Roos, M. D., Yang, X., Han, I., Paterson, A. J., and Kudlow, J. E. (1999) J. Biol. Chem. 274, 15194-15202[Abstract/Free Full Text]
64. Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. (1995) J. Biol. Chem. 270, 25879-25884[Abstract/Free Full Text]
65. Zutter, M. M., Ryan, E. E., and Painter, A. D. (1997) Blood 90, 678-689[Abstract/Free Full Text]
66. Rohlff, C., Ahmad, S., Borellini, F., Lei, J., and Glazer, R. I. (1997) J. Biol. Chem. 272, 21137-21141[Abstract/Free Full Text]
67. Hagen, G., Müller, S., Beato, M., and Suske, G. (1994) EMBO J. 13, 3843-3851[Abstract]
68. Ulven, S. M., Natarajan, V., Holven, K. B., Lovdal, T., Berg, T., and Blomhoff, R. (1998) Eur. J. Cell Biol. 77, 111-116[Medline] [Order article via Infotrieve]
69. Hata, Y., Duh, E., Zhang, K., Robinson, G. S., and Aiello, L. P. (1998) J. Biol. Chem. 273, 19294-19303[Abstract/Free Full Text]
70. Strahle, U., Schmid, W., and Schutz, G. (1988) EMBO J. 7, 3389-3395[Abstract]
71. Schule, R., Muller, M., Kaltschmidt, C., and Renkawitz, R. (1988) Science 242, 1418-1420[Medline] [Order article via Infotrieve]
72. Duan, R., Porter, W., and Safe, S. (1998) Endocrinology 139, 1981-1990[Abstract/Free Full Text]
73. Giguere, V. (1994) Endocr. Rev. 15, 61-77[Medline] [Order article via Infotrieve]
74. Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841-850[Medline] [Order article via Infotrieve]
75. Gronemeyer, H., and Laudet, V. (1995) Protein Profile 2, 1173-1308[Medline] [Order article via Infotrieve]


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