Isoform Variable Action among Thyroid Hormone Receptor Mutants Provides Insight into Pituitary Resistance to Thyroid Hormone

J. D. Safer, M. F. Langlois, R. Cohen, T. Monden, D. John-Hope, J. Madura, A. N. Hollenberg and F. E. Wondisford

Thyroid Unit Department of Medicine Beth Israel Hospital and Harvard Medical School Boston, Massachusetts 02215


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
Resistance to thyroid hormone (RTH) is due to mutations in the ß-isoform of the thyroid hormone receptor (TR-ß). The mutant TR interferes with the action of normal TR to cause the clinical syndrome. Selective pituitary resistance to thyroid hormone (PRTH) results in inappropriate TSH secretion and peripheral sensitivity to elevated thyroid hormone levels. Association of the PRTH phenotype with in vitro behavior of the mutant TR has proved elusive. Alternative exon utilization results in two TR-ß isoforms, TR-ß1 and TR-ß2, which differ only in their amino termini. Although the TR-ß1 isoform is ubiquitous, the TR-ß2 isoform is found predominantly in the anterior pituitary and brain. To date, in vitro evaluation of RTH mutations has focused on the TR-ß1 isoform. Site-directed mutagenesis was used to create several PRTH (R338L, R338W, V349M, R429Q, I431T) and generalized RTH ({Delta}337T, P453H) mutations in both TR-ß isoforms. The ability of mutant TRs to act as dominant negative inhibitors of wild type TR-ß function on positive and negative thyroid hormone response elements (pTREs and nTREs, respectively) was evaluated in transient transfection assays. PRTH mutants had no significant dominant negative activity as TR-ß1 isoforms on pTREs found in peripheral tissues or on nTREs found on genes regulating TSH synthesis. PRTH mutants, in contrast, had strong dominant negative activity on these same nTREs as TR-ß2 isoforms. Cotransfected retinoid X receptor-{alpha} was required for negative T3 regulation via the TR-ß1 isoform but was not necessary for negative regulation via the TR-ß2 isoform in CV-1 cells. The differing need for retinoid X receptor cotransfection demonstrates two distinct negative T3-regulatory pathways, one mediated by the TR-ß1 and the other mediated by TR-ß2. The selective effect of PRTH mutations on the TR-ß2 isoform found in the hypothalamus and pituitary vs. the TR-ß1 isoform found in peripheral tissues suggests a molecular mechanism for the PRTH disorder.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
Resistance to thyroid hormone (RTH) is the result of mutations in the carboxyl terminus of the ß-thyroid hormone receptor (TR-ß) (1, 2, 3, 4). Individuals with the disorder require greater thyroid hormone (T3) concentrations to achieve T3-dependent actions. RTH is a dominant disorder in which most individuals are heterozygous for the mutant allele. In a phenomenon called dominant negative activity, the mutant allele interferes with the activity of the normal allele (5, 6, 7, 8).

Clinically, thyroid hormone resistance can be divided into two entities: generalized resistance to thyroid hormone (GRTH) and central or pituitary resistance to thyroid hormone (PRTH) (4). In both syn-dromes there is thyroid hormone resistance at the level of the pituitary and hypothalamus causing inappropriate TSH secretion and, in turn, elevated thyroid hormone levels. In GRTH there is also peripheral resistance, often resulting in a hypothyroid- or euthyroid-appearing patient. In PRTH, however, peripheral sensitivity to thyroid hormone is preserved and the individual suffers thyrotoxic symptomatology from the elevated levels of circulating T3 (4a). A molecular mechanism to explain these two clinical phenotypes has proved elusive, and many authors have concluded that they are part of a spectrum of the same disorder (9, 10, 11).

Both GRTH and PRTH mutations congregate in two major hot spots in the ligand-binding domain of TR-ß (Fig. 1Go) (12, 13). GRTH mutations have also been reported in the hinge region, suggesting a third hot spot for mutations at the TR-ß locus (11, 14, 15). Alternative promoter utilization at the TR-ß locus yields two isoforms of the TR-ß: TR-ß1, which is ubiquitously expressed, and TR-ß2, which is expressed almost exclusively in the anterior pituitary, hypothalamus, and elsewhere in the brain (16, 17, 18, 19, 20). While the TR-ß isoforms are identical in their DNA- and ligand-binding domains, they differ significantly in their N-terminal A/B domains. A functional difference between the two TR-ß isoforms in thyroid hormone regulation would provide a molecular mechanism for tissue-specific regulation of gene expression (21). In this study, therefore, the role of the TR-ß2 isoform in mediating the PRTH syndrome was evaluated.



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Figure 1. Schematic Representation of the Location of TR-ß Mutations Used in This Study

Mutations cluster in two hot spots in the TR-ß C terminus and surround a region with nine heptad repeats.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
TR-ß Mutations Used in This Study
As shown in Fig. 1Go, seven TR-ß mutations were evaluated in this study. Two of the mutations ({Delta}337T and P453H) significantly reduce T3 binding and cause GRTH (3, 22). One of the mutations (R338W) preserves T3 binding and is reported to cause both GRTH and PRTH, although reports of this mutation causing PRTH predominate (11, 23, 24). The latter studies have included specific measurements of thyroid hormone action in the periphery. The remaining four mutations (R338L, V349M, R429Q, and I431T) are reported to cause PRTH (11, 25). Patients with R338L (25a) and R429Q (25) mutations have had detailed measurements of thyroid hormone action in the periphery, indicating they indeed have PRTH. Measurements of peripheral thyroid hormone action in patients with V349M and I431T mutations have not been described, making the diagnosis of PRTH less certain in these patients. With the exception of I431T, TRs containing these mutations bind well to T3. These mutations were introduced into either a TR-ß1 or TR-ß2 cDNA, using site-directed mutagenesis, and inserted into a viral expression vector (pSG5) for use in transient transfection studies. All transfections in this study were performed in a TR-deficient cell line (CV-1 cells), in the absence of a suitable pituitary cell line, which is also TR deficient.

Certain PRTH Mutants Have Weak Dominant Negative Activity on Positive Thyroid Hormone Response Elements
We first sought to determine the function of each of these mutations on thyroid hormone-regulated genes expressed in peripheral tissues. In peripheral tissues such as the kidney, heart, and liver, TR-ß1 and TR-{alpha}1 mRNAs are readily detected by Northern blot analysis and together represent 82–90% of total T3 binding activity (25b). In contrast, TR-ß2 mRNA is not detected by Northern analysis, and TR-ß2 protein represents a small fraction of T3-binding capacity in these same tissues (10–18%). Because TR-ß1 is the major TR-ß isoform expressed in peripheral tissues, each mutation was only tested in the context of the TR-ß1 isoform. As a model for thyroid hormone action in peripheral tissues, two copies of positive thyroid hormone response elements (pTREs) were fused upstream of a heterologous promoter luciferase construct (pTK109-Luc) for use in transient transfection assays. Shown in Fig. 2AGo are the dominant negative activities of each mutant on a well characterized pTRE found in a number of thyroid hormone-regulated genes (Direct repeat with a 4-bp gap [Dr+4]). Dominant negative activity is plotted with 100% meaning complete interference with WT TR-ß1 T3-mediated stimulation. The I431T, V349M, P453H, and {Delta}337T mutations all had significant dominant negative activity on this element at 2.5 nM T3. Essentially equivalent results were obtained at 10 nM T3 (data not shown). In contrast, the PRTH mutants, R338L, R338W, and R429Q, were unable to block wild type (WT) TR-ß1 function on this element at 2.5 nM (Fig. 2AGo) or 10 nM T3 (data not shown). Similar results were obtained with another naturally occurring pTRE [inverted palindrome (F2)] except that the putative PRTH mutant I431T was without effect, and the GRTH mutant P453H had less dominant negative activity (Fig. 2BGo). Others have also reported the lack of dominant negative activity of PRTH mutants as TR-ß1 isoforms on pTREs (27, 28).



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Figure 2. Dominant Negative Activity of Mutant TR-ß1 Expression Vectors on a 2xDr+4 Reporter Construct (A), a 2xF2 Reporter Construct (B), and a 2xPal Reporter Construct (C and D)

A 10-fold excess of mutant to WT TR-ß1 expression vector (1 µg to 100 ng) was transfected with the indicated reporter construct in CV-1 cells and compared with transfection of the WT TR-ß1 expression vector alone (100 ng). Dominant negative activity is plotted on the x-axis, where 100% represents complete blockade and 0% represents no change in T3 stimulation [at 2.5 nM T3 (panels A, B, and C) and 10 nM T3 (panel D)] relative to the WT TR-ß1 transfection alone.

 
We next tested the effect of these mutants on the palindromic element, an artificial pTRE. GRTH mutants have significant dominant negative activity (>75%) on the element at either 2.5 nM T3 (Fig. 2CGo) or 10 nM T3 (Fig. 2DGo). The I431T and V349M mutants had between 50% and 70% dominant negative activity on the WT TR at either 2.5 or 10 nM T3. Finally, R429Q, R338W, and R338L had weak dominant negative activity on this element (<40%) at 2.5 nM T3 (Fig. 2CGo) and even less activity (<30%) at 10 nM T3 (Fig. 2DGo). Taken together, the I431T and V349M putative PRTH mutations function like GRTH mutants on pTREs found in the periphery. In contrast, three PRTH mutations (R429Q, R338W, and R338L) have weak or no dominant negative activity on these same elements.

The Dominant Negative Activity of PRTH Mutants on Negative Thyroid Hormone Response Elements Depends on the TR-ß Isoform
Dominant negative activity on genes negatively regulated by thyroid hormone was next evaluated. In contrast to peripheral tissues, TR-ß2 is a major isoform in the anterior pituitary and hypothalamus. We therefore tested the function of both TR-ß isoforms on genes expressed in the anterior pituitary and hypothalamus that are responsible for control of thyroid hormone synthesis. The human TRH and common {alpha}-subunit gene promoters were inserted into a luciferase reporter construct and used in transient transfection assays of CV-1 cells. The TSH-ß promoter was not used in this study because of its very low expression in this cell line. On the TRH promoter, the {Delta}337T mutation had complete dominant negative activity in the context of the TR-ß1 isoform (Fig. 3Go). Dominant negative activity is plotted with 100% meaning complete interference with WT TR-ß1 T3-mediated inhibition. The P453H, V349M, and I431T mutations had approximately 50% dominant negative activity as TR-ß1 isoforms. In contrast the PRTH mutants, R429Q, R338W, and R338L had little or no activity on the same reporter as TR-ß1 isoforms even at 10-fold excess of mutant vs. WT TR-ß1 expression vector.



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Figure 3. Dominant Negative Activity of Mutant TR-ß Isoform Expression Vectors on a TRH Reporter Construct

A 10-fold excess of mutant to WT TR-ß isoform expression vector (1 µg to 100 ng) was transfected with the indicated reporter construct in CV-1 cells and compared with transfection of the WT TR-ß1 or WT TR-ß2 expression vector alone (100 ng). Dominant negative activity is plotted on the x-axis, where 100% represents complete blockade and 0% represents no change in T3 inhibition (at 2.5 nM T3) relative to the WT TR-ß1 or WT TR-ß2 transfection alone.

 
Interestingly, these same PRTH mutants, which had weak dominant negative activity as TR-ß1 isoforms on the TRH gene, had strong dominant negative activity on the TRH gene as TR-ß2 isoforms at 2.5 nM T3 (Fig. 3Go). The PRTH mutants, R429Q, R338W, and R338L, as TR-ß2 isoforms were converted into strong dominant negative inhibitors on the TRH gene; and the dominant negative activity of the I431T and V349M mutants was greater as TR-ß2 vs. TR-ß1 isoforms. This was not true, however, of the GRTH mutants that had essentially similar dominant negative activity on the TRH promoter as either TR-ß1 or TR-ß2 isoforms, suggesting that the isoform differences on the TRH promoter were due to specific TR-ß mutations.

Identical experiments were performed using the common {alpha}-subunit gene promoter as the reporter construct and a 10-fold excess of mutant vs. WT TR-ß1 or TR-ß2 expression vector (Fig. 4Go). Note that the PRTH mutants, R429Q, R338W, and R338L, had no significant dominant negative activity on this promoter in the context of the TR-ß1 isoform and near 100% activity as TR-ß2 isoforms. The putative PRTH mutants, I431T and V349M, competed as effectively as either TR-ß1 or TR-ß2 isoforms, similar to data obtained with the TRH promoter (Fig. 3Go). Likewise, the GRTH mutants were effective competitors in either isoform. These results indicate that the R429Q, R338W, and R338L mutations display consistent and dramatic TR-ß isoform differences on two negatively regulated genes involved in the central control of thyroid hormone synthesis.



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Figure 4. Dominant Negative Activity of Mutant TR-ß Isoform Expression Vectors on the Common Glycoprotein {alpha}-Subunit Reporter Construct

A 10-fold excess of mutant to WT TR-ß isoform expression vector (1 µg to 100 ng) was transfected with the indicated reporter construct in CV-1 cells and compared with transfection of the WT TR-ß1 or WT TR-ß2 expression vector alone (100 ng). Dominant negative activity is plotted on the x-axis, where 100% represents complete blockade and 0% represents no change in T3 inhibition (at 2.5 nM T3) relative to the WT TR-ß1 or WT TR-ß2 transfection alone.

 
PRTH Mutants Have Significant Dominant Negative Effects on the TRH Gene over a Wide Range of T3 Concentrations
To determine whether changes in T3 concentration would alter the dominant negative effect of the TR-ß2 isoform on the TRH gene, T3 concentrations were varied from 1–10 nM in transfection experiments in which a 10-fold excess of mutant vs. WT TR-ß2 expression vector was used. Similar to data obtained in Fig. 3Go, both GRTH and PRTH mutants had significant dominant negative activity as TR-ß2 isoforms at all concentrations of T3 tested (Fig. 5Go). Interestingly, however, the dominant negative activity for all of the PRTH mutations (except for I431T and V349M) and one of the GRTH mutants (P453H) was reduced at higher T3 concentration from approximately 80% to 100% at 1 nM T3 to 40% to 60% at 10 nM T3. Other investigators have noted that supraphysiological T3 concentrations can reduce dominant negative activity of RTH mutations presumably by increasing T3 binding to less avid receptors (26). Parallel experiments were also performed using the TR-ß1 isoform and varying the T3 concentration from 1–10 nM. At 10 nM T3, none of the mutations in this study had dominant negative activity on the TRH promoter except for {Delta}337T, which retained 100% activity (data not shown). Thus, PRTH mutations in the TR-ß2 isoform display significant dominant negative activity on the TRH promoter over a wide range of T3 concentrations.



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Figure 5. Dominant Negative Activity of Mutant TR-ß2 Expression Vectors on a TRH Reporter Construct at Various T3 Concentrations

Experiment identical to that described in Fig. 3Go except that it was performed at the indicated T3 concentration.

 
PRTH Mutants in the TR-ß2 Isoform Inhibit the Function of Both WT TR-ß1 and TR-ß2 on the TRH and Common {alpha}-Subunit Genes
Since all of the transfections on the TRH and common {alpha}-subunit genes had been performed at a 10-fold excess of the mutant vs. WT TR-ß, experiments were repeated at a 1:1 ratio of WT to mutant TRß expression vector. Data obtained from the TRH promoter are shown in Fig. 6AGo. PRTH mutants in the TR-ß1 isoform were weak dominant negative inhibitors of WT TR-ß1 at a 1:1 ratio as noted previously at a 10-fold excess of mutant receptor. In contrast, the GRTH mutant ({Delta}337T) in the TR-ß1 isoform had strong activity on the TRH promoter. When these mutants were expressed in the TR-ß2 isoform, stronger dominant negative activity on the TRH promoter was observed with the PRTH mutants, and weaker dominant negative activity was found with the GRTH mutant. Since the anterior pituitary contains both TR-ß1 and TR-ß2, we also wanted to determined whether PRTH mutants in the TR-ß2 isoform could compete against WT TR-ß1 function. Note that each of the PRTH mutants in the TR-ß2 isoform (stippled bars) had significant dominant negative activity against WT TR-ß1, although they were better competitors against WT TR-ß2 function (black bars). Conversely, the GRTH mutant in the TR-ß2 isoform (stippled bar) had significant dominant negative activity against either WT TR-ß1 or TR-ß2, although it was a better competitor as a TR-ß1 isoform against WT TR-ß1 function (white bar). In data not shown, these PRTH mutants in the TR-ß1 isoform had no significant activity against WT TR-ß2 function on the TRH promoter. Similar results were obtained at a 1:1 ratio of WT vs. mutant TR-ß on the common {alpha}-subunit gene promoter as shown in Fig. 6BGo. Thus, PRTH mutants in the TR-ß2, but not TR-ß1, isoform were effective competitors against both WT TR isoforms in the pituitary. The GRTH mutant ({Delta}337T), in contrast, competed against WT TR-ß function in both the TR-ß1 and TR-ß2 isoform but was distinctly superior as a competitor in the TR-ß1 isoform.



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Figure 6. Dominant Negative Activity of Mutant TR-ß Isoform Expression Vectors on a TRH Reporter Construct (A) and on a Common Glycoprotein {alpha}-Subunit Reporter Construct (B)

An equal quantity of mutant and WT TR-ß isoform expression vector (100 ng and 100 ng) was transfected with the indicated reporter construct in CV-1 cells and compared with transfection of the WT TR-ß1 or WT TR-ß2 expression vector alone (100 ng). Dominant negative activity is plotted on the x-axis, where 100% represents complete blockade and 0% represents no change in T3 inhibition (at 2.5 nM T3) relative to the WT TR-ß1 (open and stippled bars) or WT TR-ß2 (solid bars) transfection alone.

 
Pathways for Negative Regulation by Thyroid Hormone Are TR-ß Isoform-Specific
A possible mechanism for this isoform-specific phenomenon came from our interesting observation that retinoid X receptor (RXR) is a necessary cofactor for negative regulation with the TR-ß1 isoform. The CV-1 cell line used in this study is relatively deficient in RXRs and does not support transcriptional activation by RXR-specific ligands of RXR-responsive promoters in the absence of cotransfected RXRs (29, 30). We tested the effect of cotransfected RXR-{alpha} on T3 inhibition of the TRH and common {alpha}-subunit genes and a negative control promoter from the thymidine kinase gene (TK199). As noted in Fig. 7Go, TR-ß1 at 100 ng transfected was able to negatively regulate the TRH and common {alpha}-subunit genes only in the presence of cotransfected RXR-{alpha}. We have previously demonstrated on the TRH promoter that TR-ß1 could negatively regulate the TRH promoter in CV-1 cells but only at higher amounts of TR-ß1 cotransfected (500 ng)(31). Negative T3 regulation under these conditions was also increased (from 2- to 4.5-fold) by cotransfected RXR-{alpha}, consistent with an RXR dependence for T3 regulation by the TR-ß1 isoform. In contrast, TR-ß2 was able to regulate both promoters in the absence or presence of cotransfected RXR. As expected, TK199 was not negatively regulated in this cell line. These data could not be explained by TR-ß isoform differences in binding to a TRH response element, as TR-ß1 and TR-ß2 bound equally well as monomers, homodimers, or heterodimers with RXR-{alpha} to this response element (data not shown). Regardless of the mechanism, these results are consistent with previous reports demonstrating that both of these promoters are strongly negatively regulated by TR-ß1 in JEG-3 cells, which have abundant RXR-{alpha} (32), and suggest that pathways for negative regulation by TR-ß1 and TR-ß2 vary in their response to cellular RXR. The relative contribution of TR-ß isoforms in T3 inhibition may, therefore, depend on either the type or quantity of RXR expressed in cells.



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Figure 7. Thyroid Hormone Inhibition (T3) of Reporter Constructs in CV-1 Cells with and without Cotransfected RXR-{alpha}

T3 inhibition of gene expression was measured in CV-1 cells after the indicated reporter was transfected with the WT TR-ß1 or WT TR-ß2 expression vector (100 ng). In some experiments, an RXR-{alpha} expression vector was cotransfected (100 ng) as indicated. Fold T3 inhibition is plotted on the x-axis and is calculated by dividing luciferase activity in the absence of T3 treatment by luciferase activity in the presence of T3 treatment.

 
Because TR-ß1 was unable to negatively regulate the TRH and common {alpha}- subunit genes in CV-1 cells in the absence of cotransfected RXR, comparisons between the two TR-ß isoforms was performed in the presence of cotransfected RXR in all studies described above. We wanted to determine, therefore, whether the dominant negative activity of the PRTH mutants was altered in the absence of cotransfected RXR. Results obtained with three PRTH mutants (R429Q, R338W, R338L) and one GRTH mutant ({Delta}337T) in the TR-ß2 isoform in the absence of cotransfected RXR are shown in Fig. 8Go. Importantly, dominant negative activity by the PRTH mutants in the TR-ß2 isoform was 100%, indicating that PRTH mutants were effective competitors in the TR-ß2 isoform in either the presence or absence of cotransfected RXR.



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Figure 8. Dominant Negative Activity of Mutant TR-ß2 Isoform Expression Vectors on a TRH Reporter Construct in the Absence of Cotransfected RXR-{alpha}

An equal quantity of mutant and WT TR-ß2 isoform expression vector (100 ng and 100 ng) was transfected with the indicated reporter construct in CV-1 cells and compared with transfection of the WT TR-ß2 expression vector alone (100 ng). Dominant negative activity is plotted on the x-axis, where 100% represents complete blockade and 0% represents no change in T3 inhibition (at 2.5 nM T3) relative to a WT TR-ß2 transfection alone.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
The data presented here represent a plausible explanation for PRTH and its molecular distinction form GRTH. All RTH mutations described to date, except for deletion of the TR-ß locus, are point mutations within a common region of the receptor (4). Thus, both the TR-ß1 and TR-ß2 isoforms found in the anterior pituitary and other regions of the brain would carry this mutation. Previous studies have extensively explored the function of the TR-ß1 isoform on a number of natural or artificial thyroid hormone response elements (5, 6, 8, 26, 27, 33, 34, 35) and concluded that most PRTH mutants would be expected to cause a mild form of RTH, which could not be distinguished from GRTH in in vitro studies. The role of the TR-ß2 isoform in the genesis of PRTH is less well known even though it is highly expressed in the anterior pituitary. We propose that PRTH mutants function as dominant inhibitors of thyroid hormone action in the TR-ß2 isoform, but not the TR-ß1 isoform, and RTH caused by these mutations is therefore limited to tissues that express this isoform, most notably the anterior pituitary and hypothalamus.

In order for PRTH to represent a distinct clinical and biochemical entity, PRTH mutants should have the following two in vitro characteristics: 1) lack of dominant negative activity on genes expressed in peripheral tissues; and 2) significant dominant negative activity on genes involved in regulating TSH secretion. Three of the PRTH mutants have these characteristics: R338L, R338W, and R429Q. Each has weak dominant negative activity on positive TREs found in peripheral tissues via the TR-ß1 isoform expressed in those tissues. This is in part due to the fact that these mutations do not interfere with T3 binding. Two PRTH mutations (V349M and I431T), in contrast, behaved biochemically like GRTH mutations on these elements. In addition, R338L, R338W, and R429Q are all strong dominant inhibitors on the TRH and common {alpha}-subunit genes in the TR-ß2, but not the TR-ß1, isoform. These PRTH mutants should, however, lack dominant negative activity on other genes negatively regulated by T3 in the periphery (e.g. myosin heavy chain ß), since TR-ß2 is not expressed in peripheral tissues. Thus, TR-ß isoforms mediate negative T3 regulation via different mechanisms. The TR-ß1 isoform acts by an RXR-dependent pathway in which the PRTH mutants have little effect in antagonizing normal T3 inhibition by TR-ß1. On the other hand, the TR-ß2 isoform acts through a distinct pathway, which is not dependent on cotransfected RXR, such that PRTH mutants, as TR-ß2 isoforms, exert significant dominant negative activity against normal T3 inhibition by either isoform.

GRTH mutants, by contrast, interfere with WT TR function as either TR-ß1 or TR-ß2 isoforms. Both {Delta}337T and P453H have significant dominant negative activity against pTREs as TR-ß1 isoforms and negative TREs via either isoform. On certain TREs (F2, and TRH), {Delta}337T was a stronger dominant inhibitor than P453H, consistent with previous reports. In addition, {Delta}337T was a superior competitor on negative TREs (TRH and common {alpha}-subunit, Fig. 6Go) as a TR-ß1 vs. TR-ß2 isoform, in contradistinction to PRTH mutants. These data suggest that GRTH mutants can act either through the TR-ß1 or TR-ß2 pathway to cause central resistance to thyroid hormone.

Attempts have been made to correlate phenotype with location of mutation and/or T3-binding capacity of the mutant receptor (5, 7, 36). Previous authors have been unable to use these receptor characteristics to distinguish RTH syndromes. Our data confirm and extend these observations. Mutations found in either major hot spot caused clinical PRTH and have similar biochemical characteristics (R338L and R338W vs. R429Q) in our study. In addition, each of these PRTH mutations preserved near-normal T3 binding as did the V349M mutation, which biochemically resembles a GRTH mutation. Our data suggest that tissue-specific expression of TR-ß isoforms, coupled with a distinct effect of PRTH mutants on T3 inhibition via the TR-ß2 isoform, explains PRTH syndrome.

How PRTH mutants selectively alter the function of the TR-ß2 isoform is unclear. Electrophoretic mobility shift assay studies reveal that R338L, R338W, and R429Q are all homodimer deficient but heterodimerize with RXR normally on both TRH and DR+4 elements as either isoform, while V349M and I431T both homo- and heterodimerize on these elements (data not shown and Ref.25). Perhaps the TR homodimer is critical for T3 inhibition and mutations that disrupt homodimerization cause PRTH. This is unlikely because TR homodimers are not thought to be important for negative T3 regulation of the common {alpha}-subunit gene. In addition, it would not explain why PRTH mutants, only as TR-ß2 isoforms, act as dominant negative inhibitors on the TRH and common {alpha}-subunit genes. Alternatively, the homodimer defect may be a marker for a conformation change in the receptor that causes PRTH via the TR-ß2 isoform. Finally, the homodimer defect may be unrelated to the pathogenesis of the PRTH syndrome.

In summary, certain TR-ß mutations cause PRTH by converting the centrally expressed TRß2 isoform into a dominant inhibitor of genes that regulate TSH secretion. Importantly, these mutations do not significantly alter T3 binding or the function of the TR-ß1 isoform on thyroid hormone-responsive genes, thus limiting resistance to only those tissues that express the TR-ß2. We, therefore, propose the following model (Fig. 9Go) to explain our findings and to provide a molecular framework for future study of GRTH and PRTH syndromes. Two discrete pathways mediate negative regulation of gene expression by thyroid hormone in the anterior pituitary and hypothalamus. PRTH mutants interfere with the TR-ß2 pathway to cause selective resistance in these tissues, inappropriate TSH secretion, and elevated thyroid hormone levels. GRTH mutants interfere with both pathways, causing both central and peripheral resistance to thyroid hormone on negative TREs.



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Figure 9. Model of RTH Syndromes Based on the Location of TR-ß Isoforms

Both TR-ß isoforms (open circles, TR-ß1; cross-hatched circles, TR-ß2) are expressed in the anterior pituitary and hypothalamus (upper panel), while the TR-ß1 isoform is the predominant form expressed in peripheral tissues (lower panel). Transcription of the TRH and TSH subunit genes is stimulated by the unliganded TR (thick arrow), and inhibited by T3 binding to TR (dashed arrow) via negative thyroid hormone response elements (nTREs). Transcription in peripheral tissues is either stimulated or inhibited by T3. Shown in the model is a positive TRE (pTRE). Dominant negative activity by GRTH mutants, and lack of activity by PRTH mutants, as TR-ß1 isoforms on genes expressed in the periphery and negatively regulated by T3 (e.g. MHC-ß) is inferred from this study but was not directly tested. TRs with GRTH mutations usually bind T3 poorly, while TRs with PRTH mutations usually bind T3 well (shown in this figure), but there are exceptions to this generalization. The orientation and DNA binding of RXR on negative TREs, unlike pTREs, are unknown and are illustrated in this way for conceptual reasons only.

 

    MATERIALS and METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 
The following naturally occurring mutations were evaluated: the GRTH mutations {Delta}337T and P453H (3, 22), the PRTH mutation R429Q (25), and the putative PRTH mutations R338L, R338W, V349M, and I431T (11) (Fig. 1Go). Mutations chosen included two that bind T3 poorly ({Delta}337T, I431T) and five that bind T3 in the same order of magnitude as WT (R338L, R338W, V349M, R429Q, P453H) to remove poor binding as an explanation for observed phenomena. Site-directed mutagenesis (CLONTECH, Palo Alto, CA) was used to create the mutations in the context of the human TR-ß1 isoform (37). All mutations were confirmed by DNA sequencing of the TR-ß1 C terminus. The common region of TR-ß was transferred into either the TR-ß1 or TR-ß2 expression vector as a PstI/BamHI fragment forming the mutant receptor isoforms. The expression vector used in this study was pSG5, containing either WT or mutant TR-ß cDNAs as EcoRI fragments. The human TR-ß1 cDNA was used as the WT TR-ß1. The WT TR-ß2 was fashioned by inserting the amino terminus of the rat TR-ß2 as an EcoRI/SacI fragment into the TR-ß1 cDNA in place of the amino terminus of the TR-ß1. The human RXR-{alpha} as an EcoRI fragment was inserted into the same expression vector (38). Expression vector plasmid preparations used in this study were carefully quantitated by agarose gel electrophoresis. To confirm the integrity and quality of each expression vector, plasmid DNA preparation, in vitro translation with [35S]methionine, was performed using T7 polymerase, and the products were analyzed by SDS-PAGE.

Reporter constructs included two copies of idealized pTREs: palindrome (Pal, Ref.11), inverted palindrome (chicken lysozyme F2, Ref.39), and direct repeat with a 4-bp interval (DR+4, Ref.11). The negative response elements included the 5'-flanking sequences for the TRH and the common glycoprotein {alpha}-subunit gene (5, 31). In the case of the positive reporters, constructs contained the pTRE element fused upstream of a -109 thymidine kinase promoter and the luciferase gene to measure activity. The negative reporter constructs included their own promoters and were also fused upstream of the luciferase gene. The luciferase reporter gene was derived from pSV0 and contains two transcriptional stop sequences upstream of the promoter to prevent read-through transcription. The reporter was documented to have neither positive nor negative thyroid hormone responses in the absence of response elements.

Transfections were performed in CV-1 cells, which are relatively RXR deficient (29, 30), and thereby provided an opportunity to examine the importance of limiting concentrations of RXR. Each transfection included a WT TR-ß expression vector (100 ng), a mutant or WT TR-ß expression vector (100 ng or 1 µg), an RXR-{alpha} expression vector except where indicated (100 ng), and a response element reporter construct (10 µg). Cell cultures were transfected using a calcium-phosphate precipitation method, and precipitate was applied for 16 h in DMEM containing 10% FBS, glutamine, and appropriate antimicrobials. The next day, medium was removed and DMEM containing both anion exchange and charcoal-stripped FBS (10%) was added ± T3. Most of the experiments used 2.5 nM T3, although other concentrations were used as noted. Data were pooled from at least three independent experiments and displayed as mean ± SE.

Gel-shift studies were performed with proteins derived from a coupled in vitro transcription/translation reaction in reticulocyte lysate. Three to four microliters of either TR-ß1, TR-ß2, or RXR-{alpha} lysate were used in binding studies on either a radiolabeled Dr+4 or TRH element according to published methods (31).


    ACKNOWLEDGMENTS
 
We would like to thank Dr. Steve Usala for certain mutant TRs, Dr. Larry Jameson for the common {alpha}-glycoprotein subunit gene promoter, Dr. Mitchell Lazar for the rat TR-ß2 cDNA, and Dr. Ronald Evans for the RXR-{alpha} cDNA.


    FOOTNOTES
 
Address requests for reprints to: Fredric E. Wondisford, M.D., Thyroid Unit, Beth Israel Hospital, Research North, Room 330C, 99 Brookline Avenue, Boston, Massachusetts 02215.

This work was supported by NIH Grants DK-02423 (to J.D.S), DK-02354 (to A.N.H.), and DK-43653 and DK-49126 (to F.E.W.) and the McLaughlin Foundation (to M.F.L.).

Received for publication August 2, 1996. Revision received October 9, 1996. Accepted for publication October 10, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS and METHODS
 REFERENCES
 

  1. Usala SJ, Bale AE, Gesundheit N, Weinberger C, Lash RW, Wondisford FE, McBride OW, Weintraub BD 1988 Tight linkage between the syndrome of generalized thyroid hormone resistance and the human c-erbA beta gene. Mol Endocrinol 2:1217–1220[Abstract]
  2. Sakurai A, Takeda K, Ain K, Ceccarelli P, Nakai A, Seino S, Bell GI, Refetoff S, Degroot LJ 1989 Generalized resistance to thyroid hormone associated with a mutation in the ligand-binding domain of the human thyroid hormone receptor ß. Proc Natl Acad Sci USA 86:8977–8981[Abstract]
  3. Usala SJ, Tennyson GE, Bale AE, Lash RW, Gesundheit N, Wondisford FE, Accili D, Hauser P, Weintraub BD 1990 A base mutation of the c-erbAß thyroid hormone receptor in a kindred with generalized thyroid hormone resistance. J Clin Invest 85:93–100[Medline]
  4. Refetoff S, Weiss RE, Usala SJ 1993 The syndromes of resistance to thyroid hormone. Endocr Rev 14:348–399[Medline]
  5. Gershengorn MC, Weintraub BD 1975 Thyrotropin-induced hyperthyroidism caused by selective pituitary resistance to thyroid hormone. A new syndrome of inappropriate secretion of TSH. J Clin Invest 56:633–642[Medline]
  6. Chatterjee VK, Nagaya T, Madison LD, Datta S, Rentoumis A, Jameson JL 1991 Thyroid hormone resistance syndrome. Inhibition of normal receptor function by mutant thyroid hormone receptors. J Clin Invest 87:1977–1984[Medline]
  7. Yen PM, Darling DS, Carter RL, Forgione M, Umeda PK, Chin WW 1992 Triiodothyronine (T3) decreases binding to DNA by T3-receptor homodimers but not receptor-auxiliary protein heterodimers. J Biol Chem 267:3565–3568[Abstract/Free Full Text]
  8. Nagaya T, Eberhardt NL, Jameson JL 1993 Thyroid hormone resistance syndrome: correlation of dominant negative activity an location of mutations. J Clin Endocrinol Metab 77:982–990[Abstract]
  9. Zavacki AM, Harney JW, Brent GA, Larsen PR 1993 Dominant negative inhibition by mutant thyroid hormone receptors is thyroid hormone response element and receptor isoform specific. Mol Endocrinol 7:1319–1330[Abstract]
  10. Beck-Peccoz P, Chatterjee VK, Chin WW, DeGroot LJ, Jameson JL, Nakamura H, Refetoff S, Usala SJ, Weintraub BD 1994 Nomenclature of thyroid hormone receptor beta gene mutations in resistance to thyroid hormone. First workshop on thyroid hormone resistance, July 10–11, 1993, Cambridge, UK. J Endocrinol Invest 17:283–287[Medline]
  11. Beck-Peccoz P, Chatterjee VK 1994 The variable clinical phenotype in thyroid hormone resistance syndrome. Thyroid 4:225–232[Medline]
  12. Adams M, Matthews C, Collingwood, TN, Tone Y, Beck-Peccoz P, Chatterjee VK 1994 Genetic analysis of 29 kindreds with generalized and pituitary resistance to thyroid hormone. J Clin Invest 94:506–515[Medline]
  13. Parrilla R, Mixson AJ, McPherson JA, McClaskey JH, Weintraub BD 1991 Characterization of seven novel mutations of the c-erbAß gene in unrelated kindreds with generalized thyroid hormone resistance. Evidence for two "hot spot" regions of the ligand binding domain. J Clin Invest 88:2123–2130[Medline]
  14. Takeda K, Weiss RE, Refetoff S 1992 Rapid localisation of mutations in the thyroid hormone receptor ß gene by denaturing gradient gel electrophoresis in 18 families with thyroid hormone resistance. J Clin Endocrinol Metab 74:712–719[Abstract]
  15. Behr M, Loos U 1992 A point mutation (Ala 229 to Thr) in the hinge domain of the c-erbAß thyroid hormone receptor gene in a family with generalized thyroid hormone resistance. Mol Endocrinol 6:1119–1126[Abstract]
  16. Onigata K, Yagi H, Sakuri A, Nagashima T, Nomura Y, Nagashima K, Hashizume K, Morikawa A 1995 A novel point mutation (R243Q) in exon 7 of the c-erbA beta thyroid hormone receptor gene in a family with resistance to thyroid hormone. Thyroid 5:355–358[Medline]
  17. Hodin RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen PR, Moore DD, Chin WW 1989 Identification of a thyroid hormone receptor that is pituitary specific. Science 244:76–79[Medline]
  18. Wood WM, Ocran KW, Gordon DF, Ridgway EC 1991 Isolation and characterization of mouse complementary DNAs encoding alpha and beta thyroid hormone receptors from thyrotrope cells: the mouse pituitary specific beta 2 isoform differs at the amino terminus from the corresponding species from rat pituitary tumor cells. Mol Endocrinol 5:1049–1061[Abstract]
  19. Lazar M 1993 Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14:184–193[Medline]
  20. Cook CB, Kakucska I, Lechan RM, Koenig RJ 1992 Expression of thyroid hormone receptor ß2 in rat hypothalamus. Mol Cell Endocrinol 130:1077–1079
  21. Li M, Boyages SC 1996 Detection of extended distribution of ß2-thyroid hormone receptor messenger ribonucleic acid (RNA) in adult rat brain using complementary RNA in Situ hybridization histochemistry. Endocrinology 137:1272–1275[Abstract]
  22. Ng L, Forrest D, Haugen BR, Wood WM, Curran T 1995 N-terminal variants of thyroid hormone receptor ß: differential function and potential contribution to syndrome of resistance to thyroid hormone. Mol Endocrinol 9:1202–1213[Abstract]
  23. Usala SJ, Menke JB, Watson TL, Wondisford FE, Weintraub BD, Berard J, Bradley WEC, Ono S, Mueller OT, Bercu BB 1991 A homozygous deletion in the c-erbAß thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor. Mol Endocrinol 5:327–335[Abstract]
  24. Mixson AJ, Renault JC, Ransom S, Bodenner DL, Weintraub BD 1993 Identification of a novel mutation in the gene encoding the beta-triiodothyronine receptor in a patient with apparent selective pituitary resistance to thyroid hormone. Clin Endocrinol (Oxf) 38:227–234[Medline]
  25. Sasaki S, Nakamura H, Tagami T, Miyoshi Y, Nogimori T, Mitsma T, Imura H 1993 Pituitary resistance to thyroid hormone associated with a base mutation in the hormone binding domain of the human 3,5,3'-triiodothyronine receptor beta. J Clin Endocrinol Metab 76:1254–1258[Abstract]
  26. Flynn TR, Hollenberg AN, Cohen O, Menke JB, Usala SJ, Tollin S, Hegarty MK, Wondisford FE 1994 A novel C-terminal domain in the thyroid hormone receptor selectively mediates thyroid hormone inhibition. J Biol Chem 269:32713–32716[Abstract/Free Full Text]
  27. Crino A, Borrelli P, Salvatroi R, Cortelazzi, Roncoroni R, Beck-Peccoz P 1992 Anti-iodothyronine autoantibodies in a girl with hyperthyroidism due to pituitary resistance to thyroid hormones. J Endocrinol Invest 15:113–120[Medline]
  28. Schwartz HL, Lazar MA, Oppenheimer JH 1994 Widespread distribution of immunoreactive thyroid hormone ß2 receptor (Trß2) in the nuclei of extrapituitary rat tissues. J Biol Chem 269:24777–24782[Abstract/Free Full Text]
  29. Meier CA, Dickstein BM, Ashizawa K, McClaskey JH, Muchmore P, Ransom SC, Menke JB, Hao EH, Usala SJ, Bercu BB, Cheng SY, Weintraub BD 1992 Variable transcriptional activity and ligand binding of mutant ß1 3,5,3'-triiodothyronine receptors from four families with generalized resistance to thyroid hormones. Mol Endocrinol 6:248–258[Abstract]
  30. Collinwood TN, Adams M, Tone Y, Chatterjee VKK 1994 Spectrum of transcriptional, dimerization, and dominant negative properties of twenty different mutant thyroid hormone ß receptors in thyroid hormone resistance syndrome. Mol Endocrinol 8:1262–1277[Abstract]
  31. Hayashi Y, Sunthornthepvrakul T, Refetoff S 1994 Mutations of CpG dinucleotides located in the triiodothyronine (T3)-binding domain of the thyroid hormone receptor (TR) ß gene that appears to be devoid of natural mutations may not be detected because they are unlikely to produce the clinical phenotype of resistance to thyroid hormone. J Clin Invest 94:607–615[Medline]
  32. Lehmann JM, Jong L, Fanjul A, Cameron JF, Lu XP, Haefner P, Dawson MI, Pfahl M 1992 Retinoids selective for retinoid X receptor response pathways. Science 258:1944–1946[Medline]
  33. Zhang X, Lehmann J, Hoffmann B, Dawson MI, Cameron J, Graupner G, Hermann, Tran P, Pfahl M 1992 Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid. Nature 358:587–591[CrossRef][Medline]
  34. Hollenberg AN, Monden T, Flynn TR, Boers ME, Cohen O, Wondisford FE 1995 The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements. Mol Endocrinol 9:540–550[Abstract]
  35. Hsu JH, Zavacki AM, Harney JW, Brent GA 1995 Retinoid-X receptor (RXR) differentially augments thyroid hormone response in cell lines as a function of the response element and endogenous RXR content. Endocrinology 136:421–430[Abstract]
  36. Banahmad A, Tsai SY, O’Malley BW, Tsai MJ 1992 Kindred S thyroid hormone receptor is an active and constitutive silencer and a repressor for thyroid hormone and retinoic acid responses. Proc Natl Acad Sci USA 89:10633–10637[Abstract]
  37. Nagaya T, Jameson JL 1993 Thyroid hormone receptor dimerization is required for dominant negative inhibition by mutations that cause thyroid hormone resistance. J Biol Chem 268:15766–15771[Abstract/Free Full Text]
  38. Hao E, Menke JB, Smith AM, Jones C, Geffner ME, Hershman JM, Wuerth JP, Samuels HH, Ways DK, Usala SJ 1994 Divergent dimerization properties of mutant ß1 thyroid hormone receptors are associated wit different dominant negative activities. Mol Endocrinol 8:841–851[Abstract]
  39. Hayashi Y, Weiss RE, Sarne DH, Yen PM, Sunthornthepvarakul T, Marcocci C, Chin WW, Refetoff S 1995 Do clinical manifestations of resistance to thyroid hormone correlate with the functional alteration of the corresponding mutant thyroid hormone-ß receptors? J Clin Endocrinol Metab 80:3246–3256[Abstract]
  40. Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A gene encodes a thyroid hormone receptor. Nature 324:641–646[Medline]
  41. Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM 1990 Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 345:224–229[CrossRef][Medline]
  42. Baniahmad A, Steiner C, Kohne AC, Renkawitz R 1990 Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site. Cell 61:505–514[Medline]