TRβ1-mediated transcriptional activity is cell type dependent.
We hypothesized that the diverse effects of T3 could be mediated by the interaction of TR with a network of tissue-dependent factors and that the diverse effects reflect the cellular context. To test this hypothesis, we evaluated the transactivational responses of TRβ1 in six cultured cell lines which were derived from different tissues. They were monkey kidney cells, rat pituitary growth hormone-producing GC cells, human cervical carcinoma HeLa cells, human epidermoid carcinoma A431 cells, human breast cancer MCF-7 cells, and human colon carcinoma RKO cells. Based on the T3-independent TRβ1-mediated transcriptional activities, these six cell lines can be grouped into three classes. CV1 and GC cells belonged to the first class in that TRβ1 was a strong repressor in the absence of T3; as shown in Fig. A, about 90% of the basal transcriptional activity was repressed (bars 3 and 7 versus bars 1 and 5, respectively). HeLa, A431, and MCF-7 cells belonged to the second class in that TRβ1 had no effect on basal transcriptional activity in the absence of T3 (Fig. B, bars 3, 7, and 11 versus bars 1, 5, and 9, respectively). RKO cells belonged to the third class, in which strong T3-independent transactivation was seen (Fig. C, bar 3 versus bar 1).
FIG. 1 Cell type-dependent transcriptional activity of TRβ1. Cells (4 × 105 to 8 × 105/60-mm dish) were plated 24 h before transfection in DMEM containing 10% fetal bovine serum. The cells were transfected with the appropriate (more ...)
In all these cells, however, the addition of T3 led to transcriptional activation (bar 4 versus bar 3 and bar 8 versus bar 7 in Fig. A; bar 4 versus bar 3, bar 8 versus bar 7, and bar 12 versus bar 11 in Fig. B; and bar 4 versus bar 3 in Fig. C). Fold T3 activation, however, varied with cell type in that the highest activation was seen in CV1 cells (~40-fold) and the lowest was detected in RKO cells (Fig. ). These results indicate that the transcriptional activity of TRβ1 was cell type dependent.
Identification of Ear-2 as a TR-interacting protein and its expression in tissues.
The dramatic differences in the T3-independent transcriptional activities between RKO cells and other cell lines prompted us to search for TR-interacting proteins in RKO cells using a yeast two-hybrid system. Intact TRβ1 was fused to the GAL4 DNA binding domain to be used as bait to search the cDNA library prepared from RKO cells for interacting proteins. After repeated screening, three positive clones were identified. One of them was identified as the orphan nuclear receptor, Ear-2, by DNA sequencing. Human Ear-2 was cloned from human embryo fibroblasts by Miyajima et al. (23
). It shares 59% sequence identity with TRβ1 in the DNA binding domain and 26 and 35% identities in regions II and III of the hormone binding domain, respectively (32
). However, the Ear-2 gene has not been very well studied, and its functions are yet to be elucidated.
We first examined Ear-2 expression in human tissues and cultured cells by Northern blot analysis. In human tissues, Ear-2 was expressed as a single mRNA species of 2.5 kb (Fig. A). It was, however, differentially expressed in the heart, placenta, liver, skeletal muscle, kidney, and pancreas, with the most abundant message being found in the heart, liver, and pancreas. It was not detectable in the brain and lung. Using the same human tissue blot (Fig. B), we found that TRβ1 mRNA was expressed in the heart, brain, placenta, liver, skeletal muscle, kidney, and pancreas. A comparison of the expression patterns in Fig. A and B indicates that except for the brain, Ear-2 and TRβ were coexpressed in the same tissues.
FIG. 2 Expression of Ear-2 in tissues and cultured cells, as determined by Northern blot analysis. (A and B) An RNA human tissue blot was purchased from Clontech and probed with human Ear-2 cDNA (A) and TRβ1 cDNA (B) labeled with [32P]dCTP. (more ...)
We also examined the expression of Ear-2 mRNA in several cultured cell lines (Fig. C). Lanes 1 and 2 of Fig. C show that in addition to the 2.5-kb mRNA, a second mRNA species, of 4.8 kb, was detected in human hepatocellular carcinoma SK-Hep-1 and RKO cells. In CV1 cells, however, only a single mRNA of the same size as that seen in the tissues was detected (Fig. C, lane 4). Ear-2 was not expressed in GC cells (Fig. C, lane 3). These results indicate that additional splicing products of mRNA were present in SK-Hep-1 and RKO cells. However, the nature of the protein encoded is unknown.
The hormone binding domain of TRβ1 is the binding site for Ear-2.
Ear-2 was shown to interact with intact TRβ1 by the yeast two-hybrid system. To identify the domain of TRβ1 to which Ear-2 bound, we used a GST pull-down assay. 35S-labeled Ear-2 was prepared by in vitro transcription-translation. Analysis by SDS-PAGE showed that it had an apparent molecular mass of 44 kDa (Fig. A, lane 2). For comparison, lane 1 of Fig. A shows the size of TRβ1 prepared by in vitro transcription-translation. Increasing concentrations of 35S-labeled Ear-2 were incubated with a constant amount of GST-TRβ1 or GST alone. As shown in Fig. B, 35S-labeled Ear-2 bound to GST-TRβ1 in a concentration-dependent manner (lanes 4, 5, and 6), whereas no 35S-labeled Ear-2 at the corresponding concentrations bound to the control (GST) (lanes 1, 2, and 3). The amounts of GST used in lanes 1, 2, and 3 corresponded to those used in lanes 4, 5, and 6, respectively. Lane 7 shows the input of 35S-labeled Ear-2 for comparison. These results provided additional evidence that Ear-2 physically interacted with TRβ1.
FIG. 3 The C-terminal region of TRβ1 is critical for the interaction of TRβ1 with Ear-2. (A) Molecular size of Ear-2. Ear-2 (lane 2) and TRβ1 (lane 1) were prepared by in vitro transcription-translation in the presence of [35 (more ...)
To understand whether the interaction of Ear-2 with TRβ1 was T3 dependent, we carried out a GST pull-down assay in which Ear-2 was fused to GST (Fig. C). Consistent with the results shown in Fig. B, we found that 35S-labeled TRβ1 bound to GST–Ear-2 (Fig. C, lanes 3 and 4) but not to the control (GST) (lanes 1 and 2). The binding, however, was T3 independent, because no differences in the amounts of 35S-labeled TRβ1 bound were detected with T3 or without T3 (Fig. C, lane 3 versus lane 4).
TRβ1 consists of the amino-terminal A/B domain, the DNA binding domain (domain C), and the hormone binding domains (domains D and E). To identify the domain to which Ear-2 bound, we used truncated TRβ1 in which domain A/B, domains A/B and C, domains A/B, C, and D, and domains A/B, C, and D and amino acids 420 to 461 were deleted (16
). We prepared various 35
S-labeled truncated TRβ1 species and determined their binding to GST–Ear-2 (Fig. D). We found that domains C, D, and E (Fig. D, lane 6), domains D and E (lane 9), and domain E (lane 12) bound to GST–Ear-2 but not to the control (GST) (lanes 5, 8, and 11, respectively), indicating that domain E of TRβ1 was the binding site for Ear-2. Lanes 4, 7, and 10 of Fig. D show 1/10 the inputs of 35
S-labeled domains C, D, and E, domains D and E, and domain E used in the binding experiments, respectively.
We next examined whether the binding site of TRβ1 for Ear-2 was localized at the C-terminal region of domain E. We therefore further truncated domain E of TRβ1 by deleting all of helix 12 and most of helix 11 (amino acids 420 to 461) (33
). Lane 15 of Fig. D shows that upon deletion of this region, no binding to GST–Ear-2 was detected, suggesting the important role of amino acids 420 to 461 in the binding of Ear-2 to the hormone binding domain of TRβ1.
Development of anti–Ear-2 antibodies and detection of the interaction of Ear-2 with TRβ1 in cells by coimmunoprecipitation.
We prepared antibodies to human Ear-2 with the use of the DNA immunization technique, in which an antigen-specific immune response is elicited by injection of nonreplicative transcription units (30
). We adopted the protocol described by Chowdhury et al. (5
), using the cytomegalovirus promoter to drive the expression in mice of the human Ear-2 protein (pCDM-Ear-2), which is processed by the immune system to elicit the immune response (8
). All five mice injected with pCDM-Ear-2 developed anti–Ear-2 antibodies after 6 to 8 weeks. A representative result from the screening of the mouse antisera is shown in Fig. A. Lane 1 shows 35
S-labeled Ear-2 prepared by in vitro transcription-translation as a marker. Lane 2 shows that 35
S-labeled Ear-2 was immunoprecipitated by anti–Ear-2 sera designated PC-9. Lane 3 shows that only a minor background signal was detected when the preimmune sera from the same mouse were used in the immunoprecipitation. These results demonstrate that the injection of mice with pCDM-Ear-2 led to the successful production of anti–human Ear-2 antibodies.
FIG. 4 TRβ1 interacts with Ear-2 in cells. (A) Preparation of anti-Ear-2 antibody PC-9. PC-9 was prepared by injection of mice with expression plasmid pCDM-Ear-2 as described in Materials and Methods. Antisera (1 μl; lane 2) and preimmune sera (more ...)
Using the yeast two-hybrid system and the GST pull-down assay, we have already shown that Ear-2 physically interacts with TRβ1 (see above). To demonstrate their physical interaction in cells, we expressed both proteins in CV1 cells by transfection with their expression plasmids. After metabolically labeling the cells with 35
S-labeled methionine, we immunoprecipitated Ear-2 with PC-9. As shown in Fig. B, lane 3, three bands with molecular masses of 55, 44, and 35 kDa were immunoprecipitated with PC-9. The immunoprecipitation of these three proteins was specific because, as shown in Fig. B, lane 4, when an unrelated antibody (MOPC) was used, no immunoprecipitable bands were detected. The 55-kDa band was TRβ1, because when cells were transfected only with a TRβ1 expression plasmid and subsequently immunoprecipitated with an anti-TRβ1 MAb (C4) (36
S-labeled TRβ1 was detected (Fig. B, lane 2). Lane 1 of Fig. B shows the control, in which in vitro-translated 35
S-labeled TRβ1 was directly loaded as a marker (17
). The 44-kDa band observed in Fig. B, lane 3, represented Ear-2, because it had the same molecular mass as the control, in which in vitro-translated 35
S-labeled Ear-2 was similarly immunoprecipitated with PC-9 (lane 5). However, the nature of the 35-kDa band was unclear. It could represent a degradation product of Ear-2. As shown in lane 6 of Fig. B, the same 44- and 35-kDa proteins were observed when the cells were transfected only with an Ear-2 expression plasmid and immunoprecipitated with PC-9. The two proteins immunoprecipitated in lane 6 of Fig. B were specific because, when an unrelated antibody (MOPC) was used (lane 7), no such proteins were immunoprecipitated. These results indicate that TRβ1 was coimmunoprecipitated with Ear-2 and thus provide additional evidence to demonstrate the interaction of these two receptor proteins in cells.
The binding of TRβ1 to TRE is inhibited by Ear-2.
To understand the functional consequences of the physical interaction between TRβ1 and Ear-2, we first evaluated whether the binding of TRβ1 to TRE was affected by Ear-2. We carried out an EMSA with constant amounts of Lys-TRE and TRβ1 in the presence of increasing concentrations of Ear-2. Lane 1 of Fig. A shows the control, in which unprogrammed lysates were used. No signals were detected. Lane 2 of Fig. A shows the binding of TRβ1 to Lys-TRE in the absence of Ear-2. In the presence of Ear-2, however, the binding of TRβ1 to Lys-TRE was reduced (Fig. A, lane 3). The extent of reduction was Ear-2 dependent, because in the presence of increasing concentrations of Ear-2, the intensities of Lys-TRE-bound TRβ1 were gradually decreased (band intensity in Fig. A lanes: 3 > 5 > 7 > 9). Furthermore, the binding of TRβ1 to Lys-TRE was completely inhibited when the ratio of Ear-2 to TRβ1 reached 12 (Fig. A, lane 11).
FIG. 5 The binding of TRβ1 or Ear-2 to TRE is modulated by TRβ1 or Ear-2. 35S-labeled Lys-TRE, TRβ1, and Ear-2 receptors were prepared as described in Materials and Methods. (A) A constant amount of 32P-labeled Lys-TRE was incubated with (more ...)
Interestingly, Ear-2 was found to bind to Lys-TRE (Fig. A, lane 4). However, in contrast to the effect of Ear-2 on the binding of TRβ1 to Lys-TRE, the binding of Ear-2 to Lys-TRE was enhanced by TRβ1. Equal amounts of Ear-2 were present in lane pairs 3-4, 5-6, 7-8, 9-10, and 11-12 of Fig. A. However, the TRE-bound Ear-2 band was stronger when TRβ1 was present (intensity of Ear-2 band in Fig. A lanes: 3 > 4, 5 > 6, 7 > 8, 9 > 10). Ear-2 was also found to bind to Pal-TRE and DR4-TRE, and the binding of TRβ1 to these TRE was inhibited by Ear-2 (data not shown). These results indicate that the physical interaction between Ear-2 and TRβ1 affected their binding to DNA. These data further indicate that Ear-2 and TRβ1 competed for binding to TRE.
That the slower-mobility bands shown in Fig. A were TRE-bound Ear-2 was further confirmed by supershift experiments (Fig. B). Lane 2 of Fig. B shows Lys-bound TRβ1, which was supershifted by anti-TRβ1 MAb C4 (lane 3). The binding of Ear-2 to Lys-TRE (Fig. B, lane 4), however, was blocked by anti–Ear-2 sera designated PC-9 (compare lane 5 with lane 4). Lane 6 of Fig. B shows that both Ear-2 and TRβ1 bound to Lys-TRE. In the presence of MAb C4, Lys-bound TRβ1 was supershifted, whereas Ear-2 was unaffected (Fig. B, compare lane 7 with lane 6). When PC-9 was present, consistent with the results shown in Fig. B, lane 5, the binding of Ear-2 was blocked, whereas no effect on the binding of TRβ1 was detected (compare lane 8 with lane 6). Lane 9 of Fig. B is a control used to indicate that the presence of an unrelated antibody (MOPC) did not affect the gel mobility of Lys-bound Ear-2 or TRβ1 (compare lane 9 with lane 6). These results clearly indicate that the slower-mobility bands shown in Fig. A were Lys-bound Ear-2.
Whether the binding of Ear-2 to TRβ1 affected the formation of TRβ1-retinoid X receptor (RXR) heterodimers is addressed in the experiments shown in Fig. C. Lanes 2 and 3 are the controls used to show that TRβ1 bound to Lys-TRE as a homodimer and as a heterodimer with RXR subtype β (RXRβ), respectively. Lanes 5 and 6 show Lys-bound Ear-2 in the absence and presence of RXRβ, respectively, demonstrating that Ear-2 did not form a heterodimer with RXRβ. Lane 7 shows that in the presence of both Ear-2 and TRβ1, consistent with the results shown in Fig. A, the binding of TRβ1 to Lys was reduced (cf. lane 2) and the binding of Ear-2 to Lys was intensified (cf. lanes 5 and 6). However, whether the binding of TRβ1 with RXRβ as a heterodimer was also inhibited by Ear-2 could not be discerned in lane 8, because TRβ1-RXRβ migrated in a position similar to that of Lys-bound Ear-2 (compare the Lys-bound Ear-2 migration position in lane 5 with the position of the TRβ1-RXRβ heterodimer in lane 3). We therefore supershifted the TRβ1-RXRβ heterodimer with MAb C4 to a more retarded position, as shown in lane 9. In lane 9, Lys-bound Ear-2 was not affected by MAb C4; however, the intensity of the supershifted TRβ1-RXRβ complex was clearly lower than that in lane 4, which was supershifted TRβ1-RXRβ complex in the absence of Ear-2. These results indicate that the binding of TRβ1 to Lys-TRE as a TRβ1-RXRβ heterodimer was inhibited by Ear-2. Lane 10 shows the negative control, in which a control antibody (MOPC) was used and no supershifted TRβ1-RXRβ complex was detected. Taken together, these results indicate that the binding of TRβ1 to Lys-TRE both as a homodimer and as a heterodimer with RXRβ was inhibited by its physical interaction with Ear-2. Similar results were observed when the TRE were DR4 and Pal (data not shown).
Hormone-dependent differential repression of TRβ1 transcriptional activity by Ear-2.
The above results prompted us to further evaluate the effect of Ear-2 on the transcriptional activity of TRβ1. Figure A shows that in RKO cells, both T3-independent (lanes 11 to 16 versus lane 3) and T3-dependent (lanes 17 to 22 versus lane 4) transcriptional activities were repressed in the presence of increasing concentrations of Ear-2. Lanes 5 to 10 of Fig. A were controls used to indicate that Ear-2 could not mediate transcription via TRE. Quantitation of the extent of the repression caused by Ear-2 shows that T3-independent activation was more sensitive to the repression effect of Ear-2 than T3-dependent activation at Ear-2/TRβ1 ratios of 0.125 to 0.5 (Fig. B). At higher ratios, i.e., 1 to 4, both transcriptional activities were completely repressed by Ear-2 (Fig. A and B).
FIG. 6 The differential transcriptional repression by Ear-2 is cell type and T3 dependent. (A) RKO cells were transfected with the CAT reporter plasmid (pTK28m; 0.2 μg) and the TRβ1 expression plasmid (pCDM-TRβ1; 0.2 μg) in the (more ...)
To assess whether the basal repression effect of TRβ1 was also affected by Ear-2, we used CV1 cells. In these experiments, the repression of basal activity by unliganded TRβ1 was ~70% (Fig. C, bar 3 versus bar 1). Figure C shows not only that T3-dependent activation was repressed by Ear-2 (lanes 7 and 8 versus lane 4) but also that the basal repression effect of TRβ1 was further affected (lanes 5 and 6 versus lane 3). These results indicate that Ear-2 was a potent repressor of TR-mediated transcriptional activity.
Ear-2 represses the promoter activity of the S14 T3-targeted gene.
To establish that Ear-2 plays a role in TR-mediated signaling pathways, we examined whether Ear-2 modulated the promoter activity of a T3-targeted gene. We chose to use the S14 gene because it is an important T3-targeted gene in the lipid metabolism pathway of liver and its TRE have been extensively characterized (27
). Using a promoter-luciferase reporter system, we found that the promoter activity of S14 was also repressed by Ear-2 (Fig. ). Bars 2 and 4 of Fig. show the extent of T3-dependent activation mediated by the endogenous and transfected TR in a primary liver culture, respectively. These activities were repressed by Ear-2 (Fig. , bar 6 versus bar 2 and bar 8 versus bar 4). Consistent with the results obtained with CV1 cells, the extent of the basal repression effect of TR was also further enhanced by Ear-2 (Fig. , bar 5 versus bar 1 and bar 7 versus bar 3). These results indicate that Ear-2 could play an important role in TR-mediated signaling pathways.
FIG. 7 Ear-2 suppresses basal and T3-induced responses of the human S14 promoter. The data are from a representative experiment that was repeated three times. Each bar is the mean + standard deviation of three plates with the indicated treatment. Bars (more ...) Mutation of the DNA binding domain of Ear-2 leads to a loss of TRE binding.
The above results indicated that Ear-2 repressed TR-mediated transcriptional activity. Since Ear-2 also bound to TRE (Fig. ), the repression could be the result of the competition of Ear-2 with TRβ1 for binding to TRE. To understand if this was the case, we mutated the DNA binding domain (domain C) of Ear-2 from 73CysGluGlyCysLysSer80 (wild-type Ear-2) to 73CysGlySerCysLysVal80 (Ear-2 mutant). This mutant (1 and 2 μl of lysates were loaded in lanes 5 and 6 of Fig. A, respectively) had the same molecular mass as wild-type Ear-2 (1 and 2 μl of lysates were loaded in lanes 3 and 4 of Fig. A, respectively), as indicated by the proteins prepared with an in vitro transcription-translation system. This mutant, however, had lost the ability to bind to Lys-TRE in either the absence or the presence of RXRβ (lanes 6 and 7 versus lanes 4 and 5, respectively, of Fig. B). Lanes 2 and 3 of Fig. B were controls used to indicate that TRβ1 bound to Lys-TRE as a homodimer and as a heterodimer with RXRβ, respectively. Lanes 4 and 5 of Fig. B were also controls used to show that Ear-2 bound to Lys-TRE but did not form a heterodimer with RXRβ. The lack of binding of the mutant to Lys-TRE was not a result of smaller amounts of the Ear-2 mutant being used in the binding experiments. Equal amounts of Ear-2 and Ear-2 mutant proteins were used in the experiments.
FIG. 8 Lack of binding of the Ear-2 mutant to TRE and its partial repression of TRβ1-mediated transcriptional activity. (A) The Ear-2 mutant has the same molecular size as wild-type Ear-2. 35S-labeled TRβ1 (lanes 1 and 2), Ear-2 (lanes 3 and (more ...)
We further evaluated whether this Ear-2 mutant, which had lost DNA binding activity, could act as a repressor in CV1 cells. We transfected a constant amount of TRβ1 expression plasmid in the presence of increasing concentrations of wild-type Ear-2 or mutant Ear-2 plasmids. We concurrently determined both the CAT activities and the levels of expression of wild-type Ear-2 and mutant Ear-2 proteins by Western blotting. Figure C shows the levels of expression of wild-type Ear-2 and mutant Ear-2 proteins in cellular lysates, as determined by Western blotting with antibody PC-9. Lane 1 shows Ear-2 from the in vitro transcription-translation as a marker. Lanes 2 and 3 show the protein expression levels from the transfection of 1 and 2 μg of Ear-2 expression plasmid, respectively. Lanes 4 and 5 show the protein expression levels from the transfection of 1 and 2 μg of Ear-2 mutant expression plasmid, respectively. Quantitative comparison of the intensities of the bands indicated that relative ratios for the protein expression levels of the Ear-2 bands in lanes 2, 3, 4, and 5 were ~1, 2, 0.5, and 1. Since the expression of both proteins was driven by the same CMV promoter, it is unclear why the expression of the Ear-2 mutant was lower than that of wild-type Ear-2 when the same amounts of plasmids were transfected (e.g., lane 2 versus lane 4 and lane 3 versus lane 5). The lower expression was not due to the lower reactivity of PC-9 with the Ear-2 mutant because, when in vitro-translated receptors were used, PC-9 reacted with wild-type Ear-2 and mutant Ear-2 with similar avidities (data not shown).
Figure D compares the repression effect of wild-type Ear-2 and mutant Ear-2 on TRβ1-mediated transcriptional activity. Bars 5 and 6 of Fig. D show that when 1 and 2 μg of wild-type Ear-2 expression plasmid were cotransfected with a constant concentration of TRβ1 expression plasmid, 92 and 95% of T3-dependent transcriptional activities were repressed, respectively. When 1 and 2 μg of mutant Ear-2 expression plasmid were cotransfected with TRβ1 (Fig. D, bars 7 and 8), ~57 and ~80% of the transcriptional activities of TRβ1 were repressed, respectively. The repression effect of wild-type Ear-2 in bar 5 of Fig. D and that of mutant Ear-2 in bar 8 were mediated by similar levels of receptor proteins (see lanes 2 and 5 of Fig. C). Therefore, the extents of repression mediated by wild-type Ear-2 and mutant Ear-2 could be compared. Because mutant Ear-2 had lost the ability to bind to TRE, the repression seen for mutant Ear-2 was unlikely to be mediated by competition with TRβ1 for binding to TRE. These results suggest that the repression of the transcriptional activity of TRβ1 by wild-type Ear-2 was a combination of a protein-protein interaction and competition for binding to TRE.
The repression of the T3-dependent transcriptional activity of TRβ1 by Ear-2 was reversed by SRC-1 in CV1 cells.
SRC-1 is a coactivator for many members of the receptor superfamily, including TR (9
). It interacts with the activation function 2 (AF2) region of the receptors via its LXXLL motifs (9
). We transfected SRC-1 into CV1 cells to determine if SRC-1 could lead to a reversal of the repression caused by Ear-2 (Fig. ). Bar 5 of Fig. was a control used to indicate that SRC-1 enhanced T3-dependent TRβ1-mediated transcriptional activity. Under the experimental conditions, the enhancement was about twofold (compare bars 5 and 4 of Fig. ). Compared to bar 4, bars 7, 15, and 23 of Fig. show the Ear-2 concentration-dependent repression of TRβ1-mediated transcription. When a small amount of Ear-2 expression plasmid (Ear-2/TRβ1 ratio, 0.125) was transfected, SRC-1 not only derepressed the repression effect of Ear-2 but also enhanced TRβ1-mediated transcriptional activity (Fig. , bar 9 versus bars 7 and 5). When more Ear-2 expression plasmid (Ear-2/TRβ1 ratio, 0.25 or 0.5) was transfected into cells, SRC-1 was sufficient to compete with Ear-2 for TRβ1 to reverse the repression (Fig. , bar 17 versus bar 15 and bar 25 versus bar 23) but not sufficient for additional activation (bars 17 and 25 versus bar 5). In addition, when an Ear-2/TRβ1 ratio of >2 was used, SRC-1 was not able to reverse the repression effect of Ear-2 (data not shown).
FIG. 9 Reversal of the Ear-2 repression of the T3-dependent transcriptional activity of TRβ1 by SRC-1 in CV1 cells. CV1 cells were cotransfected with the CAT reporter plasmid (pTK28m; 0.2 μg), the TRβ1 expression plasmid (pCDM-TRβ1; (more ...) The repression of the T3-independent transcriptional activity of TRβ1 by Ear-2 was reversed by SRC-1 in RKO cells.
In RKO cells, both T3-dependent and T3-independent transcriptional activities were repressed by Ear-2 (Fig. A and B). We first examined whether the Ear-2 mutant could repress both transcriptional activities in these cells. Figure A shows that, as in CV1 cells, the Ear-2 mutant was able to repress both T3-independent (bar 7 versus bar 3) and T3-dependent (bar 8 versus bar 4) transcriptional activities. Figure B shows that transfection of equal amounts of expression plasmids for wild-type Ear-2 and mutant Ear-2 in RKO cells led to the expression of the same levels of Ear-2 proteins (duplicates in lanes 2 and 3 versus lanes 4 and 5 of Fig. B). Unlike the results obtained for CV1 cells, no 35-kDa protein band was detected in RKO cells (Fig. C). The reasons for such differences are not clear. Thus, the extents of repression shown in bars 5 and 6 and in bars 7 and 8 of Fig. C were mediated by the same levels of wild-type Ear-2 and mutant Ear-2, respectively. The finding that the Ear-2 mutant retained the ability to repress TRβ1-mediated transcriptional activity suggested that competition for DNA binding was not the only mechanism for the repression effect of Ear-2 in RKO cells.
FIG. 10 Repression of the T3-independent and T3-dependent transcriptional activities of TRβ1 by wild-type Ear-2 and mutant Ear-2 and its reversal by SRC-1 in RKO cells. (A) Repression by wild-type Ear-2 and mutant Ear-2. RKO cells (8 × 105 cells/60 (more ...)
We also ascertained if the repression effect of Ear-2 in RKO cells could be reversed by SRC-1. SRC-1 enhanced both T3-independent and T3-dependent transcriptional activities 2.6- and 2.3-fold, respectively (compare bars 5 and 3 and bars 6 and 4 in Fig. C). In the presence of Ear-2 (plasmid Ear-2/TRβ1 ratio, 2), both T3-independent and T3-dependent transcriptional activities were completely repressed (Fig. C, bar 7 versus bar 3 and bar 8 versus bar 4). In the presence of SRC-1, ~60 and ~50% of T3-independent (bar 9 versus bar 3) and T3-dependent (bar 10 versus bar 4) transcriptional activities were restored, respectively. These results indicate that SRC-1 was able to reverse the repression mediated by Ear-2 in RKO cells.
The reversal of the Ear-2-mediated repression of the transcriptional activity of TRβ1 by SRC-1 could be due to competition of TRβ1 with SRC-1 for binding to Ear-2. To test this possibility, we carried out a GST pull-down assay. As shown in Fig. , lane 3, 35S-labeled SRC-1 bound to GST–Ear-2, whereas no 35S-labeled SRC-1 was detected when GST alone was used (lane 2). Lane 1 of Fig. shows the input of SRC-1 as a marker. These results indicated that Ear-2 physically interacted with SRC-1. This interaction raises the possibility that the reversal of the Ear-2 repression effect by SRC-1 could be due to the recruitment of Ear-2 by SRC-1, leading to the reduction of TRβ1–Ear-2 complexes.
FIG. 11 Ear-2 binds to SRC-1, GR, and ER. E. coli-expressed GST (4 μg; lanes 2, 5, and 8) or GST–Ear-2 (4 μg; lanes 3, 6, and 9) noncovalently bound to GSH-Sepharose beads was incubated with 10 μl of in vitro-translated 35S-labeled (more ...) Ear-2 is also a negative regulator for the transcriptional activities of GR and ER.
To address the question of whether Ear-2 also functions as a negative coregulator for other members of the steroid hormone receptors, we first evaluated whether Ear-2 physically interacted with GR or ER in a GST pull-down assay. Lanes 6 and 9 of Fig. show that GR and ER, respectively, bound to GST–Ear-2. Lanes 5 and 8 of Fig. were the corresponding negative controls used to indicate that GR and ER, respectively, did not bind to GST alone. Lanes 4 and 7 of Fig. show the 10% inputs of GR and ER, respectively. These results indicated that Ear-2 also physically interacted with other members of the steroid hormone receptors.
We further evaluated whether Ear-2 functions as a negative regulator in GR- or ER-mediated transactivation activity (Fig. ). Like that of TRβ1, the Dex-dependent transactivation activity mediated by GR was repressed by Ear-2 (Fig. A, lane 6 versus lane 4). At a plasmid Ear-2/GR ratio of 1, the extent of repression of the Dex-dependent transactivation activity was 85%. Lanes 1 and 2 of Fig. A were the controls used to show the basal transcriptional activities in the absence and presence of Dex, respectively. Ear-2 also was a negative regulator for the transcriptional activity mediated by ER. A comparison of bars 6 and 4 of Fig. B indicates that the estrogen (E2)-dependent transactivation of ER was repressed 85% by Ear-2. Therefore, Ear-2 is a negative regulator not only for the transactivation activity of TR but also for that of other members of the steroid hormone receptors.
FIG. 12 Ear-2 represses the hormone-dependent transcriptional activity of GR and ER. (A) CV1 cells (4 × 105 to 8 × 105/60-mm dish) were cotransfected with the CAT reporter plasmid (pMSG-CAT; 0.2 μg), pCMV-GR (0.2 μg), and pCH110 (more ...)