- Original Articles
- Open Access
Cell Type-Dependent Modulation of the Dominant Negative Action of Human Mutant Thyroid Hormone β1 Receptors
© Molecular Medicine 1995
- Published: 1 March 1995
Mutations in the ligand-binding domain of the thyroid hormone receptor β (TRβ) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TRα and the non-mutated TRβ alleles by the TRβ1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TRβ1 (h-TRβ1) mutants.
Materials and Methods
Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TRβ1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TRβ1 expression in these two cell types. In order to determine how TRβ1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed.
Transfection studies using WT h-TRβ1 in HeLa and NIH3T3 cells, showed that the 3,3′,5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TRβ1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif.
The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.
Resistance to thyroid hormone (RTH) is a syndrome characterized by refractoriness of the pituitary and peripheral tissues to the action of thyroid hormone and is usually transmitted in an autosomal dominant fashion. There is an inappropriately elevated level of thyroid-stimulating hormone in the face of elevated levels of circulating free thyroid hormones, together with clinical features of variable degrees of thyroid hormone resistance action in peripheral tissues and the associated condition of attention-deficit hyperactivity disorder (1–3). The disease is caused by mutations in the ligand-binding domain of the thyroid hormone receptor β (h-TRβ) gene, which result in variable reductions in the affinity of h-TRβ1 for its ligand, 3,3′, 5-triiodothyronine (T3), and impaired transcriptional capacity (4,5). The mutant h-TRβ1 inhibits the function of normal h-TRβ1 and α1 via a dominant negative effect, thereby mediating the abnormal phenotype (6–9). Kindreds with RTH display a remarkable heterogeneity in organ resistance within an individual and among kindreds harboring identical mutations (10), and there are ongoing attempts to correlate genotype with the phenotype of these patients.
The difficulty in establishing such correlations stems from the complex molecular nature of the interactions of the thyroid hormone receptor. Not only are there four isoforms of the thyroid hormone receptor, which are expressed in a tissue-specific and development-stage-specific manner, but they may also heterodimerize with multiple cellular partners, most prominently the retinoid X-receptors (RXRs), to create unique transcriptional responses of T3-regulated genes (11–16). Published examples of genotype-phenotype correlations include an association between language abnormalities with mutations in exon 9 (17), and a high ratio of mutantmormal h-TRβ mRNA during a period of bone resistance with growth retardation, attenuated in teenage years with a concomitant improvement in growth rate (18). In general, however, neither the T3-binding impairment of h-TRβ1 mutants, as reflected by blood levels of thyroid hormones, nor the location of mutations predicts a phenotype.
Until recently, studies of mutant h-TRβ1 have employed mostly artificial thyroid response elements (TREs) (7,19–21). Moreover, there has not been a systematic comparison of the effects of mutant h-TRβ1 on various natural positive TREs in different cell types. Furthermore, there have been conflicting data regarding the relative roles of heterodimers versus homodimers in mediating the dominant negative effect of these mutant receptors (22,23). These studies have employed transfections in a single cell type, utilizing mostly artificial TREs and exogenous RXRs to characterize the nature of protein-protein complexes. We were interested in determining the extent to which the cellular environment contributes to the dominant negative action of mutant receptors. Thus, we performed transient transfection assays using naturally occurring TREs in two different cell lines, HeLa and NIH3T3, and gel-shift studies using these nuclear extracts to establish if there are differences in protein-protein interactions. These proteins may include those that interact either directly with mutant h-TRβ1, so called thyroid hormone receptor auxiliary proteins (TRAPs), or indirectly to bring the TR-complex into contact with the transcriptional machinery, so called adaptor or coactivator proteins (24). Our results demonstrated that the extent of the transactivation by wild-type (WT) h-TRβ1 on the three natural TREs studied is different in the two cell types and that the three mutant receptors display different dominant negative potencies in the two cell types despite equivalent expression levels of receptor in these cells, as demonstrated by immunocytochemistry. Furthermore, the pattern of hetero- and homodimerization differs when nuclear extracts from these two cell types are used in gel-shift studies. The cell type in which the mutant receptors act, together with the type of TRE motif, most probably determines the patterns of hetero- and homodimerization of these receptors, the relative abundance of which, in turn, contributes to the strength of their dominant negative effect.
Construction of Plasmids
Construction of the WT h-TRβ1 and mutant pSV2 expression vectors-ED, -OK, and -PV, and the corresponding pGEM 3Z-vectors for in vitro translation has been described before (19,20,25). The TK-TRE constructs, -Lys, -ME, and -GH, were a kind gift of Dr. G. Brent, Harvard Medical School, Boston, MA, U.S.A.; the construction of these has been described previously (26).
Cells (HeLa, NIH3T3) were plated 24 hr before transfection in Dulbecco’s modified Eagle’s medium containing 10% (v/v) hormone-depleted fetal calf serum (27), 100 µ/ml penicillin, 100 µg/ml streptomycin and 0.25 µl/ml amphotericin B in 10-cm petri dishes. The medium was changed 4 hr prior to transfection. Transfections were performed in pairs (CellPhect kit; Pharmacia-LKB, Piscataway, NJ, U.S.A.) using the CaPO4 method. Each plate received 5.0 µg TRE-containing TK-CAT plasmid, 3.0 µg pXGH5 plasmid as a transfection efficiency control, 1.0 µg pSV2-WT h-TRβ1, 0–5.0 µg mutant h-TRβ1, and 0–5.0 µg pSV2 to keep the amount of transfected DNA constant. After 24 hr, the plates were washed once with phosphate-buffered saline and fresh medium added together with the appropriate T3 concentration. CAT assays were performed 24 hr later, after harvesting and lysing the cells, as previously described (28). CAT activity was normalized for protein concentration as measured by the Coomassie blue method. No substantial differences in transfection efficiency were present as assessed by the cotransfection of a growth hormone expression vector pXGH5.
In Vitro Transcription and Translation of Receptors
For in vitro transcription, WT and mutant h-TRβ1 cDNAs were linearized with HindIII and used as a template for transcription with T7-RNA Polymerase. [35S]-labeled and unlabeled receptor proteins were synthesized from the transcribed RNA using the rabbit lysate kit according to the manufacturer’s manual (Promega, Madison, WI, U.S.A.). The translated proteins were analyzed for their molecular weights by 10% SDS-PAGE, and quantitation was performed by the trichloroacetic acid precipitation method.
Electrophoretic Gel Mobility Shift Assay
Complementary oligonucleotides with 5′ overhangs were annealed and filled-in in the presence of 32P-dCTP with Klenow DNA Polymerase. The labeled double-stranded oligonucleotides were purified on a 12% Polyacrylamide gel. The gel-mobility shift assay was carried out in a 10 µl volume: equal amounts of receptor were incubated with the purified double-stranded oligonucleotides with or without T3 in binding the buffer (25 mM Hepes/pH 7.5, 5 mM MgCl2, 4 mM EDTA, 10 mM DTT, 0.11 M NaCl, and 0.8 µg/µl sheared salmon DNA). Where appropriate, RXRβ was added. After incubation for 30 min at room temperature, the reaction mixture was loaded onto a 5% Polyacrylamide gel and electrophoresed at 4°C. for 2.5 hr. The gel was dried and autoradiographed.
Preparation of Nuclear Extracts
HeLa and NIH3T3 cells were grown as above. Nuclear extracts were prepared according to the Schaffner method (29). Briefly, the scraped cells were pooled, spun at 1500 rpm, 4°C. for 5 min and then resuspended in cold Buffer A (10 mM HEPES/pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). The cells were then lysed with Nonidet-40 at a final concentration of 0.5%. After inverting the tube several times, it was spun at 1500 rpm, 4°C. for 5 min, and the supernatant decanted leaving a whitish nuclear pellet, which was resuspended in 400 µl Buffer C (20 mM HEPES/pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). This was thoroughly vortexed for 5 min at 4°C., placed on a rotating shaker for 2 hr, then spun at full speed, precipitating the nuclear membranes and leaving nuclear extract in the supernatant. Protein concentration was determined by the Bradford method.
Cultured HeLa and NIH3T3 cells were transfected as described above. After 2 days, the cells were processed for immunological studies as previously described (30). Briefly, the cells were fixed with 3.7% formaldehyde in PBS for 5 min at 23°C. and incubated with 10 µg/ml monoclonal antibody C4 (30) in PBS containing 0.1% saponin and 4 mg/ml normal goat globulin for 30 min at room temperature. After being washed with PBS, the cells were incubated with affinity-purified goat antimouse immunoglobulin G conjugated to rhodamine (25 µg/ml) for 30 min. The cells were viewed, and representative fields were photographed using a microscope equipped with rhodamine epifluorescence optics.
Transfection experiments were performed at least twice, with duplicate determinations. Statistical analyses were performed by Student’s t test; p < 0.05 was considered significant. Note that in Figs. 3 and 4, separate values were obtained for the dominant negative potencies in both HeLa and NIH3T3 cells at the designated WT:mutant receptor ratio and the resultant values obtained for each cell were then compared by t test to obtain a p value. Thus, the asterisk refers to this second determination.
Characteristics of wild-type and mutant h-TR β 1
Affinity Constant of T3 Binding (Ka × 109 M−1)
Transcriptional Capacity of WT h-TRβ1 on Natural TREs Was Cell Type Specific
Sequences of natural TREs
Rat Malic Enzyme (ME-TRE)
Rat Growth Hormone (GH-TRE)
Chicken Lysozyme Silencer (Lys-TRE)
Transcriptional Capacity of h-TRβ1 Mutants
Dominant Negative Effect of h-TRβ1 Mutants
Relative Abundance of Hetero- and Homodimers
In contrast, WT h-TRβ1 did not bind to the ME-TRE probe (Fig. 6B, Lane 1). Strong heterodimer bands, as with Lys-TRE, formed with the HeLa nuclear extract (Lane 5). A similar band of increased electrophoretic mobility formed, which was partially competed away by specific competitor. The heterodimer bands formed with the 3T3 nuclear extract (Lane 9) were much weaker than with HeLa nuclear extract, indicating that the quantity of RXRβ/α or RXR-like TRAPs may be lower in 3T3 cells.
Using the ME-TRE in HeLa cells, prominent heterodimers formed when either the WT h-TRβ1 or mutant receptors were used (Fig. 6B, Lane 5 versus Fig. 7B, Lane 5). No homodimers were seen using either HeLa or 3T3 nuclear extract on the ME-TRE. In 3T3 cells, upper heterodimers of weaker intensities formed when either WT or mutant h-TRβ1 were used (Fig. 6B, Lane 9 versus Fig. 7B, Lane 9). Thus, depending on the cell type and the TRE, both mutant hetero- and homodimers may contribute to the dominant negative activities of mutant receptors. Our findings also suggested that on the identical TRE, the same mutation could mediate varying strengths of dominant negative inhibition depending on whether homodimers or heterodimers form. It could further be postulated that, since transactivation is thought to occur mostly via the stable heterodimer, the greater the ability to form heterodimers, the less potent the dominant negative effect of a mutant receptor will be. Since h-TRβ1 was only able to form heterodimers on the ME-TRE, the dominant negative potencies of mutant receptors on this element could be predicted to be less than on the Lys-TRE, where both heterodimers and homodimers formed. This approach of using nuclear extract to identify potential hetero- and/or homodimerization complexes could be useful in further studies to assess the relative roles of these complexes in the dominant negative activities of mutant receptors.
Expression of TRβ1 Protein
The molecular mechanisms underlying the variable organ resistance within an individual and between kindreds with RTH, sharing identical mutations, have remained unresolved. We characterized positive TREs to investigate the role of cell type in modulating the dominant negative effect of mutant h-TRβ1. Three different naturally occurring mutations from kindreds with RTH, which had previously been shown to have three distinct types of functional impairment on an idealized TRE (19,20,31), were used. Mutant ED has a 5-fold reduction in T3-binding affinity, a correspondingly shifted T3-dose response curve in transient transfection assays and, as expected, high levels of T3 restored its transcriptional capacity to wild-type levels and obliterated its dominant negative effect. Despite having a similar 5-fold reduction in T3-binding affinity, mutant OK was only partially transactivated at high T3 levels, and its dominant negative potency was likewise not completely reversed. It was speculated that this point mutation not only affected T3-binding but also the transactivation domain (20). PV has a frame shifted carboxy terminus, which abolished T3-binding, predictably failed to transactivate with T3, and exhibited the most potent dominant negative effect on the Lap-TRE.
In this study, we systematically compared two different cell types and found that, in addition to the TRE motif and the nature of the mutation in h-TRβ1 (20,33), the dominant negative potencies of mutant receptors were modulated by the cellular environment. This may be the result of whether heterodimers or homodimers can form in the particular cell type in question.
Sugawara et al. (32) have recently demonstrated a heterogeneity of TRAPs in different rat tissues and cell lines that were differentially distributed and which were able to heterodimerize with TRs. Petty (34) also demonstrated, by Far-Western Blotting, multiple bands of different molecular sizes, many of which differed from that of the RXRs, that formed between TRβ1 and nuclear extracts of rat brain, kidney, liver, GH3 cells, and HeLa cells. Suen and Chin (35) also showed that distinct, cell-specific TR-DNA complexes formed when GH3 and HeLa nuclear extracts were used in cell-free in vitro transcription assays. In this paper, we showed that the interactions of WT h-TRβ1 and mutant OK on the Lys- and ME-TREs differed in HeLa and 3T3 cells, as seen by gel-mobility shift assays. Heterodimers between WT h-TRβ1 and RXRβ/α could mediate a 10-fold transcription from the Lys-TRE observed in HeLa cells, whereas, in 3T3 cells, there was a weaker 5-fold transcriptional response, together with weaker heterodimer bands and the presence of homodimers. Thus, the strength of the transcriptional capacity of wild-type and mutant receptors may be dependent on the degree of heterodimerization, which, in turn, is dependent on the amount of TRAPs present in different tissues.
Almost all of the mutations that cause RTH cluster in two “hot spot” regions on either side of the dimerization domain (5). This domain consists of nine heptad repeats, which are thought to be important for both dominant negative activity and transcription (36,37); furthermore, a dimerization domain encompassing codons 281–300 has been defined by in vitro mutagenesis (38–41). Nagaya et al. (6) showed that receptor heterodimerization was important for dominant negative inhibition and that double mutants that formed only homodimers lost their dominant negative capacity in JEG3 cells. They studied two naturally occurring RTH mutant receptors that formed homodimers and heterodimers with RXRα, as well as an artificial mutation in one of the hydrophobic heptad repeats of the putative receptor dimerization domain, which impaired heterodimerization without altering the formation of homodimers. Upon introducing this dimerization mutant into either of the RTH receptor mutants, the dominant negative activity of the RTH mutant was abolished, suggesting the importance of heterodimerization for the dominant negative effect. Conversely, Hao et al. (22) were able to show, using several artificial TREs, preferential homodimerization with the S-mutant receptor, a powerful dominant negative receptor (42,19), and predominant heterodimerization with the GH receptor, which has compromised dominant negative function and was found in both normal individuals and in a patient with severe pituitary resistance to thyroid hormone (42,43). The latter finding with the GH receptor also supports one of our contentions that the greater the degree of heterodimerization versus homodimerization, the lesser the dominant negative potency of the receptor. The findings of Hao et al. (22) are also in contrast to those of Au-Fleigner et al. (23), where mutational analysis of the ninth heptad repeat of the c-erbAal receptor demonstrated that this region was required for heterodimerization but not homodimerization and, furthermore, that the chick c-erbAα1 mutant with intact homodimerization lacked dominant negative activity. Our present study, however, suggests the possibility that both hetero- and homodimers in different amounts are capable of mediating the dominant negative effect depending on the contributions of cell type, mutation, and the TRE motif. Using h-TRβ1 mutants in NIH3T3 cells on the Lys-TRE, homodimers were the predominant species; however, in HeLa cells, only heterodimers formed on the Lys- and ME-TREs, and this species may thus be responsible for dominant negative action in this cell.
All the studies to date have mostly focused on the TR-TRE relationships within one particular cell type. We present the first systematic study of mutant h-TRβ1 interactions on natural TREs using two different cell types in order to assess the contribution of cell type to dominant negative potencies of mutant receptors. Although the cells used here may not necessarily be physiologically relevant, and the TREs used are not in the context of their full-length native promoters, certain preliminary conclusions can be drawn. Not only does the TRE motif and the isoform of the endogenously active receptor determine the degree of inhibition of a specific gene in RTH individuals as has been suggested by others (19,20), but we showed here that the cellular milieu in which a particular mutation operates could have important influences on the outcomes of these receptor-DNA interactions. Firstly, the magnitudes of the transcriptional activities on various natural TREs were different in the two cell types. Secondly, the dominant negative effect of a particular mutant receptor also differed between the two cell types when the identical TRE was used. Thirdly, the nature of the protein-protein complexes formed as assessed by gel-shift assays differed depending on the TRE and cell used. Fourthly, we have established that h-TRβ1 expression was equal in the two cells studied here, confirming that the differences in functional activities of the receptors observed herein were truly cell type specific. Thus, the cell type in which a mutant receptor is found could determine the relative amounts of hetero- and homodimers, and this, together with the nature of the mutation and the TRE motif, could influence the strength of the dominant negative action of mutant receptors and potentially contribute to the degree of organ resistance. Studies are underway to extend these observations to characterize the behavior of mutant h-TRβ1 on various natural TREs in the respective cell types in which they normally function. It is clear that more physiological approaches are required to further elucidate the mechanisms by which the dominant negative effect of mutant h-TRβ1 correlates with the clinical phenotypes, and, to this end, we have developed a transgenic mouse model of RTH using the PV mutant receptor (44).
We are grateful to Dr. G. Brent (Harvard Medical School, Boston, MA, U.S.A.) for providing us with the TK-TRE-CAT plasmids and Dr. C. Meier for helpful discussions.
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