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Printed in U.S.A.
Molecular Endocrinology 15(9):1529–1538
Copyright © 2001 by The Endocrine Society
Aberrant Alternative Splicing of Thyroid Hormone
Receptor in a TSH-Secreting Pituitary Tumor Is
A Mechanism for Hormone Resistance
SHINICHIRO ANDO, NICHOLAS J. SARLIS, JAY KRISHNAN, XU FENG, SAMUEL REFETOFF,
MICHAEL Q. ZHANG, EDWARD H. OLDFIELD, AND PAUL M. YEN
Molecular Regulation and Neuroendocrinology Section (S.A., N.J.S., J.K., X.F., P.M.Y.), Clinical
Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and
Surgical Neurology Branch (E.H.O.), National Institute of Neurological Disorders and Stroke, National
Institutes of Health, Bethesda, Maryland 20892; Departments of Pediatrics and Medicine (S.R.),
University of Chicago, Chicago, Illinois 60637; and Cold Spring Harbor Laboratory (M.Q.Z.), Cold
Spring Harbor, New York 11724
Patients with TSH-secreting pituitary tumors
(TSHomas) have high serum TSH levels despite
elevated thyroid hormone levels. The mechanism
for this defect in the negative regulation of TSH
secretion is not known. We performed RT-PCR to
detect mutations in TR␤ from a surgically resected
TSHoma. Analyses of the RT-PCR products revealed a 135-bp deletion within the sixth exon that
encodes the ligand-binding domain of TR␤2. This
deletion was caused by alternative splicing of
TR␤2 mRNA, as near-consensus splice sequences
were found at the junction site and no deletion or
mutations were detected in the tumoral genomic
DNA. This TR␤ variant (TR␤2spl) lacked thyroid
hormone binding and had impaired T3-dependent
negative regulation of both TSH␤ and glycoprotein
hormone ␣-subunit genes in cotransfection studies. Furthermore, TR␤2spl showed dominant negative activity against the wild-type TR␤2. These
findings strongly suggest that aberrant alternative
splicing of TR␤2 mRNA generated an abnormal TR
protein that accounted for the defective negative
regulation of TSH in the TSHoma. This is the first
example of aberrant alternative splicing of a nuclear hormone receptor causing hormonal dysregulation. This novel posttranscriptional mechanism for generating abnormal receptors may occur
in other hormone-resistant states or tumors in
which no receptor mutation is detected in genomic
DNA. (Molecular Endocrinology 15: 1529–1538,
2001)
T
TRs are members of a family of nuclear hormone
receptors that include the steroid hormone, vitamin D,
and retinoic acid receptors (3, 4). These receptors
have a variable amino terminus, a central DNA-binding
domain, and a carboxy-terminal ligand-binding domain. TRs bind to their thyroid hormone response
elements, usually located in the promoter regions of
target genes, and regulate transcription by interacting
with coactivators and corepressors (5). In positively
regulated target genes, coactivators mediate hormone-induced activation, while corepressors, nuclear
receptor corepressor (NCoR) and silencing mediator
of retinoic acid and thyroid hormone receptor (SMRT),
mediate transcriptional silencing. In negatively regulated target genes, which are not as well characterized, corepressors activate the glycoprotein hormone
␣-subunit and TSH␤ genes, whereas coactivators
such as steroid receptor coactivator-1 (SRC-1), glucocorticoid receptor interacting protein 1, thyroid receptor activator molecule 1, and activator of thyroid
and retinoic acid receptors enhance the T3-dependent
negative regulation of glycoprotein hormone ␣-subunit
gene (6, 7).
There are two distinct TR genes, TR␣ and TR␤. The
TR␣ gene generates two proteins, TR␣1 and a car-
HYROID HORMONE SYNTHESIS is tightly regulated by a negative feedback system involving the
hypothalamus, pituitary, and thyroid gland. In most
forms of hyperthyroidism, elevation of T3 and T4 represses secretion of TSH by the pituitary gland. However, in patients with TSH-secreting pituitary tumors
(TSHomas), this negative feedback is disrupted, as
TSHomas overproduce TSH in the face of elevated
serum T3 and T4 levels (1, 2). Furthermore, TSH secretion cannot be stimulated by TRH. These patients
typically have physical signs and symptoms of thyroid
hormone excess. Additionally, TSHomas present almost invariably as macroadenomas and can cause
visual and vascular complications. The mechanism for
this loss of negative regulation of TSH secretion by
thyroid hormone in TSHomas is not known but may
involve a defect in signaling via thyroid hormone receptors (TRs).
Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; NCoR, nuclear receptor corepressor; RTH, resistance to thyroid hormone; SMRT, silencing mediator of
retinoic acid and thyroid hormone receptor; SRC-1, steroid
receptor coactivator-1; ss, splice site; TIF2, transcription intermediary factor 2.
1529
1530 Mol Endocrinol, September 2001, 15(9):1529–1538
boxy-terminal splice variant, c-erbA␣2, that does not
bind thyroid hormone. The TR␤ gene generates two
isoforms, TR␤1 and TR␤2, via promoter choice (8).
TR␤1 and TR␤2 proteins have identical DNA- and
ligand-binding domains, but differ at their amino termini. Although TR␣1, TR␤1, and c-erbA␣2 have widespread expression, TR␤2 has tissue-selective expression in the anterior pituitary gland, hypothalamus, and
developing brain (9, 10). Recently, TR␤2-selective
knockout mice have been generated, which manifest
elevated serum TSH and thyroid hormone levels (11).
This finding suggests that TR␤2 may be the critical TR
isoform that negatively regulates TSH secretion.
Patients with the inherited syndrome of resistance to
thyroid hormone (RTH) also have elevated serum T3
and T4 concentrations and normal or elevated TSH
level (12–14). These patients generally have point mutations in one of the alleles of the TR␤ gene that results
in a mutant TR that cannot bind T3 or regulate transcription but can nonetheless bind to thyroid hormone
response elements of target genes. The dominant
negative activity of the mutant TR␤ on wild-type TR
reduces thyroid hormone suppression of the two
genes that generate TSH subunits: glycoprotein hormone ␣-subunit and TSH␤ (15, 16). Given the precedence for TR␤ mutations in RTH patients, we performed mutational analysis of TR␤ isolated from a
surgically resected TSHoma. Surprisingly, we detected a splice variant of TR␤ mRNA that translates
into an abnormal TR␤2 protein that is unable to bind
T3. This abnormal TR␤2 was transcriptionally inactive
and disrupted the negative regulation of TSH by T3 in
cotransfection studies. These findings suggest that a
novel, posttranscriptional mechanism caused central
thyroid hormone resistance in this patient with a
TSHoma. Similar posttranscriptional mechanisms may
explain other hormone-resistant states or tumors in
which no hormone receptor mutations can be detected from genomic DNA.
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
be completely removed due to dural invasion, and the
patient was started on octreotide therapy. The patient’s symptoms abated and her thyroid function tests
normalized. A TRH stimulation test showed normal
TSH secretory response (data not shown). A T3 suppression test performed 4 yr after surgery (Fig. 1) failed
to suppress the patient’s serum TSH to less than 10%
of the baseline, suggesting that at least part of the
patient’s TSH secretion was due to residual tumor (1).
Annual pituitary magnetic resonance imagings have
shown a persistent 7-mm residual adenoma with no
further growth. The patient’s clinical status has remained unchanged.
Finding of TR␤2 mRNA Splice Variant from
a TSHoma
RNA from the patient’s TSHoma and pooled normal
pituitaries were used to amplify full-length TR␤1,
TR␤2, and an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA by RT-PCR.
Similar amounts of GAPDH mRNA were detected in
the samples, suggesting that similar amounts of RNA
were analyzed from the TSHoma and the pooled normal pituitaries (Fig. 2A, lanes 5 and 6). Additionally,
similar amounts of TR␤1 mRNA were measured in the
TSHoma and pooled normal pituitaries (Fig. 2A, lanes
1 and 2). Surprisingly, a short variant TR␤2 mRNA
(TR␤2spl) was the major amplified product from the
TSHoma, as only a small amount of amplified wildtype TR␤2 product was observed (Fig. 2A, lane 4). In
RESULTS
Clinical Studies
A 79-yr-old Caucasian female presented at NIH with
goiter and palpitations due to hyperthyroidism. The
patient had a serum TSH concentration of 10.5 ␮U/ml
(normal, 0.43–4.60), free T4 concentration of 7.4 ng/dl
(normal, 0.9–1.6), and glycoprotein hormone ␣-subunit
concentration of 34.1 ␮g/liter (normal, ⬍5). Serum GH
and PRL levels were normal. A magnetic resonance
image of pituitary revealed an 18-mm pituitary adenoma with extension into the left cavernous sinus.
Transsphenoidal surgery was performed, and immunohistochemical analysis of the tumor showed
strongly positive staining for TSH and glycoprotein
hormone ␣-subunit, as well as staining for both GH
and PRL. Postoperatively, the patient’s serum TSH
decreased to 3.1 ␮U/ml. However, the tumor could not
Fig. 1. T3 Suppression Test of the Patient
T3 (300 ␮g) was administered orally to the patient, and
serum samples were obtained just before, and 48 h after, T3
administration for measurement of serum TSH. Normal basal
serum TSH ranges from 0.43 to 4.60 ␮U/liter. Normal controls
suppress to less than 10% of the baseline 48 h after T3
administration. Shaded areas represent normal ranges for
baseline serum TSH and expected ranges for T3-suppressed
serum TSH 48 h after T3 administration, respectively.
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
Mol Endocrinol, September 2001, 15(9):1529–1538
1531
Fig. 2. TR␤2spl Detection in a TSHoma
A, RT-PCR was performed using mRNA from a TSHoma (lanes 2, 4, and 6) or pooled normal pituitary RNA from 87 individuals
(lanes 1, 3, and 5). Primers were specific for the amplification of TR␤1 (lanes 1 and 2), TR␤2 (lanes 3 and 4), and GAPDH (lanes
5 and 6). Lanes 7 and 8 are products of exon 6 amplification of genomic DNA of TSHoma or circulating peripheral leukocytes of
the same patient. B, Ratio of TR isoforms mRNA expression normalized with respect to GAPDH mRNA expression.
contrast, only the wild-type TR␤2 product was amplified from the pooled normal pituitary RNA (Fig. 2A,
lane 3). TR␤2spl mRNA was detected only in the
TSHoma RNA when RT-PCR was performed using
TR␤2 exon 6 primers (data not shown). Taken together, these findings suggested that TR␤2spl mRNA
was specific for the TSHoma and contained a deletion
within exon 6. Furthermore, since the primers used for
amplification detected full-length TR␤2 cDNA, they
enabled measurement of the relative expression of
both wild-type TR␤2 and TR␤2spl mRNA within the
same PCR reaction. These semiquantitative RT-PCR
results showed that there is much higher expression of
TR␤2spl mRNA than wild-type TR␤2 mRNA in the
TSHoma, although the total amount of TR␤2 mRNA is
not significantly different than TR␤2mRNA from the
pooled normal pituitaries (Fig. 2B). Similar findings
were observed in three repeat experiments. The intensities of the bands in Fig. 2A, lanes 3 and 4, corrected
for GADPH mRNA expression, are shown in Fig. 2B.
The RT-PCR products then were sequenced, and
the TR␤2spl mRNA was found to have a 135 base
in-frame deletion in exon 6 (Fig. 3). This deleted region
was identical in TR␤1 and TR␤2 and encoded a part of
the ligand-binding domain. Additionally, TR␤2spl protein had a newly created isoleucine in place of the
deleted 46 amino acids in the ligand-binding domain
(Fig. 3). PCR was performed using genomic DNA from
1532 Mol Endocrinol, September 2001, 15(9):1529–1538
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
by aberrant splicing via latent splice sites that existed
within exon 6 of wild-type TR␤2 mRNA.
Functional Properties of TR␤2 Splice Variant
Fig. 3. Structure of TR␤2 and Location of the Deletion of
TR␤2spl
Receptor domains and the three exons encoding the ligand-binding domain of TR␤2 are indicated. TR␤2spl has a
deletion from amino acids 340 to 385 substituted by an
isoleucine in the ligand-binding domain. Note that exons 5, 6,
and 7 of TR␤2 are the same as exons 8, 9, and 10 of TR␤1;
amino acids 340–385 of TR␤2 are the same as amino acids
325–340 of TR␤1.
the patient’s TSHoma and peripheral leukocytes and
did not show any deletion in exon 6 (Fig. 2A, lanes 7
and 8).
Analysis of Splice Sites
Examination of both 5⬘- and 3⬘-sequences flanking the
junction site of the deletion of TR␤2spl mRNA showed
near-consensus splicing sequences that were located
within exon 6 of wild-type TR␤2 (Fig. 4 A). These
sequences conformed to several rules that are important for efficient splicing (17, 18). Most splice junctions
are bordered by GT/AG at the 5⬘- and 3⬘-ends of the
spliced sequences, as was the case for TR␤2spl
mRNA. Additionally, there are consensus sequences
for the 5⬘-splice site (ss), 3⬘-ss, and branch sequence,
which are located 20 to 50 bases upstream of the
3⬘-ss. TR␤2 mRNA had near-consensus 5⬘- and 3⬘splice sequences, as well as a consensus branch sequence within exon 6 (Fig. 4B). Using an internal coding exon predicting program, Michael Zhang’s Exon
Finder (19), the 5⬘-ss and 3⬘-ss used by TR␤2spl had
scores indicating their high potential for alternative
splicing (AAAGTGAGA, 5⬘-ss score ⫽ 46.77; TGGATGACACTGAAGTA, 3⬘-ss score ⫽75.47), although their
scores were slightly lower than those of the wild-type
TR␤ ss (CAGGTGAGT, 5⬘-ss score ⫽ 49.93; ACTGGTTCTTTTCAGCT, 3⬘-ss score ⫽83.75).
Aberrant alternative splicing theoretically could be
due to somatic mutations in the genomic DNA of the
TSHoma, which, in turn, might generate a new cryptic
splice site or create an unfavorable splice sequence in
the intron/exon junctions of exon 6. However, sequencing of the PCR products from genomic DNA of
the TSHoma showed no mutation in exon 6 and or in
the flanking intronic sequences (data not shown).
These findings demonstrate that TR␤2spl was created
Most mutations identified in patients with RTH are
located in three clusters of the ligand-binding domain of TR␤ (cluster 1: TR␤1 234–282, cluster 2:
TR␤1 310–383, cluster 3: TR␤1 429–460) (20). The
deletion of TR␤2spl is located within cluster 2 of the
ligand-binding domain of TR␤2. Since most RTH
mutants reduced binding to T3, the T3-binding activity of TR␤2spl was measured and compared with
the T3-binding activities of wild-type TR␤2 and
TR␤2G360R (TR␤2Mf), a cluster 2 mutant from a
patient with RTH previously shown to have minimal
T3 binding (20a). As expected from the location of
the deletion in the ligand-binding domain, both
TR␤2spl and TR␤2Mf showed minimal T3 binding
(Fig. 5). We then examined the functional activity of
the wild-type TR␤2 and TR␤2spl on the regulation of
the genes encoding the two subunits of TSH: glycoprotein hormone ␣-subunit and TSH␤. It has been
previously shown that wild-type TR can cause T3independent activation and T3-dependent negative
regulation of transcription for these two genes
(6, 15). As shown in Fig. 6, TR␤2spl lost both T3independent activation and T3-dependent negative
regulation on glycoprotein hormone ␣-subunit and
TSH␤ genes. In the presence of T3, TR␤2spl also
showed higher transcriptional activity of the glycoprotein hormone ␣-subunit and TSH␤ genes than
wild-type TR␤2, consistent with the clinical observation of nonsuppressible TSH in the face of elevated T3 observed in the patient (Fig. 1). Cotransfection of the wild-type TR␤2 with TR␤2spl, as well
as TR␤2Mf, resulted in diminished T3-dependent
negative regulation of both glycoprotein hormone
␣-subunit and TSH␤ genes (Fig. 7). These results
show that both TR␤2spl and TR␤2Mf have strong
dominant negative activity against the wild-type
TR␤2 on the transcription of glycoprotein hormone
␣-subunit and TSH␤ genes.
To study the interaction between TR␤2spl and
transcriptional cofactors, the LexA yeast two-hybrid
system was used. cDNAs of the ligand-binding
domains of wild-type TR␤ and TR␤2spl were subcloned into LexA expression vector, and coactivators, SRC-1 and transcription intermediary factor 2
(TIF2), and corepressors (NCoR and SMRT) were
subcloned into B42AD expression vector. As expected, the ligand-binding domain of wild-type TR␤
interacted with both SRC-1 and TIF2 in the presence
of ligand and interacted with both NCoR and SMRT
in the absence of ligand (Table 1). In contrast, the
ligand-binding domain of TR␤2spl did not interact
with any of these cofactors either in the presence or
absence of ligand.
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
Mol Endocrinol, September 2001, 15(9):1529–1538
1533
Fig. 4. Analysis of Splice Sites
A, Splice sites used for TR␤2spl (red) and normal splice sites (blue and black) are depicted. B, 3⬘-ss, 5⬘-ss, and branch site of
TR␤2spl were compared with naturally occurring splice sequences flanking exon 6 of TR␤2, and consensus sequences. The
branch site of the deletion of TR␤2spl mRNA is located 48 bases upstream of the 3⬘-ss of the deletion. Underlined nucleotides
are absolutely required for splicing. Capitalized nucleotides of the consensus splice sequences are important, whereas lower case
nucleotides are less important. Note that TR␤2 exon 6 is the same as TR␤1 exon 9.
DISCUSSION
We have found a novel alternatively spliced variant of
thyroid hormone receptor, TR␤2spl, from a TSHoma.
This is also the first example of abnormal TR in TSHomas. TR␤2spl lacked T3-binding activity and was unable to mediate T3-dependent negative regulation of
glycoprotein ␣-subunit and TSH␤ genes. Furthermore,
TR␤2spl showed dominant negative activity, as it
blocked T3-dependent regulation of both these genes
by wild-type TR␤2. These findings strongly implicate
TR␤2spl in the defective negative regulation of TSH
secretion exhibited by the tumor. This dysregulation in
TSH secretion is similar to that observed in patients
with RTH who have germline mutations in one of their
TR␤ alleles (12). Additionally, transgenic mice that
overexpress dominant negative mutant TR␤s in the
pituitary have similar defects in the negative regulation
of TSH (21–23). In this connection, we recently examined TR␤ in five other TSHomas. Although we did not
find another example of aberrant alternative splicing of
TRs, we identified a somatic mutation in the ligandbinding domain of TR␤1 in one TSHoma (24). Gittoes
et al. (25) recently reported decreased expression of
1534
Mol Endocrinol, September 2001, 15(9):1529–1538
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
Fig. 5. T3-Binding Activity of the Wild-Type TR␤2, TR␤2spl,
and TR␤2Mf
Wild-type TR␤2, TR␤2spl, or TR␤2Mf was transfected into
CV-1 cells. Nuclear extracts were prepared and incubated
with 125I-T3 as described in Materials and Methods. Bound
and free 125I-T3 were separated, and the ratio between the
bound and total T3 (B/T) was calculated. The specific T3
binding was obtained by subtracting the nonspecific binding
determined in the incubation mixture containing a 10,000fold excess of nonlabeled T3. The value of T3-binding activity
of the wild-type TR␤2 is expressed as 100%. The data are
represented as the mean ⫾ SD of four samples.
TR␣ and TR␤ in two TSHomas; therefore, downregulation of TRs may be an additional mechanism for
defective negative regulation of TSH by thyroid hormone. Furthermore, unlike GH-secreting adenomas,
which can harbor G protein ␣ chain (Gs␣) mutations
that inhibit GTPase activity (26), no such mutations or
TRH receptor mutations have been implicated in the
constitutive expression of TSH in TSHomas (27).
Taken together with our study, these findings suggest
that defective negative regulation by the TR signaling
pathway, rather than overactivity of the TRH signaling
pathway, may be operative in some TSHomas.
TR␤2spl had a 46-amino acid deletion replaced by a
newly created isoleucine, which maintained the original sequence of the remaining amino acids. The deletion is located in a region of the TR␤ ligand-binding
domain where a cluster of mutations has been reported for RTH patients (12). This region also is known
to contribute to the formation of helices 7, 8, and 9 of
TR␤, which compose part of the ligand-binding pocket
of TR (28). Thus, the lack of T3 binding and transcriptional activity, as well as the dominant negative activity
observed for TR␤2spl, are fully consistent with a deletion in this important region of the TR␤2 ligandbinding domain. The inability of the ligand-binding domain of TR␤2spl to interact with corepressors and
coactivators in the yeast two-hybrid system is consistent with the loss of both T3-independent activation
and T3-dependent negative regulation on glycoprotein
hormone ␣-subunit and TSH␤ genes observed in our
cotransfection studies. Notably, helices 3, 4, 5, and 6
of TR␤ and helices 3, 5, 6, and 12 of TR␤ have been
reported to contact surfaces with corepressors and
coactivators, respectively (29–31). Although the dele-
Fig. 6. Transcriptional Activity of TR␤2 and TR␤2spl on
Genes Encoding TSH Subunits
Equal amounts of pcDNA expression vectors for TR␤2 and
TR␤2spl were cotransfected with glycoprotein ␣-subunit or
TSH␤ luciferase reporter vectors into TSA 201 cells as described in Materials and Methods. The cells were incubated
with 50 nM T3 for 40 h and harvested, and luciferase activity
was measured. Data are represented as the mean ⫾ SD from
nine individual samples. The asterisk indicates significant
difference between TR␤2spl-dependent and TR␤2-dependent luciferase activity in the presence of T3 (P ⬍ 0.01)
determined by unpaired t tests. A, Glycoprotein hormone
␣-subunit luciferase activity. B, TSH␤ luciferase activity.
tion of TR␤2spl is not within these areas, tertiary structure changes, presumably due to the large conformational changes caused by the deletion, are likely
responsible for the loss of interaction with corepressors. Tertiary structure changes also likely affected
T3-binding activity and/or coactivator interactions of
TR␤2spl.
In our study, only a small amount of wild-type TR␤2
mRNA was detected in the TSHoma, suggesting that
TR␤2spl mRNA may have been spliced after wild-type
TR␤2 mRNA was generated. Indeed, the 5⬘-ss score
and 3⬘-ss score of the TR␤2spl ss were weaker than
those of the naturally occurring ss flanking exon 6 of
TR␤2. It is interesting that the variant form of TR␤1
mRNA was not observed in the TSHoma, particularly
since TR␤1 and TR␤2 have identical sequences coding for the ligand-binding domain. It is possible that
TR␤1 and TR␤2 mRNA have different higher order
structures or differences in the turnover and/or export
rates from the splicesome to account for this difference in splicing of the RNA between the two TR␤
isoforms. Moreover, although TR␤2 and TR␣1 have
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
Fig. 7. Dominant Negative Activity of TR␤2spl and TR␤2Mf
on TR␤2 Regulation of Genes Encoding TSH Subunits
TSA 201 cells were transfected with equal amounts of
TR␤2 and TR␤2spl or TR␤2Mf with glycoprotein hormone
␣-subunit or TSH␤ luciferase vector. The cells were incubated with 50 nM T3 and harvested, and luciferase activity
was measured. Fold T3 inhibition is calculated as luciferase
activity in the absence of T3 divided by luciferase activity in
the presence of T3. Data are represented as the mean ⫾ SD
from nine individual samples. A, Glycoprotein hormone
␣-subunit luciferase activity. B, TSH␤ luciferase activity.
high homology in the mRNA sequences encoding their
respective ligand-binding domains, it is unlikely that
TR␣1 has a corresponding splice variant, since TR␣1
has GC/AG instead of GT/AG in the critical sequences
that flank the splice junction of TR␤2spl. TR␣ mRNA
can undergo alternative splicing to generate mRNA for
a non-T3 binding protein, c-erbA␣-2 (3). However, the
location of the ss of TR␤2spl mRNA is more upstream
than the 5⬘-ss of c-erbA␣-2 mRNA; therefore, the generation of TR␤2spl mRNA is distinct from this naturally
occurring process.
There are several potential mechanisms by which
abnormal alternative splicing could generate TR␤2spl
mRNA (32, 33). One possibility is the creation of a new
cryptic splice site by a mutation(s) in the genomic
DNA. However, this was not observed, as genomic
DNA from the patient’s TSHoma did not have any
mutations in the sequences flanking the splice junction
or within the spliced sequence. Another possibility is
that a mutation(s) in the natural ss of the flanking
introns might create a less favorable splicing site, and
thus allow splicing via the intraexonic ss. However, the
intraexonic splicing pattern, which maintains portions
of exon 6, and the absence of mutations in the naturally occurring ss contained in the intronic sequences
immediately flanking exon 6, argue against this possibility. Thus, the most likely explanation for the aberrant splicing is increased activity of splicing factors in
the TSHoma. The basis for this phenomenon could be
Mol Endocrinol, September 2001, 15(9):1529–1538
1535
a mutation in a splicing factor(s), or increased splicing
activity as a consequence of overexpression or altered
function (perhaps by phosphorylation or other regulatory events) of splicing factor(s) (34). Of note, aberrant
alternative splicing and overactivity of splicing factors
has been recently reported in breast cancer (35–38).
Additionally, aberrant splicing has been reported for
ER mRNA in breast cancer (39, 40). Given the aberrant
splicing of TR␤2 mRNA in the TSHoma, it is possible
that other mRNAs may be aberrantly spliced in the
tumor and perhaps contribute to the growth of the
tumor. Recently, an incidental pituitary adenoma in a
patient with RTH was reported (41). In addition, there
is one case report of pituitary enlargement in a patient
with RTH in whom pituitary regression occurred after
thyroid hormone treatment (42). However, TR␤ knockout mice and transgenic mice overexpressing mutant
TR␤ showed defective TSH regulation but no increase
in tendency to form TSHomas (11, 21, 22, 43). Additionally, there have been no reported cases of TSHomas in RTH patients who have TR␤ mutations (12, 14),
and patients with RTH do not seem to have a higher
risk for TSHomas. Mutant TR␤s per se may not be
sufficient for the abnormal growth characteristics of
TSHomas. Hence, the defective TR␤ splicing could be
a consequence rather than a cause of tumorigenesis.
Several families with RTH have been described who
do not have mutations in their TR␣ and TR␤ genes
(44). Although knockout mice that lack a coactivator
have mild central thyroid hormone resistance (45), no
such postreceptor defects have been detected in
these patients. Thus, it is possible that aberrantly
spliced TRs may account for thyroid hormone resistance in some of these families. Hormone resistance in
humans has been described for estrogen, glucocorticoid, androgen, and vitamin D, as well as peptide
hormones, such as insulin, vasopressin, and PTH (46–
52). In many cases, mutations in the nuclear hormone
receptors or peptide hormone receptors were detected in genomic DNA and accounted for the clinical
picture of hormone resistance. However, in rare instances in which no receptor mutation was found,
receptor down-regulation or postreceptor defects
have been described or hypothesized (44, 51, 53, 54).
Our findings suggest that aberrant alternative splicing
is another mechanism for hormone resistance in signaling pathways in which no receptor mutation is
found in the genomic DNA.
In conclusion, we describe a splicing variant of TR
from a TSH-secreting pituitary tumor that caused defective T3-dependent negative regulation of glycoprotein hormone ␣-subunit and TSH␤ genes in a transient
transfection system. Since the TR␤ gene did not contain any mutations that accounted for the deletion, this
abnormal TR was caused by aberrant alternative splicing of TR␤2 mRNA. This example enlarges our understanding of the possible mechanisms for hormone
resistance and suggests that posttranscriptional
causes need to be considered whenever genomic mutations are not detected in hormone receptors.
1536 Mol Endocrinol, September 2001, 15(9):1529–1538
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
Table 1. Interactions between TR and Cofactors in a Yeast Two-Hybrid System
B42AD
B42AD-SRC1
B42AD-TIF2
B42AD-SMRT
B42AD-NCoR
LexA
⫺T3
⫹T3
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
LexA-TR␤LBD
⫺T3
⫹T3
⫺
⫺
⫺
⫹
⫺
⫹
⫹
⫺
⫹
⫺
LexA-TR␤2splLBD
⫺T3
⫹T3
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
The expression plasmids producing either LexA DNA-binding-domain fusion proteins (listed at left) or pB42AD that encode B42
transactivation-domain fusion proteins (listed at top) were introduced into EGY48 cells, and the resulting transformants were
grown on plates lacking leucine and containing X-Gal in the presence (⫹T3) or absence (⫺T3) of T3 (1 ␮M). Two-hybrid plasmids
that resulted in production of blue colored colonies are indicated as (⫹).
MATERIALS AND METHODS
Analyses of RNA and Genomic DNA
Genomic DNA was isolated from the patient’s TSHoma and
peripheral blood leukocytes. Total RNA was extracted from
the TSHoma by Trizol reagent (Life Technologies, Inc., Gaithersburg, MD). Pituitary gland polyA RNA (pool of 87 normal
tissue specimens) was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). cDNA was synthesized by Moloney murine leukemia virus-reverse transcriptase. DNA was
amplified by PCR with the following specific oligonucleotide
primers: TR␤1 sense primer: 5⬘-GGATCCAGAATGATTACTAACCTATGACTC-3⬘; TR␤2 sense primer: 5⬘-ACCAGGGAAACAAAATGAACTACTGTATGC-3⬘; TR␤1 and TR␤2
common antisense primer: 5⬘-GGAATTATAGGAAGGAATCCAGTCAGTCTA-3⬘; TR␤2 exon 6 sense primer (corresponding to exon 9 of TR␤1): 5⬘-CTGCCATGTGAAGACCAGATCATCCT-3⬘; TR␤2 exon 6 antisense primer: 5⬘CTGAAGACATCAGCAGGACGGCCTGA-3⬘; Sense primer
sequence for TR␤2 exon 6 and flanking introns: 5⬘-TCACAGAAGGTTATTCCTATT-3⬘; antisense primer sequence for
TR␤2 exon 6 and flanking introns: 5⬘-ACTCAAGTGATTGGAATTAG-3⬘; GAPDH sense primer: 5⬘-CATCACGCCACAGTTTCCCGGAGG-3⬘; GAPDH antisense primer: 5⬘-TTTCTAGACGGCAGGTCAGGTCCACC-3⬘. For semiquantitative RTPCR, PCR conditions for temperature were optimized for
each primer pair, and comparative kinetic analyses were
performed to determine that the selected PCR cycle number
for each primer pair was within the exponential phase of
product generation. PCR products were visualized on 1%
Tris-borate-EDTA agarose gels stained with ethidium bromide. Ethidium bromide-stained gels were digitized, and
densitometry was performed on the product bands using
Image Quant (Molecular Dynamics, Inc., Sunnyvale, CA) (55,
56). Quantification of RT-PCR by analysis of ethidium-stained
gels yielded consistent results. PCR products were subcloned into pCR4TOPO plasmid (Invitrogen, Carlsbad, CA),
and then sequenced.
ss Profile Analysis
ss Profiles were analyzed by Michael Zhang’s Exon Finder, a
software program designed to predict ss sequences (19).
Construction of TR cDNA Expression Vectors
The full-length human wild-type TR␤2 cDNA was cloned into
pcDNA. The DNA fragment carrying the deletion mutation
identified in the patient’s TSHoma was digested with Tth111I
and BglII and then subcloned into the corresponding TR␤2
region. A natural mutant TR␤1G345R from a patient with RTH
was also digested with Tth111I and BglII and replaced with
the corresponding TR␤2 region to create TR␤2G360R
(TR␤2Mf). The final constructs were verified by sequencing.
T3 Binding Assay
T3 binding affinity of wild-type TR␤2 and TR␤2spl protein was
measured in transfected CV-1 cells as described previously
(57). CV-1 cells were transiently transfected with wild-type
TR␤2, TR␤2spl, or TR␤2Mf using Lipofectamine Plus (Life
Technologies, Inc.). Cells were homogenized in 0.25 M sucrose and 3 mM MgCl2 (SM). The crude nuclear pellet was
suspended once with and once without 0.5% Triton X-100 in
SM. Nuclear proteins were extracted by stirring isolated nuclei at 4 C for 1 h in 0.4 M KCl, 5 mM MgCl2, 1 mM dithiothreitol, and 0.05 M Tris-glycine HCl (pH 8.5). After nuclear extracts were centrifuged at 117,000 ⫻ g for 45 min, T3-binding
activities in the supernatants were assayed. Nuclear proteins
(125 ␮g) were incubated with 1 nM 125I-T3 (NEN Life Science
Products, Boston, MA) and 4 nM non-labeled T3 at 4 C overnight. 125I-T3 was separated from the bound form using
AG1-X8 resin (Bio-Rad Laboratories, Inc., Richmond, CA).
Nonspecific binding was determined in the incubation mixture containing a 10,000-fold amount of nonlabeled T3.
Cotransfection Studies of TR
TSA 201 cells (a strain of HEK293 cells transformed with T
antigen) were maintained in Opti-MEM (Life Technologies,
Inc.) containing 4% FCS pretreated with AG1-X8 resin to
remove endogenous thyroid hormones. The cells were plated
in six-well dishes and transfected 24 h later by the calcium
phosphate precipitation method with 250 ng of TR expression vector and 500 ng of human glycoprotein hormone
␣-subunit promoter (⫺846 to ⫹44) linked to pA3 luciferase
reporter gene (6). Additionally, TSA201 cells were transfected
with 400 ng of TR expression vector, 200 ng of human TSH␤
promoter (⫺1,192 to ⫹37) gene linked to pA3 luciferase
reporter gene (15), and 200 ng of human thyrotroph embryonic factor expression vector (obtained by RT-PCR) with
Lipofectamine Plus. Eight hours after transfection, the cells
were washed and incubated for 40 h with Opti-MEM media
containing 1% of resin-treated FCS with and without 50 nM
T3. The cells were lysed and assayed for luciferase activity.
Luciferase activity was normalized according to protein
concentration.
Yeast Two-Hybrid System
EGY48 yeast cells, p8op-lacZ, and LexA-parental vectors
(pGilda) and B42-parental vectors (pB42AD) were purchased
Ando et al. • Aberrant Splicing of TR mRNA in a TSHoma
from CLONTECH Laboratories, Inc. Fragments of human
wild-type TR␤2 and TR␤2spl ligand-binding domain (amino
acids 189–476) were subcloned into EcoRI and SalI sites of
pGilda to make LexA-TR␤LBD and LexATRb2spl LBD. Fragments of human F-SRC-1 (1–1440), human TIF2 (1–1464),
human NCoR (982–1495), and human SMRT (982–1495)
were subcloned into pB42AD to make B42AD-SRC-1,
B42AD-TIF2, B42AD-NCoR, and B42AD-SMRT. LexA fusion
vectors, B42AD fusion vectors, and p8op-lacZ were transformed into EGY48 according to the manufacturer’s manual.
Interactions of fusion proteins were tested using selection for
leucine auxotrophy and Lac Z reporter gene on plates in the
presence and absence of 1 ␮M T3.
Acknowledgments
We thank Dr. Frederic Wondisford (University of Chicago,
Chicago, IL) for human TR␤2 expression and TSH␤ reporter
vectors, Dr. Laird Madison, Dr. Larry Jameson, Mr. Kevin
Long (Northwestern University, Chicago, IL) for glycoprotein
hormone ␣-subunit reporter vector and TSA 201 cells, Dr.
Ronald Evans (Salk Institute, La Jolla, CA) for human SMRT
expression vector, Dr. Anthony Hollenberg (Beth Israel Hospital, Boston, MA) for human NCoR (NCoRi) expression vector, and Dr. Pierre Chambon (INSERM, Strasbourg, France)
for TIF2 expression vector. We also thank Drs. Thomas
Misteli and Carl Baker (National Cancer Institute, Bethesda,
MD) for helpful discussions on splicing mechanisms.
Received February 12, 2001. Accepted May 23, 2001.
Address requests for reprints to: Paul M. Yen, M.D. Molecular Regulation and Neuroendocrinology Section, Clinical
Endocrinology Branch, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892. E-mail: [email protected].
nih.gov.
This work was supported in part by NIH Grants HG-01696
(to M.Q.Z.) and DK-15070 (to S.R.).
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