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Endocrinology Vol. 139, No. 8 3423-3431
Copyright © 1998 by The Endocrine Society

ARTICLES

Characterization of a Negative Thyroid Hormone Response Element in the Rat Sodium, Potassium-Adenosine Triphosphatase {alpha}3 Gene Promoter1

Shing Chin, James Apriletti and Gregory Gick

Department of Biochemistry, State University of New York Health Science Center, Brooklyn, New York 11203; and Metabolic Research Unit, University of California (J.A.), San Francisco, California 94143

Address all correspondence and requests for reprints to: Gregory Gick, Ph.D., Department of Biochemistry, State University of New York Health Science Center, Brooklyn, New York 11203. E-mail: gickg11{at}hscbklyn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The thyroid hormone L-T3 elicits either a stimulatory or an inhibitory effect on expression of the Na,K-adenosine triphosphatase {alpha}3-subunit gene in primary cultures of neonatal rat cardiac myocytes. The present study was undertaken to characterize a negative thyroid hormone response element present within the rat Na,K-adenosine triphosphatase {alpha}3-subunit gene proximal promoter. Transient transfection assays indicated that the DNA-binding domain of thyroid hormone receptor was essential for mediating repression of {alpha}3 gene transcription by thyroid hormone. This negative effect of thyroid hormone was enhanced in the presence of cotransfected retinoid X receptor and its ligand 9-cis-retinoic acid. Inhibition of {alpha}3 chimeric gene expression by thyroid hormone was dependent on the initial cell plating density. The negative thyroid hormone response element was localized to a region between nucleotides -68 to -6 of the {alpha}3 gene. Electrophoretic mobility shift assays showed that thyroid hormone receptor binds in a synergistic manner as a heterodimer with retinoid X receptor to two sites at positions -62 to -41 and -39 to -17 of the {alpha}3 gene promoter. The upstream and downstream heterodimer binding sites coexist with CAAT and TATA elements, respectively.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Na,K-ADENOSINE triphosphatase (Na,K-ATPase) is a membrane-associated enzyme that catalyzes transport of Na+ and K+ ions across the plasma membrane of all animal cells (1). The activity of Na,K-ATPase is integral for the establishment of an electrochemical gradient, which is essential for maintaining the resting potential, cell volume, and osmotic balance (2). As the only known receptor for cardiac glycosides such as ouabain and digitalis, Na,K-ATPase is a target for treatment of congestive heart failure and cardiac arrhythmias (3). Na,K-ATPase consists of two subunits, {alpha} and ß, and each subunit has several isoforms (4). Expression of {alpha} isoform genes {alpha}1, {alpha}2, and {alpha}3 is tissue specific. In the adult rat, for instance, {alpha}1 subunit expression is nearly constitutive, whereas the {alpha}2 isoform is mainly present in neural and muscle tissue. Na,K-ATPase {alpha}3-subunit is limited primarily to neural tissues. Major changes in Na,K-ATPase {alpha} isoform content also occur during development. For example, {alpha}1 is constitutively expressed throughout development in rat heart. In contrast, {alpha}2 is predominantly found in adult heart, whereas {alpha}3 is selectively up-regulated in fetal and neonatal myocardium (5). In addition, each {alpha} isoform exhibits differential ouabain sensitivity and affinity for Na+ ions (6).

Regulation of Na,K-ATPase activity and subunit gene expression has been of great interest because of its vital physiological and therapeutic significance. L-T3 is the major active form of thyroid hormone and stimulates Na,K-ATPase activity and isoform messenger RNA (mRNA) content in a variety of mammalian tissues (7, 8, 9). Both transcriptional and posttranscriptional mechanisms have been implicated in the regulation of Na,K-ATPase subunit gene expression by T3 (8, 10, 11, 12, 13). T3 regulation of Na,K-ATPase {alpha}3 subunit gene expression has been studied in primary cultures of neonatal rat cardiac myocytes. In this cell culture system, Kamitani et al. (11) observed a 3-fold stimulation of {alpha}3 mRNA content by T3 at 1 day and a 50% decrease with prolonged exposure to hormone. Furthermore, in this study, a 2.6-kb (-2500/+136) portion of the {alpha}3 gene conferred a 3-fold stimulation of reporter gene expression in transient transfection assays (11). In contrast, we found that the region between nucleotides -116 to -6 of the {alpha}3 gene promoter suppressed chimeric gene expression about 50% in primary cultures of cardiac myocytes incubated in the presence of T3 and cotransfected T3 receptor (T3R) expression vector (14).

T3 exerts its transcriptional function through an interaction with T3R, which is preassociated with a specific DNA sequence, a T3 response element (TRE). A hexanucleotide half-site, AGGTCA, has been identified as a consensus sequence for binding of T3R (15). Naturally occurring TREs usually are arranged as direct repeats (DRs) of a half-site, a palindrome, or an inverted palindrome (16, 17, 18). Interestingly, DRs with different spacings are also recognized by retinoic acid receptor (RAR), vitamin D receptor, and retinoid X receptor (RXR), which, along with T3R, comprise a nuclear receptor superfamily (19). In addition, RXR is capable of enhancing DNA binding of T3R, RAR, and vitamin D receptor by formation of a heterodimer (20, 21). The effect of T3 on gene transcription can be either stimulatory or inhibitory, depending on the cell type and the specific sequence and arrangement of the individual TRE (22). A palindrome of AGGTCA or DRs with a four-nucleotide spacing has been widely accepted as a positive TRE (pTRE) (22), whereas the nature of a negative TRE (nTRE) has not been well defined.

In a previous study we identified a nTRE in the -116 to -6 bp region of the rat Na,K-ATPase {alpha}3-subunit gene (14). To continue our characterization of this nTRE, transient transfection assays were conducted to evaluate the molecular mechanism underlying T3-mediated repression of {alpha}3 gene transcription. T3R binding sites within the {alpha}3 gene proximal promoter region were localized by electrophoretic mobility shift assays (EMSAs). We report here the identification of two binding sites for T3R/RXR heterodimers within a 63-bp region of the {alpha}3 gene promoter containing a functional nTRE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Neonatal rat cardiac myocytes were prepared by a trypsin/deoxyribonuclease I digestion method as previously described (13). Cells were maintained for 1 day in DMEM containing 7% FBS and antibiotics (100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 250 ng/ml amphotericin B as fungizone). After transfection on day 2 of culture, cardiac myocytes were incubated in serum-free DMEM supplemented with 10 µg/ml insulin, 10 µg/ml transferrin, and antibiotics for 1 day and then treated with T3 and/or 9-cis-retinoic acid (9-cis-RA).

Construction of {alpha}3/luciferase chimeric genes
pLUC36 is an {alpha}3/luciferase gene construct containing {alpha}3 sequence spanning nucleotides -116 to -6 in a promoterless firefly luciferase plasmid pXP1 (14). Two additional deletion constructs, pLUC38 and pLUC39, which contain the {alpha}3 gene -68/-6 and -39/-6 regions, respectively, were derived from pLUC36. To generate pLUC38, pLUC36 was digested with BsrBI and HindIII to release a fragment containing the {alpha}3 gene -68/-6 region, which was subsequently ligated with SmaI- and HindIII-digested pXP1. To generate pLUC39, pLUC36 was digested with SacI and SacII to remove the {alpha}3 gene -116/-40 region. The remaining fragment containing nucleotides between -39 and -6 within the pXP1 vector was treated with T4 polymerase to produce blunt ends and then religated with T4 ligase. These constructs were subjected to both restriction enzyme digestion and double stranded DNA sequencing.

Transient transfection
Cardiac myocytes (4.0 x 106 cells/6-cm plate) were transfected via a calcium phosphate coprecipitation method as previously described (13). For T3 studies, 5.0 µg pLUC36, pLUC38, or pLUC39 were cotransfected with 5.0 µg of either a rat T3Rß1 expression vector (23) or a mutant rat T3Rß1 expression plasmid that has an internal deletion of the DNA-binding domain at amino acids 100–171 (24). To assess the effect of cell density on T3-mediated repression, pLUC36 and T3Rß1 plasmids were cotransfected into cells at both 4.0 and 8.0 x 106 cells/6-cm plate. For RXR cotransfection studies, cells were cotransfected with 2.0 µg of either pLUC36 or positive retinoid X response element (pRXRE)/LUC and 2 µg of a mouse RXRß expression vector (25). pRXRE/LUC is a luciferase reporter gene containing three pRXREs in front of the thymidine kinase promoter (26). For activating protein-2 (AP2) and Sp1 cotransfection studies, cells were transfected with 2 µg pLUC36 along with 2 µg of either pRSVAP2, an AP2 expression vector (27), or pPACSP1, an Sp1 expression vector (28). Cells were treated with 100 nM T3, 1 µM 9-cis-RA, or both for 1 day. Cells were lysed by incubation for 15 min at room temperature with 200 µl reporter lysis buffer (Promega, Madison, WI) containing 1% Triton-X 100, 10% glycerol, 25 mM Tris-HCl (pH 7.8), 2 mM EDTA, and 20 mM dithiothreitol (DTT). To measure luciferase activity, 25 µl of cell lysate were combined with 100 µl substrate [470 µM luciferin, 270 µM coenzyme A, 530 µM ATP, 20 mM tricine (pH 7.8), 1.1 mM (MgCO3)4 Mg(OH)25H2O, 2.7 mM MgSO4, 0.1 mM EDTA, and 33.3 mM DTT], and photon emission was counted in a liquid scintillation counter within 20 sec. Luciferase activity was expressed per total cellular protein as previously described (14).

Bacterial expression and purification of receptors
Escherichia coli-expressed human T3Rß1 was purified to approximately 7% homogeneity by phenol hydrophobic interaction chromatagraphy and heparin affinity chromatography steps (29). Plasmids pGEXKG-rRXRß and pGEXKG-hT3Rß1 express rat RXRß-glutathione-S-transferase (GST) and human T3Rß1-GST fusion proteins, respectively (20). These plasmids were transformed into bacterial strain JM 83. GST fusion protein was prepared according to a protocol provided by Pharmacia (Piscataway, NJ). Briefly, the recombinants were grown at 37 C in 500 ml Luria Beroni medium containing ampicillin until an OD at 600 nm of 0.8–1.0 was reached. Cells were induced by incubation with 0.5 mM isopropylthiogalactoside for 3 h, centrifuged, and lysed by three cycles of sonication for 30 sec each. Supernatants of cell lysates were incubated with 1 ml glutathione agarose beads [50% (vol/vol) in 1 x PBS and 1% Triton-X 100] with slow shaking at 4 C for 30 min. Finally, GST fusion protein was eluted by resuspension of the beads in 1 ml of a buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM glutathione, 1 mM phenylmethylsulfonylfluoride, and 20% glycerol and stored at -70 C. The purity of GST fusion proteins was assessed by SDS-PAGE, and the concentration was estimated by determination of OD at 600 nm.

EMSAs
Oligonucleotides that span the {alpha}3 gene regions -116/-72, -62/-21, -62/-41, -44/-21, -39/-17, and -28/-6 were synthesized, as were mutation-containing -62/-41 and -39/-6 oligonucleotides (Life Technologies, Grand Island, NY). The fragments spanning the -116/-6 and -39/-6 regions were generated by digestion of pUC19(-116/-6), which contains the -116 to -6 bp region of the {alpha}3 gene with PvuII/XbaIII and SacII/XbaIII, respectively. DNA was labeled as previously described (13). Briefly, 50 ng of either annealed oligonucleotide or DNA fragments produced by restriction enzyme digestion were incubated with 6 U Klenow fragment in the presence of 1 mM deoxy (d)-ATP, dTTP, and dGTP and 50 µCi [32P]dCTP (3000 Ci/mmol) at room temperature for 20 min. Unincorporated [32P]dCTP was removed by chromatography on Sephadex G-25 spin columns. To detect T3R and RXR binding to {alpha}3 gene fragments in a 15-µl reaction, each labeled DNA (2.0–5.0 x 105 cpm, 1.0 ng) was incubated with 6 ng human T3Rß1 or 0.4 µg human T3Rß1-GST and/or 1.3 µg rat RXRß-GST in the presence of 1.5 µg polyd(I-C), 1 mM DTT, 10% glycerol, 10 mM HEPES (pH 7.9), 75 mM KCl, and 1 mM EDTA for 20 min at room temperature. For competition experiments, unlabeled 10-, 100-, or 1000-fold excesses of wild-type or mutant -62/-41 oligonucleotides were incubated with T3Rß1 and RXRß for 10 min before addition of labeled wild-type -62/-41 oligonucleotide. The reaction was continued for another 20 min. Electrophoresis in 5–8% polyacrylamide gels was carried out under low ionic strength conditions (0.5 x TBE) at 4 C. Gels were dried and either exposed to x-ray film at -70 C or subjected to phosphorimaging analysis.

Statistical analysis
Results are expressed as the mean ± SEM. Statistical significance was determined by unpaired Student’s t test (two tailed).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA-binding domain of T3R and lower cell density are required for T3-mediated repression of {alpha}3 gene transcription
Both ligand- and DNA-binding domains of T3R are essential for mediating the stimulatory transcriptional effect of T3 (22); however, the role of binding of T3R to nTREs is less well defined. To examine this issue, a mutant T3Rß1 expression vector that has its DNA-binding domain deleted was cotransfected with {alpha}3/luciferase gene construct pLUC36 (-116/-6 bp) in transient transfection experiments with primary cultures of neonatal rat cardiac myocytes. As shown in Fig. 1Go, pLUC36 was repressed 60% by T3 in the presence of wild-type T3Rß1. In the presence of cotransfected mutant T3Rß1, however, T3 did not repress pLUC36 expression. This indicated that DNA binding of T3Rß1 is required for T3-mediated repression of {alpha}3 gene transcription.



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  		Figure 1. DNA-binding domain of T3Rß1 is required for T3-mediated repression of {alpha}3 gene transcription. Neonatal rat cardiac myocytes were maintained under conditions defined in Materials and Methods. pLUC36 (2 µg) was cotransfected with equivalent amounts of either a wild-type or a mutant T3Rß1 (T3Rmut) expression vector. The mutant T3Rß1 has a deletion of its DNA-binding domain. After treatment with 100 nM T3 for 1 day, cells were lysed, and luciferase activity was measured and normalized to protein content. Data (n = 6) are presented as the ratio of the normalized luciferase activity in the presence of T3 relative to that in the absence T3, which is set at 1.0. *, P < 0.05, with T3 vs. without T3.

 
As cell to cell interactions have been implicated as important parameters in hormone responsiveness (30, 31), we evaluated the effect of cell density on T3-mediated inhibition of {alpha}3 gene transcription (Fig. 2Go). Neonatal rat cardiac myocytes were plated at two different densities and transfected with pLUC36 and a T3Rß1 expression vector. At a lower cell density (4 x 106 cells/6-cm plate), pLUC36 conferred a T3-mediated repression of 60%. By contrast, T3 had no effect on pLUC36 expression when cardiac myocytes were plated at a higher cell density (8 x 106 cells/6-cm plate).



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  		Figure 2. The effect of cell density on repression of {alpha}3 gene promoter activity by T3. Neonatal rat cardiac myocytes were cultured at two different cell densities as described in Materials and Methods. Cells were transfected with 2 µg pLUC36 and an equivalent amount of a T3Rß1 expression vector. After cells were treated with 100 nM T3 for 1 day, luciferase activity was measured as in Fig. 1Go. Data (n = 13–16) are presented as the ratio of luciferase activity in the presence of T3 relative to that in the absence of T3. *, P < 0.05, with T3 vs. with-out T3.

 
Effect of RXR on T3-mediated inhibition of {alpha}3 gene expression
Our previous in vitro binding studies demonstrated that RXRß was capable of producing a synergistic increase in binding of T3Rß1 to the {alpha}3 gene region between nucleotides -116 and -6, which contained a functional nTRE (14). It was intriguing to ask whether a similar synergistic effect of T3Rß1 and RXRß could be observed in vivo. To this end, neonatal rat cardiac myocytes were transfected with pLUC36 with or without cotransfection of an equivalent amount of either a T3Rß1 or RXRß expression vector, or both. After cells were treated with 100 nM T3, 1 µM 9-cis-RA, or both for 1 day, cells were lysed, and luciferase activity was measured (Fig. 3Go). Consistent with our earlier results (14), T3Rß1 in the presence of T3 suppressed pLUC36 activity 63% (lane 2). Interestingly, a combination of RXRß and 9-cis-RA repressed pLUC36 expression 40% (lane 3), indicating that a negative RXRE (nRXRE) is present in the {alpha}3 gene -116/-6 region. The transcriptional activity of pLUC36 was further decreased if cells were cotransfected with T3Rß1 and RXRß and treated with both T3 and 9-cis-RA (lane 4). As a positive control, pRXRE/LUC, containing three pRXREs was stimulated by 9-cis-RA in the absence (lane 7) and presence of cotransfected RXRß (lane 8). These observations suggest that a functional interaction may occur between T3Rß1 and RXRß in mediating repression of {alpha}3 gene transcription via binding to a nTRE within the proximal promoter.



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  		Figure 3. Role of RXR in T3-mediated repression of {alpha}3 gene transcription. Cardiac myocytes cultured as described in Fig. 1Go were transfected with 2 µg of either pLUC36 (left) or pRXRE/LUC (right) with or without cotransfection of 2 µg of either T3Rß1 or RXRß expression vectors. Cells were treated with 100 nM T3 and/or 1 µM 9-cis-RA for 1 day. Luciferase activity was assayed as detailed in Fig. 1Go. Luciferase activities of pLUC36 and pRXRE/LUC were expressed, respectively, relative to the basal activities of pLUC36 (lane 1) and pRXRE/LUC (lane 5), which were set at 1.0 (n = 10). *, P < 0.05; **, P < 0.05 (lane 4 vs. 3).

 
Neither Sp1 nor AP2 mediates the inhibitory action of T3 on {alpha}3 gene transcription
The transcription factors Sp1 and AP2 have been suggested to play a role in the T3 responsiveness of several genes (20, 32, 33). As the -116 to -6 bp region of the {alpha}3 gene promoter contains potential binding sites for both Sp1 and AP2 (34, 35), we investigated whether either of these trans-activators contributes to T3-mediated repression of {alpha}3 gene expression. To address this issue, pLUC36 expression was evaluated in the presence of either cotransfected Sp1 or AP2 expression vectors. As illustrated in Fig. 4Go, cotransfection of AP2 did not affect pLUC36 activity (lane 2), whereas Sp1 stimulated pLUC36 expression 2-fold (lane 3). However, neither AP2 nor Sp1 counteracted T3-mediated repression of pLUC36 expression (lane 5 vs. lane 6 or 7).



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  		Figure 4. Effects of AP2 and Sp1 on inhibition of {alpha}3 gene promoter activity by T3. Neonatal rat cardiac myocytes were maintained as defined in Fig. 1Go and transfected with 2 µg pLUC36 with or without cotransfection of 2 µg of a T3Rß1, Sp1, or AP2 expression vector. Cells were exposed to 100 nM T3 for 1 day, and luciferase activity was quantitated as in Fig. 1Go. Values of the control groups (lanes 1 and 4) were set at 1.0, and values of experimental groups were expressed relative to that of the control group (n = 8). *, P < 0.05 (lane 3 vs. 1, lane 5, 6, or 7 vs. 4).

 
nTRE is localized to the -68/-6 region of the {alpha}3 gene promoter
To further localize the nTRE present within the -116/-6 bp region of the {alpha}3 gene promoter, we prepared two additional 5'-deletion constructs containing the -68 to -6 bp and -39 to -6 bp regions of the {alpha}3 gene. Before analysis of the effect of T3, we evaluated the basal activity of these deletion constructs (Fig. 5AGo). In construct pLUC38, deletion of {alpha}3 sequence from -116 to -69 bp lead to an approximately 65% reduction of basal gene transcription compared with the activity of pLUC36. Expression of construct pLUC39 containing 34 bp of 5'-flanking DNA was only marginally higher than the background expression observed in nontransfected cell lysates. Thus, we did not continue our characterization of the nTRE in the {alpha}3 gene proximal promoter with the pLUC39 construct. Transient transfection assays were conducted, however, with the new 5'-deletion construct pLUC38 (Fig. 5BGo). In primary cultures of neonatal rat cardiac myocytes cotransfected with an expression vector encoding T3Rß1, expressions of pLUC36 and pLUC38 were repressed 44% and 39%, respectively, in response to T3. These data indicate that the region of the {alpha}3 promoter between nucleotides -68 and -6 contains a functional nTRE.



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  		Figure 5. A nTRE exists in the -68 to -6 bp region of the {alpha}3 gene promoter. A, Primary cultures of cardiac myocytes were incubated as described in Fig. 1Go and transfected with 5 µg of pLUC36, pLUC38, or pLUC39 in the presence of 5 µg cotransfected T3Rß1 expression vector. Luciferase activity associated with pLUC36 was set at 1.0, and the activities of pLUC38 and pLUC39 were expressed relative to pLUC36 (n = 8–10). The promoter activity of any of these three constructs is statistically significantly different from the other two, as indicated by asterisks (P < 0.05). B, Cardiac myocytes cultured as described in Fig. 1Go were transfected with 5 µg of either pLUC36 or pLUC38 and 5 µg of a T3Rß1 expression vector. After treatment with 100 nM T3 for 1 day, cells were lysed, and luciferase activity was measured as described in Fig. 1Go. Data (n = 8–14) are presented as the ratio of the normalized luciferase activity in the presence of T3 relative to that in the absence of T3. *, P < 0.05, with T3/without T3.

 
T3R/RXR heterodimers bind to the -62/-21 region of the {alpha}3 gene
Extensive studies on T3-responsive genes have identified a hexanucleotide sequence, AGGTCA, in the form of DRs, inverted repeats, or a palindrome as a pTRE (19). Binding of T3R as a monomer to this hexanucleotide motif has been implicated in repression of rat TSH gene transcription by T3 (36). A computer-assisted search of the {alpha}3 gene promoter between nucleotides -68 to -6 revealed three regions, located at -60/-55, -34/-29, and -20/-15 bp, with a four of six match to the AGGTCA motif (Fig. 6Go).



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  		Figure 6. Potential T3R-binding sites in the -68/-6 region of the {alpha}3 gene. Sequences with a four of six match to the T3R binding site consensus AGGTCA motif are indicated by overhead arrows. The TATA box is in boldface.

 
To determine whether the three potential half-sites are indeed T3R-binding sites, EMSAs were performed using DNA fragments that span the subregion of the {alpha}3 gene promoter from -116 to -6 bps (Fig. 7Go). The {alpha}3 gene regions -116/-6, -116/-72, and -62/-21 were labeled with {alpha}[32P]dCTP and incubated with T3Rß1, RXRß, or both receptors. Samples were resolved by 5% PAGE, and DNA-protein complexes were detected by autoradiography (Fig. 7AGo). In the presence of T3Rß1 alone, weak binding of T3R monomer was detected to the {alpha}3 region -62/-21 (lane 7), consistent with our earlier observation (14) (lane 1). In the presence of both T3Rß1 and RXRß, however, strong binding was observed with the {alpha}3 gene -62/-21 region, indicating the formation of a T3Rß1/RXRß heterodimer (lane 9). By contrast, heterodimer binding was not evident in the {alpha}3 gene fragment -116/-72. Therefore, a specific binding site for T3Rß1/RXRß heterodimers is present in the -62/-21 region of the {alpha}3 gene.



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  		Figure 7. Formation of T3Rß1/RXRß heterodimers on the {alpha}3 gene -62/-21 region. A, {alpha}3 gene fragments -116/-6, -116/-72, and -62/-21 were labeled with [32P]dCTP. Probes were incubated with T3Rß1, RXRß-GST, or both receptors as described in Materials and Methods. Electrophoresis was carried out in a 5% polyacrylamide gel under lower ionic strength (0.5 x TBE) at 15 mA. This result is representative of three experiments. B, 32P-Labeled -62/-21, -62/-41, and -44/-21 regions of the {alpha}3 gene were incubated with either T3Rß1 or T3Rß1-GST with or without RXRß-GST. The reaction products were resolved using 6% PAGE, and DNA-protein complexes were detected by autoradiography. This result is representative of three experiments.

 
Oligonucleotides spanning nucleotides -62 to -41 and -44 to -21 were used in EMSAs to further localize T3Rß1 and RXRß binding within the region of the {alpha}3 gene containing a nTRE (Fig. 7BGo). T3Rß1 associated with RXRß-GST and formed complexes with the -62/-41 oligonucleotide (lanes 7 and 8), but did not bind to the -44/-21 oligonucleotide (lanes 11 and 12). A selective effect of T3Rß1 is indicated by the observation that both native T3Rß1 and T3Rß1-GST proteins participated in heterodimer formation with RXR. In addition, there was an approximately 25-fold decrease in the intensity of T3Rß1/RXRß binding to the -62/-41 oligonucleotide compared with that of the -62/-21 region probe (lane 3 vs. 7 and lane 4 vs. 8).

To examine whether the potential T3R binding site between nucleotides -60 and -55 (Fig. 6Go) was indeed associated with T3Rß1/RXRß binding to the -62/-41 oligonucleotide, mutations were produced at three positions that are homologous to the T3R-binding site hexamer AGGTCA. Both wild-type and mutation-containing -62/-41 oligonucleotides were tested for binding of T3Rß1/RXRß in EMSAs (Fig. 8Go, left panel). T3Rß1/RXRß heterodimer formation was evident only on the wild-type -62/-41 oligonucleotide (lane 2 vs. 4). The specificity of T3Rß1/RXRß binding to the -62/-41 region was examined in a competition experiment (Fig. 8Go, right panel). A 10-fold excess of the wild-type -62/-41 oligonucleotide competitor did not reduce T3Rß1/RXRß formation (lane 6); however, a 100-fold excess of wild-type competitor lead to a 69% decrease in binding of heterodimers (lane 7), and a 1000-fold excess of unlabeled wild-type oligonucleotide abolished binding (lane 8). In contrast, the mutant -62/-41 oligonucleotide did not compete efficiently (lanes 9–11). These observations indicate that T3Rß1/RXRß heterodimers bind in a specific manner to the -62 to -41 bp region of the {alpha}3 gene.



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  		Figure 8. T3Rß1/RXRß heterodimers bind to a site within the -62/-41 region of the {alpha}3 gene. In the left panel, 32P-labeled {alpha}3 gene oligonucleotide -62/-41 and mutant -62/-41 were incubated with either GST or T3Rß1 and RXRß-GST as indicated in Fig. 7Go. Three nucleotides were changed (lower case) in the potential T3R-binding site (arrow) present in the complementary strand that has a four of six match to the AGGTCA motif to generate the mutant -62/-41 region (mut -62/-41). In the right panel, unlabeled oligonucleotides -62/-41 or mut -62/-41 were incubated with T3Rß1 and RXRß-GST for 10 min before addition of 32P-labeled -62/-41. Reaction samples were resolved by 6% PAGE, and DNA-protein complexes were detected by autoradiography. This result is representative of three experiments.

 
The striking difference in the apparent binding affinity of T3Rß1/RXRß to oligonucleotides comprising the -62/-21 and -62/-41 regions (Fig. 7BGo) raised the possibility that an additional T3Rß1/RXRß binding site(s) is present downstream of the -62/-41 region. Indeed, two regions exist between nucleotides -34 and -29 and -20 and -15 with partial homology to the consensus T3R binding motif AGGTCA (Fig. 6Go). To test this hypothesis, a fragment between nucleotides -39 to -6 of the {alpha}3 gene was labeled and examined in EMSAs (Fig. 9Go). Binding of T3Rß1/RXRß heterodimers to the -39 to -6 bp region was detected (lane 4). Heterodimer formation was also evident in EMSAs using an oligonucleotide spanning the -39 to -17 region (lane 12). In contrast, no binding was detected to an oligonucleotide containing {alpha}3 sequence from -28 to -6 bps (lane 16). Mutation of four conserved nucleotides within the potential T3R-binding site between -34 and -29 bp abolished heterodimer binding (lane 8). Taken together, these data suggest that a second T3Rß1/RXRß-binding site exists within the proximal promoter of the {alpha}3 gene.



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  		Figure 9. Localization of T3Rß1/RXRß heterodimer binding to nucleotides between -39 and -17 of the {alpha}3 gene promoter. 32P-Labeled {alpha}3 gene regions -39/-6, mutant -39/-6, -39/-17, and -28/-6 were incubated with GST, T3Rß1, RXRß-GST, or both T3Rß1 and RXRß-GST as indicated in Fig. 7Go. Four nucleotides were changed (lower case) in the potential T3R-binding site (arrow), which has a four of six match to the AGGTCA motif to generate the mutant -39/-6 region (mut -39/-6). Reaction samples were resolved by 6% PAGE, and DNA-protein complexes were detected by autoradiography. This result is representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our earlier studies identified a nTRE in the region of the rat Na,K-ATPase {alpha}3 gene between nucleotides -116 and -6 (14). In this report, we have characterized functional aspects of this nTRE and have defined binding sites for T3Rß1 and RXRß. The results indicate that the DNA-binding domain of T3Rß1 was necessary to produce repression of {alpha}3 gene transcription. Similarly, T3-mediated inhibition of transcription of the epidermal growth factor receptor gene was abrogated in the presence of the identical mutant T3Rß1 used in our studies (24). In contrast, repression of GHF-1/Pit-1 gene expression by T3 did not require direct DNA binding of T3R (37). Instead, protein-protein interactions between T3R and a cAMP-responsive element-binding protein were postulated to contribute to T3-mediated repression.

Repression of {alpha}3 gene transcription by T3 was abolished at a high cell density, which yielded an approximately 70% confluent culture of neonatal rat cardiac myocytes. To our knowledge, this is the first demonstration that cell density influences T3 responsiveness of a mammalian gene. Our findings are consistent with previous observations that an alteration of cell density may affect the responsiveness of cells to other hormones (30, 31). For example, 17ß-estradiol increased PRL mRNA levels in low and intermediate density cultures of pituitary tumor GH4C1 cells, whereas it did not affect PRL mRNA content in high density cultures (30). In contrast, clone 5 mRNA was inducible by glucocorticoid hormone only in a high density culture of adipogenic TA1 cells (31). Although the molecular events associated with loss of T3-mediated repression of {alpha}3 gene transcription at high cell density have not been delineated, we speculate that the underlying mechanism may involve either the cell density-dependent activation or accumulation of an antagonist of T3R.

As we previously demonstrated a synergistic effect of RXRß on T3Rß1 binding to the nTRE located between nucleotides -116 and -6 of the {alpha}3 gene (14), we initiated an investigation of the functional effect of RXRß on T3-mediated repression of {alpha}3 gene transcription. Cotransfection of T3Rß1 and RXRß in the presence of T3 and 9-cis-RA yielded the greatest degree of repression of {alpha}3 chimeric gene expression, and the difference was statistically significant compared with the inhibition seen in the presence of RXRß and its ligand. These results suggest that a functional interaction between T3Rß1 and RXRß may occur in the in vivo regulation of {alpha}3 gene transcription. This finding is also consistent with the results reported by Hollenberg et al. (38) in their examination of the nTRE present in the human TRH gene. The functional response of nTREs to RXR is not uniform, as the inhibitory effect of T3 on rat and mouse TSHß gene transcription was abrogated in the presence of cotransfected RXR and exposure to 9-cis-RA (39).

Interestingly, we observed a T3R-independent inhibitory effect of RXRß and 9-cis-RA on {alpha}3 transcription in transient transfection studies, suggesting that a nRXRE exists within the -116 to -6 bp region of the {alpha}3 gene. Consistent with this potential functional role of RXRß, we detected binding of RXRß to nucleotides between -62 to -21. The suppression of murine TSHß promoter activity by 9-cis-RA and the thyrotrope-specific {gamma}1 isoform of RXR represents the only other example of RXR-mediated repression of gene transcription (40). In this study a nRXRE was localized to nucleotides between -200 and -149 of the TSHß gene promoter region, distant from the known TSHß nTRE, which is found close to the transcription start site. Whether the {alpha}3 gene nRXRE is colocalized or distinct from the nTRE will require further investigation.

Our data indicate that the {alpha}3 gene nTRE is localized to a region of the proximal promoter between nucleotides -68 to -6. Within this 63-bp region, there exist two specific T3Rß1/RXRß-binding sites between nucleotides -62 and -41 and nucleotides -39 and -17. To determine whether one or both of these receptor binding sites were necessary for T3-mediated repression of {alpha}3 gene transcription, we prepared a deletion construct containing nucleotides between -39 to -6 of the {alpha}3 gene promoter and synthesized chimeric constructs with mutations that abolished T3Rß1/RXRß heterodimer binding at both sites (data not shown). Unfortunately, both of these strategies lead to basal luciferase expression that was too low to proceed with further functional characterization of the {alpha}3 gene nTRE. It is interesting to note that a deletion of the nTRE-containing region between -244 to -180 bp in the human glycoprotein hormone {alpha} gene resulted in a reduction of its basal activity to background levels (41). Similarly, mutations in the two repeats of a composite hormone response element of the human apolipoprotein AI gene that abolished the binding of RAR, RXR, T3R, and hepatic nuclear factor 4 also abrogated baseline promoter activity (42).

To investigate the molecular mechanism underlying repression of {alpha}3 gene transcription by T3, we evaluated whether T3R might interfere with the stimulatory action of either AP2 or Sp1. We found that neither cotransfected AP2 nor Sp1 counteracted the inhibitory effect of T3 on {alpha}3 gene expression. These results suggest that interference between T3Rß1 and either AP2 or Sp1 is not likely to account for the suppression of {alpha}3 promoter activity in response to T3. In contrast, binding of T3R and Sp1 to mutually exclusive overlapping sites was implicated in the mechanism of T3 repression of both epidermal growth factor receptor and human immunodeficiency virus gene expression (24, 43). Given the close proximity of the downstream T3Rß1/RXRß-binding site and the TATA element of the {alpha}3 gene, it is possible that T3-mediated inhibition may involve direct functional interference between T3Rß1/RXRß and TATA binding protein. A similar interference mode of action might involve the upstream T3Rß1/RXRß-binding site and a recently defined functional CAAT element in this region (35). Alternatively, T3Rß1/RXRß heterodimer formation may act directly to repress initiation of {alpha}3 gene transcription.

Thyroidal regulation of rat Na,K-ATPase {alpha}3 gene expression appears to involve the selective utilization of both stimulatory and inhibitory pathways. For example, a pTRE has been proposed to be responsible for the up-regulation of {alpha}3 mRNA content observed after a 1-day exposure of primary cultures of neonatal rat cardiac myocytes to T3 (11). In this study exposure to T3 for either 2 or 3 days, however, yielded a 50% decrease in {alpha}3 mRNA, suggesting that the pTRE was inactivated, and an inhibitory mechanism was functional. We propose that the nTRE present in the -68 to -6 bp region of the {alpha}3 promoter plays a fundamental role in this T3 inhibitory pathway. A similar switch of T3 responsiveness was evident in our demonstration that T3-mediated repression of {alpha}3 gene transcription was abolished when neonatal rat cardiac myocytes were cultured at a high cell density. Finally, {alpha}3 mRNA content was unresponsive to T3 in a rat telencephalon organotypic cell culture system (10). Taken together, these findings indicate that T3 can stimulate, repress, or have no effect on {alpha}3 gene expression and suggests that an interplay between positive and negative transcriptional events contributes to this complex response.


   Acknowledgments
 
We thank Drs. D. Moore and G. Gill for providing rat T3Rß1 and mutant rat T3Rß1 expression vectors, respectively. The authors are also grateful to Dr. P. Chambon for providing an expression plasmid for mouse RXRß. We thank Dr. M. Rosenfeld for rat RXRß-GST fusion protein and Dr. W. Solomon for the gift of GST. The authors thank Drs. R. Gaynor and R. Tjian for providing AP2 and Sp1 expression vectors, respectively. We also thank Dr. L. Freedman for the gift of a luciferase reporter construct containing a pRXRE.


   Footnotes
 
1 This work was supported by grants from the National Science Foundation and an Investigatorship and Grant-in-Aid from the American Heart Association, New York City Affiliate (to G.G.), and Grant DK-41842 (to J.A). Back

Received January 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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