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Endocrinology Vol. 140, No. 11 5004-5013
Copyright © 1999 by The Endocrine Society

ARTICLES

Regulation of the Mouse Preprothyrotropin-Releasing Hormone Gene by Retinoic Acid Receptor

Teturou Satoh, Takahiro Ishizuka, Tsuyoshi Monden, Nobuyuki Shibusawa, Tetsu Hashida, Mikiko Kishi, Masanobu Yamada and Masatomo Mori

First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan

Address all correspondence and requests for reprints to: Teturou Satoh, M.D., Ph.D., First Department of Internal Medicine, Gunma University School of Medicine, 3–39-15 Showa-machi, Maebashi 371-8511, Japan. E-mail: tsato{at}sb.gunma-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Retinoic acid (RA) has been reported to inhibit the secretion and synthesis of the pituitary TSH in vivo and in vitro. However, little is known about the influence of RA on the expression of the prepro-TRH gene. We therefore investigated whether the promoter activity of the mouse TRH gene is directly regulated by RA using a transient transfection assay into CV-1 cells. In the absence of cotransfected RA receptor (RAR), all-trans-RA did not affect the promoter activity. In contrast, the cotransfected RAR{alpha} significantly stimulated promoter activity in the absence of ligand, and all-trans-RA reversed basal promoter activation. The cotransfected thyroid hormone receptor-ß (TRß), but not 9-cis-RA receptor (RXR), had an additive effect on the RAR-dependent stimulation. TR and RAR can similarly interact with the corepressor proteins, and the cotransfected nuclear receptor corepressor (N-CoR) has been demonstrated to augment the transcriptional stimulation of the TRH gene by unliganded TR. As observed with TR, the coexpression of a N-CoR variant significantly enhanced the ligand-independent stimulation by RAR. A mutant RAR (RAR403) lacking the C-terminal activation function-2 (AF-2) activation domain that was essential for ligand-induced corepressor release constitutively stimulated the promoter activity. The constitutive stimulation by RAR403 was augmented by the cotransfected N-CoR variant. A deletion analysis of the 5'-flanking region of the TRH gene revealed that the minimal promoter region for the regulation by RAR was -83 to +53, with a consensus half-site motif for the thyroid hormone response element at -57. In contrast to the strong binding of TR to the thyroid hormone response element half-site in gel retardation assays, no binding of RAR homodimer, RAR/RXR heterodimer, or RAR/TR heterodimer was observed to the minimal promoter region. These results collectively suggest that RAR without heterodimerization with RXR and TR regulates transcription of the mouse TRH gene in cooperation with the corepressor, and that the DNA binding of RAR appeared to be unnecessary for regulation of the TRH gene promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VITAMIN A and its biologically active derivatives, such as all-trans-retinoic acid (atRA) and 9-cis-RA have profound effects on embryonal morphogenesis and cell differentiation as well as growth, vision, and reproduction (1). The biological action of retinoids is mediated by the nuclear receptors that belong to the superfamily of steroid/thyroid hormone receptors (2). Retinoic acid receptor (RAR) regulates gene transcription in both ligand-dependent and -independent manners by binding to the specific DNA element [retinoic acid-responsive element (RARE)] located within the promoter region of target genes (2). The established RARE consists of a direct repeat of the hexamer sequence (AGGTCA) that is identical to the consensus half-site sequence for the binding site for thyroid hormone receptor (TR) (3, 4). RAR binds to RARE as a heterodimer with 9-cis-RA receptor (RXR) and with TR (2, 5). That there is close interplay between RAR and TR is stressed by the similarity of structure of their DNA-binding domains and by the ability of these receptors to activate gene transcription through a common hormone-responsive element such as the palindromic thyroid hormone response element (TRE) (6). It has therefore been speculated that RA and thyroid hormone positively regulate the expression of some common genes, such as the GH gene (7). In contrast, functional antagonism between RAR and TR has been demonstrated in certain positive TRE, such as that in the {alpha}-myosin heavy chain gene promoter (8).

A recent in vivo study demonstrated that atRA plays an inhibitory role in basal and TRH-stimulated secretion of pituitary TSH in both euthyroid and hypothyroid rats (9). Suppression of TSH ß-subunit gene expression by atRA has previously been shown in rats, A transient transfection study using the rat TSH ß-subunit gene promoter revealed that the cotransfected RAR stimulated promoter activity in the absence of ligand, and the addition of atRA reversed its activity in CV-1 cells (10). A recent study using a thyrotropic tumor cell line documented the presence of a negative 9-cis-RA response element (RXRE) in the promoter of the mouse TSH ß-subunit gene that was separate from the established negative TRE in its promoter (11). These findings raise the possibility that atRA and 9-cis-RA, in addition to thyroid hormone (12), play inhibitory roles in the synthesis of pituitary TSH at the level of gene transcription.

TRH is the central regulator of thyroid function, stimulating TSH synthesis and secretion in the pituitary gland (13). Via a feedback mechanism, the expression of the hypothalamic TRH gene is negatively regulated by thyroid hormone (14, 15). In transient transfection assays performed in TR-deficient cell lines such as CV-1 cells, the cotransfected TR stimulated transcription of the human and mouse TRH gene promoters in the absence of T3, and the addition of T3 suppressed basal promoter stimulation (16, 17, 18). TR binds to the conserved TRE half-site located in the proximal promoter of the human and mouse TRH genes in electrophoretic mobility shift assays (EMSA), and the site-directed mutation of the TRE half-site significantly reduced the ligand-independent stimulation by TR (17, 19). Recent studies demonstrated that the cotransfection of the nuclear receptor corepressor (N-CoR) and its putative splicing variant (N-CoRI), which lacks the N-terminal repression domain, enhanced the ligand-independent stimulation by TR of the TRH gene promoters from different species (19, 20, 21), suggesting the paradoxical involvement of corepressors in the ligand-independent activation of the gene negatively regulated by T3.

Although the inhibitory regulation of TRH gene expression by thyroid hormone has been well studied, as noted above, no in vivo and in vitro data are currently available regarding the influence of atRA on the expression of the TRH gene. It is possible that the suppression of pituitary TSH secretion and synthesis by atRA observed in vivo (9, 10) is mediated in part by inhibition of the synthesis and release of the hypothalamic TRH. RAR and TR possess the same ability to interact with corepressor proteins, such as N-CoR and the silencing mediator of retinoid and thyroid hormone receptor (22, 23), suggesting that RAR and TR control gene transcription via a common mechanism in cooperation with the corepressor proteins. We therefore evaluated the effect of RA on the direct control of mouse TRH gene transcription as well as the role of a corepressor protein in RAR-mediated regulation by transient transfection assay and EMSA. In the present study, we found that RAR regulates mouse TRH gene transcription in a manner similar to that of TR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
CV-1 cells were maintained in DMEM supplemented with 10% FBS, penicillin, streptomycin, and fungizone at 37 C in a 5% CO2 atmosphere as previously described (18).

Reporter plasmids
Mouse TRH-Luc plasmids were constructed, in which -254/+87, -177/+83, -130/+83, -83/+53, and -36/+127 fragments of the 5'-flanking region of the mouse TRH gene were inserted into a firefly luciferase reporter plasmid, pA3Luc (18, 24). A luciferase reporter plasmid carrying RXRE upstream of the minimal thymidine kinase gene promoter (DR1-TK Luc) was previously described (25).

Expression vectors
Expression vectors for the human RAR{alpha} (pCMV-RAR{alpha}), mutant RAR (pCMV-RAR403), and pCMV-mRXR{alpha}, -ß, and -{gamma} were described previously (26, 27). A complementary DNA (cDNA) fragment encompassing the hinge and ligand-binding domains of human RAR{alpha} (amino acid residues 153–462) (28) was amplified by PCR using a sense primer (RARS1; 5'-GAAGTGCTTTGAATTCGGCATGTC-3') and an antisense primer (RARAS; 5'-GCGAGGGCTGAATTCATGTGGCGT-3') and was subcloned into an EcoRI site in an expression vector, pKCR2 (pRARLBD). The same cDNA fragment was inserted in-frame also into pCMV-GAL4DBD (amino acids 1–147; pGAL4-RARLBD) (29). A mutant RAR (amino acid residues 87–462) that lacks the A/B domain (28) was created by PCR using a sense primer (RARS2; 5'-TCGAATTCGCACCATGCCTTGCTTTGTCTG-3') that contained the Kozak consensus translation initiation site (30) and the RARAS primer, and was subcloned into pKCR2 (pRAR{Delta}N). The PCR-amplified fragments were subcloned also into pGEM-T Easy (Promega Corp., Madison, WI), and the nucleotide sequences were verified by the dideoxy termination method. Expression vectors for human TRß (pKCR2TRß) and human N-CoRI (pKCR2N-CoRI) were previously described (20).

Transient transfection and luciferase assay
Transient transfection was performed in 60-mm culture dishes using a calcium phosphate precipitation method with 3 µg reporter constructs and 150 ng expression vectors. The total amounts of plasmids transfected to each plate were adjusted by adding the empty pKCR2 in all experiments. Sixteen hours after transfection, media were changed to phenol red-free DMEM containing 10% FBS treated with AG1-X8 resin (Bio-Rad Laboratories, Inc., Hercules, CA) and activated charcoal (Sigma Chemical Co., St. Louis, MO). Cells were incubated for an additional 48 h in the presence and absence of 10 nM T3 and/or 1 µM atRA (Sigma Chemical Co.) as described in the figure legends. The luciferase assay was carried out as previously described (18, 31). Luciferase activities were normalized by the protein concentration measured by Bradford’s method and were expressed as light units per µg protein as previously described (18, 31). All transfection experiments performed with triplicate plates were repeated at least twice with similar results.

Oligonucleotides
The nucleotide sequences of the upper strand of oligonucleotides used in EMSA were as follows (lowercase letters indicate linker sequences, and underlining indicate a TRE half-site sequence at -57); -90/-60, 5'-agctCGCCCCTGCATTCGGCCTGCGCCCCCTC CCa-3'; -73/-47, 5'-agctTGCGCCCCCTCCCCGCTGACCTCACA-3'; -62/-32, 5'-agcttCCCGCTGACCTCACAGGGGCCGCTGTCTCGA-3'; -36/+10, 5'-ag-CTCGAGCGCATATAAGCCTCGGCCCCTCCGAGGAGC GCGCAG-TCGA-3'; +6/+60, 5'-agctGTCGACTCTGGATTCTGGAGCC TTGCAG-ACTCTACCCAGCCAGTTA-3'; and DR5, 5'-agctGGTTCACCGA AAG-TTCA-3'.

EMSA
In vitro translated human RAR{alpha}, TRß, and RXR{alpha} were prepared with a TNT-coupled rabbit reticulocyte lysate system (Promega Corp.) using pCMVRAR, peA101 plasmid, and pBShRXR, respectively, as previously described (18). In vitro translated human N-CoRI was prepared using pKCR2N-CoRI and T7 RNA polymerase. Synthesis of proteins of the expected mol wt was confirmed by labeling of in vitro translated products with [35S]methionine (DuPont, NEN Life Science Products, Boston, MA), followed by SDS-PAGE analysis (data not shown). Double stranded oligonucleotides, as described above, were labeled with [{alpha}-32P]deoxy-CTP (ICN Biomedicals, Inc., Costa Mesa, CA) by a fill-in reaction using a Klenow fragment of DNA polymerase I. The binding reaction was performed as previously described (18). A specific antibody for human RAR{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used in supershift experiments. Gel electrophoreses and autoradiographies were carried out as previously described (18).

Statistical analysis
Statistical analyses were performed using Duncan’s multiple range test among multiple groups. The level of significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ligand-independent and -dependent regulation of the mouse TRH gene by RAR
To study the direct effect of atRA on transcription of the TRH gene, the 5'-flanking region of the mouse TRH gene (-254/+83) fused to a luciferase reporter vector was transiently transfected into CV-1 cells in the presence and absence of a vector expressing RAR{alpha}. CV-1 cells have been reported to express a minimal amount of endogenous RAR (7). In the absence of cotransfected RAR, 1 µM atRA did not influence promoter activity. In contrast, the cotransfected RAR significantly stimulated promoter activity in the absence of ligand, and the addition of atRA reversed basal promoter stimulation (Fig. 1Go). The ligand-independent and -dependent regulations by RAR were similar to those by cotransfected TRß in the absence and presence of T3 (Fig. 1Go).



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  		Figure 1. Ligand-independent and -dependent regulation of the mouse TRH gene promoter by RAR and TR. A luciferase reporter driven by the promoter region (-254/+87) of the mouse TRH gene was cotransfected into CV-1 cells with an empty expression vector (pKCR2) or a vector expressing human RAR{alpha} or human TRß1. Sixteen hours after transfection, cells were incubated in the absence and presence of 1 µM atRA or 10 nM T3, respectively, for 48 h. Luciferase activities were measured as described in Materials and Methods. The data represent the mean ± SE from triplicate plates.

 
Effect of cotransfection of RXR on regulation of the TRH gene by RAR
We next examined whether the cotransfection of RXR, a heterodimer partner of RAR (2), affects the ligand-independent stimulation of the TRH gene promoter by RAR. As shown in Fig. 2AGo, the activity of the reporter plasmid possessing RXRE (DR1-TK) was not affected by 1 µM 9-cis-RA in the absence of cotransfected RXR, suggesting that the level of expression of endogenous RXR in CV-1 cells was not sufficient to mediate the 9-cis-RA action. In contrast, in the presence of the cotransfected RXR{alpha}, -ß, and -{gamma}, DR1-TK Luc activity was significantly stimulated by 9-cis-RA (Fig. 2AGo), indicating that the cotransfected RXR was expressed and functioned in the cells. However, the transfection of either isotype of RXR in the presence of RAR did not enhance the ligand-independent stimulation of the TRH gene promoter (Fig. 2BGo). These results suggest that RXR is not required in the RAR-mediated TRH gene regulation.



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  		Figure 2. Effect of cotransfection of the three RXR isoforms on regulation of the promoter carrying RXRE (A) and the TRH gene promoter (B). TK gene promoter carrying RXRE (DR1-TK Luc) was cotransfected with expression vectors for mouse RXR{alpha}, -ß, and -{gamma}. After transfection, cells were incubated in the presence and absence of 1 µM 9-cis-RA for 48 h. Mouse TRH Luc (-254/+87) was cotransfected with three isoforms of RXR in the presence of the cotransfected RAR and incubated in the presence and absence of atRA. The data represent the mean ± SE from triplicate plates.

 
TR and RAR stimulate TRH gene transcription in an additive manner
We next tested whether the cotransfection of TR in the presence of RAR affects regulation of the TRH gene. Transfection of the same amount of RAR or TR expression vector (300 ng/plate) similarly stimulated TRH gene promoter activity in the absence of ligand. When 150 ng TR and RAR were simultaneously transfected, promoter activity was stimulated in a manner similar to when 300 ng individual receptor were transfected. A separate addition of T3 or atRA did not completely repress the promoter activity stimulated by both RAR and TR, but incubation with both T3 and atRA reversed it to the basal level (Fig. 3Go). These results indicate that TR and RAR can additively stimulate TRH promoter activity in the absence of ligands.



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  		Figure 3. The cotransfected TR and RAR additively stimulated TRH gene promoter in the absence of ligand. The amounts (nanograms) of expression vectors for TR and RAR transfected to CV-1 cells are indicated in parentheses. After transfection, cells were incubated in the presence and absence of 10 nM T3 and/or 1 µM atRA as indicated. The data represent the mean ± SE from triplicate plates.

 
N-CoRI augments the ligand-independent stimulation by RAR
Cotransfection of N-CoR and a putative splicing variant of N-CoR (N-CoRI) that lacks two N-terminal repression domains has been reported to enhance ligand-independent stimulation of the TRH gene promoter by TR (19, 20, 21). Because unliganded RAR has been shown to bind to the receptor interaction domain of the corepressors in the same manner as unliganded TR in vitro (22, 23), we examined whether the cotransfected N-CoRI can enhance ligand-independent stimulation of the TRH gene promoter by RAR. As shown in Fig. 4Go, cotransfection of N-CoRI in the absence of RAR and TR but in the presence of RXR did not modulate the promoter activities. In the absence of ligand, cotransfection of N-CoRI significantly augmented promoter activity in the presence of RAR in a manner similar to that observed in the presence of TR (Fig. 4Go). The addition of atRA completely reversed the promoter activity stimulated by RAR and N-CoRI to the basal level (Fig. 4Go). These results suggest that the interaction of unliganded RAR with N-CoRI was required for augmentation of the ligand-independent regulation of the TRH gene promoter.



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  		Figure 4. The cotransfected N-CoRI augmented the ligand-independent stimulation by RAR of the TRH gene promoter. TRH Luc (-254/+87) was transfected together with TR, RAR, or RXR expression vector (150 ng) in the presence and absence of a vector expressing N-CoRI (1500 ng). One micromolar concentration of atRA, 10 nM T3, and 1 µM 9-cis-RA were used as ligands for RAR, TR, and RXR, respectively. The data represent the mean ± SE from triplicate plates.

 
The AF-2 activation domain of RAR is necessary for the ligand-dependent inhibition
The corepressor proteins have been demonstrated to be released from TR after ligand binding (22, 23), and the C-terminal AF-2 activation domain of TR has been shown to be necessary for the ligand-induced corepressor dissociation (32, 33). To gain additional evidence that corepressors are involved in the ligand-independent stimulation of the TRH gene promoter by RAR, the ability for ligand-independent and -dependent regulation of a mutant RAR (RAR403) that lacks the AF-2 domain was evaluated. This mutant has been reported to bind atRA with an affinity similar to that of the wild-type receptor and to constitutively repress activity of the positively regulated promoter in the absence and presence of atRA in CV-1 cells (27). As shown in Fig. 5Go, RAR403 significantly stimulated TRH gene promoter activity in the absence of ligand. In contrast to the wild-type receptor, the addition of atRA did not reverse the stimulated promoter activity by RAR403. Moreover, cotransfected N-CoRI could augment the transcriptional stimulation by RAR403, but, again, atRA did not reverse the enhancement (Fig. 5Go). These results indicate that the dissociation of the corepressor from liganded RAR might be necessary for mediation of ligand-dependent inhibition of the TRH gene promoter.



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  		Figure 5. The ligand-independent and -dependent regulation of the TRH gene promoter by RAR403. A mutant RAR lacking the C-terminal AF-2 domain (RAR403) was cotransfected with TRH Luc (-254/+87) in the presence and absence of N-CoRI and was incubated with or without 1 µM atRA for 48 h. The data represent the mean ± SE from triplicate plates.

 
A putative RA response element is located between -83 and +53 in the mouse TRH gene promoter
Using a series of deletion mutant promoters, we determined the minimal promoter region in the mouse TRH gene that was responsible for the ligand-independent and -dependent regulation by RAR using a transfection assay. The ligand-independent stimulation and -dependent inhibition by RAR as well as that by TR were observed in the -177/+83, -130/+83, and -83/+53 constructs similar to that in the -254/+83 construct (Fig. 6Go). Ligand-independent stimulation of the -83/+53 construct by RAR was augmented by the cotransfected N-CoRI (data not shown). Further deletion of the 5'-region of the promoter (-32/+126) resulted in no luciferase activity. These results indicated that the proximal promoter region between -83 and +53 contains the regulatory element necessary for the RA-independent and -dependent regulation of the mouse TRH gene.



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  		Figure 6. Delineation of a putative RARE in the TRH gene promoter. Deletion mutants of the 5'-flanking region of the mouse TRH gene fused to the luciferase cDNA (-177/+83, -130/+83, and -83/+53) were transfected with TR or RAR and were incubated with or without 10 nM T3 or 1 µM atRA, respectively, for 48 h. Cells transfected with an empty pKCR2 were incubated with both atRA and T3. The data represent the mean ± SE from triplicate plates.

 
RAR does not bind to the -83/+53 region of the TRH gene promoter in EMSA
We previously reported that in vitro translated TR can bind to the TRE half-site sequence (TGACCT) positioned at -57 in the mouse TRH gene promoter by EMSA (18). We therefore examined whether RAR could bind to the element containing the TRE half-site by EMSA. In vitro translated RAR{alpha} strongly bound as heterodimer with RXR{alpha} to an established RARE (DR5), and a specific antibody for RAR supershifted the heterodimer complex (Fig. 7Go, lanes 1–3). As we recently reported (19), in vitro translated TR bound strongly as homodimer and weakly as heterodimer with RXR to the element containing the TRE half-site at -57 in the mouse TRH gene (-62/-32). An addition of in vitro translated N-CoRI supershifted the TR homodimer complex over TR/RXR heterodimer in the absence of T3 (Fig. 7Go, lanes 10–13). In striking contrast to TR, in vitro translated RAR did not bind to the -62/-32 element as either monomer or homodimer, and an incubation with neither RXR nor N-CoRI promoted RAR binding to the element (Fig. 7Go, lanes 4–9). Moreover, no formation of RAR/TR heterodimer on the -62/-32 element was detected (Fig. 7Go, lanes 14–17). We also evaluated RAR binding to the overlapping oligonucleotide probes (-90/-60, -73/-47, -36/+10, and +6/+60) that covered the minimal promoter region (-83/+53) responsible for the regulation by RAR and found that RAR did not bind to these probes in the absence or presence of RXR, TR, and N-CoRI (data not shown). These results together with the data obtained by the transfection studies suggest that the direct DNA binding of RAR is unnecessary for regulation of the mouse TRH gene promoter by RAR.



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  		Figure 7. Binding of in vitro translated RAR and TR to the TRE-containing region of the mouse TRH gene and to DR5. An oligonucleotide corresponding to the region between -62 and -32 of the mouse TRH gene that contained a consensus TRE half-site sequence (TGACCT) at -57 (-62/-32) and an established RARE oligonucleotide (DR5) were radiolabeled and were incubated with in vitro translated RAR in the presence and absence of in vitro translated RXR, TR, and N-CoRI as indicated. The incubation mixtures were electrophoresed in a nondenaturing polyacrylamide gel. Autoradiography was performed for 16 h. The positions of TR homodimer, TR/RXR heterodimer, RAR/RXR heterodimer, and TR homodimer/N-CoRI complex are indicated by arrows. SS indicates the complex supershifted by an antibody specific for RAR (RARAb). Asterisks indicate nonspecific binding.

 
An intact DNA-binding domain of RAR is necessary for the ligand-independent and -dependent regulation of the TRH gene promoter
We lastly examined whether the DNA-binding domain (DBD) of RAR was required for regulation of the TRH gene promoter. To assess this issue, we examined the ability for ligand-independent and -dependent regulation of the mutant RARs in which the A/B domain ({Delta}N) or the A/B and C domains (RARLBD) was deleted. The ability of a chimeric mutant receptor in which the ligand-binding domain (LBD) of RAR was fused to the DBD of a yeast transcriptional activator, GAL4 (GAL4-RARLBD), was also evaluated. As shown in Fig. 8AGo, the {Delta}N receptor showed ligand-independent and -dependent regulation similar to the wild-type receptor, whereas no stimulation or inhibition was observed with RARLBD. Interestingly, GAL4-RARLBD, but not GAL4DBD by itself, strongly stimulated the promoter activity, and the addition of atRA reversed the stimulation (Fig. 8BGo). The cotransfected RARLBD inhibited the trans-activation of a positive RARE by the wild-type receptor in a dose-dependent manner, confirming the expression and nuclear trans-location of RARLBD (Fig. 8CGo). These results indicated that the DBD, but not the A/B domain, of RAR was necessary for regulation of the TRH gene despite the fact that RAR did not bind to the proximal promoter region in EMSA. Moreover, the DBD of GAL4 might mimic the function of the DBD of RAR in regulation of the mouse TRH gene.



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  		Figure 8. A, Ligand-independent and -dependent regulation of the TRH gene promoter by mutant RARs lacking the A/B domain and the A/B/C domain. An expression vector for RAR lacking the A/B domain ({Delta}N) or the A/B/C domain (LBD) was transfected with TRH Luc (-254/+87) and incubated in the presence and absence of atRA. Data represent the mean ± SE from triplicate plates. Asterisks indicate a significant difference (P < 0.01) from the luciferase activity transfected with an empty pKCR2 in the absence and presence of atRA. B, Ligand-independent and -dependent regulation by GAL4RARLBD. TRH Luc (-254/+87) was cotransfected with an expression vector in which RARLBD was fused to GAL4DBD or a vector expressing GAL4DBD. Data represent the mean ± SE from triplicate plates. C, The cotransfected RARLBD inhibits the trans-activation function of the wild-type receptor. A luciferase reporter possessing a positive RARE (DR5) upstream of the TK gene promoter was cotransfected with the wild-type RAR in the absence and presence of increasing amounts of RARLBD. The luciferase activity was measured after 48-h incubation with 1 µM atRA. Data represent the mean ± SE from triplicate plates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present transfection study provided new evidence that RAR regulates transcription of the mouse TRH gene in a manner similar to TR in CV-1 cells. In contrast to the TR-mediated regulation of the TRH gene promoter (17, 19), the present results suggest that the direct binding of RAR to the specific DNA element in the proximal TRH gene promoter is unnecessary for the regulation. Moreover, the RAR-mediated regulation was likely to be independent of heterodimerization with RXR and TR. The ligand-independent and -dependent regulatory pattern of the TRH gene by TR and RAR was similar to that of the rat TSH ß-subunit gene (8). However, the DNA binding and heterodimerization with RXR of RAR have recently been shown to be required for TSH gene regulation in CV-1 cells (34). The promoter activity of the rabbit keratin gene has been demonstrated to be stimulated by unliganded TR, but not by unliganded RAR, although both T3 and atRA suppressed keratin gene promoter activity (35). The regulation by RAR of mouse TRH gene transcription, therefore, seems to be characteristic compared with that of other promoters negatively regulated by T3.

Without binding to their cognitive response elements located in the target gene promoters, nuclear hormone receptors can regulate gene transcription by modulating the function of other transcription factors through protein-protein interactions. For instance, liganded RAR has been shown to prevent c-Jun phosphorylation and, consequently, activating protein-1 activation by blocking the Jun amino-terminal kinase signaling cascade (36). Peroxisome proliferator-activated receptor has recently been shown to activate the PRL promoter without DNA binding through protein-protein interaction with GH factor-1 and coactivator proteins (37). In certain cell types, such as GH4C1 cells, unliganded TR has been demonstrated to bind to a tumor suppressor, p53, and to inhibit p53 function, resulting in transcriptional activation (38). These results together with the present findings suggest that unliganded RAR interferes with the function of some inhibitory factor that directly or indirectly suppresses TRH gene transcription through a protein-protein interaction and thus stimulates the activity of the TRH gene promoter. Ligand binding to RAR might abrogate such RAR/putative inhibitor interaction, resulting in reversal of the activated transcription. It is also possible that the unliganded RAR recruits some coactivator protein to stimulate the transcription, and the coactivator is released by ligand binding. However, the unliganded RAR403 lacking the AF-2 activation domain could stimulate TRH promoter activity in the present study, suggesting that known coactivators interacting with the AF-2 domain of RAR in the presence of ligands (39, 40, 41, 25) are not involved in the ligand-independent stimulation.

A DBD-truncated RAR (RARLBD), but not an A/B domain-truncated receptor ({Delta}N), failed to stimulate TRH gene promoter activity in the absence of ligand, suggesting that the integrity of RARDBD was required to exert the ligand-independent stimulation by RAR. The nuclear trans-location of RARLBD fused to the green fluorescent protein in the presence and absence of ligand was similar to that of the wild-type receptor when analyzed by confocal microscopy (our unpublished observation). Although the DBD of nuclear hormone receptors appears to be mainly involved in DNA binding and receptor dimerization (2), there is evidence that this domain also serves as an interaction interface for other proteins (38, 42, 43, 44, 45). These results together with the present findings raise the possibility that the RAR/putative cofactor interaction involved in TRH gene regulation occurs through the DBD of RAR. A fusion of GAL4DBD restored the regulatory function to the RARLBD in the present study, suggesting that GAL4DBD contains a structural domain similar to that in the RARDBD necessary for the interaction with the putative cofactor. Alternatively, GAL4-RARLBD may stimulate TRH promoter activity via a mechanism different from that used by the wild-type receptor.

The cotransfection of N-CoRI, which binds to the CoR box in the hinge region of RAR (22), augmented RAR-mediated stimulation of the TRH gene promoter, and the addition of atRA completely reversed the enhanced promoter activity to the basal level in the present study. A mutant RAR (RAR403) defective in ligand-induced corepressor release (27) stimulated the TRH gene promoter even in the presence of atRA, and such constitutive activation by RAR403 was further augmented by the cotransfection of N-CoRI. These results strongly suggest that the direct interaction of unliganded RAR with N-CoRI was necessary for the enhancement of ligand-independent stimulation of TRH gene transcription by RAR. However, the molecular mechanism underlying the enhancement by N-CoRI remains to be elucidated.

As the present transfection studies were performed using a kidney cell line, the physiological relevance of RA regulation of the TRH gene expression remains to be determined. High levels of expression of RAR have been demonstrated in the mouse brain (46). RAR-deficient mice exhibited an impaired locomotion and dopamine signaling pathway resulting from the reduced expression of the D1 and D2 dopamine receptors in the ventral striatum (47) where abundant expression of the TRH gene has been found (48). TRH and its receptor genes were also shown to be expressed in the retina, a tissue well known to be responsive to vitamin A (49, 50). Further studies regarding the physiological importance of RA regulation of the TRH gene expression in vivo are on-going using the TRH-deficient mouse recently established in this laboratory (13).


   Acknowledgments
 
We thank Dr. Ronald M. Evans (Howard Hughes Medical Center, The Salk Institute for Biological Studies, La Jolla, CA), Dr. William M. Wood (University of Colorado Health Sciences Center, Denver, CO) and Dr. Kazuhiko Umesono (Kyoto University, Kyoto, Japan) for supplying materials.

Received April 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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