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Clinical Studies |
Departments of Pediatrics (H.Y., J.P., S.R.) and Medicine (Y.H., S.R.), and the J. P. Kennedy, Jr., Mental Retardation Research Center (S.R.), University of Chicago, Chicago, Illinois 60637; and the Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University (Y.H.), Nagoya; and the Department of Geriatrics, Endocrinology, and Metabolism, Shinshu University School of Medicine (A.S.), Matsumoto, Japan
Address all correspondence and requests for reprints to: Samuel Refetoff, M.D., University of Chicago (MC3090), 5841 South Maryland Avenue, Chicago, Illinois 60637-1470.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We recently identified two families in which RTH was associated with the mutations R243Q3 and R243W in the TRß gene. (7, 8). However, the recombinant mutant TRßs 243Q and 243W caused no significant impairment of T3 binding affinity despite the relatively severe clinical manifestations of RTH associated with these mutant TRßs. As a consequence, the molecular mechanism by which they cause RTH is unclear. Thus, we examined other functional properties of these mutant TRßs.
The results show that 243Q and 243W translocate to the nucleus, where they exert a normal ligand-independent repression and have impaired transactivation and DNE that are more potent than those of a mutant TRß 320H with impaired T3 binding. These mutant TRßs require larger concentrations of T3 to prevent the formation of mutant TRß homodimers on thyroid response elements (TRE). Our data suggest that the substitution of R243 in the WT TRß produces RTH by increasing the propensity for the formation of tightly bound homodimers or by reduction of the receptor affinity for T3 only after it binds to DNA.
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Subjects and Methods |
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Data from 26 unaffected normal controls and 23 patients with RTH were used for the correlation of serum free T4 and TSH concentrations. Seventeen of the 23 subjects with RTH were heterozygous for the TRß mutation R320H and belonged to 4 unrelated families (F67, F95, F136, and F165) (9, 10, 11, 12),4 2 were heterozygous for the R243Q mutation (7), 2 were heterozygous for the R243W mutation (8), and 2 were heterozygous for the G345R mutation (F44) (13). When determinations on more than 1 serum sample obtained at different times from the same individual were available, results were averaged. Free T4 values are expressed as a percentage of the upper limit of normal, as previously described (5, 14).
Cell culture
Simian virus 40-transformed African green monkey kidney fibroblasts (COS-7) and human hepatoblastoma (HepG2) cell lines were maintained and propagated in DMEM supplemented with 10% FBS (Life Technologies, Gaithersburg, MD) at 37 C under 100% humidity in 90% room air and 10% CO2.
Construction of the plasmids
A plasmid expressing the WT TRß was constructed and subcloned
into the mammalian expression vector, pcDNAI/Amp (Invitrogen, San
Diego, CA). The mutant TRßs were constructed by either site-directed
mutagenesis (R243Q and R320H) using pSelect (Promega Corp., Madison,
WI) or by swapping PCR-amplified DNA fragments containing the mutations
(R243W and G345R) with the corresponding DNA fragments in the
pcDNAI/Amp plasmid containing the WT TRß insert. These methods have
been previously described in detail (13, 15). Methods for construction
of the reporter vectors, TRE Pal x3-Luc and F2 x3-Luc, have also been
described (15). These reporters are driven by three copies of TRE-pal
(AGGTCA-TGACCT) and TRE-F2 (ttatTGACCCcagctgAGGTCAagttacg),
respectively, fused to the herpes simplex virus thymidine kinase
promoter. The sequences of all PCR-amplified fragments inserted into
vectors were verified by sequencing. Human retinoid X receptor (RXR)
, cDNA was obtained from R. M. Evans and transferred into pcDNAI/Amp
expression vector.
Expression of TRßs
For T3 binding, TRßs were expressed in COS-7 cells and in reticulocyte lysate as described previously (5). Briefly, COS-7 cells were transfected with 10 µg pcDNA/Amp-TRß and 10 µg carrier DNA (Bluescript, Stratagene, La Jolla, CA) by the calcium phosphate coprecipitation method. After overnight incubation in medium containing 10% T3-stripped FBS (16) at 5% rather than 10% CO2, cells were washed twice with HBSS (Life Technologies) and further incubated for 72 h in the same culture conditions. Cells were harvested, and whole cell extracts were prepared. Alternatively, TRßs were synthesized by in vitro transcription and translation using T7-coupled TNT lysate (Promega Corp.).
For in vitro functional analysis of TRßs, expression vectors were transiently transfected into HepG2 cells. Cells were transferred to 12-well plastic plates and cultured for 24 h in DMEM containing T3-stripped FBS in the conditions described above. Twenty-four hours later, cells in each well were transfected with 1 µg of the reporter vector, 25 ng of the various TRß expression vectors alone or in combination with an equal amount of the WT TRß vector, and enough carrier plasmid DNA to adjust the total amount of DNA in the calcium phosphate precipitate to 2 µg. Cells were incubated for 16–20 h with DNA-calcium phosphate coprecipitate, washed twice with HBSS, and then incubated for an additional 48 h with the complete medium containing T3-stripped FBS in the absence or presence of different amounts of T3. To asses the level of ligand-independent repression, cells were transfected with 1 µg of the reporter vector, 0.1 µg of the various TRß expression vectors or carrier plasmid DNA, and 0.9 µg of pSV40 ß-galactosidase (Promega Corp.). Cells were handled as described above, but without the addition of T3 to the medium.
For the intracellular localization of expressed TRßs by immunocytochemistry, COS-7 cells were transferred onto four-chamber microscope slides and transfected with 1 µg of each TRß expression vector using the calcium phosphate precipitate method described above. Twenty-four hours after transfection, cells were processed as described below.
For electrophoresis mobility shift assays (EMSA), TRßs and human
RXR were synthesized by transcription coupled translation using TNT
lysate (Promega Corp.).
T3 binding to TRßs assessed by displacement analysis
T3 binding affinity was determined by the filter binding assay (17) and the anion exchange method (18) to separate TRß-bound from free T3. Briefly, 1–3 µL COS-7 cell extract or reticulocyte lysate containing transcribed TRßs were incubated at 4 C for 18 h with 14 fmol [125I]T3 (DuPont, Boston, MA; 2200 Ci/mmol) in the presence of 0–7000 fmol unlabeled T3 (Sigma Chemical Co., St. Louis, MO). The protein-bound fractions were collected onto nitrocellulose membranes, or the free T3 was adsorbed onto 200- to 400-mesh Dowex 1-X8 Cl- (Bio-Rad, Richmond, CA) resin before determination of 125I activity. Scatchard analysis was used to determine the T3 association constant (Ka). In independent binding assays, the Ka values of the mutant TRßs were normalized by dividing their value by that of the WT TRß Ka that was determined with each assay. Two determinations were carried out in the presence of twice the amount of in vitro synthesized RXR. Values are expressed as the mean ± SD Ka mutant/Ka WT obtained from four to eight independent binding assays, and the statistical significance of differences was analyzed by Student’s t test.
In vitro functional analysis of WT and mutant TRßs
After transfection into HepG2 cells and incubation for 48 h in medium containing various concentrations of T3, the culture medium was removed, the cells were lysed, and an aliquot of the lysate was assayed for luciferase activity using luciferase assay reagents (Promega Corp.) as previously described (5). Individual data points are either means of duplicates or the mean ± SD for transfections of six culture wells under identical conditions. Data are expressed as the fold induction by T3, which is the multiple of the baseline level of luciferase activity in the absence of T3, or as a percentage of the luciferase activity measured in cells expressing the reporter alone and corrected for transfection efficiency using the ß-galactosidase assay (Promega Corp.).
Immunocytochemistry
Immunocytochemisty was carried out as previously described (19). Briefly, after transfection, cells were fixed with 1% paraformaldehyde and then permeabilized with 0.1% Triton X-100. Slides were blocked with normal goat serum and incubated with rabbit antibody specific for the N-terminal domain of TRß1 protein (20). This was followed by incubation with goat antirabbit fluorescein-conjugated antibody. Controls included mock-transfected cells and incubation with nonspecific rabbit IgG processed in the same manner.
EMSA
TRs and RXR, synthesized in vitro as described above,
were incubated for 10 min in 20 mmol/L HEPES (pH 7.5), 50 mmol/L KCl, 1
mmol/L ethylenediamine tetraacetate, 10% glycerol, and 50 µg/mL
poly(dI-dC) (Pharmacia, Piscataway, NJ) in the absence or presence of
various concentrations of T3. Inverted palindromic (lap)
TRE (agcttTGACCTgacgtcAGGTCAc; 1 x 10-5 cpm), end
labeled with [-32P]CTP using Klenow, was added and
incubated for another 15 min. The DNA-protein mixture was run, at room
temperature, through 5% PAGE (polyacrylamide-bisacrylamide, 37.5:1)
containing 2.5% glycerol using 0.5 x Tris-borate-ethylenediamine
tetraacetate buffer. Gels were dried and exposed to X-AR film (Eastman
Kodak, Rochester, NY). For quantitative analysis, gels were analyzed by
Molecular Imager GS-363 (Bio-Rad).
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Results |
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TRß affinity for T3
Results were not different with the two methods of separation of
bound from free T3 or whether the receptors were
synthesized in transfected COS-7 cells or in reticulocyte lysate in the
absence and presence of RXR. Thus, data from eight independent assays
were combined and submitted to statistical analysis. As previously
reported (7), the slight reduction in the mean Ka
of 243Q failed to reach statistical significance (Table 1
). We now report similar results with the TRß 243W, a
mutation in the same codon reported in a family with RTH (8). Addition
of RXR did not affect the binding affinity for T3. The
mean ± range of duplicate Ka determinations
without and with RXR, respectively, were 0.98 ± 0.10 x
1010 and 1.04 ± 0.12 x 1010 for the
WT TRß, and 0.83 ± 0.18 x 1010 and 0.90
± 0.20 x 1010 for TRß 243Q. In contrast, the
mutant TRßs 320H and 345R showed significant impairment of
T3 binding.
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The sensitivity of the pituitary thyrotrophs to thyroid hormone
was used to assess the relative severity of RTH in vivo.
This was achieved by correlating the serum free T4
concentration to that of TSH and by determination of the thyrotroph
T4 resistance index, which is the product of the free
T4 and TSH values. Both methods quantitate the sensitivity
of the thyrotrophs to the feedback regulation by thyroid hormone. As
shown in Fig. 1, all subjects with RTH had higher serum
free T4 levels for the corresponding TSH concentrations.
However, heterozygous individuals for the mutant TRßs R243Q and R243W
had relatively higher free T4 levels than subjects
expressing R320H despite the lower affinity of 320H for T3.
The mean ± SD thyrotroph T4 resistance
index was 664 ± 231 for the combined individuals with R243Q and
R243W compared to 308 ± 138 for those with R320H
(P < 0.001). The mean thyrotroph T4
resistance index in normal individuals of 136 ± 73 was
significantly lower than the corresponding value in all subjects with
RTH (P < 0.0001).
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Amino acid 243 is located in the hinge or D domain of the receptor
molecule, a region that interacts with nuclear localization proteins
(21). Thus, we examined the possibility of defective nuclear targeting
by immunocytochemistry using an antibody specific for the human TRß1.
As shown in Fig. 2, the mutant TRßs transfected into
COS-7 cells were expressed and transferred into the nucleus as well as
the WT TRß. Furthermore, there was no difference in the total binding
capacity, which ranged from 90–125 fmol/106 cells.
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The functional properties of the mutant TRßs were examined in cultured cells. Cells were transfected with vectors expressing the WT TRß and each of the mutant TRßs, and their basal and T3-dependent transactivation functions were determined using the reporter constructs TRE-F2 (inverted palindrome) and TRE-pal (15).
Transfection of the WT TRß and mutant TRßs 243Q and 243W produced a
similar ligand-independent repression of the basal expression of both
reporter constructs transfected into HepG2 cells (Fig. 3).
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, T3-dependent
transactivation regulated by TRE-F2 was more severely impaired by the
mutant TRß 243Q than 320H, and 243Q showed a stronger DNE, which was
evident at 1 and 10 nmol, but not at 100 nmol T3. These
properties were also tested using TRE-pal with 10 nmol T3
and in sextuplicate to generate data for statistical analysis. As
depicted in Fig. 5A, the mutant TRßs 243Q and 243W
showed impaired T3-dependent transactivation (16 ±
2% and 13 ± 3%, respectively) compared to the WT TRß and also
significantly reduced function relative to that of the TRß 320H
(50 ± 8%). However, the transactivation function of TRß 345G
was more severely impaired (<5%) than that of TRßs 243Q and 243W.
These results correlate well with the clinical severity of RTH and the
relative resistance of the pituitary thyrotrophs to thyroid hormone
depicted in Fig. 1
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). These results are in agreement with
the nuclear localization studies, as a faulty nuclear targeting of
TRßs 243Q and 243W should have diminished, if not abrogated, their
DNE on the WT TRß. Protein and DNA interactions
The ability of the mutant TRßs to form homodimers and
heterodimers bound to TRE lap (inverted palindrome) in the presence of
various amounts of T3 was tested using EMSA. The WT TRß
as well as the mutant TRßs (320H, 243Q, and 243W) bound to the TRE as
homodimers and T3 reduced homodimer formation in a
dose-dependent manner (Fig. 6). However, the relative
potency of T3 to produce this effect was WT >
320H > 243Q = 243W (Figs. 6 and 7A).
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(Fig. 6B). However, concomitant binding as
homodimer in the presence of 100 nmol/L T3, determined from
the ratio of homodimer/heterodimer quantitated by densitometry, was
greater for TRß 243Q than 320H and was the lowest for the WT TRß
(Fig. 7). These results, as previously reported by us (22) and others
(23, 24) for different mutant TRßs, indicate that preferential
formation of homodimers correlates with the DNE and that the reduced
potency of T3 to dissociate them roughly correlates with
the clinical severity of RTH, as assessed by the sensitivity of the
thyrotrophs to T3. |
Discussion |
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Sixty-two different mutations, excluding the two under investigation
herein, have been now identified in 102 families expressing the RTH
phenotype (2, 30). With the exception of three, all are located in
areas functionally relevant for T3 binding to TRß and are
clustered in two regions of the ligand-binding domain (11, 14). The
three exceptions are a complete deletion of the TRß gene resulting in
recessive inheritance of RTH (31) and two mutations in the hinge region
of the TRß [A234T (see Footnote 1) and V264D] (11, 32). However,
the latter mutations produce an impairment of T3 binding.
This contrasts with the mutations in the hinge region investigated
herein that failed to produce a significant alteration in
T3 binding. Indeed, based on a recent analysis of the TR
crystal structure, amino acid 243, which is located at the junction of
-helix 2 and ß-strand 1, is not in direct contact with
T3 (33). Yet, affected subjects showed all the
characteristic findings of RTH, including goiter, elevated levels of
free T4 and T3 with nonsuppressed TSH, and no
clinical or laboratory manifestations of thyroid hormone excess.
Furthermore, RTH at the level of the thyrotrophs was more severe than
that in individuals with R320H, a mutation with more than 50%
reduction in T3 binding affinity. Thus, the mechanism
involved in the clinical manifestation of RTH was not readily apparent.
It should be noted that, in general, the clinical severity of RTH
correlates with the degree of impairment of T3 binding to
the mutant TRß (5, 34). Exceptions to this rule involve mutant TRßs
with severe impairment of T3 binding despite mild clinical
manifestations of RTH that have been attributed to reduced potency of
the DNE (5, 23, 24).
The present studies demonstrate that impaired T3-mediated transactivation is involved in the mediation of RTH in subjects expressing R243Q and R243W. The defect in this process was more marked with the mutant TRß 243Q and 243W than with 320H and is, thus, in agreement with the relatively greater degree of resistance to T3 observed clinically. Several hypotheses were considered to explain this finding based on current information concerning the mode of thyroid hormone action: 1) decreased nuclear localization, 2) inability of T3 to produce conformational changes in the TR necessary for the induction of transactivation, 3) prevention of T3 binding when the mutant TRß is associated with DNA, 4) impaired interaction with cofactors, and 5) impaired ability of T3 to dissociate a corepressor that binds to the hinge domain two amino acids upstream of R243 (35, 36, 37).
We have ruled out a defect in nuclear localization by the
demonstration of unimpaired nuclear targeting of the mutant receptors
expressed in COS-7 cells. This conclusion is supported by the basal
repression and DNE of the mutant receptors, as this effect requires
entry of the receptor into the nucleus. Our results cannot exclude
faulty conformational changes in the receptor and tight association of
the mutant receptor with a corepressor. The EMSA results, showing that
larger concentrations of T3 were required to prevent the
formation of mutant TRß homodimers, can be due to a tighter
interaction with DNA or impaired T3 binding when the
receptors are associated with DNA. A higher ratio of homodimer to
heterodimer formation with RXR in the presence of 100 nmol
T3 also supports this conclusion.
Our results are in agreement with the observation that preferential formation of homodimers, with reduced T3-mediated dissociation, correlates with the DNE as well as the relative clinical severity of RTH (22, 23, 24). It remains unclear whether the substitution of R243 increases the propensity for the formation of tightly bound homodimers or reduces the receptor affinity for T3 only after it binds to DNA.
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Acknowledgments |
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cDNA, and to Drs. L. H. Schwartz and J. H. Oppenheimer for
provision of the TRß antiserum. We thank Dr. Graeme I. Bell, Kenneth
S. Polonsky, and Roy E. Weiss for revision of the manuscript. |
Footnotes |
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2 Supported by a Japan Society for the Promotion of Science Research
Fellowship for Young Scientists.
3 Mutant TRßs are identified using single letter
amino acid code. Wild-type amino acid precedes and the substituted
amino acid follows the codon number, corrected as recommended (6 ). Both
appear to indicate heterozygotes.
4 Families are identified by "F" number as
listed in the registry of patients with RTH, the content of which and
mode of accession have been published (3 ).
Received July 25, 1996.
Revised December 11, 1996.
Accepted January 17, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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1 specifies positive and negative transcriptional
regulation functions. J Biol Chem. 268:2021–2028.
4 activation domain of the thyroid
hormone receptor is required for release of a putative corepressor(s)
necessary for transcriptional silencing. Mol Cell Biol. 15:76–86.[Abstract]
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