Differential Regulation of Direct Repeat 3 Vitamin D3 and Direct Repeat 4 Thyroid Hormone Signaling Pathways by the Human TR4 Orphan Receptor*

Yi-Fen LeeDagger , Win-Jing YoungDagger , Wen-Jye Lin, Chih-Rong Shyr, and Chawnshang Chang§

From the George Whipple Laboratory for Cancer Research, Department of Pathology, Urology, Radiation Oncology, and The Cancer Center, University of Rochester Medical Center, Rochester, New York 14642

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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In situ hybridization analysis demonstrated that abundant testicular orphan receptor (TR4) transcripts were detected in kidney, intestine, and bone, which are vitamin D3 target organs. Cell transfection studies also demonstrated that the expression of the vitamin D3 target gene, 25-hydroxyvitamin D3 24-hydroxylase, can be repressed by TR4 through high affinity binding (Kd = 1.32 nM) to the direct repeat 3 vitamin D3 receptor response element (DR3VDRE). This TR4-mediated repression of DR3VDRE is in contrast to our earlier report that TR4 could induce thyroid hormone target genes containing a direct repeat 4 thyroid hormone response element (DR4T3RE). Electrophoretic mobility shift assay using several TR4 monoclonal antibodies when combined with either TR4-DR3VDRE or TR4-DR4T3RE showed a distinct supershifted pattern, and proteolytic analysis further demonstrated distinct digested peptides with either TR4-DR3VDRE or TR4-DR4T3RE. These results may therefore suggest that TR4 can adapt to different conformations once bound to DR3VDRE or DR4T3RE. The consequence of these different conformations of TR4-DR3VDRE and TR4-DR4T3RE may allow each of them to recruit different coregulators. The differential repression of TR4-mediated DR3VDRE and DR4T3RE transactivation by the receptor interacting protein 140, a TR4 coregulator, further strengthens our hypothesis that the specificity of gene regulation by TR4 can be modulated by protein-DNA and protein-protein interactions.

    INTRODUCTION
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Steroid hormones are key physiological mediators of development and homeostasis (1-4). Understanding the crosstalk between steroid hormone-dependent and -independent signaling pathways is critical for gaining further insight into the integration of cellular regulatory cues that modulate development and tissue-specific gene expression. The biological effects of steroids and related hormones, including derivatives of vitamins A and D3, are mediated through their cognate receptors (1-4). These receptors are members of a large group of ligand-activated proteins that act as transcriptional activators or repressors. However, there is another group of nuclear receptors that shares the same molecular structure as steroid hormone receptors but has no known ligands. The members of this group have therefore been named orphan receptors (5). A common characteristic of many of these orphan receptors is that they exert at least part of their function as regulators of other receptors. This may occur by several different mechanisms: competition for the same response element, heterodimer formation with the regulated receptor, or heterodimer formation with the retinoid X receptor (RXR),1 thus titrating out available RXR protein. For instance, chicken ovalbumin upstream promoter transcription factors have been demonstrated to suppress ligand-induced gene activation, including that of vitamin A, vitamin D3, and thyroid hormone target genes (6). This interfering effect might involve the formation of a transcriptional silencing complex with RXR or competition with steroid hormone receptors for DNA binding sites (7).

The human TR4 orphan receptor (TR4) was first identified by using degenerate polymerase chain reaction cloning. The open reading frames of TR4 cDNA encode 615 amino acids with a calculated molecular mass of 67 kDa (8). On the basis of sequence similarities, TR4 is classified as a member of the steroid hormone receptor superfamily, very close to the TR2 orphan receptor (9, 10). A comparison of the amino acid sequence in the p-box of the DNA binding domain groups TR4 in the estrogen receptor/thyroid hormone receptor (T3R) subfamily, which recognizes the AGGTCA core consensus motif. From this information we were able to identify several target genes that are up-regulated by TR4, including the fifth intron of the ciliary neurotrophic factor receptor alpha  (11, 12) and DR4T3RE (13). In contrast, TR4 represses the SV40 major late promoter (14) and retinoic acid receptor/RXR target genes (15). Similar results were also obtained with TR2 (16, 17). Molecular mechanisms of the differential regulation by TR4 remain unclear. Determining whether TR4 is capable of interfering with gene regulation by binding to other AGGTCA-like motifs and/or interacting with cofactor(s) might provide us more detailed information about this orphan receptor. A vitamin D3 target gene is a potential candidate of interest for more study, because its receptor functions through interaction with the vitamin D3 response element, which contains two AGGTCA repeat motifs with a 3-nucleotide space between repeats (DR3-VDRE). These response element motifs may also be recognized by TR4. Despite the similarity of hormone response elements (HRE) recognized by both VDR and TR4, these two receptors have also been found to interact in vitro with the receptor interacting protein 140 (RIP140) cofactor (18, 19). Investigating the regulation of TR4 in the vitamin D signaling pathway provides us with a molecular mechanism to explain the way TR4 regulates the gene expression in terms of protein-DNA and protein-protein interactions.

Vitamin D3 is hydroxylated first in the liver at carbon 25 to yield 25-hydroxyvitamin D3 and then in the kidney at the alpha -position of carbon 1 to generate 1,25-(OH)2D3, the active form of vitamin D. This has various biological effects, including the maintenance of calcium homeostasis, regulation of bone remodeling, and modulation of cell growth and differentiation (20-23). The 25-hydroxyvitamin D3 24-hydroxylase gene (P450cc24) encodes a key enzyme involved in vitamin D metabolism, which is responsible for the conversion of 25-(OH)D3 and 1,25-(OH)2D3 to 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and 1,24,25-trihydroxyvitamin D3, respectively (24). These metabolites are thought to be inactive forms of vitamin D (25). However, 1,25-(OH)2D3 induces 24-hydroxylase activity in its target cells, and thus its presence may play a crucial role in eliminating hormone activity of the vitamin D compound (25). Two vitamin D-responsive elements (VDRE-1 and VDRE-2) responsible for 1,25-(OH)2D3 stimulation of transcription were identified at nucleotides -151 to -137 and nucleotides -259 to -245 of the 5'-flanking region of the rat P450cc24 gene (26-28). Both VDREs contain two AGGTCA-like repeat motifs with a 3-nucleotide space in sense or antisense orientation and are similar to the VDREs found in the human P450cc24 gene (29). Examination of regulation via the 24-hydroxylase induction mechanism at the molecular level may contribute to the understanding of vitamin D in the endocrine system.

In this study, we analyzed the regulation by TR4 on the vitamin D3 signaling pathway and compared the expression pattern of TR4 to that in vitamin D3 target organs. The differential regulation of target genes containing DR3VDRE and DR4T3RE by TR4 was further investigated, and the results suggested that conformational changes because of DNA-protein and protein-protein interactions might play major roles in this regulation.

    MATERIALS AND METHODS
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Plasmid Construction-- For the transient transfection or coupled in vitro transcription/translation of the full-length TR4 protein, the pCMX-TR4 and pET14b-TR4 plasmids were constructed as described previously (14). The chimeric receptor pCMX4A4 was constructed as described previously (13). The reporter plasmid P450cc24-CAT, the 5'-flanking region (nucleotide -2200 to +188) of the rat vitamin D3 24-hydroxylase gene, was kindly provided by Dr. Y. Kato (26). The glutathione S-transferase (GST) fusion protein was constructed by inserting the full-length TR4 to the SmaI site of pGEX-3X (Amersham Pharmacia Biotech). Full-length RIP140 was cloned into the pET-28a(+) vector at the NotI site for in vitro transcription/translation reaction. The coupled in vitro transcription and translation expression plasmids, pET14b-TR4, pCMX-RXRalpha , and pSG5-VDR (29), were in vitro transcribed and translated by the TNT system (Promega) as described previously (14).

GST Pull-down Assay-- A GST pull-down assay was performed according to the methods described previously (30). Briefly, the GST fusion protein and GST control protein were expressed in Escherichia coli BL21 (DE3) pLys bacterial culture and recovered on glutathione-Sepharose-4B beads. GST fusion protein bound to glutathione-Sepharose-4B beads was incubated for 2 h at 4 °C with 5 µl of in vitro translated [35S]methionine-labeled protein in a total volume of 100 µl of incubation buffer (20 mM HEPES, pH 7.9, 150 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, 1 mg/ml bovine serum albumin, 10% glycerol, and protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, leupeptin, and pepstatin)). The glutathione-Sepharose-4B beads were then washed three times with wash buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). The beads were boiled in 2× SDS sample buffer, loaded onto SDS-polyacrylamide gels, and visualized by autoradiography.

Other Methods-- In situ hybridization, electrophoretic mobility shift assay (EMSA), Scatchard analysis, and transient transfection were performed according to the methods described previously (11, 14, 31, 32).

    RESULTS
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TR4 Specifically Binds to DR3VDRE, the AGGTCA Motif with 3-nucleotide Spacing-- EMSA was used to determine the binding specificity of TR4 to DR3VDRE. In vitro translated TR4 protein was incubated with 32P-labeled DR3VDRE and analyzed on a 5% polyacrylamide gel. As shown in Fig. 1, free probe was detected at the bottom of the gel (lane 1), and a specific DNA·protein complex was identified when 1 µl of in vitro translated TR4 protein was added (indicated by an arrow, lane 2). This complex was competed out by adding 100-fold excess of unlabeled DR3VDRE (lane 3). This DNA·protein complex was abolished when anti-TR4 monoclonal antibody 4 was added (lane 4). In contrast, when the anti-TR4 monoclonal antibody 2 was added to the reaction, a DNA·receptor·antibody complex supershifted band was visible (indicated by an arrowhead, lane 5).


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Fig. 1.   Binding of in vitro expressed TR4 to DR3-VDRE. 32P-labeled DR3VDRE oligomers were used in EMSA without or with 1 µl of in vitro translated TR4 (lanes 2-5). A 100-fold excess of unlabeled DR3VDRE (lane 3) was added as a competitor. One microliter of anti-TR4 monoclonal antibodies 4 and 2 were added (lanes 4 and 5). The migration position of the specific binding formed by the DNA·protein complex and the supershifted band formed by adding monoclonal antibodies are indicated with an arrow and an arrowhead, respectively.

DR3VDRE Binds to TR4 with Higher Affinity than to the RXR/VDR Heterodimer-- Vitamin D3 decreases the affinity of the VDR/VDR homodimer for DNA targets, enhances the formation of RXR/VDR heterodimers, and potentiates RXR/VDR affinity for VDREs (33, 34). Comparing the different affinities of these receptors and TR4 for DR3VDRE might provide more information about how these receptors regulate their target genes. EMSA was performed to determine receptor response element Kd. Fixed amounts of in vitro translated TR4 protein (1 µl) or RXR/VDR (1 µl of each) were incubated with an increasing amount of 32P-DR3VDRE (from 0.1 to 12.8 ng) and analyzed by EMSA. As shown in Fig. 2, Scatchard plot analysis of the DNA·protein complexes in the EMSA demonstrated that the Kd values of TR4 for DR3VDRE and RXR/VDR for DR3VDRE were 1.32 nM and 7.31 nM, respectively. Therefore TR4 has an affinity for DR3VDRE that is 5.5-fold higher than that of the RXR/VDR heterodimer. These data suggest that TR4 was able to compete with RXR/VDR for the binding to DR3VDRE.


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Fig. 2.   Scatchard analysis of RXR/VDR and TR4 affinity for DR3VDRE. One microliter of in vitro translated TR4, VDR, and RXR was incubated with serially diluted 32P-labeled DR3VDRE (concentrations from 0.1 to 12.8 ng) and resolved by EMSA. After autoradiography, the respective bands were excised, placed in scintillation fluid, and quantified directly in a scintillation counter. The ratio of activity between specific DNA protein binding (Bound) and free DNA probe with respect to specific DNA protein binding (bound/free (B/F)) was plotted. The dissociation constant (Kd) and Bmax values were generated using the EBDA program (Biosoft).

TR4 Represses the Vitamin D3-induced Rat P450cc24 Gene Expression-- The consequence of high affinity binding between TR4 and DR3VDRE was then investigated by reporter assay. The target gene used here was the 5'-flanking region (nucleotide -2200 to +188) of the rat P450cc24, containing VDREs that are responsible for the 1,25-dihydroxyvitamin D3 enhancement and are located at nucleotides -167 to -102 and nucleotides -204 to -129 (22). Co-transfection of 2.5 µg of pSG5VDR with 3 µg of P450cc24-CAT into CHO cells enhanced the transactivation of P450cc24-CAT to 32-fold in the presence of 10-7 M 1,25-(OH)2D3 (Fig. 3B, lane 2 versus 3). However, there is no CAT activity with the co-transfection of 2.5 µg of pCMX-TR4 or pCMX-4A4 either in the absence or presence of 1,25-(OH)2D3 (Fig. 3B, lanes 4-7). The chimeric receptor pCMX-4A4, which replaces the DNA binding domain of TR4 with that of an androgen receptor (Fig. 3A), was unable to bind to the AGGTCA DR motif sequence and served here as a negative control. The transcriptional activity of P450cc24-CAT induced by vitamin D3 was repressed when co-transfected with pCMX-TR4, but not when co-transfected with the chimeric receptor, pCMX-4A4 (Fig. 3B, lane 3 versus 8 and 9). These results suggest that the DNA binding domain of TR4 is essential for the TR4-mediated repression of the vitamin D3 responsive enhancement of the P450cc24 gene. To examine the expression levels of both wild-type and chimeric receptor TR4 proteins after transfection, polyclonal antibodies against both proteins were produced and examined. This repression effect was further proven by co-transfection of pSG5VDR with different amounts of pCMX-TR4 (from 1 to 5 µg) into CHO cells. As shown in Fig. 4, the repression effect mediated by TR4 was gradually increased when an increasing amount of TR4 was co-transfected (lanes 4-8). This result clearly demonstrated that TR4 could suppress the vitamin D3-induced P450cc24 gene promoter activity in a TR4 dose-dependent manner.


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Fig. 3.   Repression of vitamin D3-induced rat 25-hydroxyvitamin D3 24-hydroxylase gene activity by TR4. A, the molecular structure of wild-type TR4 (pCMX-TR4) and chimeric receptor 4A4 (pCMX-4A4) plasmids. CMV, cytomegalovirus promotor. N, DBD, and LBD indicate the N terminus, DNA-binding domain, and ligand-binding domain of TR4, respectively. ARDBD indicates the DNA-binding domain of the androgen receptor. B, P450cc24-CAT reporter plasmids (3 µg of each) were transfected into CHO cells with or without co-transfection of a VDR expression vector (pSG5-VDR, 2.5 µg), a TR4 expression vector (pCMX-TR4, 2.5 µg), or a chimeric receptor (pCMX-4A4, 2.5 µg). Twenty-four hours after transfection, 10-7 M 1,25-(OH)2 vitamin D3 was added (lanes 3, 5, 7-10), and an equal volume of ethanol was added in the control experiment. Cells were harvested 3 days after transfection for CAT analysis. Significant differences were determined by the Student's t test and are marked with an asterisk. *, p < 0.005 compared with lane 3. Each value represents the mean ± S.D. of four independent experiments.


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Fig. 4.   Dose response of P450cc24-CAT repression by TR4. CHO cells were transfected with P450cc24-CAT reporter plasmid (3 µg of each) and pSG5VDR (2.5 µg of each) and co-transfected with increasing amounts of pCMX-TR4 (from 0 to 5 µg). The percentage of CAT conversion was plotted. All CAT assays were normalized relative to beta -galactosidase activity. Significant differences (*, p < 0.005) of CAT activity reduced by TR4 at different doses were determined by one-way analysis of variance. Each value represents the mean ± S.D. of three independent experiments.

Localization of TR4 Transcripts in Vitamin D3 Target Organs during Mouse Embryogenesis-- To determine if TR4 is expressed in vitamin D target tissues, we applied in situ hybridization analysis to mouse embryos. As shown in Fig. 5, high levels of TR4 transcripts were detected in the perichondrium, which contains cells active in bone formation (Fig. 5B). Distinct TR4 distribution was also observed in the developing glomeruli and tubule structures of the kidney as well as the intestinal villi (Fig. 5, C and D). Fig. 5A shows strong TR4 expression detected in certain nonclassical vitamin D target tissues, such as lung and hair follicles (35, 36). Active TR4 expression in these tissues with the known expression domains of VDR (37) and 24-hydroxylase (38, 39) suggests TR4 may interact with the in vivo vitamin D signaling pathway.


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Fig. 5.   Localization of TR4 transcripts in vitamin D3 target organs during mouse embryogenesis. Saggital sections of mouse embryos at gestation day 15 (A) and day 16 (B-D) were analyzed by in situ hybridization with a 35S-UTP-labeled mouse TR4 antisense riboprobe. Autoradiograms were photographed under light field illumination with (B) or without (A, C, and D) hematoxylin staining. Tissues with strong hybridization signals (dark areas) are labeled. High magnification (B-D) shows an intensive TR4 signal in the perichondrium (PC), kidney (K), and intestine (I). g, glomerulus; t, renal tubules; v, villi; HF, hair follicles; L, lung. The bars represent 1 and 0.1 mm of length in A and B-D, respectively.

The Conformational Differences between TR4·DR3 Complex and TR4·DR4 Complex-- The above results suggest that TR4 may exert a repression effect on vitamin D3 responsive target gene expression by binding to DR3VDRE. In our previous studies, we concluded that TR4 activates the expression of the genes that contain DR4T3RE in both HRE sequence- and TR4 dose-dependent manners (13). This contrasting and differential regulation by TR4 could be because of different DNA-protein or protein-protein interactions. When compared, we found the Kd values of TR4 to DR3VDRE and TR4 to DR4T3RE to be very similar (1.32 versus 2.0 nM). This result eliminates the possibility that a different binding capacity between TR4 and DR3 or DR4 results in the distinctive regulation.

The second possibility is that the mechanism of regulation may be through different protein-protein interactions that are dependent on the distinct conformations of TR4 once bound to either DR3VDRE or DR4T3RE. To test this second possibility, EMSA was used to examine a series of monoclonal antibodies raised against TR4, which are able to recognize conformational epitopes. Four monoclonal antibodies (mAbs) were initially characterized by EMSA on the basis of their ability to supershift or abolish the TR4·DNA complex. As shown in Fig. 6A, we observed a specific DR3·TR4 complex (lane 2) that was distinct from mock-translated protein (lane 1); with the addition of mAbs 1, 2, and 3, supershifted bands were found (lanes 3-5). In contrast, the specific band formed by DR3·TR4 was abolished when the mAb 4 was added (lane 6). The same band-shifted pattern was observed when DR3 was replaced with DR4 (Fig. 6B, lane 1-6). Interestingly, different band-shifted patterns were observed with various combinations of different antibodies. As shown in Fig. 6A, an enhanced supershifted band shows migration positions that are different from the supershifted band (lane 8). This suggests that mAbs 1 and 3 recognize different epitopes because their simultaneous addition resulted in an increased mobility shift beyond that of either antibody alone. In contrast, the addition of mAbs 1 and 2 did not lead to an enhanced supershifted band (lanes 3 and 4 versus 7). These results suggest that the mAb 1 recognizes the same epitope of TR4 as the mAb 2 when TR4 is bound to DR3. However, a supershifted band was detected when the mAb 1 was added with the mAb 3 simultaneously to TR4·DR4 complex (Fig. 6B, lane 8). These results indicate that TR4 might fold into different conformations upon binding to diverse HREs and that different antibodies can recognize these conformations.


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Fig. 6.   Conformational differences of TR4 induced by binding to DR3-VDRE and DR4-T3RE. EMSAs were performed by incubation of 1 µl of in vitro translated TR4 with various combinations of mAbs prior to adding 32P-DR3VDRE (A) and 32P-DR4T3RE (B). The combinations of mAbs are indicated. C, 5 µl of [35S]methionine-incorporated in vitro translated TR4 was incubated with 100 ng of DR3-VDRE and DR4-T3RE at 25 °C for 1 h. 1 µl of different concentrations of trypsin (25 and 50 µg/µl) and alpha -chymotrypsin (50 and 100 µg/µl) was incubated with the TR4·DNA complex in 20 µl for another 15 min at 25 °C, and the peptide profiles were analyzed in 12.5% SDS-polyacrylamide gel electrophoresis.

This second hypothesis was further proven by proteolytic analysis. [35S]methionine-incorporated, in vitro translated TR4 was incubated in the absence or presence of HRE (DR4 or DR3) at 25 °C for 1 h. As shown in Fig. 4C, TR4-DR4T3RE has a similar trypsin-resisting fragment pattern to that of unbound TR4 control. In contrast, the TR4·DR3VDRE complex was more sensitive to trypsin when 2.5 µg/ml trypsin was applied. As results showed, the full-length of TR4 was completely degraded and some trypsin-resisting fragments disappeared compared to that with TR4·DR4T3RE at the same concentration of protease treatment. Similar results were also obtained when we replaced trypsin with alpha -chymotrypsin. We concluded that TR4-bound DR4T3RE or DR3V3RE had a different sensitivity to protease digestion and different protease-resisting fragments could be obtained with a higher concentration of protease digestion. These results further confirm our hypothesis that, to exert its proper function, TR4 may adopt distinct conformations when bound to DR3VDRE or DR4T3RE, leading to different protein environments.

RIP140 Interacts with TR4 and Differentially Modulates the TR4-mediated DR3VDRE-CAT and DR4T3RE-CAT Activities-- In vitro interaction of TR4 and RIP140 was performed by GST pull-down assay. As shown in Fig. 7A, TR4 could interact with RIP140 but not RXR (lane 3 versus 4). No interaction could be detected when GST-TR4 was replaced with GST (lane 5 and 6). The effects of RIP140 on TR4-mediated DR3VDRE-CAT and DR4T3TE-CAT activities were also investigated. As shown in Fig. 7B, RIP140 can further repress the TR4-mediated DR3VDRE-CAT suppression (lane 8 versus 9). In contrast, RIP140 can repress both TR4 and T3R-mediated DR4-T3RE-CAT activities (Fig. 7C, lane 5 versus 6 and lane 7 versus 10) significantly, but RIP140 has no significant effect on the DR4T3RE-CAT induction when TR4 and liganded T3R were co-transfected (Fig. 7C, lane 8 versus 9). It is also worth noting that whereas RIP140 can further enhance the TR4-mediated DR3VDRE-CAT suppression (lanes 8 versus 9), RIP140 has no significant effect on DR3VDRE-CAT transactivation in the absence of TR4 (Fig. 7B, lane 7 versus 10).


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Fig. 7.   RIP140 interacts with TR4 and exerts repressive control over DR3VDRE-CAT and DR4T3RE-CAT. 35S-labeled RIP140 and RXRalpha were incubated with GST-TR4 or GST bound to glutathione-Sepharose beads in a pull-down assay. The input represents 20% of the amount of labeled protein used in the pull-down assay (A). CHO cells were co-transfected with 1 µg of pCMX-TR4, 2 µg of pSG5VDR (B) or pCD-T3Ralpha 1 (C), 1 µg PEF-RIP140, and 3.5 µg DR3-VDRE-CAT or DR4-T3RE-CAT in different combination sets. Twenty-four hours after transfection, the cells were treated with 100 nM vitamin D3 (B) or T3 (C). All CAT assays were normalized relative to beta -galactosidase activity. Each value represents the mean ± S.D. of three independent experiments.


    DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
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According to the sequence of the TR4 DNA binding domain and our previous results from EMSA, we conclude that TR4 binds to the AGGTCA core consensus motif arranged in a direct repeat orientation with various numbers of nucleotide spacings. In this paper, we demonstrated that TR4 binds to DR3VDRE with high affinity and thus suppresses vitamin D3-induced P450cc24 gene activation. However, in our previous studies, we demonstrated that TR4 activates the genes that contain DR4-T3RE or nonclassical T3RE by binding to their response elements (13). The mechanisms of different binding responses of TR4 to the same sequence in core motifs with different spacings remain unclear. The responsiveness of genes to steroid hormones involves both the binding of regulatory proteins to specific DNA sequences and the formation of critical protein-protein associations. Throughout the past decade, a number of nuclear receptor coregulators has been characterized and provides us a more detailed molecular model of how nuclear receptors regulate their target genes. Because TR4 binds to DR3VDRE and DR4T3RE with similar affinities (1.32 versus 2.0 nM), we propose that whether TR4 works as a transcriptional enhancer or a silencer might be mediated not only by direct DNA binding but also by protein-protein interactions. This indicates that in addition to the receptor and the DNA, other factors may also contribute to the selectivity of receptors in the recognition of their target genes.

To test this hypothesis, EMSAs and proteolytic analyses were performed. In Fig. 4, A and B, distinct EMSA patterns were observed when TR4 was bound 32P-DR3VDRE and 32P-DR4T3RE in the presence of different combinations of various TR4 monoclonal antibodies. Meanwhile, different peptide patterns were obtained when DR4- or DR3-bound TR4 was digested with trypsin. Taken together, these data suggest that TR4 binding to diverse HREs may result in distinct conformational changes that can then trigger differential regulation. This finding supports the existence of a unique spacing of the direct repeat, which serves as a binding site for an auxiliary protein that modifies receptor activity (39). Similar approaches have also been used to study a thyroid receptor response element, RSVT3RE, which contains an inverted repeat with a 6-nucleotide space. RSVT3RE allows strong activation by c-ErbAalpha in the absence of thyroid hormone, and the results of antibody-induced supershift experiments indicate that binding to this element may result in a different conformation as compared with binding to a typical DR4T3RE (40). These results suggest that different conformational changes may be involved in determining whether TR4 would function as a positive or negative regulatory factor.

As previous studies indicated that RIP140 could interact with VDR, T3R, and TR4 (18, 41), we were interested in determining if RIP140 could differentially affect TR4-mediated gene induction and repression. Using the GST pull-down assay, we demonstrated that TR4 could interact in vitro with RIP140. We then proved that RIP140 could also enhance the trans-repressive effect of TR4 on the vitamin D3-signaling pathway. In contrast, RIP140 has only marginal effects on the vitamin D3-induced VDRE-CAT activity, although it might interact with VDR (18). On the other hand, RIP140 repressed the transactivation mediated by both TR4 (Fig. 7B, lane 5 versus 6) and liganded T3R (Fig. 7B, lane 7 versus 10) on DR4T3RE-CAT. However, RIP140 had only very marginal repressive effects on DR4T3RE-CAT once we overexpressed TR4 and liganded T3R simultaneously. A reasonable explanation for the effect of RIP140 on DR4T3RE-CAT is that TR4 and liganded T3R may both interact with RIP140 and sequester RIP140 blocking the repressive effect of RIP140 on DR4-T3RE-CAT (Fig. 8). Because RIP140 can repress both pathways mediated by TR4, it might not be recruited differently by TR4 when it is bound to DR4T3RE or DR3VDRE. However, RIP140 did show differential effects in the regulation of DR4T3RE-CAT and DR3VDRE-CAT when receptors, TR4 and VDR versus TR4 and T3R, are co-transfected. These data support the role of the protein environment surrounding the DNA as a key factor in determining gene regulation. Therefore, the dynamic interaction between the receptors and cofactors in response to their target DNA may determine the specificity of gene regulation.


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Fig. 8.   The molecular model for the action of TR4 and RIP140 on DR3-VDRE (A) and DR4-T3RE (B).

Although the functional reporter assay presented here was carried out in vitro, the expression patterns of TR4 in the vitamin D target organs support the potential role of TR4 in regulation of the in vivo vitamin D pathway in the endocrine system. Vitamin D and its receptor play essential roles in the regulation of calcium homeostasis and bone formation. Bone, kidney, and intestine are three major targets for such action. In addition, vitamin D is involved in the regulation of cell proliferation, differentiation, and the immune response. At present, the nonclassic targets reported include lung (35) and hair follicles (36). It has been demonstrated that high levels of VDR transcripts are present in bone, kidney, and all identified nonclassic target organs. Throughout the entire process of bone differentiation, VDR transcripts were detected in osteoblasts and osteocytes of both normal and actively remodeling bone tissue (42). In the kidney, VDR and P450cc24 were co-localized in the distal renal tubules, the collecting ducts, the proximal tubes, and the parietal epithelial cells of the glomerulus (37, 43). Our data show TR4 transcripts in perichondrium, kidney, hair follicles, and lung, suggesting TR4 and VDR may be co-localized during the development of these tissues. Among the vitamin D target genes, P450cc24 has been shown to respond to vitamin D treatment in the kidney and intestine. The distribution of this enzyme in the kidney is exclusive to the proximal tubules (38), whereas its location in the intestine was detected in the lower part of the villi and columnar epithelium of the crypt. Our in situ data showed TR4 expression in renal tubules and intestinal villi, indicating TR4 and P450cc24 may be co-expressed in these places, thus providing physiological significance for the interaction between TR4 and VDR on the regulation of the P450cc24 gene. Moreover, immunostaining for VDR revealed that VDR was apparent in rat fetuses as early as embryonic days 9 and 11. In developing limbs at embryonic days 13-15, VDR epitopes were present in the skin, the cytoplasm of condensing mesenchymal cells, and the chondrocytes (44). Whether TR4 could also interact with VDR in regulating other vitamin D target genes, such as osteocalcin and alkaline phosphatase, is an intriguing question to ask.

On the other hand, vitamin D3 decreases VDR/VDR homodimer formation and enhances the formation of RXR/VDR heterodimers. Thus, it potentiates RXR/VDR action by enhancement of the RXR/VDR binding affinity to VDREs and activation of target gene expression (33, 34). We then compared the binding affinity of TR4 and RXR/VDR to DR3VDRE and found that TR4 bound to DR3VDRE with a 5.5-fold higher affinity than the RXR/VDR heterodimer. These binding affinity data suggest that TR4 may be able to compete with RXR/VDR to bind to DR3VDRE and in this way, exert its repressive effect. The ratio of functional TR4·DR3VDRE complex to RXR/VDR·DR3VDRE might also be important in determining target gene action. Additionally, the failure of the repression effect observed when PCMX-TR4 was replaced with the chimeric receptor PCMX-4A4 in a reporter gene assay further supports the idea that DNA binding is essential for TR4 to exert its proper function.

The responsiveness of genes to a steroid hormone receptor is principally mediated by functional interactions between DNA-bound receptors and components of the transcription initiation machinery. In this paper we demonstrated that TR4 represses the P450cc24 gene activation induced by vitamin D3 via in vitro and in vivo evidence. Both DNA binding and proper protein-protein interactions may be the key factors in determining the specific function of TR4. This study may lead to an understanding of the role of DNA binding in altering the conformation of TR4 and allowing different protein interactions resulting in a complex that is capable of mediating differential regulation.

    ACKNOWLEDGEMENTS

We thank Dr. Y. Kato for providing the P450cc24-CAT plasmids.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK47258, CA68568, and CA71570.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 Contributed equally to this work.

§ To whom correspondence should be addressed. Fax: 716-756-4133; E-mail: chang{at}pathology.rochester.edu.

    ABBREVIATIONS

The abbreviations used are: RXR, retinoid X receptor; TR4, testicular orphan receptor; HRE, hormone response elements; VDRE, vitamin D response element; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; CHO, Chinese hamster ovary; mAb, monoclonal antibody; CAT, chloramphenicol acetyltransferase; DR, direct repeat; VDR, vitamin D3 receptor; T3RE, thyroid response element; RIP140, receptor interacting protein 140; T3R, thyroid receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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