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
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ABSTRACT
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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 TRß 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 TRß2. This deletion was caused by alternative splicing of
TRß2 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 TRß variant (TRß2spl) lacked thyroid hormone
binding and had impaired T3-dependent negative regulation
of both TSHß and glycoprotein hormone
-subunit genes in
cotransfection studies. Furthermore, TRß2spl showed dominant negative
activity against the wild-type TRß2. These findings strongly suggest
that aberrant alternative splicing of TRß2 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.
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INTRODUCTION
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THYROID 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).
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 TSHß 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 TRß. The TR
gene
generates two proteins, TR
1 and a carboxy-terminal splice variant,
c-erbA
2, that does not bind thyroid hormone. The TRß gene
generates two isoforms, TRß1 and TRß2, via promoter choice
(8). TRß1 and TRß2 proteins have identical DNA- and
ligand-binding domains, but differ at their amino termini. Although
TR
1, TRß1, and c-erbA
2 have widespread expression, TRß2 has
tissue-selective expression in the anterior pituitary gland,
hypothalamus, and developing brain (9, 10). Recently,
TRß2-selective knockout mice have been generated, which manifest
elevated serum TSH and thyroid hormone levels (11). This
finding suggests that TRß2 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, 13, 14). These patients generally have point
mutations in one of the alleles of the TRß 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
TRß on wild-type TR reduces thyroid hormone suppression of the two
genes that generate TSH subunits: glycoprotein hormone
-subunit and
TSHß (15, 16). Given the precedence for TRß mutations
in RTH patients, we performed mutational analysis of TRß isolated
from a surgically resected TSHoma. Surprisingly, we detected a splice
variant of TRß mRNA that translates into an abnormal TRß2 protein
that is unable to bind T3. This abnormal TRß2
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.
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RESULTS
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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 µU/ml (normal, 0.434.60), free
T4 concentration of 7.4 ng/dl (normal, 0.91.6),
and glycoprotein hormone
-subunit concentration of 34.1 µg/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 patients serum TSH decreased to 3.1 µU/ml. However, the tumor
could not be completely removed due to dural invasion, and the patient
was started on octreotide therapy. The patients 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
patients serum TSH to less than 10% of the baseline, suggesting that
at least part of the patients 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
patients clinical status has remained unchanged.

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Figure 1. T3 Suppression Test of the Patient
T3 (300 µg) 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 µU/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.
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Finding of TRß2 mRNA Splice Variant from a TSHoma
RNA from the patients TSHoma and pooled normal pituitaries were
used to amplify full-length TRß1, TRß2, 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 TRß1 mRNA were measured in
the TSHoma and pooled normal pituitaries (Fig. 2A
, lanes 1 and 2).
Surprisingly, a short variant TRß2 mRNA (TRß2spl) was the major
amplified product from the TSHoma, as only a small amount of amplified
wild-type TRß2 product was observed (Fig. 2A
, lane 4). In contrast,
only the wild-type TRß2 product was amplified from the pooled normal
pituitary RNA (Fig. 2A
, lane 3). TRß2spl mRNA was detected only in
the TSHoma RNA when RT-PCR was performed using TRß2 exon 6 primers
(data not shown). Taken together, these findings suggested that
TRß2spl mRNA was specific for the TSHoma and contained a deletion
within exon 6. Furthermore, since the primers used for amplification
detected full-length TRß2 cDNA, they enabled measurement of the
relative expression of both wild-type TRß2 and TRß2spl mRNA within
the same PCR reaction. These semiquantitative RT-PCR results showed
that there is much higher expression of TRß2spl mRNA than wild-type
TRß2 mRNA in the TSHoma, although the total amount of TRß2 mRNA is
not significantly different than TRß2mRNA 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
.

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Figure 2. TRß2spl 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 TRß1 (lanes 1 and 2),
TRß2 (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.
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The RT-PCR products then were sequenced, and the TRß2spl mRNA was
found to have a 135 base in-frame deletion in exon 6 (Fig. 3
). This deleted region was identical in
TRß1 and TRß2 and encoded a part of the ligand-binding domain.
Additionally, TRß2spl 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 the patients TSHoma and
peripheral leukocytes and did not show any deletion in exon 6 (Fig. 2A
, lanes 7 and 8).

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Figure 3. Structure of TRß2 and Location of the Deletion of
TRß2spl
Receptor domains and the three exons encoding the ligand-binding domain
of TRß2 are indicated. TRß2spl 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 TRß2 are the same as exons 8, 9, and 10 of
TRß1; amino acids 340385 of TRß2 are the same as amino acids
325340 of TRß1.
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Analysis of Splice Sites
Examination of both 5'- and 3'-sequences flanking the junction
site of the deletion of TRß2spl mRNA showed near-consensus splicing
sequences that were located within exon 6 of wild-type TRß2 (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 TRß2spl 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. TRß2 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 Zhangs
Exon Finder (19), the 5'-ss and 3'-ss used by TRß2spl
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 TRß ss (CAGGTGAGT, 5'-ss score = 49.93;
ACTGGTTCTTTTCAGCT, 3'-ss score =83.75).

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Figure 4. Analysis of Splice Sites
A, Splice sites used for TRß2spl (red) and normal
splice sites (blue and black) are
depicted. B, 3'-ss, 5'-ss, and branch site of TRß2spl were compared
with naturally occurring splice sequences flanking exon 6 of TRß2,
and consensus sequences. The branch site of the deletion of TRß2spl
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 TRß2 exon 6 is the same as
TRß1 exon 9.
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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 TRß2spl was created by aberrant
splicing via latent splice sites that existed within exon 6 of
wild-type TRß2 mRNA.
Functional Properties of TRß2 Splice Variant
Most mutations identified in patients with RTH are located
in three clusters of the ligand-binding domain of TRß (cluster 1:
TRß1 234282, cluster 2: TRß1 310383, cluster 3: TRß1
429460) (20). The deletion of TRß2spl is located
within cluster 2 of the ligand-binding domain of TRß2. Since most RTH
mutants reduced binding to T3, the
T3-binding activity of TRß2spl was measured and
compared with the T3-binding activities of
wild-type TRß2 and TRß2G360R (TRß2Mf), 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
TRß2spl and TRß2Mf showed minimal T3 binding
(Fig. 5
). We then examined the
functional activity of the wild-type TRß2 and TRß2spl on the
regulation of the genes encoding the two subunits of TSH:
glycoprotein hormone
-subunit and TSHß. It has been previously
shown that wild-type TR can cause T3-independent
activation and T3-dependent negative regulation
of transcription for these two genes (6, 15). As
shown in Fig. 6
, TRß2spl lost both
T3-independent activation and
T3-dependent negative regulation on glycoprotein
hormone
-subunit and TSHß genes. In the presence of
T3, TRß2spl also showed higher transcriptional
activity of the glycoprotein hormone
-subunit and TSHß genes than
wild-type TRß2, 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
TRß2 with TRß2spl, as well as TRß2Mf, resulted in diminished
T3-dependent negative regulation of both
glycoprotein hormone
-subunit and TSHß genes (Fig. 7
). These results show that both
TRß2spl and TRß2Mf have strong dominant negative activity against
the wild-type TRß2 on the transcription of glycoprotein hormone
-subunit and TSHß genes.

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Figure 5. T3-Binding Activity of the Wild-Type
TRß2, TRß2spl, and TRß2Mf
Wild-type TRß2, TRß2spl, or TRß2Mf 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 10,000-fold excess of nonlabeled T3.
The value of T3-binding activity of the wild-type TRß2 is
expressed as 100%. The data are represented as the mean ±
SD of four samples.
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Figure 6. Transcriptional Activity of TRß2 and TRß2spl on
Genes Encoding TSH Subunits
Equal amounts of pcDNA expression vectors for TRß2 and TRß2spl were
cotransfected with glycoprotein -subunit or TSHß 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 TRß2spl-dependent and TRß2-dependent
luciferase activity in the presence of T3
(P < 0.01) determined by unpaired t
tests. A, Glycoprotein hormone -subunit luciferase activity. B,
TSHß luciferase activity.
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Figure 7. Dominant Negative Activity of TRß2spl and
TRß2Mf on TRß2 Regulation of Genes Encoding TSH Subunits
TSA 201 cells were transfected with equal amounts of TRß2 and
TRß2spl or TRß2Mf with glycoprotein hormone -subunit or TSHß
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, TSHß luciferase activity.
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To study the interaction between TRß2spl and transcriptional
cofactors, the LexA yeast two-hybrid system was used. cDNAs of the
ligand-binding domains of wild-type TRß and TRß2spl 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 TRß 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 TRß2spl did not interact with
any of these cofactors either in the presence or absence of ligand.
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DISCUSSION
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We have found a novel alternatively spliced variant of thyroid
hormone receptor, TRß2spl, from a TSHoma. This is also the first
example of abnormal TR in TSHomas. TRß2spl lacked
T3-binding activity and was unable to mediate
T3-dependent negative regulation of glycoprotein
-subunit and TSHß genes. Furthermore, TRß2spl showed dominant
negative activity, as it blocked T3-dependent
regulation of both these genes by wild-type TRß2. These findings
strongly implicate TRß2spl 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 TRß alleles (12).
Additionally, transgenic mice that overexpress dominant negative mutant
TRßs in the pituitary have similar defects in the negative
regulation of TSH (21, 22, 23). In this connection, we
recently examined TRß in five other TSHomas. Although we did not find
another example of aberrant alternative splicing of TRs, we identified
a somatic mutation in the ligand-binding domain of TRß1 in one TSHoma
(24). Gittoes et al. (25) recently
reported decreased expression of TR
and TRß in two TSHomas;
therefore, down-regulation 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.
TRß2spl 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 TRß
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 TRß, 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
TRß2spl, are fully consistent with a deletion in this important
region of the TRß2 ligand-binding domain. The inability of the
ligand-binding domain of TRß2spl 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 TSHß genes observed in our cotransfection
studies. Notably, helices 3, 4, 5, and 6 of TRß and helices 3, 5, 6,
and 12 of TRß have been reported to contact surfaces with
corepressors and coactivators, respectively (29, 30, 31).
Although the deletion of TRß2spl 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 TRß2spl.
In our study, only a small amount of wild-type TRß2 mRNA was detected
in the TSHoma, suggesting that TRß2spl mRNA may have been spliced
after wild-type TRß2 mRNA was generated. Indeed, the 5'-ss score and
3'-ss score of the TRß2spl ss were weaker than those of the naturally
occurring ss flanking exon 6 of TRß2. It is interesting that the
variant form of TRß1 mRNA was not observed in the TSHoma,
particularly since TRß1 and TRß2 have identical sequences coding
for the ligand-binding domain. It is possible that TRß1 and TRß2
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 TRß isoforms.
Moreover, although TRß2 and TR
1 have 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 TRß2spl. 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 TRß2spl mRNA is more upstream than the 5'-ss of c-erbA
-2 mRNA;
therefore, the generation of TRß2spl mRNA is distinct from this
naturally occurring process.
There are several potential mechanisms by which abnormal alternative
splicing could generate TRß2spl 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 patients 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 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, 36, 37, 38). Additionally,
aberrant splicing has been reported for ER mRNA in breast cancer
(39, 40). Given the aberrant splicing of TRß2 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, TRß knockout mice and transgenic mice overexpressing mutant
TRß 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 TRß
mutations (12, 14), and patients with RTH do not seem to
have a higher risk for TSHomas. Mutant TRßs per se may not be
sufficient for the abnormal growth characteristics of TSHomas. Hence,
the defective TRß 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 TRß 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, 47, 48, 49, 50, 51, 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 TSHß genes in a transient transfection
system. Since the TRß gene did not contain any mutations that
accounted for the deletion, this abnormal TR was caused by aberrant
alternative splicing of TRß2 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.
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MATERIALS AND METHODS
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Analyses of RNA and Genomic DNA
Genomic DNA was isolated from the patients 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: TRß1 sense primer:
5'-GGATCCAGAATGATTACTAACCTATGACTC-3'; TRß2 sense primer:
5'-ACCAGGGAAACAAAATGAACTACTGTATGC-3';
TRß1 and TRß2 common antisense primer:
5'-GGAATTATAGGAAGGAATCCAGTCAGTCTA-3'; TRß2 exon 6 sense
primer (corresponding to exon 9 of TRß1):
5'-CTGCCATGTGAAGACCAGATCATCCT-3'; TRß2 exon 6 antisense primer:
5'-CTGAAGACATCAGCAGGACGGCCTGA-3'; Sense primer sequence for TRß2 exon
6 and flanking introns: 5'-TCACAGAAGGTTATTCCTATT-3'; antisense
primer sequence for TRß2 exon 6 and flanking introns:
5'-ACTCAAGTGATTGGAATTAG-3'; GAPDH sense primer:
5'-CATCACGCCACAGTTTCCCGGAGG-3'; GAPDH antisense primer:
5'-TTTCTAGACGGCAGGTCAGGTCCACC-3'. For semiquantitative RT-PCR, 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 Zhangs Exon Finder, a
software program designed to predict ss sequences
(19).
Construction of TR cDNA Expression Vectors
The full-length human wild-type TRß2 cDNA was cloned into
pcDNA. The DNA fragment carrying the deletion mutation identified in
the patients TSHoma was digested with Tth111I and BglII
and then subcloned into the corresponding TRß2 region. A natural
mutant TRß1G345R from a patient with RTH was also digested with
Tth111I and BglII and replaced with the corresponding TRß2
region to create TRß2G360R (TRß2Mf). The final constructs were
verified by sequencing.
T3 Binding Assay
T3 binding affinity of wild-type TRß2
and TRß2spl protein was measured in transfected CV-1 cells as
described previously (57). CV-1 cells were transiently
transfected with wild-type TRß2, TRß2spl, or TRß2Mf 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
x g for 45 min, T3-binding activities
in the supernatants were assayed. Nuclear proteins (125 µg) 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
TSHß 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 from CLONTECH Laboratories, Inc. Fragments of human wild-type TRß2 and
TRß2spl ligand-binding domain (amino acids 189476) were subcloned
into EcoRI and SalI sites of pGilda to make
LexA-TRßLBD and LexATRb2spl LBD. Fragments of human F-SRC-1
(11440), human TIF2 (11464), human NCoR (9821495), and human SMRT
(9821495) 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
manufacturers 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 µM
T3.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Frederic Wondisford (University of Chicago,
Chicago, IL) for human TRß2 expression and TSHß 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.
 |
FOOTNOTES
|
---|
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: pauly{at}intra.niddk.nih.gov
This work was supported in part by NIH Grants HG-01696 (to M.Q.Z.) and
DK-15070 (to S.R.).
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.
Received for publication February 12, 2001.
Accepted for publication May 23, 2001.
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