Requirements for Repression of Retinoid X Receptor by the Oncoprotein P75gag-v-erbA and the Thyroid Hormone Receptors

Gunilla M. Wahlström and Björn Vennström

Department of Cellular and Molecular Biology Laboratory for Developmental Biology Karolinska Institute S-171 77 Stockholm, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The oncogenic counterpart of thyroid hormone receptor-{alpha} (TR{alpha}), denoted P75gag-v-erbA, has served as a paradigm for the ability of TRs to repress basal levels of transcription. We show here that the retinoid X receptor (RXR), when activated by its specific ligand SR11237, is repressed by both the normal TR{alpha} and the P75gag-v-erbA. The repression caused by the two proteins is distinct and dependent on both the cell type and the hormone-response element through which RXR acts. In HeLa cells only TR repressed efficiently through the palindromic 2xIR0 element, whereas the proteins were equally efficient in JEG cells. This demonstrates that proteins distinct in the two cell types mediate the repression. RXR-dependent induction via the natural response element of the cellular retinol-binding protein (CRBPII) gene was likewise (>=50%) repressed by TR, whereas P75gag-v-erbA did not repress during the same conditions. Furthermore, P75gag-v-erbA and its variants v-erbAtd359 (lacking repressing activity on TR) and v-erbAr12 (a highly active repressor of TR) efficiently repressed induction by a hybrid protein consisting of the DNA- binding domain of Gal4 and the ligand-binding region of RXR. The viral proteins did not, however, associate with RXR unless the two partners were allowed to heterodimerize upon binding to a specific response element, such as the 2xIR0 element or that of the CRBPII gene. In conclusion, we suggest that the efficient repression seen with the the 2xIR0 element is due to heterodimerization of TR or the viral oncoproteins with RXR and a concomitant inhibition of binding of the RXR-specific ligand that results in an inability of RXR to attract a cell type-specific cofactor. In addition, the data suggest that the interaction between RXR and P75gag-v-erbA on the CRBPII element is too weak to inhibit RXR from binding a ligand and therefore also to repress.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The retinoid X receptor (RXR) is a heterodimerization partner for many members of the nuclear hormone receptor superfamily. The number of nucleotides in the spacer sequence between two half-sites in a hormone-response element of the direct repeat type (DR), determines when RXR heterodimerizes with either a retinoic acid receptor (RAR), a vitamin D3 receptor or a thyroid hormone receptor (TR) (1). Here, RXR binds to the 5'-half-site (2, 3). RXR also homodimerizes and activates transcription via synthetic DR elements spaced by one nucleotide (4, 5) or via a palindromic element (6). Only one natural promoter gene, the cellular retinol-binding protein type II (CRBPII) (7), has been shown to be induced by homodimeric RXRs as a response to ligand. This gene contains four half-sites oriented as DRs with one nucleotide in each spacer. Chen et al. (8) showed that the RXR receptor bound to this element as a cooperative high-order oligomer. Studies by magnetic resonance spectroscopy have demonstrated that a specific {alpha}-helix located immediately after the second zinc finger in the DNA-binding domain (DBD) of RXR is responsible for homodimeric protein interaction as well as for protein-DNA interaction (9). Furthermore, ligand-dependent homodimerization is abolished by both a 29-amino acid deletion in the C-terminal domain of RXR and by point mutations within this domain (10).

The RAR activates transcription as a response to both the natural all-trans- and the 9-cis-retinoic acid ligands, whereas RXR activates only after binding the latter ligand (11, 12). Recently, the importance of ligand specificity has been investigated, and several synthetic ligands have been shown to function in a very specific manner. Two RXR-specific ligands, SR11217 and SR11237 (13), have been shown to induce RXR homodimer formation as well as to repress T3 induction of a reporter construct containing the DR4 element of the myosin heavy chain gene (14).

Not only the ligand is important for activation and repression of promoter genes. Several cofactors have been found to interact with different nuclear receptors, thereby exerting a major impact on gene regulation. The coactivators have been suggested to bind to the activation domain, AF2, in the C-terminal part of the receptors, whereas the corepressors, N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), preferentially bind to the hinge region (15, 16, 17, 18).

The oncoprotein P75gag-v-erbA is the viral counterpart to TR and contains several point mutations, but it still interacts with corepressors via the hinge region. A transformation-defective mutant of v-erbA, denoted v-erbAtd359, has no transforming capacity, whereas its revertant, v-erbAr12, is more potent than the parental v-erbA gene (19). The loss of oncogenic function in v-erbAtd359 is due to a single point mutation in the hinge region (19), a mutation that also causes loss of interaction with the corepressor SMRT when present in the normal TR (17). The revertant v-erbAr12 contains a reversal of this mutation. Both the v-erbAtd359 and v-erbAr12 proteins contain, in contrast to P75gag-v-erbA, a proline at position 349 in helix 11 in the C-terminal region, an amino acid suggested to be essential for efficient heterodimerization with RXR (20, 21).

We have investigated the capacity of different TR isomers, P75gag-v-erbA and the mutant proteins of v-erbAtd359 and v-erbAr12, to repress RXR-induced transactivation. We show that transactivation induced by the RXR-specific ligand SR11237 via palindromic or natural RXR-specific response elements is efficiently repressed by both TR{alpha} and TRß. Our data thus extend those of Forman et al. (22). Furthermore, a dominant negative effect on RXR-specific activation was observed with P75gag-v-erbA and its variants but only when acting through the palindromic type of element. Of the three oncoproteins, that of v-erbAr12 repressed the most efficiently and that of v-erbAtd359 the least efficiently. This repression was dependent on DNA binding by the oncoproteins; constructs encoding only their ligand-binding domains (LBDs) failed to repress RXR, and they all failed to bind to RXR in the absence of hormone-response elements. Our studies thus highlight important functional differences between the P75gag-v-erbA and TR and therefore suggest a new role for unliganded nuclear receptors in transcriptional repression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TR Interferes with Induction Mediated by RXR Homodimers
Since RXR is important for transactivation by TR due to its role as a heterodimerization partner, we studied whether TRs could also modulate the ability of ligand-bound RXR to transactivate its target genes. The receptors and the response elements used in all the experiments shown are presented in Fig. 1Go.



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Figure 1. Schematic View of Nuclear Hormone Receptors and Response Elements Used

The TR{alpha} proteins p46 and p40 are distinct only in the N-terminal A/B domain. Two chicken TRß isomers (ß0 and ß2), RXR, P75gag-v-erbA, and two transformation-defective mutants are also shown. The P in circles indicate important phosphorylation sites, and P, S, and R indicate the amino acids proline, serine, and arginine located in the LBD of TR and v-erbA. Two differently oriented response elements with the orientation of the half-site indicated by arrows and the spacing in between shown by number, are depicted at the bottom.

 
First, we tested the capacity of the chicken TR{alpha} to repress RXR{alpha}-induced transactivation of reporter plasmids containing different response elements. For this, we transfected JEG cells with plasmids expressing RXR{alpha} and TR{alpha} along with relevant reporter plasmids. Figure 2AGo (left panel) shows that SR11237-induced transactivation via the synthetic palindromic element (2xIR0) was completely inhibited by an equal amount of cotransfected TR{alpha} plasmid. The observation that thyroid hormone (T3) could induce a transactivation similar to that seen when only RXR was active in the cell confirms that the transfection procedure did not adversely affect the transfected cells. In a second experiment we tested the RXR-specific response element found in the CRBPII gene. Figure 2AGo (right panel) shows that SR11237-dependent transactivation was blocked by approximately 50%. To rule out that the repression was due to squelching, titration of TR in cotransfection experiments was done as shown in Fig. 2BGo. Already a small proportion of co-transfected TR (20 ng) gave maximal repression on the 2xIR0 element. The best repression through the element in the CRBPII gene was achieved when the receptors were transfected at a 1:1 ratio and was at most 50% of the maximal RXR-induced activation. The 2xIR0 element can be interpreted to contain an everted repeat since the two single IR0 elements are separated by an eight-nucleotide linker. We therefore tested repression through response elements of the ER6, ER8, and also the DR4 types containing typical T3-responsive sequences. None of these response elements gave any SR11237-dependent transactivation (data not shown).



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Figure 2. Repression by TR of RXR-Induced Transactivation

Two response elements, 2xIR0 and CRBPII, cloned into the pBLCAT2 vector, were tested in cotransfection experiments with RXR in JEG cells. Panel A shows the relative RXR{alpha}-mediated induction (open bars), in the absence or the presence of SR11237 (SR). The striped bars represent the addition of TR{alpha} into the cotransfections, in the presence of SR11237 and/or T3 (SR/T3). The use of increasing amounts (20–640 ng) of cotransfected TR{alpha} plasmid along with 100 ng RXR{alpha} vector is shown in panel B on the elements 2xIR0 (left) and CRBPII (right). Panel C compares the transactivation activity of different TRs in JEG and HeLa cells. As control, the RXR{alpha} receptor was transfected with only an empty reporter (pBLCAT2). The SR11237 ligand was present as indicated (+), and, in addition, T3 was added as indicated by (++). The absence of ligands is indicated by (-). N.D. represents experiments not done.

 
To determine whether different forms of TR contain similar repressing activities on RXR, we co-transfected, into two different cell lines, equal amounts of plasmids expressing RXR and one of the four different chicken TRs. The TRs tested were the chicken TR{alpha} receptors, p40 and p46, as well as the chicken TRß0 and TRß2. Figure 2CGo (left panel) shows that all TRs tested mediated repression of RXR-induced transactivation obtained via a 2xIR0 containing reporter in JEG cells to approximately the same degree, and that addition of T3 activated the TRs. Similar results were seen when HeLa cells were transfected (Fig. 2CGo, right panel). Our data show that all forms of avian TRs can repress RXR, thus extending the data of Forman et al. (22).

Repression of RXR by the P75gag-v-erbA Oncoprotein
P75gag-v-erbA represses ligand-mediated activation of RAR and TR (21, 23, 24, 25). The mechanisms for this have been suggested to be an out-titration of the common heterodimerization partner RXR, resulting in the formation of inactive heterodimers. To determine whether P75gag-v-erbA also could repress activation by the RXR-specific ligand SR11237, we cotransfected equal amounts of plasmids encoding P75gag-v-erbA and RXR into JEG and HeLa cells along with the 2xIR0 reporter gene. The experiments show that P75gag-v-erbA abolished activation by RXR in JEG cells (Fig. 3AGo, left panel). In contrast, only a 50% reduction of SR11237-dependent transactivation was seen in HeLa cells (Fig. 3AGo, right panel). In addition, the ability of P75gag-v-erbA to repress RXR via the CRBPII element was tested. Our data demonstrate that although the viral oncoprotein showed a minor repression of basal transcription when expressed at high levels (5-fold excess), no repression was seen in any instances when RXR was activated by the ligand (Fig. 3BGo). The oncoprotein formed heterodimers with RXR on the CRBPII element, as shown by a gel retardation assay (Fig. 3CGo). Moreover, the complexes formed were supershifted with anti-v-erbA or anti-RXR antibodies, thus verifying that the receptors formed a heterodimer.



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Figure 3. Repression of RXR by the v-erbA, v-erbAtd359, and v-erbAr12 Genes

Panel A shows transfections with vectors expressing RXR along with plasmids encoding v-erbA or the variants v-erbAtd359 or v-erbAr12. The reporter contained the 2xIR0 element, and the SR11237 ligand was added as indicated (+). The transfections were done with either JEG or HeLa cells as indicated. Two representative and independent experiments are shown. Panel B shows transfections in JEG cells done with the CRBPII element as reporter and a plasmid expressing RXR. One- or 5-fold excess of a P75gag-v-erbA expressing plasmid was added (1x, 5x) in two instances. Panel C shows a gel retardation assay, demonstrating binding of the Vaccinia virus-expressed TR, RXR, or P75gag-v-erbA receptors to the CRBPII element. The complexes were supershifted with antibodies directed against either RXR or erbA as indicated. The arrowhead indicates unbound oligonucleotides.

 
The two mutants of v-erbA, v-erbAtd359 and v-erbAr12, have properties distinct from those of the wild-type oncogene. V-erbAtd359 yields a protein that has lost the ability to repress TR and RAR, whereas the v-erbAr12 protein is more effective in this than P75gag-v-erbA. We therefore tested whether these variant proteins retained their respective properties when acting on RXR through the 2xIR0 element. Figure 3AGo (left panel) shows that in JEG cells the v-erbAr12 protein was, as expected, more potent than P75gag-v-erbA (96% and 92% repression, respectively). Surprisingly, the v-erbAtd359 protein also gave a good repression, although it was less efficient than with the other two proteins (80%). To exclude that the transfected mutants expressed different amount of proteins, nuclear extract was made from the transfected cells and compared by Western blotting (data not shown). In HeLa cells the repressing activity of the mutant proteins was similar to that seen with P75gag-v-erbA (Fig. 3AGo, right panel).

Dependence of the DNA-Binding Region for Repression
Previous experiments have shown that repression of TR-mediated activation by P75gag-v-erbA is dependent on elements that allow receptor dimerization to occur (24, 26). Furthermore, heterodimerization of P75gag-v-erbA with RXR was found to be dependent on binding of the receptor proteins to specific response elements (26). We therfore determined whether the repression of RXR is dependent on the DNA-binding region of P75gag-v-erbA. For this, we compared the repressional activity of full-length P75gag-v-erbA with that of a construct that lacks the DBD. JEG cells were transfected with increasing amounts of plasmid expressing the respective viral proteins, a reporter containing the 2xIR0 response element, and an RXR-encoding vector. Figure 4BGo shows that the construct containing only the hinge region and the LBD of P75gag-v-erbA reduced the induction to about 50%, regardless of the fold excess of the viral protein. In contrast, transfection of as little as 20 ng of the P75gag-v-erbA-encoding plasmid reduced the SR11237-induced transactivation by 75%, and a 6-fold (640 ng) excess of P75gag-v-erbA expressing construct abolished all induction (Fig. 4AGo). The data show that the DBD of P75gag-v-erbA is required for efficient repression and, furthermore, indicate that the LBD/hinge region of P75gag-v-erbA weakly interferes with a cofactor for RXR or with the basal transcription machinery.



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Figure 4. Fig. 4. Repression by the Full-Length or the LBD of P75gag-v-erbA

Plasmids (100 ng) expressing full-length (panels A and B) or Gal4-fused RXR protein (panel C) was cotransfected along with increasing concentrations (20–640 ng) of either the full-length (panels A and C) or the LBD of the P75gag-v-erbA protein (panel B). As reporters the 2xIR0 (panels A and B) or Gal4 (panel C) elements were used. Fold repression is shown as percentage of RXR activation.

 
To substantiate that the efficient repression is dependent on response elements that allow heterodimerization, we investigated the capacity of the full-length P75gag-v-erbA to repress the SR11237-induced activation of a hybrid protein consisting of the DBD of Gal4 and the LBD of RXR (denoted Gal4DBD-RXRLBD). JEG cells were transfected as described above, except that a reporter containing five binding sites for the chimeric Gal4 protein was used. Figure 4CGo shows that transfection of equal amounts (100 ng) of plasmids encoding the receptor protein reduced transactivation by only about 65%, and that a 6-fold excess of P75gag-v-erbA led only to a 85% reduction. We conclude that DNA binding by the P75gag-v-erbA is required for efficient repression of the SR11237-dependent induction by RXR.

The poor repression seen could be due to an inability of P75gag-v-erbA to associate with Gal4DBD-RXRLBD in the absence of specific DNA binding (26). Such a deficiency of P75gag-v-erbA has been suggested to be due to the point mutation (P->S) at position 349 in helix 11 of TR (20, 21, 26, 27). The v-erbAtd359 and v-erbAr12 proteins do not contain this mutation. We therefore compared the ability of these viral proteins and of TR to repress the Gal4DBD-RXRLBD protein. Figure 5AGo shows that an equal amount of transfected v-erbAtd359 construct was as inefficient as P75gag-v-erbA and TR in repression, and that the cotransfected v-erbAr12 construct was only slightly more effective.



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Figure 5. Inability of the v-erbA and the Variant v-erbAtd359 and v-erbAr12 Proteins to Associate with RXR in the Absence of Specific DNA Response Elements

Panel A shows SR11237-dependent transactivation from cotransfections with a 5xGal4 reporter and expression constructs containing the Gal4DBD-RXRLBD. In addition, plasmids expressing the TR, v-erbA, or the variant v-erbAtd359 or v-erbAr12 proteins were cotransfected with the Gal4DBD-RXRLBD construct in equal amounts. The experiments was done in the presence or absence of the SR 11237 ligand. Panel B shows the interaction between RXR and P75gag-v-erbA and its variants -/+ T3 as tested by the receptor-dependent two-hybrid method. Again, the RXR LBD was fused to the DBD of Gal4 but then cotransfected with plasmids expressing a full-length P75gag-v-erbA containing an N-terminal VP16 domain, or with similar plasmids expressing VP16-v-erbAr12 or VP16-v-erbAtd359 proteins. VP16- denotes a construct to which no receptor was fused and which therefore shows only background activation. In panel C the same VP16-receptor constructs were allowed to bind to the DR4 element in an in vivo assay. The binding is shown as VP16-mediated CAT activity. The figures show both the results of one representative experiment of a total of four done.

 
The modest repression seen with the proteins could be due to a general repression of transcriptional activity in the cells or to a weak association with the Gal4DBD-RXRLBD that only marginally reduces the ability of the LBD of RXR to bind the ligand SR11237. We therefore tested whether oncoprotein constructs fused to an N-terminal VP16 transactivation domain associate with the Gal4DBD-RXRLBD construct in vivo. Figure 5BGo shows that in contrast to a VP16-TR control, none of the VP16-oncoprotein chimeras gave transactivation, indicating a lack of association with Gal4DBD-RXRLBD. This was independent of the presence of thyroid hormone. Control experiments have shown that a VP16-gag-TR construct is as efficient as VP16-TR in associating with Gal4DBD-RXRLBD (26). Moreover, the VP16 constructs used above expressed comparable amounts of proteins in the transfected cells, as demonstrated by gel retardation assay using cell extracts from the transfected cells (data not shown). In addition, they bind efficiently to DNA in vivo, since a reporter containing the DR4 element gave high transactivation levels when transfected into JEG cells along with the respective expression plasmids (Fig. 5CGo). Taken together, our data suggest that the viral oncoproteins do not associate with RXR unless they are allowed to heterodimerize on a specific response element.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Repression via the 2xIR0 Element
The dependence on RXR by other nuclear hormone receptors for ligand-induced activation or repression of target gene expression has been well studied. Some studies have also addressed how these receptors affect RXR and its specific ligands in transcriptional regulation. For instance, a heterodimerization partner prevents RXR from binding ligand when the dimer complex has bound to certain response elements (4). Moreover, such a heterodimer forms a complex with corepressors (15, 16, 17). In this paper, we show that different TRs repress SR11237-induced homodimeric RXR activation from a synthetic palindrome (2xIR0) and from the natural CRBPII response element. Repression via 2xIR0 was very efficient, and the data suggest that it is due to a heterodimerization between RXR and TR that inhibits RXR from binding its ligand. This would prevent RXR from expressing its transactivating properties e.g. through attraction of coactivators. Also the oncoprotein P75gag-v-erbA repressed RXR-mediated transactivation efficiently in JEG cells via the 2xIR0 element. The mechanism is likely to be the same as that proposed for TR, since P75gag-v-erbA heterodimerizes efficiently with RXR when binding to this type of element (26). The reason for the much less efficient repression in HeLa cells is unclear but may be due to interaction with other cofactors that have distinct affinities for TR and P75gag-v-erbA, respectively, in the two types of cells. Alternatively, the cells contain different coactivators for RXR that exhibit distinct activating functions.

The JEG cells are known to contain detectable amounts of RXR{alpha} but presumably also other endogenous factors. Recently, Teboul and collaborators (28) showed that RXR can form heterodimers with the orphan receptor OR1, and that RXR in such a heterodimer can be activated by ligand through elements of the DR4 type. We have tested whether TR or P75gag-v-erbA can repress activation of such heterodimers: our results obtained by transfection of an OR1-expressing plasmid showed that OR1 did not contribute to SR11237-dependent transactivation of RXR from the 2xIR0 element, and that OR1 did not affect the ability of P75gag-v-erbA to repress RXR (G. Wahlström, unpublished data).

Repression through the CRBPII Element
TR exhibited a modest repression via the the natural RXR element in the CRBPII gene. Our gel retardation data showed that TR forms a heterodimer with RXR on such an element. However, the RXR/TR heterodimer has a relatively low affinity to elements of the DR1 type as compared with an IR0 element [a dissociation constant (kd) of 5.8 nM vs. 1.4 nM (26, 29)]. It is thus possible that the modest repression is due to an inability of TR to form a stable heterodimer with RXR on the CRBPII element.

P75gag-v-erbA completely failed to represss RXR through the CRBPII element, even when present in vast excess over RXR within the cell (Fig. 3BGo). This could be due to distinct abilities of TR and P75gag-v-erbA to bind corepressors, or to a receptor function deficiency in P75gag-v-erbA. Our data suggest that the latter is the case. Gel mobility shift assays showed that RXR/P75gag-v-erbA heterodimers bound only weakly to the CRBPII element (Fig. 3CGo), showing that RXR and P75gag-v-erbA interact poorly. Previously published data suggested that a P->S mutation found in the C-terminal region of P75gag-v-erbA is responsible for the low heterodimerizing ability of the oncoprotein (20, 21, 27). We explored this further by testing the v-erbAtd359 and v-erbAr12 variant oncoproteins that have a proline in the C-terminal end like TR and should theoretically be able to interact with RXR. Other mutations in the LBDs of these VP16 oncoprotein constructs also contribute efficiently to the lack of interaction with RXR as suggested by our observation that none of them associated with the Gal4DBD-RXRLBD protein but yet transactivated in vivo. It is therefore likely that a RXR/P75gag-v-erbA heterodimer bound to a CRBPII element is disrupted by the SR11237 ligand, thus allowing RXR to bind ligand, homodimerize, and transactivate.

The oncoproteins gave a modest (75–50%) but consistent repression of Gal4DBD-RXRLBD-mediated transactivation despite the fact that they do not associate with the LBD of RXR. This type of repression was also seen with the hinge-LBD portion of P75gag-v-erbA (Fig. 5AGo) when RXR-dependent transactivation was measured with the 2xIR0 element. We suggest that this is due to an interference with transcriptional cofactors needed by RXR for maximal activation.

Role of Repression by TR and P75gag-v-erbA in Vivo?
The recently described corepressors SMRT and N-CoR (15, 16, 17) appear to play little role in P75gag-v-erbA-mediated repression of RXR on a 2xIR0 element, since transfections of plasmids expressing these proteins did not increase the repressing capacity of TR or any of the oncoproteins (G. Wahlström, unpublished data). Alternatively, they are already expressed at saturating levels in the cell types tested.

That TR represses transactivation by RXR both via synthetic and natural response elements in transfection experiments suggests that it could have similar properties in vivo. It is well known that TRs are expressed in several organisms before the development of the thyroid gland (30, 31, 32). Several investigators have suggested that a role for the unliganded receptor is to repress basal level transcription of target genes for TR (33, 34, 35, 36). A special example of this has been described for Xenopus development (Refs. 37 and 38 and references therein). TR{alpha} is already expressed in the oocyte and in the developing embryo, and treatment of such embryos with T3 induces the TRß gene (39), resulting in a precocious metamorphosis of the tadpole. In contrast, microinjection of a plasmid expressing P75gag-v-erbA into newly fertilized frog oocytes results in defective brain and facial development, which are abnormalitites usually associated with retinoid deficiency (40, 41). The published data on the action of TR and P75gag-v-erbA also show that they can oppose the teratogenic action of retinoids during Xenopus development (38, 42). Our data provide molecular information on how P75gag-v-erbA and unliganded TRs may interfere with the functions of the retinoid pathway mediated through RXR.

The v-erbA oncogene elicits its most prominent effects in erythroblasts. Here, the oncoprotein blocks the differentiation of immature cells to erythrocytes (for review, see Refs. 43 and 44). However, treatment of v-erbA- expressing cells with all-trans-retinoic acid causes the cells to overcome the differentiation block, and they terminally differentiate if provided with appropriate growth factors (45, 46). All-trans-retinoic acid is quickly metabolized to derivatives that bind to both RXR and RAR, and erythroblasts express high levels of both RXR and RAR. It is therefore possible that the differentiation-inducing effect of all-trans-retinoic acid alluded to above is mediated by RXR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
One copy of double-stranded oligonucleotides representing the natural CRBPII and rGH promoters was inserted into the HindIII site of the pBLCAT2 vector (47). Single elements of the IR0 and DR4 type (rGH A and B) from the rat GH were cloned individually into the pBLCAT2 vector (48). In addition, the synthetic response elements DR4, ER6, ER8, IR0 and 2xIR0 (26) were cloned inte the same reporter vector. The chicken TR{alpha}1 receptors (p40 and p46), mouse RXR{alpha}, and v-erbA were cloned into the pSG5 expression vector (49). cDNAs for v-erbA, v-erbAtd359, and v-erbAr12 were cloned into an Rous sarcoma virus (RSV) expression vector (23). Full length gag-erbA (V3) (50) or gag-v-erbA, v-erbAtd359 and v-erbAr12 were fused to the transactivating domain of VP16 by cloning into the KpnI/BamHI site of the pCMX-VP16 vector (51), placing the VP16 transactivation domain in the N terminus of the chimeric nuclear receptors. The v-erbA mutant lacking the DNA binding domain (DBD) was constructed by Casanova et al. (27). The vectors pCMX-Gal4-RXR and pCMX-VP16-TRß have previously been described by Perlmann and Jansson (51).

Gel Retardation Experiments
The gel retardation experiments were done with Vaccinia virus-expressed receptor proteins as previously described in Wahlström et al. (52). The receptor/DNA complexes were supershifted with polyclonal antibodies raised against either RXR or v-erbA.

Transfections
Human chorion carcinoma cells (JEG) were plated at a density of 2 x 105 per 3-cm dish in DMEM (Biological Industries) supplemented with 8% FCS. One day later the medium was replaced with DMEM containing 8% calf serum depleted of retinoic acid and/or T3 and T4 by ion exchange resin (53). Approximately 2 h later the cells were cotransfected with expression vectors encoding 100 ng mouse RXR{alpha}, 100–500 ng chicken TR{alpha}1, or v-erbA mutants plus 500 ng of different reporter constructs, unless indicated otherwise. A plasmid containing the cytomegalovirus (CMV) promoter driving the ß-galactosidase (ß-gal) gene was cotransfected to provide an internal control. The cells were maintained in the presence or absence of 0.5–1 mM SR 11237 and 12–100 nM T3, harvested 24 h after hormone treatment, and assayed for chloramphenicol acetyltransferase activity. The ß-galactosidase activity was assayed using o-nitrophenyl-ß-D-galactopyranoside as substrate. Quantifications were done with a Molecular Dynamics (Sunnyvale, CA) PhosphorImager (54). All transfections of JEG and HeLa cells were repeated at least three times with similar results. Duplicate sample points were used in each experiment and varied by less than 15%.


    ACKNOWLEDGMENTS
 
We are greatful to Dr. H. Samuels and Dr. T. Perlmann for receptor constructs and Dr. Louise Foley (Roche Diagnostics, Nutley, NJ) for the SR11237 ligand. The SMRT constuct was kindly provided by Dr. R. Evans, and we are greatful to Dr. J. Rosenfeldt for the N-CoR construct. We also thank Mrs. Gunnel Jönsson for secreterial help.


    FOOTNOTES
 
Address requests for reprints to: Bjorn Vennstrom, Department of Cellular and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden S-171 77.

This project was funded by grants from the Swedish Cancer Society (RMC), The Beijer Foundation, and funds at the Karolinska Institute. In addition, G. M. Wahlström was supported by the Swedish Society for Medical Research.

Received for publication May 7, 1997. Revision received December 3, 1997. Accepted for publication January 13, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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