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1 Receptors from Patients with Hepatocellular Carcinoma1
Department of Biochemistry, Chang-Gung College of Medicine and Technology, Taoyuan, Taiwan; and Gene Regulation Section, Laboratory of Molecular Biology, National Cancer Institute and Laboratory of Biochemical Pharmacology (P.M.), National Institute of Diabetes and Digestive and Kidney Diseases (X.-G.Z., S.-y.C.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Kwang-huei Lin, Department of Biochemistry, Chang-Gung College of Medicine and Technology, Taoyuan, Taiwan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 receptors
(TR1) were isolated from hepatocellular carcinomas of two patients.
Sequence analyses of the complementary DNAs showed a single
Val390Ala and double
Pro398Ser/Glu350Lys mutations in mutants H and
L, respectively. We characterized their hormone-binding, DNA-binding,
and dominant negative activities. Mutants H and L did not bind the
hormone T3. Their DNA-binding activities were analyzed
using three types of thyroid hormone response elements (TREs) in which
the half-site binding motifs are arranged in an everted repeat (Lys),
an inverted repeat (Pal), or a direct repeat separated by four
nucleotides (DR4). Compared with wild-type TR1 (w-TR1), which
bound these TREs with different homodimer/monomer ratios, binding of
mutant L to the three TREs as homodimers was reduced by 
90%.
However, binding of mutant H to these TREs was more complex. Although
it bound normally to DR4 as homodimers, its binding to Lys as
homodimers was reduced by 80%. Surprisingly, its binding to Pal was
markedly enhanced compared with w-TR1. The binding of these two
mutants to the three TREs as heterodimers with retinoid X receptors
(RXR and -ß) was not significantly affected. Consistent with the
lack of T3-binding activity, both mutants had lost their
trans-activation capacity. Mutants H and L exhibited
dominant negative activity, but differed in their TRE dependency. The
dominant negative potency of mutant H was in the rank order of Pal
> DR4 > Lys, whereas no TRE dependency was observed for mutant
L. The present study indicates that mutations of the TR gene do
occur in patients and that these novel TR1 mutants provide a
valuable tool to further understand the molecular basis of the dominant
negative action of mutant TRs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TRß, which are located on chromosomes 17 and 3,
respectively (2). Each gene gives rise to two isoforms of receptors,
1 and 2, and ß1 and ß2, due to alternative splicing of the
primary transcripts (2). The gene-regulating activity of TRs is
mediated by binding to specific DNA sequences, known as thyroid hormone
response elements (TREs), located on the promoter regions of thyroid
hormone target genes. The transcriptional activity of TRs is not only
dependent on the thyroid hormone, T3, but also on the type
of TREs. Recent studies have indicated that the transcriptional
activity of TRs is further modulated via interaction with four groups
of cellular proteins: 1) members of the nuclear receptor superfamily,
notably retinoid X receptors (1, 2); 2) corepressors, including
p270/N-CoR (3), SMRT (4), TRUP (5), SHP (6), and TRACs (7); 3)
coactivator, SRC-1 (8); and 4) the tumor suppressor p53 (9). Resistance to thyroid hormone (RTH) is a genetic disease characterized by an inappropriately normal or elevated level of TSH and elevated levels of circulating thyroid hormones. Clinical features include attention deficit hyperactivity disorder, decreased IQ, dyslexia, short stature, decreased weight, tachycardia, and cardiac disease (10, 11, 12). These features have been attributed to mutations in the TRß gene (10, 11). TRß1 mutants derived from RTH patients have reduced T3 binding affinities and transcriptional capacities and, unlike other nuclear receptor mutations causing hormone resistance syndromes, act in a dominant negative fashion to cause the clinical phenotype (10, 11, 13).
For reasons that are not clear, no TR mutants have ever been
identified in RTH patients. It has been postulated that mutations in
the TR gene could occur; however, they escape detection due to the
possibilities that the mutation is inconsequential, lethal, or may not
be associated with the abnormalities observed for RTH. Recently, we
have cloned and characterized a naturally occurring TR mutant,
J7-TR1, from a human hepatocellular carcinoma cell line (14).
J7-TR1 has a single Met259Ile mutation that abolishes
T3-binding activity. However, it binds to the three TREs as
homodimer, and it heterodimerizes equally well as the w-TR1 with the
retinoid X receptor ß (RXRß). Furthermore, it has strong dominant
negative action on wild-type TRs on all three TREs (14). These findings
indicate that mutation of the TR gene could occur naturally and
could abrogate the functions of TRs via a dominant negative effect.
Therefore, it is reasonable to postulate that mutations of the TR
gene could lead to other abnormalities not exclusively associated with
RTH. To test this possibility, we cloned TR genes from the
hepatocellular carcinomas of two patients. We identified two mutants
that had lost T3-binding activity and had unusual TRE
binding characteristics. These two mutants had strong dominant negative
activity, suggesting that mutations of the TR gene could be
associated with abnormalities other than RTH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]Deoxy (d)-CTP (3000 Ci/mmol) and
L-[35S]methionine (10 mCi/ml) were obtained
from Amersham International (Aylesbury, UK).
[125I]T3 (2206 Ci/mmol; 1 Ci = 37
gigabecquerels) was obtained from DuPont-New England Nuclear (Boston,
MA). Super reverse transcriptase and human placental ribonuclease
(RNase) inhibitor were purchased from HT Biotechnology (Cambridge, UK).
pcDNA3, a eukaryotic expression vector, and Lipofectamine were
purchased from Invitrogen (San Diego, CA) and Life Technologies
(Gaithersburg, MD), respectively. Taq DNA polymerase, dNTPs,
pGEM-T Vector Systems, and TNT Coupled Reticulocyte Systems were
obtained from Promega (Madison, WI). Restriction enzymes were obtained
from New England Biolabs (Beverly, MA).
Tumor samples
Surgically removed liver tumors were obtained from patients H
and L of Chang-Gung Memorial Hospital (Taipei, Taiwan). Male patients H
and L had grade II-III hepatocellular carcinoma, and all tested
positive for the hepatitis B viral surface antigen. The status of the
tumor suppressor p53 in these patients was unknown. Thyroid function
tests (total T3, total T4, and TSH) were
normal, and no other symptoms of RTH were observed. Normal adjacent
hepatic tissues were also obtained as controls.
Cloning of thyroid hormone receptors from tumor cells
Total RNA was prepared from tissues by the guanidinium
thiocyanate method (15). Reverse transcription of RNA followed by PCR
were carried out similarly as described by Cook et al. (16).
The complementary DNAs (cDNAs) obtained were used as templates for PCR,
which was carried out in 10 mM Tris-HCl (pH 8.3), 50
mM KCl, 1.5 mM MgCl2, and 0.1
mM dNTP with isoform-specific 5'- and 3'-primers and
Taq polymerase (0.5 µl; 2.5 U/µl). The amplified cDNAs
were purified and ligated onto pGEM-T vectors (Promega). The coding
sequences of the TR1 in these plasmids were verified by restriction
enzyme mapping, Southern blot analysis, and DNA sequencing. The cDNAs
for the hepatocellular carcinoma-derived TR1s were further subcloned
into pcDNA3 (Invitrogen) for in vitro
transcription/translation of TR1 proteins. Their cDNAs were also
subcloned into mammalian expression vectors as described previously
(14).
Detection of mutations in the TR gene
We used the nonisotopic RNase cleavage assay kit (Ambion,
Houston, TX) to screen for point mutations as first described by Myers
et al. (17). Briefly, the cDNAs encoding mutant TR1s and
the wild-type TR1 isolated from normal human liver (w-TR1) were
amplified by PCR using T7 or SP6 promoter-containing primers. After
amplification, 2 µl PCR products were used as templates for in
vitro transcription of sense and antisense strands of RNA, using
T7 and Sp6 polymerase, respectively. An equal volume (5 µl) of sense
and antisense RNAs from hepatocellular carcinoma-TR1 or w-TR1 was
hybridized with the RNAs derived from the same templates or with the
other templates at room temperature for 1 h. Four microliters of
hybridized RNAs were digested with RNase at 30 C for 30 min. The
digested products were run on a 2% agarose gel and visualized with
ethidium bromide staining under UV light.
Sequencing of cDNAs encoding TR1
The plasmids containing the cDNAs encoding TR1 were purified
using the Qiagen Maxi Kit (Qiagen, Chatsworth, CA). Both strands of the
coding sequence were sequenced. The sequencing of cDNA was carried out
using the Applied Biosystems model 373A automatic DNA sequencer
according to the manufacturer’s instructions (Applied Biosystems,
Foster City, CA). Briefly, the reaction mixture consisted of 4 µl 0.8
pmol/µl primer, which expanded the entire coding sequence (see
below), 4 µl 0.25 µg/µl plasmid DNA, and 9.5 µl dideoxy
mixture. The primer sequences used were described previously (14).
The PCR cycle was 96 C for 15 sec, 50 C for 5 sec, and 60 C for 4 min for a total of 25 cycles. The reaction mixture was purified on a CentriSep spin column (Princeton Separations, Adelphia, NJ) and dried. The mixture was redissolved in 4 µl of solution containing formamide and 50 mM EDTA in a ratio of 4:1, denatured at 90 C for 2 min, and applied to a 4.5% denatured gel.
Electrophoresis mobility shift assay (EMSA)
32P-Labeled Lys, DR4, or Pal were prepared as
previously described (18). TR proteins were synthesized by in
vitro transcription/translation using the TNT-coupled reticulocyte
kit according to the manufacturer’s instructions (Promega). The
synthesized TR proteins were quantified by measuring the intensity of
the 35S-labeled protein bands after SDS-PAGE. For EMSA,
identical amounts of TRs were incubated with the
32P-labeled TRE in the presence or absence of RXR or
RXRß. After electrophoresis, TR1 homodimers and heterodimers were
visualized by autoradiography and quantified by a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Binding of [3'-125I]T3 to TRs
Five microliters of the lysates containing the in
vitro translated TR proteins were incubated with 0.2
nM [3'-125I]T3 in the presence of
increasing concentrations of unlabeled T3. The TR-bound
[3'-125I]T3 was separated from free
[3'-125I]T3 as described by Lin et
al. (19). The binding data were analyzed using Eq I based on
direct competition between [125I]T3 and the
unlabeled T3 for a single site on the receptor. The
concentration of radioactive complex is given by the equation: [R] =
{([R]o + [h])/(Kd + [h] + [c]} (Eq
I), where [R]o is the total concentration of receptor,
[h] and [c] are the concentrations of
[125I]T3 and unlabeled T3,
respectively, and Kd is the dissociation constant of the
hormone-receptor complex. The data were fitted directly to Eq I using
the PC-MLAB program (Civilized Software, Bethesda, MD) to evaluate
Kd and [R]o. The fitted curves are shown in
Fig. 4
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1 and the TRE-containing luciferase reporter genes were
transfected into COS-1 cells with or without T3 by the
Lipofectamine method according to the manufacturer’s instructions
(Life Technologies, Grand Island, NY)). After 24 h, the cells were
harvested and lysed, and 20 µl were assayed for luciferase activity
(14). The transfection efficiency was normalized to the protein
concentration of the lysates.
Western blotting
Cell lysates (30 µg) from transient transfection experiments,
as described above, were loaded onto a 10% SDS-PAGE gel. After
electrophoresis, proteins were transferred onto a nitrocellulose
membrane (PH79 membrane, Schleicher and Schuell, Keene, NH). The
membrane was gently shaken in 5% nonfat milk in Tris-buffered saline
(25 mM Tris, pH 7.4, and 150 mM NaCl) for
20 h and subsequently washed three times with Tris-buffered
saline. The membrane was incubated with monoclonal antibody C4 (2
µg/ml) (20) for 1 h. After washing, the membrane was incubated
with affinity-purified rabbit antimouse Ig conjugated with horseradish
peroxidase (1:250 dilution). TR protein bands were visualized by
chemiluminescence using the ECL kit (Amersham Life Science, Arlington
Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 mutants from patients with hepatocellular
carcinoma gene in
cultured cell lines derived from hepatocellular carcinomas of patients
(14). To characterize TRs directly in tumor tissues, we cloned cDNAs
encoding TR1 from the hepatocellular carcinomas of two patients
(TR1-H and TR1-L) who had been diagnosed with grade II–III
hepatocellular carcinoma. As a control, w-TR1 was similarly cloned
from normal human liver. To determine whether their TR genes had
mutations, the mismatch point mutation detection analysis was used. As
a control, the hybridized sense and antisense RNAs of w-TR1 were not
treated (lane 2 of Fig. 1) or were
treated with RNase (lane 3 of Fig. 1). As expected, one RNA band with
the same size and similar intensity was detected in both conditions,
confirming a perfect complementarity between the two RNAs. However,
when the sense w-TR1 RNA was hybridized with antisense TR1-H
(lane 4) or antisense TR1-L (lane 7) and treated with RNase, several
degraded fragments with smaller sizes were detected. Degraded fragments
were also seen when the antisense w-TR1 RNA was hybridized with the
sense TR1-H (lane 5) or sense TR1-L (lane 8) and treated with
RNase. This was in contrast to that shown in lanes 6 and 9, in which
one major band with minor background bands were detected when the sense
and anitsense RNAs of TR1-H or TR1-L, respectively, were
hybridized and treated with RNase. These results indicate that the two
TR1s cloned from liver tumors of patients had mutations in the TR
gene. Because we did not detect mutant TR1 in adjacent normal
hepatic tissues or in biopsy sample of normal liver tissue, these
mutations most likely are somatic events of hepatocellular
carcinomas.
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1-H and TR1-L were sequenced. w-TR1 cloned from normal human
liver was also sequenced as a positive control. TR1-H had a point
mutation at nucleotide position 1241 from T to C, resulting in changing
of Val (GTC) to Ala (GCC) at codon 390 (see Fig. 2). TR1-L had two mutations at
nucleotide positions 1120 (G to A) and 1264 (C to T), resulting in
changing of Glu (GAG) to Lys (AAG) and of Pro (CCC) to Ser (TCC) at
codons 350 and 398, respectively (Fig. 2). It was of great interest to
note that the mutation sites for mutants H (V390A) and L (P398S) were
located in the corresponding region of "hot spot 2" of TRß1 (10, 21).
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1 mutants were
impaired
shows that the size and intensity of mutant proteins were similar to
those of w-TR1. The translated wild-type (lane 1 of Fig. 3A) and
mutant TR1s (lanes 2–3) show a full-length receptor with an
apparent molecular mass of 49 kDa and three other truncated proteins,
most likely due to initiations from the downstream ATG (22). In
addition, Fig. 3B shows that both mutants H and L were
immunoprecipitated by the anti-TR monoclonal antibody C4 (mAb C4; lanes
2 and 3, respectively) in a manner similar to that of w-TR1 (lane 1
of Fig. 3B) (20). Taken together, these results indicate that the
mutant proteins were correctly translated.
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1 were used for binding to
[125I]T3. Figure 4
shows the competitive binding
displacement curves. The data for mutant J7-TR1 (14) were also
included as a control. Analysis of the binding data indicates that
w-TR1 bound to T3 similarly to that shown previously
(Kd = 0.056 ± 0.023 nM) (14). In
contrast, mutants H and L had lost T3-binding activity,
similar to that seen for the mutant J7-TR1 (14).
The DNA-binding activity of mutants was characterized by EMSA using
three TREs: Lys, DR4, and Pal. As a control, lanes 1 of Fig. 5, A–C, show that w-TR1 bound to
these TREs with different homodimer/monomer ratios in the rank order of
Lys > DR4 > Pal. Thus, w-TR1 bound to Lys mainly as a
homodimer (lane 1 of Fig. 5A), whereas w-TR1 bound to Pal mainly as
a monomer (lane 1 of Fig. 5C). w-TR1 bound to these three TREs
equally well as a heterodimer with RXR or RXRß (lanes 2 and 3 of
Fig. 5, A–C).
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1. Lanes 7 of Fig. 5, A–C, show that binding of
mutant L to Lys, DR4, and Pal, respectively, as homodimers was greatly
impaired, in that the formation of homodimers was less than 10% of
that seen for w-TR1 (lanes 1 of Fig. 5, A and B). Binding of mutant
H to the three TREs was more complex. No effect on the binding of
mutant H to DR4 as homodimers was detected (lane 1 vs. lane
4 of Fig. 5B), but its binding to Lys as homodimers was greatly
impaired (>90% inhibition by comparing lane 1 vs. lane 4
of Fig. 5A), and that to Pal was markedly enhanced compared with that
of w-TR1 (lane 1 vs. lane 4 of Fig. 5C). Therefore,
formation of mutant homodimers was TRE dependent. However, the binding
of mutants H and L to the three TREs as heterodimers with either RXR
(lanes 5 and 8, respectively) or RXRß (lanes 6, and 9, respectively)
was not significantly altered compared with that of w-TR1.
To assess the functional consequences of these mutations in these two
mutants, we used transient transfection assays. Mammalian expression
plasmids of mutants were cotransfected with the TRE-containing reporter
genes into COS-1 cells. Figure 6 compares
the trans-activational activity of mutants H and L on the
three TREs. The trans-activational activities of the
w-TR1 and mutant J7-TR1 (14) were also included for comparison.
As expected, the trans-activational activity of w-TR1
mediated by Lys (Fig. 6A), ME (a DR4-type TRE; Fig. 6B), and Pal (Fig. 6C) was increased in a T3-dependent manner, whereas,
consistent with the previous findings, mutant J7-TR1 had no
trans-activational activity (14). However, similar to that
seen for J7-TR1, mutants H and L had little or no
trans-activation activity. These results indicate that the
trans-activation activity of the TR1s was consistent with
the hormone-binding activity. We have also evaluated the silencing
effect of mutants. Figure 6D shows that mutants H and L had a 40%
greater silencing effect than w-TR1 on Pal, whereas on ME and Lys,
the extent of silencing was similar to that of w-TR1.
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1. J7-TR1, which has been shown to be a strong
dominant negative mutant, was used as a control (14). An unusual
pattern of dominant negative potency emerged for these mutants. When
equal amounts of mutant and w-TR1 expression plasmids were
transfected, the rank order of dominant negative potency was Pal
> DR4 > Lys for mutant H (Fig. 7, A–C). This is in contrast to what has been seen for the TRß1 PV
mutant, in which Lys mediated the strongest dominant negative activity
(18, 23). For mutant L, a weaker dominant negative activity than that
of mutant H was observed, which was not significantly affected by the
type of TRE. At a higher mutant/w-TR1 plasmid ratio of 5, the
dominant negative potency of mutant H on Lys, DR4, and Pal was
increased 10-, 3-, and 2-fold, respectively, with no changes in the
rank order for these TREs. The dominant negative potency of mutant L
was also increased (3-fold) when the mutant/w-TR1 plasmid ratio
was increased to 5; however, no TRE dependency was seen. The dominant
negative effect detected for mutants H and L was not due to the
different expression levels of w-TR1, mutant H, or mutant L, because
identical amounts of w-TR1, mutant H, and mutant L were expressed in
the transfected cells, as determined by Western blotting (lanes 1
vs. 2 and 3 of Fig. 8). Thus,
among these three TREs, Pal which bound to mutant H with the most
avidity, mediated the strongest dominant negative activity. Mutant L,
which had weak binding to all three TREs, had similarly weak dominant
negative activity (see Fig. 5, A–C). These results indicate that there
was a correlation between homodimer formation and dominant negative
potency for these two mutants.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 from hepatocellular carcinomas of two patients. These
two mutant receptors, mutant H (V390A) and L (E350K/P398S), had
distinct properties. Compared with w-TR1, the binding of mutant L to
all three classes of TREs as homodimers was defective. The DNA-binding
characteristics of mutant H were TRE dependent. Homodimer binding to
DR4 was not altered, whereas that to Lys was greatly impaired, and that
to Pal was markedly enhanced. In addition, mutants H and L had lost
their abilities to bind T3 as well as their
hormone-dependent trans-activational activities. There was
no change in the abilities of these mutant receptors to bind to all
three TREs as heterodimers with RXR and RXRß.
Among the members of the steroid/thyroid hormone receptor superfamily,
TRs are unique in that their isolated DNA-binding domains (DBD) can
bind efficiently to DNA as monomers (24, 25, 26). Based on the crystal
structure of an RXR/TRß-DBD complex with a DR4 TRE, Rastinejad
et al. (27) ascribed this behavior to increased protein-DNA
interactions induced by the A helix of the TR DBD, which is absent in
other nuclear receptors. Intact TR1 and TRß1 differ markedly in
their abilities to bind to DNA as monomers or homodimers, and these
differences are TRE dependent. Using chimeric receptors made by
exchanging domains between TR1 and TRß1, we showed that these
differences were not intrinsic properties of their DBDs, but resulted
from interplay between the domains of the proteins (28). Chimeric TRs
containing domains D or E of TRß1 showed increased propensities to
form homodimers and mediated higher trans-activational
activities than TR1. However, no significant differences in the
abilities of these chimeric TRs to form heterodimers with RXR were
detected. Consequently, it is not surprising that these mutations,
which occur in domain E of TR1, have major effects on DNA binding.
These must reflect the effects of the amino acid substitutions on local
structure and domain interactions and could account for the differences
in the TRE-dependent binding to these mutants.
As a result of their mutations, mutants H and L had lost
transcriptional activity. The residues that are mutated in these two
mutant receptors are located in regions that have been shown to be
intimately involved in the various functions of TRs. Mutant L was
mutated in two separate locations. The first of these, E350, is
conserved in most TRs (29). In the crystal structure of rat w-TR1
ligand-binding domain (LBD), Wagner et al. (30) placed this
residue in helix 10, which together with helix 11 was suggested to form
a surface for homodimerization of intact receptors. At neutral pH,
glutamate and lysine are similar in their helix-forming propensities,
so little structural disruption may occur. However, the change in
charge produced by mutation E350K could have drastic effects on
intermolecular interactions. The second mutated residue, P398, is in
the same region of the molecule as residue V390, the mutated residue in
mutant H. From comparisons of the crystal structures of both apo and
holo forms of the LBDs of several members of the steroid/thyroid
hormone receptor family, a consensus mechanism of ligand-binding and
hormone-induced gene activation has been proposed (31, 32). In apo
RXR, the C-terminal helix 12 protrudes from the LBD into solution.
In the holo forms of RAR and TR1 LBDs, this helix is folded back
onto the body of the protein, providing part of the ligand-binding
cavity, realigning the AF-2 sequence, which it carries, and perturbing
the homodimerization interface via helix 11. Binding of hormone is
thought to accompany this transformation, which is dependent on the
flexibility of the loop connecting helixes 11 and 12. The two residues
in question are located in the last and first turns of helixes 11 and
12, respectively. Their replacement by helix-favoring side-chains
(V390A or P398S) can be expected to increase rigidity in this region,
impairing the hormone binding and thus the obligatory conformational
changes required for hormone-dependent transcriptional activity (3, 31, 32).
Both mutants H and L acted to inhibit the trans-activation
activity of w-TR1. The dominant negative potency of mutant H was TRE
dependent, with the rank order of Pal > DR4 > Lys, whereas
for mutant L, it was TRE independent. This rank order of dominant
negative potency of mutant H correlated well with the ability of
mutants to bind to TREs as homodimers. Binding of mutant H homodimers
to Pal was the strongest among three TREs (Pal > DR4 >
Lys), whereas formation of mutant L homodimers was defective on all
TREs. Furthermore, we have previously shown that mutant J7-TR1 binds
to TREs as homodimers in the rank order of Lys > DR4 > Pal
(14). Its dominant negative potency was found in the rank order of
Lys > DR4 > Pal (14). The correlation of dominant negative
potency with the ability of mutants to form homodimers is not limited
to the TR1 mutants. Previously, Kitajima et al. have also
shown that selective loss of homodimerization of the TRß1 mutant
(R316H) correlated with reduced dominant negative activity (33, 34).
To date, no TR1 mutants have been identified from RTH patients.
However, the present study isolated TR1 mutants from patients with
hepatocellular carcinoma. Therefore, mutations of the TR gene do
occur naturally, but most likely may not be found in RTH patients. This
raises the possibility that the mutants of TR isoforms may be involved
in mediating different abnormalities via the common mode of dominant
negative action. Although evidence is still lacking to directly link
the mutations of the TR gene to the pathogenesis of hepatocellular
carcinoma, the isolation of TR1 mutants that had dominant negative
action suggests that mutations of the TR gene could play an
important role in the tumorigenesis of liver. This possibility is
supported by the observation that transgenic mice harboring
v-erbA, an oncogenic homolog of TR1 and a dominant
negative transcription factor of TRs, develop hepatocellular carcinoma
(35). Furthermore, increasing evidence has been presented to support
isoform-specific functional roles of TR1 and TRß1. Strait et
al. showed that the gene encoding PCP-2 is regulated by TRß1,
but not by TR1 (36). T3-dependent negative regulation of
TRH promoter was shown to be mediated by TRß1, but not by TR1
(37). Lebel et al. showed that in a stably transfected
neuronal cell line, only cells overexpressing TRß1, not TR1, can
respond to T3 to exhibit morphological and functional
characteristics indicative of neural differentiation (38). The most
compelling evidence for the isoform-specific functional role of TRs was
provided by the recent TRß gene knock-out mouse model, in that a
unique role of TRß in the pituitary-thyroid axis was demonstrated
(39). Based on these findings, it is reasonable to postulate that
mutations of TR subtypes may lead to different phenotypic
manifestations through a common mechanism of dominant negative action.
How this is accomplished in vivo will have to await future
studies.
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Acknowledgments |
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and RXRß plasmids. |
Footnotes |
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Received May 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and ß1 receptors in
a human hepatocellular carcinoma cell line. Endocrinology 137:4073–4081[Abstract]
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K.-h. Lin and Y.-h. Wu shen-liang chen Impaired Interaction of Mutant Thyroid Hormone Receptors Associated with Human Hepatocellular Carcinoma with Transcriptional Coregulators Endocrinology, February 1, 2001; 142(2): 653 - 662. [Abstract] [Full Text] [PDF]
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K.-h. Lin, H.-y. Shieh, and H.-C. Hsu Negative Regulation of the Antimetastatic Gene Nm23-H1 by Thyroid Hormone Receptors Endocrinology, July 1, 2000; 141(7): 2540 - 2547. [Abstract] [Full Text] [PDF]
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w-TR
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