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PHYSIOLOGICAL REVIEWS

Vol. 81, No. 3, July 2001


Printed in U.S.A.

Physiological and Molecular Basis


of Thyroid Hormone Action
PAUL M. YEN

Molecular Regulation and Neuroendocrinology Section, Clinical Endocrinology Branch, National Institute
of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

I.Introduction 1097
II.Background: Thyroid Hormone Synthesis 1098
III.Multiple Thyroid Hormone Receptor Isoforms 1099
IV. Thyroid Hormone Receptor Functional Domains 1102
A. DNA-binding domain 1102
B. Ligand-binding domain 1102
C. Hinge region 1104
D. Amino-terminal (A/B) domain 1104
V. Thyroid Hormone Response Elements 1105
VI. Thyroid Hormone Receptor Complexes 1106
VII. Phosphorylation of Thyroid Hormone Receptors 1107
VIII. Molecular Mechanisms of Thyroid Hormone Receptor Action 1108
A. Corepressors/basal repression 1108
B. Coactivators/transcriptional activation 1111
C. Cross-talk with other nuclear hormone receptors 1114
D. Nongenomic effects of TH 1115
IX. Thyroid Hormone Effects on Target Tissues 1116
A. Bone 1116
B. Heart 1117
C. Fat 1118
D. Liver 1119
E. Pituitary 1120
F. Brain 1120
X. Resistance to Thyroid Hormone 1121
XI. Genetically Engineered Mouse Models of Thyroid Hormone Action 1124
XII. Conclusion 1126

Yen, Paul M. Physiological and Molecular Basis of Thyroid Hormone Action. Physiol Rev 81: 10971142, 2001.
Thyroid hormones (THs) play critical roles in the differentiation, growth, metabolism, and physiological function of
virtually all tissues. TH binds to receptors that are ligand-regulatable transcription factors belonging to the nuclear
hormone receptor superfamily. Tremendous progress has been made recently in our understanding of the molecular
mechanisms that underlie TH action. In this review, we present the major advances in our knowledge of the
molecular mechanisms of TH action and their implications for TH action in specific tissues, resistance to thyroid
hormone syndrome, and genetically engineered mouse models.

I. INTRODUCTION ism due to iodine deficiency remains a public health prob-


lem in developing countries at the advent of the third
Thyroid hormones (THs) play critical roles in differ- millennium. Thus the study of TH action has important
entiation, growth, and metabolism. Indeed, TH is required biological and medical implications.
for the normal function of nearly all tissues, with major The story of TH action is interwoven with many of
effects on oxygen consumption and metabolic rate (375). the major advances in biomedical science during the past
Disorders of the thyroid gland are among the most com- century. Contributions from clinical medicine, physiol-
mon endocrine maladies. Furthermore, endemic cretin- ogy, biochemistry, and molecular genetics have had major

http://physrev.physiology.org 0031-9333/01 $15.00 Copyright 2001 the American Physiological Society 1097
1098 PAUL M. YEN Volume 81

impacts on our understanding of TH action (376, 551). The ular mechanisms of TR action. The power of molecular
following outline sketches only some of the many early genetics has greatly aided our understanding of the roles
contributions to our knowledge. of unliganded and liganded TRs in regulating target genes.
In 1888, the Clinical Society of London published the We have learned that there are multiple TR isoforms that
definitive report that first linked cretinism and adult hy- bind to TREs with variable orientation, spacing, and se-
pothyroidism to the destruction of the thyroid gland quences for TRE half-sites. TRs also interact with other
(97a). Soon afterward, thyroid extracts from sheep were nuclear proteins such as corepressors or coactivators to
used for the treatment of hypothyroidism. Around this form complexes that regulate local histone acetylation
time, Emil Kocher performed some of his pioneering stud- and interact with the basal transcriptional machinery.
ies on the pathology and surgery of the thyroid gland for Additionally, the solution of the crystal structures of the
which he was awarded the Nobel prize in medicine in TR ligand-binding domain (LBD) and other nuclear hor-
1909. In 1914, Kendall (241) isolated 3,5,39,59-tetraiodo-L- mone receptors have provided insight into some of these
thyronine (T4) from thyroid extracts, and almost 40 years complex interactions at the molecular level. The develop-
later, Gross and Pitt-Rivers (178) synthesized 3,5,39-tri- ment of transgenic and knockout mouse models have
iodo-L-thyronine (T3) and demonstrated its presence in shed light on the roles of TRs in the regulation of specific
human plasma and its ability to prevent goiter in thioura- target genes and development. These findings have
cil-treated rats. Over the ensuing years, the metabolic and greatly aided our understanding of the molecular mecha-
oxygen consumption effects of THs as well as its effects nisms of TH action in normal and disease states. In par-
on development, particularly in amphibians, were appre- ticular, much has been learned about the pathogenesis of
ciated (59, 79, 463). the human genetic disorder of resistance to thyroid hor-
In the 1960s, Tata and co-workers (509, 510) first mone (RTH). We review some of the major advances in
suggested that THs might be involved in the transcrip- these areas by initially focusing on what is known about
tional regulation of target genes. These investigators ob- the molecular mechanisms of TH action and then discuss
served that T3 treatment of hypothyroid rats induced a their implications for TH action in specific tissues, RTH,
rapid increase in RNA synthesis in the liver which pre- and genetically engineered mouse models.
ceded new protein formation and mitochondrial oxida-
tion (509, 510). The groups of Oppenheimer (372) and
Samuels (441) then used radiolabeled TH to demonstrate II. BACKGROUND: THYROID HORMONE
specific nuclear binding sites in different T3-sensitive tis- SYNTHESIS
sues, and thus provided the first evidence for TH recep-
tors (TRs). Moreover, T3 binding was observed in almost TH synthesis and secretion is exquisitely regulated
all tissues (377). Attempts to purify these receptors bio- by a negative-feedback system that involves the hypothal-
chemically were only partially successful; however, pho- amus, pituitary, and thyroid gland [hypothalamic/pitu-
toaffinity labeling of nuclear extracts demonstrated dif- itary/thyroid (HPT) axis] (467). Thyrotropin releasing hor-
ferent-sized receptors and raised the possibility of mone (TRH) is a tripeptide (PyroGlu-His-Pro) synthesized
multiple TR isoforms (123, 384). Studies on T3 induction in the paraventricular nucleus of the hypothalamus. It is
of the rat growth hormone (GH) gene transcription sug- transported via axons to the median eminence and then to
gested that TRs recognized enhancer sequences or TH the anterior pituitary via the portal capillary plexus. TRH
response elements (TREs), similar to steroid hormone binds to TRH receptors in pituitary thyrotropes, a sub-
receptors (101, 281, 282, 440). Thus TRs behaved similar population of pituitary cells that secrete thyroid stimulat-
to steroid hormone receptors with respect to nuclear site ing hormone (TSH). TRH receptors are members of the
of action, recognition of specific DNA sequences, and seven-transmembrane spanning receptor family and are
ligand-dependent regulation of transcription. In 1985, the coupled to Gq11. TRH stimulation leads to release and
glucocorticoid receptor was cloned and, surprisingly, had synthesis of new TSH in thyrotropes. TSH is a 28-kDa
homology with a known viral oncogene product, v-erbA, glycoprotein composed of a- and b-subunits designated
that in conjunction with v-erbB, can cause erythroblasto- as glycoprotein hormone a- and TSH b-subunits. The
sis in chicks (209). Subsequent cloning of the estrogen a-subunit also is shared with other hormones such as
receptor suggested that there was a family of nuclear luteinizing hormone, follicle stimulating hormone, and
hormone receptors (174). A year later, the laboratories of chorionic gonadotropin. Both TRH and TSH secretion are
Evans (545) and Vennstrom (444) ushered in the molec- negatively regulated by TH. An important mechanism for
ular era of TH action when they cloned two different TR the negative regulation of TSH may be the intrapituitary
isoforms and showed they were the cellular homologs of conversion of circulating T4 to T3 by type II deiodinase.
v-erbA. Additionally, somatostatin and dopamine from the hypo-
Since the molecular cloning of TRs 15 years ago, thalamus can negatively regulate TSH secretion.
there has been an explosion of information on the molec- TSH is the primary regulator of TH release and se-
July 2001 THYROID HORMONE ACTION 1099

cretion. It also has a critical role in thyroid growth and Type I deioidinase is found in peripheral tissues such as
development. TSH binds to the TSH receptor (TSHr), liver and kidney and is responsible for the conversion of
which also is a seven-transmembrane spanning receptor the majority of T4 to T3 in circulation. Type II deiodinase
coupled to Gs (255, 382). Activation of TSHr by TSH or is found in brain, pituitary, and brown adipose tissue and
autoantibodies in Graves disease leads to an increase in primarily converts T4 to T3 for intracellular use. These
intracellular cAMP and stimulation of protein kinase A- deiodinases recently have been cloned and demonstrated
mediated pathways. A number of thyroid genes, including to be selenoproteins (280). 59-Deiodination by type I deio-
Na1/I2 symporter (NIS), thyroglobulin (Tg), and thyroid dinase and type III deioidinase, which is found primarily
peroxidase (TPO), are stimulated by TSH and promote in placenta, brain, and skin, leads to the generation of rT3,
the synthesis of TH. Of note, activating mutations in TSHr the key step in the inactivation of TH. rT3 and T3 can be
and Gs have been described in autonomously functioning further deiodinated in the liver and are sulfo- and gluc-
thyroid nodules and familial congenital hyperthyroidism uronide-conjugated before excretion in the bile (124).
(381, 383). There also is an enterohepatic circulation of TH as intes-
The THs, T4 and the more potent T3, are synthesized tinal flora deconjugates some of these compounds and
in the thyroid gland (Fig. 1). Iodide is actively transported promotes the reuptake of TH.
and concentrated into the thyroid by NIS (102, 475). The Although THs may exert their effects on a number of
trapped iodide is oxidized by TPO in the presence of intracellular loci, their primary effect is on the transcrip-
hydrogen peroxide and incorporated into the tyrosine tional regulation of target genes. Early studies showed
residues of a 660-kDa glycoprotein, Tg. This iodination of that the effects of THs at the genomic level are mediated
specific tyrosines located on Tg yields monoiodinated and by nuclear TRs, which are intimately associated with
diiodinated residues (MIT, monoiodo-tyrosines; DIT, di- chromatin and bind TH with high affinity and specificity
iodo-tyrosines) that are enzymatically coupled to form T4 (375, 440). Similar to steroid hormones that also bind to
and T3. The iodinated Tg containing MIT, DIT, T4, and T3, nuclear receptors, TH enters the cell and proceeds to the
then is stored as an extracellular storage polypeptide in nucleus (Fig. 2). It then binds to TRs, which may already
the colloid within the lumen of thyroid follicular cells. be prebound to TREs located in promoter regions of
Genetic defects along the synthetic pathway of THs have target genes. The formation of ligand-bound TR com-
been described in humans and are major causes of con- plexes that are also bound to TREs is the critical first step
genital hypothyroidism in iodine-replete environments in the positive or negative regulation of target genes and
(117, 261). the subsequent regulation of protein synthesis. Given
The secretion of THs requires endocytosis of the their abilities to bind both ligand and DNA as well as their
stored iodinated Tg from the apical surface of the thyroid ability to regulate transcription, TRs can be regarded as
follicular cell (511). The internalized Tg is incorporated in ligand-regulatable transcription factors.
phagolysosomes and undergoes proteolytic digestion, re-
capture of MIT and DIT, and release of T4 and T3 into the
circulation via the basal surface. The majority of released III. MULTIPLE THYROID HORMONE
TH is in the form of T4, as total serum T4 is 40-fold higher RECEPTOR ISOFORMS
than serum T3 (90 vs. 2 nM). Only 0.03% of the total serum
T4 is free (unbound), with the remainder bound to carrier In 1986, the laboratories of Vennstrom (444) and
proteins such as thyroxine binding globulin (TBG), albu- Evans (545) independently cloned cDNAs encoding two
min, and thyroid binding prealbumin. Approximately 0.3% different TRs from embryonal chicken and human placen-
of the total serum T3 is free, with the remainder bound to tal cDNA libraries. Several unexpected findings stemmed
TBG and albumin. It is the free TH that enters target cells from their landmark work. First, they demonstrated by
and generates a biological response. amino acid sequence comparison that TRs are the cellular
The major pathway for the production of T3 is via homologs of the viral oncogene product v-erbA. Second,
59-deiodination of the outer ring of T4 by deiodinases and TRs were shown to have amino sequence homology with
accounts for the majority of the circulating T3 (50, 256). steroid hormone receptors. This was initially surprising

FIG. 1. Structure of thyroid hor-


mones.
1100 PAUL M. YEN Volume 81

FIG. 2. General model for thyroid hor-


mone action in the nucleus. TR, thyroid hor-
mone receptor; RXR, retinoid X receptors.

since T3 and cholesterol-derived steroids are structurally cies such as amphibians, chick, mouse, rat, and human
different ligands. However, in the ensuing years, TRs have (284). Both TR isoforms bind T3 (reported dissociation
been shown to belong to a large superfamily of nuclear constant values between 1029 and 10210 M) and mediate
hormone receptors that include the steroid, vitamin D, TH-regulated gene expression (148, 340, 449). In mamma-
and retinoic acid receptors as well as orphan receptors lian species, TRa-1 and TRb-1s range from 400 to slightly
for which there are no known ligand or function (35, 285). over 500 amino acids in size (284, 289) and contain highly
TRs share a similar domain organization with other family homologous DNA-binding domains and LBD (Fig. 3).
members as they have a central DNA-binding domain In addition to two separate genes that encode TRs,
containing two zinc fingers and a carboxy-terminal LBD. there is additional heterogeneity of TRs due to alternative
These initial studies also suggested that there were mul- splicing (220, 292, 330, 349). Alternative splicing of the
tiple TR isoforms. Subsequent work by many groups has initial RNA transcript of the TRa gene generates two
confirmed that there are two major TR isoforms encoded mature mRNAs that each encode two proteins: TRa-1 and
on separate genes, designated as TRa and TRb, encoded c-erbAa-2. In the rat, these proteins are identical from
on human chromosomes 17 and 3, respectively (284). amino acid residues 1370, but their respective sequences
Moreover, these multiple isoforms exist in different spe- diverge markedly thereafter (Fig. 3). Consequently,

FIG. 3. Comparison of amino acid homologies and their functional properties among TR isoforms. The length of
receptors is indicated just above the receptor diagrams, and the percent amino acid homology with TRb-2 is included
in the receptor diagrams.
July 2001 THYROID HORMONE ACTION 1101

c-erbAa-2 cannot bind T3 because it contains a 122-amino Careful low-stringency hybridization studies so far
acid carboxy terminus that replaces a region in TRa-1 that have not yielded any additional TR isoforms. Double TRa
is critical for TH binding. Additionally, c-erbAa-2 binds and TRb knockout mice are viable, and these mice did not
TREs weakly but cannot transactivate TH-responsive have detectable [125I]T3 binding in nuclear extracts of
genes. Thus TRa-1, but not c-erbAa-2, is an authentic TR. several tissues (171, 316a). However, a number of short
Indeed, c-erbAa-2 may act as an inhibitor of TH action forms of TRa and TRb generated by alternative splicing of
possibly by competing for binding to TREs (253, 291). The mRNA or by use of internal translational start sites have
TRa-1 and c-erbAa-2 system, then, represents one of the been found in embryonic stem cells and in fetal bone cells
first examples in which multiple mRNAs generated by and may have biological significance (41, 75, 553, 567).
alternative splicing encode proteins that may be antago- The identification of a novel estrogen receptor isoform
nistic to each other. Mitsuhashi et al. (330) also have (ERb) 10 years after the discovery of ERa serves as a
described a second TRa variant, c-erbAa-2V, in which the
cautionary warning to remain open to the possibility of
first 39 amino acids of the divergent sequence are missing
novel TR isoforms, particularly in restricted tissues or
(330). Its function currently is unknown. Yet another in-
during transient periods in fetal development (265).
teresting feature of the TRa gene is the employment of the
The regulation of the TR mRNAs is isoform and cell
opposite strand to encode a gene product, rev-erbA. Rev-
type dependent. In the intact rat pituitary, T3 decreases
erbA mRNA contains a 269-nucleotide stretch which is
complementary to the c-erbAa-2 mRNA due to its tran- TRb-2 mRNA, modestly decreases TRa-1 mRNA, and
scription from the DNA strand opposite of that used to slightly increases rat TRb-1 mRNA (204). Despite these
generate TRa-1 and c-erbAa-2 (293, 331). This protein also opposing effects, the total T3 binding decreases by 30% in
is a member of the nuclear hormone receptor superfam- the T3-treated rat pituitary. Similar findings also were
ily. It is expressed in adipocytes and muscle cells, and can observed in GH3 cells, a somatolactotropic rat cell line
bind to TREs and retinoic acid response elements (205). In other tissues, T3 slightly decreases TRa-1 and
(RAREs) and repress gene transcription (190, 477, 597). c-erbAa-2 mRNA except in the brain where c-erbAa-2
However, rev-erbA should be considered an orphan re- levels are unaffected. TRb-1 mRNA is minimally affected
ceptor since its cognate ligand and function are not in nonpituitary tissues. Additionally, the hypothalamic tri-
known. One potential role for rev-erbA may be to regulate peptide TRH decreases TRb-2 mRNA, slightly decreases
the splicing that generates c-erbAa-2 as increased levels TRa-1 mRNA, and minimally affects TRb-1 mRNA in GH3
of rev-erbA mRNA correlate with increased TRa-1 mRNA cells (230). Retinoic acid blunts the negative regulation by
relative to c-erbAa-2 (80, 224, 290). T3 in these cells (114, 231). Additionally, in patients with
There also are two TRs derived from the TRb gene nonthyroidal illness in which their circulating free T3 and
(205, 284). This gene contains two promoter regions each T4 levels were decreased, TRa and TRb mRNAs were
of which is vital for the transcription of an mRNA coding increased in peripheral mononuclear cells and liver bi-
for a distinctive protein. By the use of alternate promoter opsy specimens (556). Thus induction of TR expression
choice, one or both of the coding mRNAs are generated may compensate for decreased circulating TH levels in
(566). The resultant TRb isoforms are designated as these patients.
TRb-1 and TRb-2. The amino acid sequences of the DNA Each of the TR isoforms found in human, rat, and
binding, hinge region, and LBDs of these two TRbs are mouse are highly homologous with respect to their amino
identical, but the amino-terminal regions bear no homol-
acid sequences (284). This conservation among species
ogy (Fig. 3). Both are authentic receptors as they bind
suggests that there may be important specialized func-
TREs and TH with high affinity and specificity and can
tions for each isoform (109). However, the evidence for
mediate TH-dependent transcription. The expression of
isoform-specific functions has been scant since most co-
the two TRb isoforms may be regulated by pituitary-
specific transcription factors such as Pit-1 (566). transfection experiments have failed to show important
Both TRa-1 and TRb-1 mRNAs and proteins are functional differences. Nonetheless, recent studies have
ubiquitously expressed in rat tissues (204). However, suggested that TRb-1 may exhibit isoform-specific regu-
TRa-1 mRNA has highest expression in skeletal muscle lation of the TRH and myelin basic protein genes, and
and brown fat, whereas TRb-1 mRNA has highest ex- TRb-2 may play an important role in the regulation of the
pression in brain, liver, and kidney. In contrast to the GH and TSHb gene expression in the pituitary (4, 129, 208,
other TR isoforms, TRb-2 mRNA and protein have tis- 283, 305). Future studies with TR knockout mice, anti-
sue-specific expression in the anterior pituitary gland sense oligonucleotides in tissue culture, and isoform-spe-
and specific areas of the hypothalamus as well as the cific ligands, perhaps in conjunction with cDNA microar-
developing brain and inner ear (47, 48, 98, 204, 590). In rays, should shed more light on the respective roles of TR
the chick, TRb-2 mRNA also is expressed in the devel- isoforms in regulating specific target genes (24, 86, 136,
oping retina (473). 151).
1102 PAUL M. YEN Volume 81

FIG. 4. General organization of major


TR domains and functional subregions.

IV. THYROID HORMONE RECEPTOR tors (VDRs) (106, 317, 531). This critical region has been
FUNCTIONAL DOMAINS shown to be important in sequence-specific recognition of
hormone response elements by different members of the
Mutational analyses of TRs and comparisons with nuclear hormone superfamily and contacts nucleic acids
other members of the nuclear hormone receptor super- and phosphate groups within the major groove of the TRE
family have yielded much information on the structural (353, 413). Additionally, there are other important contact
features of TRs (284, 583). All TRs have a similar domain points within the minor groove of the TRE just downstream
organization as that found in all nuclear hormone recep- from the second zinc finger (A-box region). Also, as dis-
tors: an amino-terminal A/B domain, a central DNA-bind- cussed below, TRs can heterodimerize with RXRs and can
ing domain containing two zinc fingers (DBD), a hinge bind to TREs that are arranged as direct repeats separated
region containing the nuclear localization signal, and a by a four nucleotide gap. These TR/RXR heterodimers bind
carboxy-terminal LBD (Fig. 4). It should be noted that to TREs with a 59 to 39 polarity with TR in the downstream
each of these domains and regions may subserve multiple position (268, 391, 586). The ability to heterodimerize with
functions, and thus their names may only reflect the first RXR is critical for TR binding to the asymmetric TRE, as
function ascribed to them. dimerization contacts stabilize the DNA binding and deter-
mine the spacing between half-sites. Within the DBD, there
are dimerization interfaces in the TR just upstream of the
A. DNA-Binding Domain
first zinc finger, within the first zinc finger, and in a subre-
gion distal to the second zinc finger (T box). The RXR
The DBD is located in the central portion of TR and has
dimerization surfaces are located in the second zinc finger
two zinc fingers, each composed of four cysteines coordi-
including an arginine located in the D box, a region which
nated with a zinc ion (Fig. 5). The integrity of each zinc
previously has been shown to be important for distinguish-
finger is critical, as deletion of zinc fingers or amino acid
ing spacing between half-sites of hormone response ele-
substitution of these cysteine residues abrogates DNA-bind-
ments (315, 531).
ing and transcriptional activity of steroid hormone receptors
and TRs (173, 345, 460, 592). Within the first zinc finger, there
is a P box, comprised of amino acids located between and B. Ligand-Binding Domain
just distal to the third and fourth cysteines, which is similar
to that of estrogen recetors (ERs), retinoic acid receptors The LBD not only is necessary for TH binding but
(RARs), retinoid X receptors (RXRs), and vitamin D recep- also plays critical roles for dimerization, transactivation,

FIG. 5. DNA-binding domains of hu-


man TRb. Schematic drawing of the two
zinc fingers of human TRb and the vari-
ous subregions within the DNA-binding
domains. Squares, TR/RXR heterodimer-
ization contacts; ovals, direct base con-
tacts; solid circles, direct phosphate con-
tacts. [From Rastinejad et al. (413).
Reprinted by permission from Nature,
copyright 1995 Macmillan Magazines
Ltd.]
July 2001 THYROID HORMONE ACTION 1103

and basal repression by unliganded TR. The recent solu- lying in a groove between helices 3 and 5. Thus the
tions of the crystal structures of the liganded TRa-1, relative positions of helix 12 and the boundary helices
unliganded RXRa, and RARg LBDs have greatly aided our may determine whether coactivators can interact with
understanding of its role on these functions and the at- TR. Indeed, studies using TR-LBD mutants based on the
tendant conformational changes that occur when T3 binds TR-LBD crystal structure have confirmed these regions
to the receptor (Fig. 6) (46, 417, 544). Ligand is buried for interacting with the coactivator GRIP-1 (107, 134).
deep within a hydrophobic pocket in the LBD formed by TRa and TRb-1 isoforms can bind T3 and various TH
discontinuous stretches that span almost the entire LBD. analogs with subtle differences in affinity. TRa binds T3
In particular, the most carboxy-terminal region (Helix 12) with slightly higher affinity than TRb-1 (449). Triac (3,5,39-
contributes its hydrophobic surface as part of the ligand- triiodothyroacetic acid) binds TRa-1 with similar affinity
binding cavity. The hydrophobic residues face inward, as T3 and binds TRb-1 with two- to threefold higher
whereas the conserved glutamate faces outward. The cav- affinity than T3. Several novel thyromimetics have been
ity also is bounded by hydrophobic surfaces from helices designed which bind TRb-1 (GC-1 and CGS 23425) with
3, 4, and 5. Although the crystal structure of unliganded 10- to 50-fold higher affinity (86, 512). The transcriptional
TR has yet to be solved, the crystal structure of unligan- activities of these isoform-specific compounds parallel
ded RXRa shows that helix 12 projects into the solvent. their binding affinities and may offer novel therapeutic
Thus it is likely that helix 12 undergoes major conforma- treatments of diseases such as hypercholesterolemia
tional changes upon ligand binding, from a more open while sparing the heart (which contains mostly TRa) from
conformation to a closed one, which has been likened to side effects. The crystal structures of hTRa and hTRb
a mouse trap mechanism. In an analogous manner, es- LBDs have been solved and may provide important infor-
trogen-bound LBD shows a similar structure as liganded- mation for designing even more selective thyromimetics
TR LBD with helix 12 facing inward (61). However, helix in the future (419).
12 of raloxifene-bound ER LBD is in a different position, The LBD also is involved in several other important
receptor functions. Scattered throughout the LBD are
discontinuous heptad repeats that have been proposed to
form hydrophobic interfaces for TR homo- and het-
erodimerization (145). Mutations in the ninth heptad re-
peat region have selectively decreased TR homo- and
heterodimer formation, suggesting that there may be dif-
ferent subregions of the LBD that are important for TR
dimerization (21, 140, 344, 592). Indeed, the TRa-1 LBD
crystal structure demonstrates that there is a hydrophobic
surface in the ninth heptad repeat region that could serve
as a potential dimerization interface (544). A natural TRb
mutation from a patient with resistance to TH at amino
acid 316 also displayed decreased homodimer formation,
suggesting that additional regions of the LBD may be
important for dimerization (189, 360, 592). The relative
contributions to dimerization by the LBD and DBD inter-
faces may depend on the receptor. A recent study sug-
gests that a region that contains the ninth heptad region
called the I box may be important for RAR heterodimer-
ization with RXR in solution and for binding to direct
repeats of variable spacing (392). On the other hand, the
DBD dimerization interface may be important for dictat-
ing binding to direct repeats of a specific spacing (in this
case, a 5-nucleotide gap). Recent studies suggest that the
ninth heptad region may be more important for het-
erodimerization of TRa-1, whereas the DBD may play the
dominant role for c-erbAa-2 because it lacks a complete
ninth heptad region due to alternative splicing (416, 568).
Baniahmad et al. (27) used a GAL4-fusion system to
FIG. 6. TRa-1 ligand-binding domain crystal structure. Dark mass
identify at least three transcriptional activation regions in
represents ligand. a-Helices are indicated. [From Wagner et al. (544).
Reprinted by permission from Nature, copyright 1995 Macmillan Maga- the LBD and designated them as t2, t3, and t4 (27).
zines Ltd.] Uppalari and Towle (534) also have used a yeast trans-
1104 PAUL M. YEN Volume 81

fection system to describe several activation regions in associated with nuclear localization (126). This lysine-rich
TRb-1 LBD as well as in the hinge region (534). In partic- sequence is highly conserved among nuclear hormone
ular, t4 located near the carboxy terminus has high ho- receptors and bears homology with the simian virus 40 T
mology with LBD sequences found in other nuclear hor- antigen nuclear localization sequence. TRs are likely im-
mone receptors previously designated as the activation ported into the nucleus shortly after synthesis as they are
function-2 (AF-2) domain (Fig. 7). This sequence located predominantly found in the nucleus and can bind DNA,
within helix 12 has been shown to be important for ligand- even in the absence of hormone. Furthermore, unlike
dependent transcriptional activation by other nuclear hor- some steroid hormone receptors, TRs do not associate
mone receptors (31, 105, 278). Recently, Chatterjee and with cytoplasmic heat shock proteins (103). Recent stud-
co-workers (521) have made point mutations in this re- ies using green fluorescent fusion proteins of wild-type
gion and have observed normal T3 binding and DNA bind- TRb and TRb hinge region mutants demonstrated this
ing, but no transcriptional activation, using a GAL4/TRb region may be important for T3-mediated translocation of
LBD fusion protein system (521). As discussed earlier, TR into the nucleus (605).
helix 12 likely undergoes major conformational changes The hinge region also has additional properties. The
upon ligand binding (419). Studies with steroid hormone laboratories of Evans (83) and Rosenfeld and co-workers
receptors and TRs have demonstrated that the AF-2 do- (211) identified corepressor proteins that can interact
main is important for interactions with coactivators such with unliganded TRs, RARs, and v-erbA and mediate re-
as SRC-1 and related family members (419). Interestingly, pression of basal transcription by these receptors and
mutations in the AF-2 region of the TR LBD had modest v-erbA (see sect. VIII). Mutations in the TRb-1 hinge region
effects on T3-dependent interaction of the coactivator abrogate the basal repression by corepressor. Addition-
TRAM-1 (SRC-3), whereas a mutation in helix 3 of the TR ally, a v-erbA mutation in the hinge region that abrogates
LBD severely impaired T3-dependent interaction with its oncogenic potential also failed to interact with a core-
TRAM-1 but had little effect on interaction with SRC-1 pressor, silencing mediator for RAR and TR (SMRT) (83).
(503). Ligand-dependent GRIP-1 (SRC-2) interactions with These findings suggest that the hinge region of unliganded
TR involve helices 3, 5, 6, and 12 (134). These findings TRs located on helix 1 may serve as a contact surface with
suggest that different subregions of TR may differentially corepressors or have allosteric effects on their interac-
contribute to interaction with specific coactivators. Addi- tion. Recent work by several groups also suggest that
tionally, several groups reported that corepressors may sequences within helices 3, 5, and 6 of the LBD contribute
interact with sequences on helices 3, 5, and 6 that overlap to corepressor binding (214, 348, 390).
sequences involved in interacting with coactivators (214,
348, 390). Several mutations from patients with resistance
to TH and artificial mutations in helices 3 and 5 do not D. Amino-Terminal (A/B) Domain
interact with coactivators or corepressors (97, 348, 390).
The amino-terminal regions have variable lengths
and divergent sequences among the TR isoforms. Even
C. Hinge Region
among different species, this region is less well conserved
for a given TR isoform, because the rat and human TRs
The hinge region between the DBD and T3-binding
are 97 and 99% identical in their DBDs and LBDs, respec-
domain likely contains an amino acid sequence that is
tively, but only 85% identical in their amino-terminal do-
mains (254). The role(s) of the amino-terminal domain is
poorly understood. Studies of the glucocorticoid receptor
have suggested that there is a major activation function
domain, t1, which has structural similarities with viral
acidic activator proteins such as VP16 (209). Previous
work by Tora and co-workers (524, 525) with progester-
one and truncated estrogen receptors also have suggested
that the amino-terminal domain may modulate cell-spe-
cific and promoter-specific transcription. Cotransfection
studies generally have demonstrated only a few examples
of isoform-specific transcriptional activation by TRs. Far-
setti et al. (129) have shown that TRb-1 has higher tran-
scriptional activity than TRa-1 via a myelin basic protein
TRE reporter in the context of the native promoter, but
FIG. 7. Comparison of AF-2 regions of nuclear hormone receptors.
not with the viral thymidine kinase promoter (129). Jean-
Conserved ffxEff sequences are underlined. nin et al. (224) observed similar TRb-1-specific effects on
July 2001 THYROID HORMONE ACTION 1105

myelin basic protein, but not on malic enzyme, gene ex- (208). Recent studies showed that the amino terminus of
pression suggesting that isoform-specific gene regulation TRb-2 may interact with the silencing domain of the core-
may occur in a subset of target genes (224). Similar TRb- pressor, SMRT, and thereby block the recruitment of
1-specific effects on the regulation of the TRH promoter other components of the corepressor complex (575). Fi-
also have been observed (181, 305). TRb-2 may have a nally, the amino-terminal domain of TR also may influ-
more potent role in the negative regulation of glycopro- ence the conformation of the DBD and the repertoire of
tein a-subunit and TSHb than other TR isoforms (208, TREs to which it can bind (184, 232).
275). In particular, TRb-2 mediates strong ligand-indepen-
dent activation of negatively regulated target genes such
as glycoprotein hormone a-subunit and TSHb (275). In- V. THYROID HORMONE RESPONSE ELEMENTS
deed, recent studies of TR knockout mice have implicated
TRb-2 as the major TR isoform in negative regulation of Steroid hormone receptors bind as homodimers to
TSH (3). conserved palindromic hormone response elements that
The role of the amino-terminal domain in transcrip- mediate hormone regulation of target genes (160). In
tional activation is still controversial. Some studies have contrast, TRs can bind to TREs as monomers, ho-
shown that deletion of the amino-terminal domain of modimers, and heterodimers in vitro. In general, most of
TRb-1 had no effect on T3-dependent transcriptional ac- these TREs are located upstream from the minimal pro-
tivation by TRb-1 (516, 592), suggesting that it does not moter, but in certain cases, also can be located in 39-
contain a major activation function domain like the glu- flanking sequences downstream from the coding region
cocorticoid receptor. On the other hand, studies of TRa-1 (40, 600). Mutational analyses of the rat growth hormone
and TRb-1 from several species have shown that the gene TRE, and sequence comparison among known TREs
amino-terminal domain may be important for transcrip- from other T3-responsive genes, have suggested a putative
tional activation and interactions with the general tran- consensus hexamer half-site sequence of (G/A)GGT(C/
scription factor TFIIB (25, 183, 520). Additionally, it has G)A (84, 555). However, there can be considerable varia-
been shown that the chick TRb-2 amino-terminal region tion found in primary nucleotide sequences of TREs as
may have two activation domains (472). A minimal sub- well as the number, spacing, and orientation of their
region of amino acids 2150 allows human TRb-2 to in- half-sites (555). In particular, TRs can bind to TREs in
teract with coactivators in the absence of ligand and may which half-sites are arranged as palindromes (TREpal),
account for the ligand-independent activation of some direct repeats (DRs), and inverted palindromes (IPs). The
positively regulated target genes (358). However, one optimal spacing for these half-site arrangements are zero,
study of the amino-terminal region suggested that rat four, and six nucleotides, respectively (TREpal0, DR4, and
TRb-2 does not appear to have a strong activation domain IP6) (Fig. 8). Almost all positively regulated target genes
in the amino-terminal region (520), whereas other studies contain two or more half-sites; however, TRs can activate
have shown that TRb-2 transactivates similar to TRb-1 transcription via an artificial single octamer half-site, per-
(205, 478). It is possible that differences in species, cell haps even as monomers (240).
types, TREs, and minimal promoters may account for Approximately 30 natural TREs have been described
these different observations (129, 491, 580). The amino- so far with DRs, followed by IPs, as the most common
terminal domain also may modulate ligand-independent motifs (555). Several groups also have observed that TR
repression via positively regulated TREs because one homo- and heterodimers bind to TREs arranged as IPs
study showed that TRa-1 is more potent than TRb-1 or and DRs better than palindromes (17, 268, 333, 588). In the
TRb-2 in mediating basal repression in the absence of T3 case of DRs, it has been shown that VDRs preferentially

FIG. 8. Half-site orientation and optimal nucleotide


spacing between half-sites. N refers to nucleotides, and
arrows show direction of half-sites on the sense strand.
TRE, thyroid hormone response element.
1106 PAUL M. YEN Volume 81

transactivate via reporter vectors containing DRs with a corepressors that link the liganded TR/RXR heterodimer
three-nucleotide gap (DR3), TRs via a four-nucleotide gap with the transcriptional machinery. In this connection,
(DR4), and RARs via a gap of five nucleotides (DR5), Kurokawa et al. (267) demonstrated that RAR/RXR het-
according to a 3 4 5 rule (160, 532). Heterodimeriza- erodimers bound to DR1 and DR5 with different polari-
tion with RXR and specific DNA contact points in the ties. In the former case, they remained bound to a core-
DBD may play important roles in determining het- pressor and mediated constitutive basal repression, and
erodimer spacing on direct repeats (153, 160, 413). How- in the latter case, they dissociated from corepressor in the
ever, the DNA binding and transcriptional activation via presence of all-trans-retinoic acid and mediated tran-
these elements do not appear to be absolutely receptor scriptional activation. Similarly, it has been shown that
specific because TRs can bind and transactivate weakly the TRE sequence can affect corepressor release from TR
on DR5 and DR6, and RARs can bind and transactivate on in the presence of ligand (368).
DR4 (557, 588). VDRs can bind to DR4 and DR5 (587) but
cannot transactivate via these HREs. However, VDR has
dominant negative activity on T3 and RA-mediated tran- VI. THYROID HORMONE RECEPTOR
scription via these elements. Additionally, the primary COMPLEXES
sequence of the half-site may be important in maintaining
receptor-specific binding to DRs as mutation of the third Early studies of TR binding to specific DNA se-
nucleotide of the half-site hexamer from a G to a T quences utilized methods such as the avidin-biotin com-
enabled VDR binding and ligand-mediated transactivation plex/DNA (ABCD) assay that did not allow direct visual-
via a DR4 (418). It also has been shown that flanking and ization of TR complexes bound to DNA (63, 161, 585).
spacing sequences in TREs can affect DNA-binding and However, successful employment of electrophoretic mo-
transcriptional activation, either by making contacts with bility shift assays (EMSAs) demonstrated that TRs could
TR or local DNA bending (216, 240, 242, 244). bind to synthetic and natural TREs as monomers, ho-
TRs can form heterodimers with RXRs, which also modimers, and heterodimers in vitro (144, 288, 557, 584).
are members of the nuclear hormone receptor superfam- TRb-1 may have a greater tendency than TRa-1 to form
ily (see below). This enables TR complexes to bind to homodimers on several different TREs, suggesting that
TREs in a specific orientation relative to the minimal these two TR isoforms may have different dimerization
promoter. Palindromic and inverted palindromic TRE potentials (109, 607). Domain swap experiments have
half-site sequences are symmetric and thus do not dictate suggested that the amino-terminal region of TRa-1 may
a particular heterodimer orientation on the TRE. On the inhibit homodimer formation via an allosteric mechanism
other hand, TRE half-sites in DR4 have a 59 to 39 polarity (208).
so the direct repeat motif could specify the heterodimer Initially, TRs were thought to mediate their effects on
orientation on the TRE. Several approaches have been transcription as homodimers, similar to steroid hormone
used to study this issue. Mutant TRs or RXRs containing receptors. However, two groups observed that TRs sur-
amino acid substitutions in the P box of the first zinc prisingly heterodimerized with proteins from pituitary
finger of the DNA-binding domain that allow preferential and liver nuclear extracts (63, 341). These proteins were
binding to a glucocorticoid hormone response element called TR auxiliary proteins (TRAPs) and enhanced TR
(GRE) half-site have been used to study TR/RXR het- binding to TREs (63, 341). Because these proteins were
erodimer binding to hybrid response elements containing expressed in nuclear extracts from many different tissues
TRE and GRE half-sites (268, 391, 586). These results and species (63, 287, 341, 494), TR/TRAP heterodimers
showed that TR binds to the downstream half-site and potentially could be formed in all cells that contain TRs.
RXR to the upstream half-site when TR/RXR heterodimer Because these heterodimers appeared to bind better to
binds to DR4. Methylation interference studies also TREs than TR homodimers, it was speculated that they
showed that TRa-1 and TRa-1/RXR preferentially bound played a role in T3-regulated transcription.
to the downstream half-sites of DR4 and the IP F2 (216). Several groups showed that TRs heterodimerize with
The apparent polarity of TR/RXR complexes on F2 may be RXRs, members of the nuclear hormone receptor super-
due to degeneracy of one of the half-site sequences or family which have high homology with RARs (250, 300,
possibly contributions by the flanking and spacing se- 321, 589, 594, 602). RXRs bind their cognate ligand, 9-cis-
quences. Cotransfection studies in which the orientation retinoic acid, with high affinity (202, 304). They can form
of these TREs were reversed also decreased T3-mediated homodimers as well as heterodimers with RARs, VDRs,
transcriptional activity (216). Taken together, these find- and peroxisome proliferator activated receptors (PPARs)
ings suggest that TR/RXR heterodimers bind with a spe- (160). Several lines of evidence suggest that RXRs are the
cific polarity which, in turn, can modulate transcriptional major TRAPs and thus play a critical role in T3-mediated
activity. This shape of the TR complex may be impor- transcription. First, they were observed to enhance TR
tant in protein-protein interactions with coactivators and binding to TREs. Second, studies using anti-RXR antibod-
July 2001 THYROID HORMONE ACTION 1107

ies showed that the major endogenous TRAPs are RXRs target genes in the absence of T3, which then dissociates
or related proteins (287, 420, 494). Third, TR/TRAP and from the TRE in the presence of T3.
TR/RXR heterodimer complexes both remained bound to Cross-linking and coimmunoprecipitation studies
TREs in the presence of T3 (17, 333, 422, 584, 589) (see suggest that TR monomers and TR/RXR heterodimers
below). Fourth, heterodimer-selective, but not ho- exist in solution, whereas TR homodimers form weakly in
modimer-selective, mutants were able to mediate tran- solution (268, 288, 581). T3 and 9-cis-retinoic acid pro-
scriptional activation of TRE-containing reporters (21, mote dimerization in solution, and thus may preform a
140, 344, 592). Mutant TRs containing deletions or amino transcriptionally active complex before DNA binding (95,
acid substitutions of amino acids at positions 290 310 or 234). It appears that dimerization promotes DNA binding
in the ninth heptad region of the LBD concomitantly (392), and DNA binding enhances dimerization (56, 366,
decreased heterodimerization and transactivation (21, 581).
140, 344, 360, 592). Fifth, RXR enhanced T3-mediated
transcription in yeast cells that do not contain endoge-
nous TRAPs (65, 187). And last, RXR enhanced T3-medi- VII. PHOSPHORYLATION OF THYROID
ated transcription in a reconstitutable in vitro transcrip- HORMONE RECEPTORS
tion system (294).
TR/RXR heterodimer formation increases the reper- Recently several groups also have observed that in-
toire of target genes that can be regulated by T3 as het- creasing the phosphorylation state of cells can enhance
erodimers bind to TREs with variable sequence and ori- T3-mediated transcriptional activation of target genes
entation of half-sites (160). Moreover, there are at least (229, 308, 496). The mechanisms for this enhanced tran-
three members of the RXR subfamily, so it is possible that scriptional activation are not known but may involve
different RXR isoforms may form TR/RXR heterodimers phosphorylation of TR, RXR, or coactivators. In support
that have different TRE-binding specificities and/or abili- of the potential role of TR phosphorylation in transcrip-
ties to transactivate target genes. Additionally, it is possi- tional activation, it recently has been demonstrated that
ble that an endogenous ligand like 9-cis-retinoic acid can TR can be phosphorylated in vitro and in vivo (165, 167,
bind and activate the heterodimer partner of the TR com- 308, 492). Chick TRa-1 has at least two serine phosphor-
plex. Of note, addition of both 9-cis-retinoic acid and T3 ylation sites in the amino-terminal A/B domain, but the
synergistically activated transcription on two different functional role(s) is not known (167). Additionally, these
TRE-containing reporters (227, 429). However, in other sites do not appear to be conserved across species. The
cases, TR can block 9-cis-binding to RXR and thereby human TRb-1 can be phosphorylated in vivo and in vitro
abrogate retinoid stimulation of target genes (91, 147, (308), although the phosphorylation sites have not been
266). Finally, TRs can heterodimerize with other members determined. Two groups have used HeLa cytosol extract
of the nuclear receptor family including RAR, PPAR, to in vitro phosphorylate Escherichia coli-expressed
chicken ovalbumin upstream promoter transcription fac- TRb-1 (39, 492). Sugawara et al. (492) examined the bind-
tor (COUP-TF), and VDRs (45, 99, 314, 448, 519, 589). The ing of phosphorylated TRb-1 to several TREs and found
functional significance of these heterodimers, most of that phosphorylation selectively enhanced TR ho-
which have been demonstrated in vitro by EMSA, is not modimer, but not TR/RXR heterodimer, binding to several
known. However, they do not appear to be major endog- different TREs. Bhat et al. (39) showed that phosphory-
enous TRAPs and may have restricted roles in particular lation enhanced DNA binding by both TR complexes.
target genes or tissues (287, 494). If they do have physi- Interestingly, phosphorylation by protein kinase A can
ological roles, though, these heterodimers increase fur- decrease v-erbA and chick TRa-1 monomer binding to
ther the diversity of TR complexes and the potential of TREs (530). These results suggest that phosphorylation
receptor cross-talk on target genes. may be another mechanism, in addition to T3 binding, that
Although TRs can form monomers, homodimers, and can modulate TR complex binding to TREs. Additionally,
heterodimers on TREs in EMSA (284, 583), the role(s) of T3 itself can modulate the phosphorylation state of TR
TR monomers and homodimers on transcription is not (518).
well understood. In contrast to steroid hormone receptors Recently, Davis et al. (113) have shown that TRb-1
in which ligand enhances receptor binding to HREs, T3 associates with mitogen-activated protein (MAP) kinase
decreases TRa-1 and TRb-1 homodimer as well as TRa- in coimmunoprecipitation studies and that ligand binding
1/TRb-1 dimer binding to several TREs arranged as DRs may stimulate TR phosphorylation by MAP kinase. Inter-
and IPs (17, 333, 422, 584). Unliganded TR homodimers estingly, previous studies have shown that MAP kinase
also interact better with corepressors than unliganded can modulate transcriptional activity of ER and PPARg by
TR/RXR heterodimers in vitro (207, 311). Thus it is pos- phosphorylation of the receptor (238, 406). Moreover,
sible that unliganded homodimers may form a complex MAP kinase phosphorylation of the steroidogenic factor-1
with corepressors that are involved in basal repression of (SF-1) and ER AF-1 regions leads to enhanced coactivator
1108 PAUL M. YEN Volume 81

recruitment and transcriptional activation (188, 528). ganded TRs bind to TREs and may modulate transcription
Stimulation of the protein kinase A pathway also poten- of target genes (Fig. 9). Several laboratories showed that
tiated T3-mediated transcription in a cell-specific manner unliganded TRs can repress basal transcription of posi-
(301), suggesting multiple kinase pathways may modulate tively regulated TREs in cotransfection studies (26, 53,
transcriptional activity of TR. Power et al. (405) have 603). It was not known initially whether these observa-
demonstrated that some nuclear hormone receptors can tions were physiologically relevant or peculiar to cotrans-
be activated by dopamine stimulation in the absence of fection systems. Early observations showing that T3 de-
ligand, mostly likely via receptor phosphorylation. It is creased TR homodimer binding to TREs led to the
possible that some ligand-independent effects by TR may hypothesis that unliganded TR homo- and/or het-
be due to receptor phosphorylation by cell-specific ki- erodimers might mediate basal repression (584). This no-
nases or phosphatases, or due to cell-specific expression tion was further supported by the demonstration that TR
of certain membrane-signaling receptors that can activate binding to TREs was important for mediating basal re-
transcription by unliganded TR. In this connection, cell- pression, as mutations in TRb-1 DBD or the TRE primary
type specific phosphorylation may stabilize TRb-1 protein sequence abrogated basal repression (26, 592). Unligan-
(517). Additionally, it has been shown that DNA binding ded TRs have been shown to interact directly with TFIIB,
by the alternative splice variant of TRa-1, c-erbAa-2, is a key component of the basal transcription machinery
regulated by casein kinase II phoshorylation of serines in (25, 183, 394, 520, 523), and potentially can interfere with
its carboxy terminus (239). This phosphorylation is criti- the assembly of a functional preinitiation complex at the
cal for determining the dominant negative activity (ability promoter. Studies of TR action in in vitro transcription
to block wild-type TR action) by c-erbAa-2. These findings systems (142) suggested that direct interaction between
suggest that phosphorylation potentially may regulate di- TRs and the basal transcriptional machinery could help
verse and important TR functions, although the precise mediate ligand-dependent basal repression. On the other
location of phosphorylation sites, their regulation, and hand, several studies also suggested that soluble core-
their functional roles remain to be elucidated. pressors may be critical for mediating basal repression
(407, 522).
The cloning and functional characterization of sev-
VIII. MOLECULAR MECHANISMS OF THYROID
eral corepressors have greatly enhanced our understand-
HORMONE RECEPTOR ACTION
ing of basal repression and shed light on these previous
issues (Fig. 10). Several laboratories used the yeast two-
A. Corepressors/Basal Repression hybrid system or biochemical purification to clone pro-
teins that exhibited decreased interaction with TR and
In contrast to steroid hormone receptors that are RAR in the presence of their cognate ligands (83, 211, 295,
transcriptionally inactive in the absence of ligand, unli- 442). One of them was a 270-kDa protein called nuclear

FIG. 9. Model for repression, dere-


pression, and transcriptional activation
by TR in the absence or presence of T3.
July 2001 THYROID HORMONE ACTION 1109

FIG. 10. Comparison of the organization and


structure of putative nuclear hormone receptor
corepressors. NCoR, nuclear receptor corepressor.

receptor corepressor (NCoR), which also was isolated as of the ligand pocket of TR. Differences in the length and
an RXR-interacting protein, RIP 13 (211, 459). It contains specific sequences of the corepressor and coactivator
three transferable repression domains and two carboxy- interaction sites coupled with the conformational
terminal a-helical interaction domains. NCoR was able to changes in the AF-2 region upon ligand binding may de-
mediate basal repression by TR and RAR, as well as termine whether corepressor or coactivator binds to TR
orphan members of the nuclear hormone receptor family (390). Additionally, corepressors can bind to the TR het-
such as rev-erbAa and COUP-TF. It had little or no inter- erodimer partner RXR. It appears that helix 12 of RXR
action with steroid hormone receptors and did not medi- masks a corepressor binding site in RXR, which is un-
ate basal repression by these receptors. NCoR also has masked upon heterodimerization with TR (599).
been shown to interact with TFIIB, TAFII32, and TAFII70, Recently, several groups have shown that corepres-
so part of its ability to repress transcription may be due to sors can complex with other repressors, Sin 3 and histone
its ability to interact with the basal transcripitonal ma- deacetylase 1 (HDAC1), that are mammalian homologs of
chinery. Recently, a truncated version of NCoR, NCoRi, well-characterized yeast transcriptional repressors RPD1
which is missing the repressor region, has been identified, and RPD3 (13, 200, 271, 347, 352). Thus local histone
which may represent an alternative-splice variant of deacetylation may play a critical role in basal repression
NCoR (207). This protein blocks basal repression by by nuclear hormone receptors. Moreover, this mechanism
NCoR and potentially may serve as a natural antagonist of basal repression may be employed by other transcrip-
for NCoR if it is expressed in signficant amounts in tis- tion factors such as Mad/Max and Myc/Mxi heterodimers
sues. Another corepressor, SMRT, has been identified and (13, 200, 271). Anti-NCoR antibodies have been shown to
has homology with NCoR (83, 295, 442). The original coimmunoprecipitate HDAC activity (200). Additionally,
sequence for SMRT was derived from a partial clone, but microinjection of specific antibodies generated against
it is now appreciated that full-length SMRT is similar in mSin3 and RPD3 were able to block basal repression by
size and has similar repression and nuclear receptor in- NCoR (200). Recent studies by Lazar (286) and Wong
teraction domains as NCoR. SMRT also is able to mediate (J. M. Wong, personal communication) suggest that
basal repression of TR and RAR in cotransfection studies. HDAC4 may interact directly with NCoR at a different
Another protein, small ubiquitious nuclear corepressor repressor site than Sin3 and HDAC1. Recent coimmuno-
(SUN-CoR), was isolated and enhanced basal repression precipitation studies also suggest that HDAC3 may be the
by TR and rev-erbA (596). This 16-kDa protein may form major HDAC associated with SMRT (286). Another com-
part of a corepressor complex as it interacts with NCoR. ponent of the corepressor may be the protooncogene
Studies of TR and v-erbA have defined the impor- c-Ski which has been shown to be involved in transcrip-
tance of the hinge region for interactions with NCoR and tional represson by Mad and TR (354). It is likely that
SMRT, because mutations in this region abrogate basal histone deacetylation by unliganded TR/corepressor com-
repression without affecting transcriptional activation plex may help maintain local chromatin structure in a
(83, 211, 324). Interestingly, rev-erbAa contains two ami- state that shuts down basal transcription. In this connec-
no-terminal regions which interact with NCoR and are tion, studies examining TRbA promoter in a Xenopus
required for basal repression, suggesting that nuclear hor- oocyte system showed that simultaneous chromatin as-
mone receptors may have different interaction sites with sembly and TR/RXR binding were required for basal re-
corepressors (597). Within the interaction domains of pression of transcription (562, 563). This repression was
NCoR and SMRT are consensus LXXI/HIXXXI/L se- relieved by addition of T3 and was accompanied by chro-
quences that resemble the LXXLL sequences that enable matin remodeling. These data demonstrate that histone
coactivators to interact with nuclear hormone receptors acetylation and deacetylation, and the consequent
(214, 348, 390). Interestingly, these motifs allow both changes in chromatin stucture and nucleosome position-
corepressors and coactivators to interact with similar ing, may be important determinants of gene transcription
amino acid residues on helices 3, 5, and 6, which are part (Fig. 11). Additionally, DNA methylation may play a role
1110 PAUL M. YEN Volume 81

FIG. 11. Molecular model for basal


repression in the absence of T3 and tran-
scriptional activation in the presence of
T3. X refers to potential unidentified co-
factors. See text for details.

in basal repression as methyl-CpG-binding proteins can rat growth hormone minimal promoter (even down-
associate with a corepressor complex containing Sin3 and stream), and still mediate negative regulation. They
HDACs (351, 543). This repression was relieved by the showed that the nTRE sequence may play a more impor-
deacetylase inhibitor trichostatin A. These findings sug- tant role than its position relative to the minimal pro-
gest that two repression processes, DNA methylation and moter. Additionally, several groups have found nTREs
histone deacetylation, may be linked via methyl-CpG- located in the 39-untranslated region of target genes (40,
binding proteins. 600). Recent studies have suggested that ligand-indepen-
TR also can activate transcription in the absence of dent activation may be mediated by TR recruitment of
ligand. Samuels and co-workers (201) showed that unli- corepressors, and this may be mediated by protein-pro-
ganded TR surprisingly could transactivate via rGH and tein interactions of DNA (500, 501). Interestingly, these
PRL TREs in pituitary cell lines (89). Additionally, several effects could be blocked by pharmacological inhibition of
groups have reported ligand-independent gene transcrip- HDAC activity. These findings suggest that histone
tion in neuroblastoma and Xenopus cells (346, 385). It is deacetylation may promote ligand-independent activation
not known whether cell-specific activators or inhibitors of a negatively regulated target gene. Another study
or other mechanisms account for these observations. showed that T3-mediated negative regulation may also
Last, TRs can activate transcription of negatively regu- require HDAC activity (445). On the other hand, NCoRi
lated genes such as RSV LTR, glycoprotein hormone (which does not contain the repression domains) en-
a-subunit, TSHb, and TRH in the absence of ligand (88, hances ligand-independent regulation in a reporter con-
206, 305, 342, 433). Brent et al. (57) showed that a negative taining the TRH promoter (207). NCoRi activation func-
TRE (nTRE) from the glycoprotein hormone a-subnunit tion is stronger than NCoR, so the ligand-independent
gene could be placed in different positions relative to the activation function may map to the carboxy terminus of
July 2001 THYROID HORMONE ACTION 1111

NCoR (93). These studies suggest a role for corepressors transcription systems highlighted the importance of min-
in ligand-independent activation of some negatively regu- imal promoters in mediating T3-regulated transcription
lated target genes, but the precise mechanism needs to be (129, 491, 580). These studies raised the possibility that
further defined. adapter proteins, or coactivators, might bridge the ligan-
The physiological role of corepressors is only par- ded TR complex with components of the basal transcrip-
tially understood. Corepressors have been implicated in tional machinery. Previously, several viral transcription
leukemias that have RAR fusions and acute myolegenous factors were shown to interact with coactivators to en-
leukemia/eight-twenty-one (ETO) fusions from chromo- hance transcription (62, 170, 310). Similarly, several po-
somal translocations (158, 176). Additionally, the TRa/ tential coactivators for hormone-regulated transcription
TRb double-knockout mice have a milder phenotype than were reported, including CREB binding protein (CBP)
congenitally hypothyroid mice, suggesting that basal re- which interacts with CREB (269). Also, several groups
pression by unliganded receptor may have more deleteri- identified proteins that interacted with ER in a ligand-
ous effects on transcription in target tissues. Lazar (286) dependent manner and thus could potentially participate
recently found that transducin B-like protein coimmuno- in estrogen-dependent transcriptional activation (72, 186,
precipitated with SMRT. This histone-binding protein was 221). These putative coactivators interacted with the AF-2
deleted in patients with congenital sensory-neural deaf- region located in helix 12 of the LBD. This region has high
ness (33). Interestingly, TRb knockout mice and affected homology among many members of the nuclear hormone
patients from a family with resistance to TH due to ab- receptor family and was shown to be important for ligand-
sence of TRb also have sensory neural deafness (149, dependent transcription for several receptors (27, 31, 105,
414). It is interesting to speculate that absence of TRb or 278, 601) (Fig. 6). This region contains a critical ffXEff
an associated repressor complex protein can cause a sequence in which f represents a hydrophobic amino
similar phenotype. Last, Feng et al. (135) have targeted a acid and X represents any amino acid.
dominant negative NCoRi to the liver in transgenic mice. Recently, OMalley and co-workers (369) used a yeast
They observed that basal transcription of spot 14 and Bcl3 two-hybrid system to clone a putative factor that en-
(a target gene identified by cDNA microarray) was higher hanced transcriptional activation for steroid hormone re-
in hypothyroid transgenic mice than in littermate con- ceptors, which they called steroid receptor coactivator-1
trols. When mice were treated with T3, both genes were (SRC-1) (Fig. 12). This protein also associates with sev-
induced to similar levels. These findings suggest that eral other members of the nuclear hormone receptor
NCoR-mediated basal repression in vivo could be re- superfamily, including TRs, and enhances their ligand-
versed by overexpression of NCoRi without effects on dependent transcription. Subsequent work has shown
transcriptional activation. Interestingly, transcription of that the original cDNA clone was only partial, and the
several other target genes was unchanged in hypothyroid full-length clone encoded a 160-kDa protein (235, 369,
transgenic mice, suggesting that there may be gene-spe- 507). Additionally, there may be alternative splicing of
cific repression by different corepressors. SRC-1 mRNA, leading to multiple SRC-1 isoforms. The
functional significance of the SRC-1 isoforms currently is
not known; however, one splice variant, SRC-1E, which
B. Coactivators/Transcriptional Activation lacked 56 amino acids of SRC-1 and had unique 14 amino
acids at the carboxy terminus, enhanced T3-mediated
Recent studies using Far-Western and coimmunopre- transcriptional activation better than SRC-1 (194, 235).
cipitation approaches showed that liganded TR may in- Another 160-kDa protein, TIFII/GRIP-1/SRC-2, has been
teract with multiple nuclear proteins that potentially can identified that interacts with liganded nuclear hormone
form a transcriptionally active complex (141, 394). Work receptors, including TRs, and has partial sequence homol-
by several groups previously showed that TRs can inter- ogy with SRC-1. These findings suggest there may be a
act with general transcription factors (25, 142, 183, 520). family of coactivators related to SRC-1 (210, 541). Indeed,
Moreover, these interactions seemed to be ligand depen- another family member, AIB1/pCIP/ACTR/TRAM1/RAC3/
dent, suggesting that direct contact between TR and gen- SRC-3, has been identified that augments transcription by
eral transcription factors could play a role in derepression nuclear hormone receptors (19, 82, 306, 503, 526). As seen
and transcriptional activation (27, 142). In support of this in Figure 12, there are several common features among
possiblity, ER was shown to interact with TBP, TAFII 30, these putative coactivators. First, there are multiple pu-
and TFIIB (217, 221, 434), and PR could interact with tative nuclear hormone receptor interaction sites that
TAFII 110. Petty et al. (396) also showed that liganded seem to bear a signature LXXLL sequence motif in which
TRb interacts with several Drosophila TAFs, particularly X represents any amino acid. This sequence has been
TAFII 60 and TAFII 110, and the latter could augment shown to be important for coactivator binding to nuclear
T3-dependent transcription (396). hormone receptors (199, 325). These coactivators also
Previous studies using cotransfection and in vitro have a polyglutamine region, similar to androgen recep-
1112 PAUL M. YEN Volume 81

FIG. 12. Comparison of the organization and structure of p160 nuclear hormone receptor coactivators.

tors. There is polymorphism observed in the length of may serve as an integrator molecule for different signaling
these trinucleotide repeats of SRC-3, but the functional inputs (262) (Fig. 10). The biological importance of CBP
signficance, if any, is not known (198). Additionally, in the in humans is exemplified by patients with Rubenstein-
amino-terminal region, there is a basic helix-loop-helix Taybi syndrome who have mental retardation, short stat-
(bHLH) motif, suggesting that these coactivators may ure, and craniofacial and other anomalies, as well as
bind to DNA. Also located in this region is the so-called mutations in CBP (393). p300 knockout mice die in utero
Per-Arnt-Sim (PAS) domain, which interestingly, also is and display defects in cell proliferation as well as neural
seen in several transcription factors that regulate the and heart development (578). CBP knockout mice also die
circadian rhythm and in the heterodimer partner of the in utero with defects in hematopoesis and angiogenesis
dioxin receptor (387, 402). Thus the PAS region may serve (363). These findings suggest that although CBP and p300
as a dimerization interface and potentially allow cross- behave similarly in enhancing nuclear hormone receptor
talk among other coactivators or transcription factors. action in cotransfection studies, they are not functionally
SRC-1 can be phosphorylated by MAP kinase, and its equivalent in vivo.
activity may be regulated by membrane-bound receptors Recent studies also have shown that CBP/p300 can
such as epidermal growth factor (431). There also are interact with PCAF (p300/CBP-associated factor), the
multiple nuclear receptor interaction domains in the mammalian homolog of a yeast transcriptional activator,
SRCs that may confer receptor specificity (122, 316, 505, general control nonrepressed protein 5, GCN5 (42, 574).
540). Among the SRC family members, there may be a Like GCN5, PCAF has intrinisic histone acetyltransferase
certain degree of functional redundancy, as SRC-1 knock- (HAT) activity. The HAT activity of PCAF is directed
out mice were viable and fertile and had only a modest primarily toward H3 and H4 histones. PCAF itself is part
decrease in growth and development in reproductive tis- of a preformed complex that contains TBP associated
sues and a mild disurbance of the hypothlamic/pituitary/ factors (TAFs) which have been shown to interact with
thyroid (HPT) axis (550, 570). Interestingly, there was a SRC-1 and SRC-3 (82, 361, 479). CBP also has been shown
concomitant increase in TIF2/SRC-2 mRNA in these mice, to be part of a stable complex with RNA polymerase (pol)
suggesting that upregulation of TIF2 may compensate for II (350). Thus PCAF and CBP can serve as adaptors of
some of the loss of SRC-1 function. Interestingly, the nuclear receptors to the basal transcriptional machinery
SRC-3 knockout mouse has a much more severe pheno- and have an enzymatic function (HAT activity). These
type with decreased growth, delayed puberty, and de- dual roles likely contribute to nuclear receptor transcrip-
creased litter size (569). The effects on the HPT axis and tional activity (Fig. 12). Subsequent studies showed that
potential contribution to the phenotype are not known at CBP/p300, as well as SRC-1 and SRC-3, have HAT activity,
the present time. although their specificity for histone substrates differed
Recently, several groups have shown that SRC-1 can (82, 262, 362, 479). Recruitment of different coactivators
interact with the CBP, the putative coactivator for cAMP- with different HAT activities may be a mechanism for
stimulated transcription as well as the related protein, promoter- or cell-specific regulation of target genes.
p300, which interacts with the viral coactivator E1A (73, Recently, Lanz et al. (277) reported that an RNA
235, 476, 577). CBP/p300 can serve as a coactivator for transcript, steroid receptor RNA activator (SRA), could
CREB, p53, AP-1, and NFkB. It is possible that CBP/p300 enhance AF-1-mediated transcription of the progesterone
July 2001 THYROID HORMONE ACTION 1113

receptor and was part of the SRC-1 complex (277). It is for nuclear hormone receptors; however, the precise
possible that SRA may be part of a ribonucleoprotein functional roles of the two different coactivator com-
scaffold that many help recruit SRC-1 to nuclear hormone plexes are not known. It is possible that the p160/SRC
receptors. Additionally, Stallcup recently showed that a complex may initiate transcriptional activity by recruiting
methyltransferase, CARM-1, can be associated with cofactors with HAT activity to ligand-bound nuclear hor-
GRIP-1 and methylate the histone H3 (81). These findings mone receptors, and DRIP/TRAP complex may then bind
suggest that histone methylation as well as acetylation to nuclear receptors that can then recruit RNA Pol II
may be employed to alter local chromatin stucture. holoenzyme to promote transcription of target genes (Fig.
In vivo foot-printing studies also have suggested con- 11). Recently, it has been shown that CBP can acetylate
formational changes in the chromatin structure near ACTR (SRC-3) and promote its dissociation from nuclear
TREs and retinoic acid response elements after ligand hormone receptors, so acetylation of components of the
addition (118, 143, 243, 562). Additionally, liganded recep- p160/SRC complex from HAT activity may facilitate the
tor binding to the hormone response element allowed exchange of complexes (82). Balasubramanian and Moore
other enhancer elements in the promoter to be foot- (22) recently showed that mammalian homologs of Sw-1/
printed (118). In studies on TRbA promoter in Xenopus Snf, BRG-1 and BRM-1, can associate with TR in vitro and
oocytes, Wolffe et al. (561, 562) have shown that chroma- activate transcription. These chromatin remodeling pro-
tin assembly is critical for basal repression by unliganded teins have ATPase activity and previously were shown to
TR/RXR heterodimers. Moreover, trichostatin A, a HDAC enhance GR-mediated transcriptional activity in yeast
inhibitor, relieves this basal repression but does not dis- (67). Recently, the sequential recruitment of coactivator
rupt chromatin structure. In contrast, liganded TR/RXR complexes and correlation with transcription has been
disrupts chromatin structure, releases the corepressor demonstrated for ER and TR (461a, 461b). Understanding
complex, and recruits coactivators such as CBP and the interplay of these coactivator complexes and the
PCAF with HAT activity. Recent studies with green fluo- mechanisms by which they activate transcription will cer-
rescent proteins have shown that TRs are predominantly tainly be the subjects of intense study in the future.
in the nucleus and that T3 can mediate intranuclear reor- In addition to the p160/SRC and DRIP/TRAP com-
ganization in the presence of ligand (34). Moreover, this plexes, a number of coacativators have been identified,
change does not require DNA binding. This reorganization but their relationship to these complexes, if any, is not
may be due to association with chromatin, active tran- known. Moore and co-workers (295, 296) have identified
scriptional complexes, and/or nuclear matrix components several other proteins that interact with TRb-1 LBD.
(34, 213, 482). These proteins, called TRIPs (TR-interacting proteins),
Recently, the groups of Freedman (411, 412) and are diverse; one of them is the human homolog of a yeast
Roeder (595) have isolated a complex of proteins (DRIPS, transcription factor, another a new member of a class of
vitamin D receptor interacting proteins; TRAPs, TR-asso- nonhistone chromosomal proteins, and yet another con-
ciated proteins) ranging from 70 to 230 kDa that inter- tains a conserved domain asssociated with ubiquitination
acted with liganded VDRs and TRs in GST-pulldown stud- of specific target proteins. Monden et al. (334) have iden-
ies. These proteins had similar sizes as a previously tified a putative coactivator, p120, that does not bear
reported complex of proteins originally isolated by coim- homology with SRC-1 but interacts with liganded TR and
munoprecipation with an antibody against epitope-tagged contains an LXXLL motif (334). Another protein called
TRa. A critical coactivator in this complex is DRIP205/ TRIP230 (TR and Rb interacting protein of 230 kDa) was
TRAP220, which contains a LXXLL motif similar to SRC intially cloned on the basis of its ability to interact with
family members and appears to anchor ;15 other pro- the retinoblastoma protein (74). This protein binds to
teins. Interestingly, none of these proteins is a member of liganded TR and augments T3-mediated transcriptional
the SRC family and their associated proteins. Instead, activation. Interestingly, cotransfection of Rb blocks the
several DRIP/TRAP components are mammalian ho- TRIP230 enhancement of transcription, suggesting cross-
mologs of the yeast Mediator complex, which associates talk between TR- and Rb-mediated pathways. Another
with RNA Pol II (179, 219). DRIP/TRAP complex is virtu- coactivator, TR-binding protein (TRBP; ASC-2; RAP250),
ally identical to a previously described SRB-MED-contain- interacts with TR via a LXXLL motif and stimulates tran-
ing cofactor complex (SMCC) that can interact with p53 scription of several nuclear hormone receptors, AP-1,
and VP16 to enhance transcription (219). These findings CREB, and NFkB (66, 252, 298). It also interacts with
suggest that TR may recruit DRIP/TRAP complex which, DRIP 130 and CBP/p300. Yet another protein, TR uncou-
in turn, may recruit or stabilize RNA Pol II holoenzyme by pling protein (TRUP), binds to TR/RXR heterodimers and
virtue of shared subunits. Additionally, DRIP/TRAP com- prevents their binding to TREs (64). In contrast to coac-
plex does not appear to have intrinisic HAT activity (411). tivators, it blocks T3-mediated transcription, presumably
Taken together, these findings suggest a model in which by the foregoing mechanism. Additionally, there are a
there may be at least two distinct coactivator complexes number of other nuclear proteins that have been identi-
1114 PAUL M. YEN Volume 81

fied by two-hybrid screening that interact with steroid with ER may be an important modulator of estrogen
hormone receptors and also may interact with TRs (212, response in this neuroendocrine tissue.
326). These findings suggest that there may be different ERs also can block T3-regulated transcription by TR
classes of coactivators in addition to the SRC and DRIP/ (579, 591). The mechanism for transcriptional blockade
TRAP complexes. However, the functional significance of may depend on the TREs of particular target genes. In one
these coactivators on TR-mediated transcription remains example, ERa blocked the T3-mediated negative regula-
to be further defined. tion of the glycoprotein hormone a-subunit gene (579).
ER bound to a hormone response element containing an
imperfect palindrome sequence. TR monomer also bound
C. Cross-talk With Other Nuclear to this composite response element and negatively regu-
Hormone Receptors lated transcription. Thus ER competition with TR for
DNA binding to the response element may block T3-me-
TRs and other nuclear hormone receptors can mod- diated negative regulation of this target gene. Addition-
ulate transcriptional activities of each other. This cross- ally, ER blocked T3-mediated transcriptional activation
talk can occur via several mechanisms: promiscuous via DR4 and F2 (591). In contrast to the glycoprotein
binding to HREs, formation of heterodimers, and compe- hormone a-subunit gene, this blockade did not require
tition for cofactors (450, 582). The first two mechanisms DNA binding since the transcriptional blockade was ob-
seem to be the most prevalent among nuclear hormone served with ER mutants that did not bind to TREs or
receptors and can result in either enhanced or decreased EREs. Similar results also were observed using GR and
transcriptional activity. As mentioned earlier, TR/RXR GR truncation mutants. Interestingly, neither ER or GR
heterodimers may mediate dual-ligand regulation of tran- had any effect on repression of basal transcription by
scription in some instances and block ligand-dependent unliganded TR, suggesting that this blockade by the ste-
transcription via RXR in others (162, 297, 314, 429, 493). roid hormone receptors had a selective effect on these
Similar mechanisms also may occur for TR blockade of two TR functions. It is likely that overexpression of ER
RAR-mediated transcription (29, 297, 589). and GR titrated limiting amounts of a cofactor(s) critical
Carlberg and co-workers (448) demonstrated that for transcriptional activation by liganded TR, but not
VDR and TR formed heterodimers on the mouse and rat basal repression, by unliganded TR. Similar results were
calbindin HREs which are arranged as DR3 and DR4, observed for PR- and TR-mediated transcription in an in
respectively, and both vitamin D and T3 could coregulate vitro transcription system (601). There also are several
transcription via these elements. VDR/RXR, but not VDR/ other examples of cross-talk between other nuclear hor-
TR, bound to several TREs, and VDR exhibited dominant mone receptors and TRs. PPARs can decrease T3-medi-
negative activity as it blocked T3-mediated transcriptional ated transcription, possibly via competition for binding to
activity on DR4 and the chick lysozyme TRE, F2 (587). HREs and/or titration of RXRs or coactivators (45, 233,
Aranda and co-workers (156) also have found that VDR 332). COUP-TF also can interfere with TR-mediated as
blocked TR-mediated transcription in the rGH TRE. Inter- well as RAR- and VDR-mediated transcriptional activity
estingly, cotransfection of SRC-1 or SRC-3 could augment (99).
VDR-mediated transcription on TRE- and RARE-contain- Nuclear hormone receptors have been shown to have
ing reporters (DR4 and DR5) (504). cross-talk with c-jun and c-fos protooncogene (AP-1) pro-
TRs and ERs also exhibit cross-talk. TRs and ERs teins (450). On the proliferin promoter, positive or nega-
have identical P box sequences and recognize the same tive glucocorticoid regulation was regulated by c-jun or
consensus half-site sequence, AGGTCA (160). However, c-fos and binding to a composite element containing over-
ERs bind as homodimers to EREs arranged as palin- lapping an AP-1 binding site and glucocorticoid response
dromes separated by three nucleotides. Although TRs can element (119). In other cases, c-jun and GR have recip-
bind to estrogen response elements (EREs), they are rocal antagonism mediated via protein-protein interac-
unable to transactivate via these elements (420, 456). tions (451, 576). Similarly, several groups have shown a
Nonetheless, TRs can block estrogen stimulation of vitel- ligand-dependent repression of AP-1 activity by TRs (312,
logenin gene expression possibly by forming transcrip- 388, 604). Interestingly, Lopez et al. (312) also have shown
tionally inactive complexes (e.g., TR/RXR heterodimers) that unliganded TR can positively regulate AP-1 activity
on the ERE (161, 456, 609). Zhu and co-workers (397, 608) on some elements (312). Additionally, c-jun can antago-
have shown that TR can bind to the EREs of pro-enkepha- nize T3-mediated transcription via positively and nega-
lin and progesterone receptor promoters in rat hypotha- tively regulated TREs (312, 388, 560, 604). The mechanism
lamic cells and decrease estrogen-mediated proenkepha- for these reciprocal effects seems to be protein-protein
lin mRNA synthesis. Because these two estrogen- interactions between TR and c-jun because several
regulated target genes are important in the hypothalamic groups have observed formation of TR/c-jun complexes in
regulation of sexual behavior in rats, coexpression of TR solution (312, 560, 604). Furthermore, TR and AP-1 com-
July 2001 THYROID HORMONE ACTION 1115

plexes can both interact with CBP, providing another a variety of tissues via a mechanism that does not require
potential point for cross-talk. Recently, Yamamoto and new protein synthesis, suggesting a direct effect on the
co-workers (426) examined TR- and GR-mediated repres- plasma membrane transport system (453 455). Addition-
sion of collagenase gene transcription by jun. Interest- ally, T3 has been shown to bind to an endoplasmic retic-
ingly, two jun mutants disrupted TR-mediated repression ulum-associated protein, prolyl hydroxylase, and also to a
but not GR-mediated repression. Also, the repression by subunit of pyruvate kinase when the enzyme is mono-
TR, but not GR, could be blocked by the histone deacety- meric but not tetrameric (20, 85, 237). It is not known
lase inhibitor trichostatin A. These findings suggest that whether T3 modulates the activities of these enzymes or
TR- and GR-mediated repression on this gene occur via whether these enzymes may subserve other functions
distinct mechanisms. related to T3 action such as transport or storage. In this
connection, it is of interest that thyroxine, T4, can inhibit
deiodinase type II activity by an allosteric mechanism and
D. Nongenomic Effects of TH may promote targeting of a substrate-binding subunit to
endosomes (132). Recently, Stensapir et al. (481) also
There is general agreement that most of the effects of have shown that deiodinase type II can be proteosomally
T3 are mediated by TR regulation of target gene transcrip- degraded in the presence of T4 and/or T3.
tion in the nucleus. However, there are a number of Sterling and colleagues (483 485) reported the pres-
reports on nongenomic effects by T3 and T4 (116). Evi- ence of specific mitochondrial receptors over 20 years ago
dence for these nongenomic effects include the lack of and thus provided an attractive unifying model for T3
dependence on nuclear TRs and structure-function rela- effects on mitochondrial activity and cellular energy state.
tionships of TH analogs that are different from their af- There is some evidence that the site of TH action in
finities for TRs. There also can be rapid onset of action mitochondria is the adenine nucleotide translocase of the
(typically seconds to minutes), and utilization of mem- inner mitochondrial membrane (428, 483). However, this
brane-signaling pathways, typically involving kinases or work has been difficult to confirm as there are conflicting
calmodulin, that have not been implicated in direct TR reports in the literature on the site of TH action in mito-
function. The putative nongenomic effects by TH are chondria (116, 185, 276). Recently, a 43-kDa protein re-
diverse. However, the biological significance is not well lated to TRa-1 LBD has been described in mitochondria
understood in many cases. that also could bind to TREs and mitochondrial DNA
Transport of T3 across plasma and nuclear mem- sequences (70, 567). Moreover, transfection of TRa-1 in
branes have been subjects of interest over the years. T3 is CV-1 cells resulted in mitochondrial localization and stim-
lipophilic and generally thought to diffuse passively ulation of mitochondrial activity. These results suggest
across the plasma and nuclear membranes. However, that there indeed may be specific mitochondrial receptors
there is some evidence for facilitated transport across for T3 which also may serve as transcription factors in
plasma membranes and high-affinity TH binding sites in mitochondria.
the plasma membranes of different cells (274, 335, 378, There also are reports of nongenomic effects on cell
421, 439). In one study of human erythrocytes, T3 is structure proteins by THs. Actin depolymerization blocks
concentrated 55-fold inside cells. There also is evidence type II deiodinase inactivation by T4 in cAMP-stimulated
for a stereo-specific transporter of T3 into the nucleus as glial cells, suggesting that an intact actin cytoskeleton is
there was a 58-fold higher concentration of L-T3 and 4-fold important for this downregulation of deiodinase activity
higher concentration of D-T3 in the nucleus than in the (131, 132). Interestingly, T4, but not T3, can promote actin
cytoplasm using isotope dilution methods, although dif- polymerization in astrocytes (468) and thus may influence
ferent affinities for TR may also contribute to this differ- the downregulation of type II deiodinase activity by a
ence (378). However, the identification of the specific secondary mechanism, perhaps by targeting to lysosomes
proteins that might be involved in T3 transport has re- (131, 132). Moreover, the regulation of actin polymeriza-
mained elusive. One potential transporter may be the tion also could contribute to the effects of TH on arboriza-
multidrug resistance P-glycoprotein that can modulate TH tion, axonal transport, and cell-cell contacts during brain
concentration when overexpressed in cells (421). Another development. In this connection, Farwell et al. (133) have
family of transporters may be the organic anion trans- shown that T4 was required for integrin clustering and
porter proteins that have been shown to import TH into attachment to laminin by integrin in astrocytes.
hepatocytes (1, 154). Finally, Davis and co-workers (307) have observed
In addition to transporters, some other potential tar- that the antiviral effects of interferon-g can be potentiated
gets of T3 in the plasma membrane include Ca21-ATPase, by T4 and T3. These effects were rapid and did not require
adenylate cyclase, and glucose transporters (112, 115, protein synthesis because they were not blocked by cy-
274, 335, 378, 439, 454, 455). In the last case, it has been cloheximide treatment and required protein kinase C and
long appreciated that T3 can enhance uptake of sugars in protein kinase A activation. These data suggest that cir-
1116 PAUL M. YEN Volume 81

culating levels of T4 may play an important role in mod- organ culture, suggesting the latter mechanism could oc-
ulating cytokine effects in early host defense. cur. Recent studies have shown that T3 also can directly
stimulate IGF-I production in osteoblasts and enhance T3
stimulation of [3H]proline incorporation, alkaline phos-
IX. THYROID HORMONE EFFECTS
phatase, and osteocalcin (215). TRs recently have been
ON TARGET ISSUES
demonstrated in osteoblast cell lines, osteoclasts derived
from an ostoclastoma, as well as in rat and human bone
TRs are expressed in virtually all tissues, although
samples (5, 6, 12, 329, 554). Both TRa-1 and TRb-1 as well
the relative expression of TR isoforms may vary among
as c-erbAa-2 are expressed in most osteoblast cell lines,
tissues (128, 204, 487). As mentioned previously, TRb-1
although there is a predominance of TRa-1 in some cell
mRNA is highly expressed in liver but also expressed in
lines (e.g., ROS25/1 and UMR 106) (554). Williams et al.
almost all other tissues, whereas TRb-2 mRNA is most
(554) have speculated that osteoblast phenotype expres-
highly expressed in the anterior pituitary. TRa-1 is ex-
sion may correlate with specific TR isoform expression as
pressed in almost all tissues. In addition to this variable
the more undifferentiated cell lines expressed predomi-
expression of TR isoforms in different tissues, the role of
nantly TRa-1. TRb-1 and c-erbAa-2 are highly expressed
TH can vary in different tissues. Indeed, the myriad ef-
in chondrocytes (5). Moreover, TH inhibits growth and
fects by a single hormone on so many different tissues is
stimulates differentiation of chondrocytes in culture
surprising and underscores THs vital role in cellular func-
(218). Recent studies have shown that TRb2 mRNA was
tion. Thus, in addition to its role on the metabolism of
unexpectedly expressed in human chondrocytes and os-
macronutrients and overall energy and oxygen consump-
teoclasts in situ (6).
tion, TH also regulates important functions in specific
Little is known about direct TH effects on osteoclasts
tissues. Recently, the transcriptional regulation of target
because of the difficulty in obtaining primary cultures or
genes in some of these tissues has been studied. We
cell lines. Recently, TR protein was detected in a human
highlight some of the effects of TH in its major target
osteoclastoma and in human bone samples by immuno-
tissues, and if known, describe their effects on the tran-
staining (5, 12), suggesting that TH might have direct
scription of target genes in those tissues.
effects on osteoclasts. However, two groups have used a
bone slice resorption assay to show that functionally
A. Bone isolated osteoclasts were unable to respond directly to T3
by increasing bone resorption, and could only do so if
TH is critical for normal bone growth and develop- other bone cells were present (9, 58). These results would
ment. In children, hypothyroidism can cause short stature suggest that TH may not have a direct effect on bone
and delayed closure of the epiphyses. Biochemical studies resorption but may mediate its effects via paracrine fac-
have shown that TH can affect the expression of various tors secreted by osteoblast cells. Indeed, TH stimulated
bone markers in serum, reflecting changes in both bone prostaglandins and IGF-I in organ culture studies (247).
formation and resorption (10, 337, 430). TH increases Additionally, there have been reports of interferon-g and
alkaline phosphatase and osteocalcin in osteoblasts. Ad- cyclosporin A inhibiting bone resorption by TH, suggest-
ditionally, osteoclast markers such as urinary hy- ing cytokines may also serve as mediators of TH effects
droxyproline, urinary pyridinium, and deoxypyridinium on osteoclasts (247, 273). Additionally, a clinical study
cross-links are increased in hyperthyroid patients. These suggests that interleukin-6 may be involved in mediating
observations suggest that both osteoblast and osteoclast bone loss in hyperthyroidism (272).
activities are stimulated by TH. Indeed, there is enhanced Although TH increases the activities of osteoblasts
calcification and bone formation coupled to increased and osteoclasts in vivo and in culture, little is known
bone resorption in hyperthyroid patients (328, 337). Ad- about its effects on the transcription of target genes in
ditionally, the time interval between formation and sub- these cells. There are a number of osteoblast proteins that
sequent mineralization of osteoid is shortened. The net are stimulated by TH. These include proteins involved in
effect on these bone cells is bone resorption and loss of matrix formation such as alkaline phosphatase, osteocal-
trabecular bone thickness in hyperthyroidism. There also cin, and collagen (10, 247). Additionally, IGF-I and IGF-
is marked increase in porosity and decreased cortical binding protein-2 mRNA are stimulated by T3 in rat pri-
thickness in cortical bone in hyperthyroid patients (30, mary cultures and osteoblastic cell lines (247). However,
175, 328, 430). These effects can lead to osteoporosis and it is not known whether TH directly regulates transcrip-
increased fractures. tion of these target genes. Varga and co-workers (163)
TH may act on bone via TH stimulation of growth have used subtractive hybridization of TH-treated mouse
hormone and insulin-like growth factor I (IGF-I) or by osteoblastic cells to isolate a cDNA that corresponded to
direct effects on target genes. Mundy et al. (339) demon- the insulin growth factor binding protein-4 (IGFBP-4)
strated that T3 had a direct effect on bone resorption in (163). IGFBP-4 mRNA was stimulated by T3 and retinoic
July 2001 THYROID HORMONE ACTION 1117

acid in mouse osteoblastic cells after 48 h of treatment There is no change in a-MHC mRNA level in the atria. This
and remained elevated after 14 days of treatment. The T3 developmental switch in the rat ventricles likely is trig-
stimulation of IGF and IGFBPs suggests that TH may gered by the surge in circulating TH levels that occurs just
participate in osteoblast differentiation and proliferation after birth. It should be noted that in humans and higher
by regulating growth factor synthesis and action. As men- mammals, there is a different developmental pattern as
tioned earlier, little is known about the direct role of T3 on myosin V3 (b/b) is predominantly expressed in the ven-
osteoclasts. Thus far, no T3-regulated target genes have tricles from fetal development to adulthood, whereas V1
been described in osteoclasts. (a/a) is expressed in the atria. Additionally, the effects of
T3 on induction of a-MHC mRNA and protein have been
small in higher mammals, suggesting that other factors in
B. Heart addition to a switch in MHC isoforms may be playing a
role in the inotropic action of TH in these species com-
TH lowers systemic vascular resistance, increases pared with rodents and rabbits. However, in one clinical
blood volume, and has inotropic and chronotropic effects study, a man with profound hypothyroidism and severe
on cardiac function (248). The combination of these ef- cardiomyopathy underwent serial ventricular biopsies be-
fects on both the circulation and the heart itself results in fore and after T4 replacement (270). The patients ventric-
increased cardiac output. Hyperthyroid patients have a ular sample showed a low a-MHC mRNA level before
high output circulation state, whereas hypothyroid pa- treatment that increased 11-fold after T4 treatment. The
tients have low cardiac output, decreased stroke volume, patient had significant improvement in several indices of
decreased vascular volume, and increased systemic vas- cardiac function, suggesting that induction of a-MHC by
cular resistance (248). These changes in cardiac function TH likely contributed to his clinical outcome.
by TH ultimately depend on the regulation of target genes The promoter and upstream regions of a- and b-MHC
within the heart and indirect effects due to hemodynamic genes have been analyzed, and putative TREs for both
changes by TH. these genes have been reported (336). In the a-MHC
TH enhances overall total protein synthesis in the promoter there are two TREs that are imperfect direct
heart (120, 121). Additionally, it regulates the transcrip- repeats separated by four nucleotides located between
tion of several specific proteins that are critical for car- 2100 and 2150. In vitro binding studies and cotransfec-
diac function such as myosin heavy chain (MHC) genes tion studies suggested that the upstream TRE may be
(120). The myosin holoenzyme has a molecular mass of more important for T3-mediated transcription. However,
500,000 kDa and is composed of two MHCs and four light transgenic studies in which the transgene contained the
chains. There are two heavy myosin genes (a and b) a-MHC promoter linked to the chloramphenicol acetyl-
whose products dimerize to form three different myosin transferase (CAT) cDNA showed that mutation of the
chain isoenzymes: myosin V1 (a/a), myosin V2 (a/b), and downstream, rather than the upstream TRE, reduced CAT
myosin V3 (b/b). The relative expression of the MHC activity in the atria and ventricles of euthyroid mice (490).
genes in ventricles is species dependent, since a-MHC b-MHC is negatively regulated by T3 (364). A putative TRE
gene is highly expressed in rodents and rabbits, whereas containing a single half-site that is located adjacent to the
b-MHC gene is predominantly expressed in humans. My- TATA box may play a role in this negative regulation
osin V1 has higher ATPase activity and increased velocity (138).
of fiber shortening than myosin V3, so the relative expres- The rate of diastolic relaxation of the heart is related
sion of isoenzymes in the heart can determine cardiac to intracellular Ca21 concentration and sarcoplasmic re-
contractility. In hypothyroid rats, myosin V3 predomi- ticulum Ca21-ATPase (SERCA2) activity. The ATPase is
nates so the less active myosin subtype participates in the an ion pump that removes calcium from the cytosol and
contractile process resulting in decreased velocity of fiber stores it in the sarcoplasmic reticulum during diastole.
shortening. In contrast, T3 treatment stimulates a-MHC This decrease in the intracellular Ca21 generated during
gene expression and decreases b-MHC gene expression, systole then leads to muscle relaxation. Hypothyroid rats
leading to increased myosin V1, and enhanced cardiac had decreased levels of SERCA2 mRNA that could be
contractility. These opposing effects on MHC gene ex- markedly stimulated by T3 administration (427). Similar
pression have been demonstrated in whole animals as findings also were observed in fetal chicken cardiac myo-
well as neonatal rat myocytes in culture (23, 120, 336). cytes (598). These findings suggest that induction of this
TH also regulates myosin isoenzyme expression dur- ATPase may account for TH enhancement of cardiac
ing development (336). In the rat fetus, a-MHC mRNA output by relaxing the heart with greater speed (lusitropic
expression is high in the atria, whereas b-MHC mRNA effect). Three different TREs that are arranged as DRs
expression is high in the ventricles. a-MHC mRNA then and IPs in the promoter region of the SERCA2 gene that
increases shortly after birth in the ventricles and almost confer T3 responsiveness (191, 471). T3 also has been
completely replaces b-MHC mRNA 7 days after birth. shown to regulate expression of several ion channels in
1118 PAUL M. YEN Volume 81

the heart such as the voltage-gated potassium channel pathway, ATP-citrate lyase, malic enzyme, and fatty acid
Kv1.5, Na1-K1-ATPase, and the hyperpolarizaton acti- synthase, are induced by T3 in differentiating adipocytes
vated cyclic nucleotide-gated channel (365, 379, 572). Ad- (43, 159, 245, 389), suggesting T3 promotes the acquisition
ditonally, TH can regulate b-adrenergic receptor number of differentiated functions in white adipocyte tissue.
in the heart and may thereby enhance sensitivity to cat- Studies in the adult rat have shown that T3 plays
echolamines (558). important roles in regulating basal oxygen consumption,
Recently, a dominant negative mutant TRb was tar- fat stores, lipogenesis, and lipolysis (374, 375). In WAT, T3
geted to the heart in transgenic mice. a-MHC and SERCA2 induces key lipogenic enzymes such as acetyl CoA car-
mRNA were decreased, but b-MHC mRNA was increased, boxylase, malic enzyme, glucose-6-phosphate dehydroge-
in the hearts of the transgenic mice (166). Cardiac muscle nase, fatty acid synthase, and spot 14 (43, 159, 245, 338,
contraction was prolonged, and the QRS interval was 389). The expression of these genes is also modulated by
prolonged on electrocardiogram (EKG). TRa 2/2 knock- other factors such as high-carbohydrate diet, insulin, and
out mice showed decreased heart rate and QRS interval cAMP (374, 375). Additionally, T3 also regulates lipolysis
prolongation on EKG, whereas TRb 2/2 knockout mice in a coordinate manner with lipogenesis (374, 404). Re-
had elevated heart rate that was unresponsive to TH cently, a member of the nuclear hormone receptor family,
administration (228, 552). These findings suggest that PPARg, has been shown to stimulate differentiation of
TRa-1 may have a major role in maintaining baseline heart adipocytes (527). Although prostaglandin J2 and thiazo-
rate, whereas TRb may mediate TH stimulation of heart lidinediones are the major ligands for PPARg, fatty acids
rate. have been shown to stimulate transcriptional activity of
Finally, a novel and potentially exciting therapeutic PPARs, either by metabolites or by induction of genes
use of T3 as an inotropic agent has been in cardiac sur- that may promote formation of endogenous ligands
gery. Novitsky (356) showed improved cardiac function within the cell (146, 251, 527). Thus TH stimulation of
and hemodynamics when brain-dead organ donors were lipolysis may activate other nuclear hormone receptor
pretreated with T3 and cardiac transplant recipients were systems, and thereby promote differentiation.
treated with T3 postoperatively. A small group of patients Recent studies also have shown that both TRa and
that underwent cardiac bypass surgery and were treated TRb-1 are differentially expressed during the develop-
postoperatively with T3 also showed some benefit (357). ment of brown adipose tissue (BAT) (529), a major con-
However, a large randomized study showed that although tributor to facultative thermogenesis in rodents. Faculta-
T3 increased cardiac output and decreased systemic vas- tive thermogenesis occurs in response to cold exposure
cular resistance in patients who underwent coronary- or overeating and depends on T3 and adrenergic stimula-
artery bypass surgery, there was no improvement in out- tion of mitochondrial uncoupling protein (UCP) synthesis
come or changes in postoperative therapy (249). (164, 203, 375, 432). There are three UCP isoforms, UCP1,
UCP2, and UCP3, that are stimulated by T3 in rat BAT (49,
279, 359, 408). UCP3 mRNA is stimulated by T3 in mouse
C. Fat BAT and skeletal muscle (168, 226). It is not known
whether these effects are directly mediated by T3 or via
TH plays important roles in the development and downstream signals such as free fatty acids generated by
function of brown and white adipose tissue (8). TH can lipolysis. The stimulation of UCP synthesis increases ther-
induce white adipose tissue (WAT) differentiation from mogenesis by uncoupling oxidative phosphorylation re-
preadipocytes in young rats as well as in preadipocyte cell sulting in energy dissipation as heat. Interestingly, BAT
lines such as Ob17 and NIH3T3-F442A cells (139, 177, 303, also contains a type II deiodinase whose activity increases
514). In these studies, T3 not only induced intracellular in response to cold, thereby enabling BAT to have the
lipid accumulation and various adipocyte-specific mark- important ability to regulate intracellular T3 concentra-
ers such as malic enzyme and glycerophosphate dehydro- tion in a tissue-specific manner (469, 470). This increase in
genase, but also stimulated adipocyte cell proliferation T3 concentration likely saturates nuclear TRs and en-
and fat cell cluster formation (139, 177). hances norepinepinephrine stimulation of UCP. The ad-
The mechanism(s) by which T3 induces WAT differ- renergic stimulation in BAT is predominantly, but not
entation currently is not known but likely involves tran- exclusively, mediated by brown fat specific adrenergic
scriptional regulation of important target genes by TRs. b3-receptors. The dual regulation of UCP by the type II
Both TRa-1 and TRb-1 are expressed in Ob17 cells, with deiodinase and the adrenergic system suggests conver-
the TRa-1 as the predominantly expressed TR isoform. gence of nuclear- and membrane-signaling systems in the
Rev-erbAa also is induced during the differentiation of transcriptional regulation of these important target genes
NIH3T3-L1 fibroblasts into adipocytes (80). This induction in BAT, but the precise relative contributions and inter-
of rev-erbAa was related to an increase in TRa-1/c- play between these regulatory systems need to be further
erbAa-2 levels. Additionally, enzymes of the lipogenic defined. Lowell et al. (313) used the UCP promoter to
July 2001 THYROID HORMONE ACTION 1119

target diptheria toxin to BAT in transgenic mice. These (lipogenic) diet (375, 376). Similar interactions between
animals had decreased amounts of BAT, resulting in de- T3 and dietary carbohydrate also occur in the gene regu-
creased cold tolerance and obesity, and suggesting a crit- lation of other lipogenic enzymes. Another T3-regulated
ical role for BAT in these functions. Recently, UCP3 gene expressed in liver that has been studied extensively
knockout mice were generated and had normal weight, has been the one encoding S14 protein (376). Its mRNA is
thermogenesis, and response to T3, suggesting that UCP3 rapidly induced by T3 after 20 min in hypothyroid rats and
is not the main mediator for TH-mediated thermogenesis precedes the expression of lipogenic enzymes. Addition-
as originally proposed, or alternatively, compensatory ally, it is coregulated by carbohydrate similar to lipogenic
mechanisms can overcome the lack of UCP3 (169, 538). enzymes. Its tissue distribution is similar to those of
Several human studies have shown that chronic hy- lipogenic enzymes as it is expressed in liver, white and
po- and hyperthyroidism as well as acute T3 treatment did brown fat, and lactating mammary tissue. Recently, it has
not affect serum leptin levels (100, 264, 319, 480). How- been shown that S14 may be localized in the nuclear
ever, one study showed that hypothyroid patients had matrix and thus may participate in regulating the tran-
increased leptin levels, but the increase correlated with scription of lipogenic enzymes (245, 246).
adiposity (399). Another study showed that hyperthyroid It has been appreciated for many years that hypothy-
patients treated with thiamazole increased their leptin roidism is associated with hypercholesterolemia with el-
levels (610). Studies in the rat have shown that T3 can evated serum intermediate and low-density lipoprotein
decrease leptin levels, but it is not known whether this is (LDL) cholesterol concentrations (52). The major mech-
due to a direct effect or due to its effects on fat mass (125, anism for these effects may be lower cholesterol clear-
302, 497). ance resulting from decreased LDL receptors. Further-
more, the genotype of the LDL receptor gene may
D. Liver influence the elevation of serum LDL cholesterol concen-
trations in hypothyroid patients and their response to
thyroxine treatment (559). An additional mechanism may
TH has multiple effects on liver function including
be decreased hepatic lipase activity in hypothyroidism
stimulation of enzymes regulating lipogenesis and lipoly-
which decreases conversion of intermediate-density li-
sis as well as oxidative processes (375, 376). Some of the
poproteins to LDL and high-density lipoprotein metabo-
lipogenic enzymes that are regulated are malic enzyme,
lism (380, 508). It is not known whether these effects are
glucose-6-phosphate dehydrogenase, and fatty acid syn-
mediated directly or indirectly by TH. Several putative
thase. In the case of malic enzyme, which has been stud-
TREs in the distal promoter region of the hepatic lipase
ied extensively, there is a biphasic induction of the malic
and apoliporotein A1 genes have been identified (457,
enzyme mRNA at 4 and 24 h, suggesting that there may be
an initial direct stimulation by T3 and a secondary effect 513). TH also has been shown to regulate the expression
due to stimulation by other gene products that are regu- of several important proteins and enzymes involved in
lated by T3 (486). Indeed, it recently has been shown that cholesterol metabolism and synthesis such as the LDL
Spot 14 (S14), a protein that originally was identified by receptor, cholesterol ester hydrolase, and cholesterol
two-dimensional gel electrophoresis of the translational acyltransferase (423, 447, 461). TH also may regulate post-
products of total hepatic tissue mRNA from T3-treated transcriptional editing of apolipoprotein mRNA (111).
rats, may regulate a number of lipogenic enzymes, includ- TRb-1 is the predominant isoform expressed in liver,
ing malic enzyme, in the liver (245, 452). Likewise, at least whereas TRa-1 is the major isoform expressed in heart
in the rat, a number of lipogenic enzymes also may be (128, 204, 487). These differences in TR isoform expres-
regulated by growth hormone, which is induced by T3 sion have spawned attempts to develop isoform-specific
(375). Interestingly, malic enzyme is very sensitive to T3 in TH analogs that may have cholesterol-lowering effects
the liver, but it is unresponsive in the brain, suggesting but minimal cardiac toxicity (86, 512, 533).
that tissue-specific factors are important in determining Recently, Hayashi and Refetoff (195) have created a
T3-mediated stimulation of transcription. Nikodem and mouse model of TH resistance in the liver by transfecting
co-workers (395) identified a putative TRE in the pro- hepatocytes with adenovirus vectors encoding a mutant
moter region of the malic enzyme gene that contains two TR (195). This mutant receptor had dominant negative
half-sites arranged as direct repeats separated by four activity (see below) on wild-type receptor function. The
nucleotides. mice that expressed the mutant TR had elevated serum
T3 regulation of malic enzyme gene transcription also cholesterol levels compared with control animals in both
can be regulated by carbohydrate intake, insulin, and hypothyroid and hyperthyroid states. Additionally, they
cAMP. For instance, T3 effects on malic enzyme gene had blunted induction of S14 protein and 59-deiodinase
transcription are minimal in fasted animals but are most mRNA by T3. These findings suggest a direct role by TRs
pronounced in animals fed a sucrose-containing fat-free in regulating these genes. This model thus may be a useful
1120 PAUL M. YEN Volume 81

tool for determining those hepatic genes that are directly tionally, these genes display ligand-independent activa-
regulated by TH. tion in cotransfection studies (207, 501). It also is possible
To identify novel hepatic target genes and examine that corepressors, rather than coactivators, may partici-
gene profiles regulated by T3, Feng et al. (136) used a pate in this activation and negative regulation (207, 445,
quantitative fluorescent cDNA microarray to identify he- 501). Additionally, it has been shown that TH decreases
patic genes regulated by TH. Fifty-five genes, 45 of which the stability of TSHb mRNA by inducing shortening of its
were not previously known to be TH responsive, were poly(A) tail (263). T3 can stimulate a cytosolic RNA-bind-
found to be regulated by TH (136). Among them, 14 were ing protein to bind to the 39-untranslated region of TSHb
positively regulated by TH, and surprisingly, 41 were neg- mRNA and may thereby regulate mRNA stability at a
atively regulated. TH had broad effects as it regulated posttranscriptional level (299).
gene expression of a diverse range of cellular pathways Recent cotransfection and knockout studies suggest
and functions such as gluconeogenesis, lipogenesis, insu- that TRb-2 isoform may be playing the predominant role
lin signaling, adenylate cyclase signaling, cell prolifera- in regulating TSH (2, 275). In situ hybridization and im-
tion, and apoptosis. This application of the microarray munostaining studies have shown that TRb-2 is highly
technique to study hormonal regulation of gene expres- expressed in thyrotropes in the pituitary (87, 590). Addi-
sion in vivo demonstrates the value of large-scale gene tionally, RXRg isoform appears to be selectively ex-
expression analyses for future studies of hormone and pressed in thyrotropes, suggesting that it also may play a
drug action. functional role in the regulation of TSH via isoform-spe-
cific TR/RXR complexes or RXRg homodimers (193, 495).
Recent findings of inappropriate TSH secretion in a RXRg
E. Pituitary knockout mice, and the suppression of TSH by RXR-
specific agonist in humans are consistent with this model
TH regulates the synthesis and secretion of several (192, 462).
pituitary hormones. Absence of GH has been observed in
the pituitaries of hypothyroid rats (440). Additionally, T3
can stimulate the transcription of GH mRNA and GH F. Brain
synthesis in rat pituitary tumor cells (322, 440, 458). Brent
et al. (54) identified a rGH TRE than contains a direct TH has major effects on the developing brain in utero
repeat separated by four nucleotides and a palindromic and during the neonatal period (38, 373). Neonatal hypo-
sequence (101, 281). This HRE can also confer sensitivity thyroidism due to genetic causes and iodine deficiency in
to retinoids and vitamin D (156, 493). However, in con- humans can cause mental retardation and neurological
trast to rodents, T3 has limited ability to regulate GH defects. Studies in hypothyroid neonatal rats have shown
synthesis in humans. For example, hypothyroid children that absence of TH causes diminished axonal growth and
have impaired growth but serum growth hormone levels dendritic arborization in the cerebral cortex, visual and
are normal (424, 537). Cotransfection studies using the auditory cortex, hippocampus, and cerebellum (409, 410).
human growth hormone promoter have not shown stim- In the cerebellum, absence of TH also delays proliferation
ulation by TH (55, 71). Studies in cultured human soma- and migration of granule cells from the external to the
totrope adenomas showed that T3 stimulated GH release internal granular layer. The critical role of TH is further
but had variable effects on transcription (90). demonstrated by a recent report in which a dominant
TH also can negatively regulate thyrotropin (TSH) negative TR was targeted to the cerebellum in transgenic
transcription by direct and indirect mechanisms (464). TH mice. The Purkinje cells showed decreased dendritic ar-
can negatively regulate TRH at the transcriptional level, borization, while the granule cells had retarded migration
which in turn decreases transcription of TSH mRNA (465, to the internal granular layer (257). The developmental
573). T3 also can downregulate prolactin mRNA by a delays in the rat brain can be reversed if TH is adminis-
similar mechanism, and also by direct effects on tran- tered within 2 wk after birth (539). These findings support
scription (565). The TRH promoter has been analyzed and the clinical observations that early T4 treatment of con-
contains several nTREs (206, 446). TH hormone also can genital hypothyroidism prevents intellectual impairment
negatively regulate TSH by decreasing transcription of the in humans and is the major impetus for neonatal screen-
glycoprotein hormone a-subunit (common to TSH, lutein- ing for congenital hypothyroidism. In utero, monodeiodi-
izing hormone, follicle-stimulating hormone, and human nation of T4 to T3 by type II deiodinase and maternal-fetal
chorionogonadotropic hormone) and the TSHb subunit transfer of T4 may help maintain normal T3 concentra-
genes (44, 51, 77, 108, 318, 464, 466). Several nTREs in the tions even when the fetus has congenital hypothyroidism
promoters of these genes have been described. In some (110, 180). Additionally, maternal transfer of thyroxine
cases, they contain single half-sites, suggesting that TR may be important, particularly during early fetal develop-
monomers may be involved in negative regulation. Addi- ment (68, 542). Recent studies have suggested that mater-
July 2001 THYROID HORMONE ACTION 1121

nal thyroid status may have significant effects on the silence T3-responsive target genes during embryogenesis.
neuropsychological outcome of children (38, 182, 403). One proposed candidate suppressor may be the orphan
The ontogeny of TR isoforms in the brain suggests receptor, COUP-TF, which binds to the suppressor region
that specific TR isoforms may be involved in transcription in the Pcp2 promoter (16). It remains controversial as to
of target genes and in brain development (373, 487). whether these cerebellar genes are specifically regulated
TRa-1 is expressed throughout the brain from early fetal by TRb-1, which is expressed in parallel. Studies in cul-
development and accounts for total T3 binding in the fetal tured oligodendrocytes have suggested that this may be
brain. TRb-1 is absent or minimally expressed, except in the case (69, 425); however, no significant developmental
a few selected areas such as the cochlea and cerebellum delays in Pcp2 and MBP expression were observed in TRb
(47, 48, 488, 489). However, there is a dramatic 40-fold knockout mice (443). Finally, it is possible that there may
increase in TRb-1 mRNA expression throughout the brain be cross-talk between TR and other transcription factors
shortly after birth that reaches maximum levels 10 days in the regulation of target genes in the brain. The knock-
afterward, and then persists until adulthood (487). In out mouse for the orphan receptor, ROR (staggerer), has
contrast, TRa-1 and c-erbAa-2 mRNA undergo a transient cerebeller defects that are almost identical to those ob-
twofold increase that decreases to adult levels 2 wk after served in hypothyroid mice (258). In this connection, ROR
birth. This early rise in TRb-1 expression coincides with has been shown to modulate transcriptional activity by
the neonatal surge in serum T3 and suggests there may be TH in cotransfection studies (260).
a coordinated temporal developmental program in which
critical target genes are regulated by specific TR isoforms.
Similar temporal patterns of expression also have been X. RESISTANCE TO THYROID HORMONE
observed in the chick and amphibian tadpole (127, 137,
473). RTH is a syndrome in which patients have hyposen-
Despite the importance of TH in brain development, sitivity to TH, elevated circulating serum T3 and T4, and
there are relatively few genes known to be directly regu- elevated or nonsuppressed TSH levels. Refetoff et al.
lated by TH, and many have only been partially charac- (414) first described this syndrome in two siblings who
terized. Farsetti and co-workers (129, 130) have shown presented with deaf-mutism, delayed bone age with stip-
that the gene for myelin basic protein (MBP) is directly pled epiphyses, goiter, and high protein-bound iodine lev-
regulated by TH and have identified a TRE at position els. Since this initial report, over 350 subjects have been
2186 to 2169 of the MBP promoter. Recent studies of described who have RTH, with ;80% of the subjects
brain-derived neutropic factor showed that TH can regu- inheriting this disorder (7, 60, 415). The clinical manifes-
late its expression in a promoter-, developmental-, and tations are variable among families with RTH and also
region-specific manner (259). TH also regulates several among affected family members. Additionally, patients
genes that are involved in a wide range of cellular func- can have clinical symptoms that have features of hypo-
tions: glutamine synthase, protein kinase C, substrate and hyperthyroidism, suggesting variable resistance in
RC3/neurogranin, prostaglandin D2 synthase, hairless (a different tissues. Some of the clinical features that have
potential transcription factor), and adhesion molecules been described include goiter, mental retardation, atten-
such as neural cell adhesion molecule and matrix proteins tion-deficit disorder, tachycardia, delayed bone growth
such as tenascin, and proteins important for neuronal and maturation, and hearing abnormalities. There also are
migration (14, 15, 155, 172, 323, 515). Nordquist et al. (355) examples of pituitary resistance to TH (PRTH), in which
have identified several genes that are expressed in the patients have resistance predominantly in the pituitary
cerebellum: calbindin, myo-inositol trisphosphate (OP-3) and have signs and symptoms of hyperthyroidism in pe-
receptor, and Purkinje cell protein-2 (Pcp2). Study of the ripheral tissues (7, 60, 415).
mRNA expression of these genes and MBP in the cerebel- With the exception of the index family, which had
lums of hypothyroid and euthyroid neonatal mice showed autosomal recessive inheritance, RTH displays autosomal
that the expression of these genes was delayed in the dominant inheritance (415). In 1988, Usala et al. (535)
absence of hormone but eventually reached similar ex- demonstrated a tight linkage between the TRb gene locus
pression levels (489). These data also suggested that there and RTH by restriction fragment length polymorphism.
may be three phases in the regulation of these particular Soon afterwards, two groups independently demon-
genes: a refractory prenatal period, a T3-responsive period strated mutations in the LBD of TRb in patients from two
typically from postnatal days 320, and a T3-independent families with RTH: glycine-345 replaced by arginine (Mf-
period. Identification of a putative silencing element in 1), or proline-453 replaced by histidine (438, 536). Subse-
the promoter of Pcp2 promoter that may be functional quent characterization of other families with RTH have
during fetal development provides support for this model shown TRb point mutations in the two major hot spots
(16). It is intriguing to speculate there may be develop- that cluster near these original mutations (Fig. 13) (36,
mentally and regionally expressed suppressors that may 415). Mutations also have been found in a third hot spot
1122 PAUL M. YEN Volume 81

FIG. 13. Diagram of hot spots in the


ligand-binding domain of TRb where nat-
ural mutations have clustered. Shown
are some representative mutations. For a
more extensive listing of mutations, see
Refs. 7, 60, 415 and resistance to thyroid
hormone (RTH) registry at the Univer-
sity of Chicago (via internet: gopher.
uchicago.edu/subfolder medicine/RTH
registry).

in the most amino-terminal portion of the LBD (37, 97, observation suggested that a single copy of TRb is suffi-
370, 400, 571). Most patients have mutations due to single cient for normal function. Furthermore, it suggests that
amino acid substitutions at a single codon, although sin- reduction of TRb (i.e., gene dosage) did not account for
gle amino acid deletions, frameshift mutations, and trun- the autosomal dominant inheritance seen in other RTH
cations due to premature termination of translation from patients. Instead, it strongly suggested that TRb point
a mutation-generated stop codon also have been identi- mutations interfere with the normal function(s) of TRs.
fied (7, 60, 415). Almost all mutations cluster around the This interference of wild-type protein function by a mu-
ligand-binding pocket observed in the TR LBD crystal tant protein has been called dominant negative activity
structure. In vitro transcription and translation of the (223, 582). The amount of dominant negative activity by a
mutant receptors typically show minimal or reduced T3- mutant TR depends in part on the level of mutant receptor
binding affinity (415). expression. For example, a patient who was homozygous
Approximately 60 different mutations in TRb have for mutations in both TRb alleles had severe RTH and
been identified in RTH patients from over 100 families (7, mental retardation (371). In contrast, his parents who had
60, 415). In some cases, the same mutations have been mutations in only one TRb allele had mild RTH.
described in different families. The clinical phenotype can Within any given cell in a RTH patient, mutant TRb as
vary among these families that harbor the same mutation well as wild-type TRb and TRa are expressed. The mo-
and also vary within a family. This suggests that there may lecular mechanism(s) for the dominant negative activity
be other genetic modifiers that determine the phenotype. by the mutant TRb on wild-type TR function has been the
In this connection, the same mutation can cause either subject of numerous studies. Transient cotransfection
GRTH or PRTH in different individuals even within the studies show that natural TRb-1 mutations not only fail to
same family. In general, there does not seem to be a mediate normal T3-regulated transcription, but also block
strong correlation between particular mutations and the the T3-regulated transcription by normal TRs (78, 327,
development of GRTH and PRTH. The R338W mutation, 415, 437). The mechanism for this dominant negative
and a ninth heptad mutant, R429Q, may be more prone to activity likely involves binding to TREs by inactive mutant
a PRTH phenotype; however, patients with these muta- homodimers or TR/RXR heterodimers that cannot bind T3
tions also can exhibit GRTH (7, 140, 415). Interestingly, no and hence cannot activate transcription of target genes.
germline TRa-1 mutants have been described thus far in Indeed, DNA binding of mutant TRs and v-erbA is required
humans. It is possible that TRa-1 mutations are lethal in for their dominant negative activity. Additionally, some
utero, silent, and/or exceedingly rare. natural mutant TRb-1s, as well as v-erbA, constitutively
Analyses of the TRb gene in the index family with repress basal transcription even in the presence of T3 (28,
RTH showed that the affected members were homozy- 104, 345, 398, 582, 592). Recent experiments also suggest
gous for the deletion of the entire coding region of TRb that dimerization may play an important role in mediating
(502). Thus complete lack of TRb expression was com- dominant negative activity (94, 196, 344). However, it is
patible with life. Interestingly, heterozygous members of not known whether a particular mutant TR complex (i.e.,
the family that contained only one deleted TRb allele had homodimer or heterodimer) mediates this activity in all
normal clinical and laboratory findings (Fig. 14). This cases. Heterodimer-specific mutants are able to mediate
July 2001 THYROID HORMONE ACTION 1123

FIG. 14. Gene dosage does not explain RTH pheno-


type. Top: point mutation in TRb gives RTH phenotype.
Bottom: deletion of one TRb allele gives no phenotype.
Deletion of both TRb alleles in patients gives severe RTH
phenotype.

dominant negative activity, albeit more weakly than other the presence of T3 (97, 370, 435, 571). These mutants also
mutants that can form heterodimers (189, 196). Amino exhibited dominant negative activity on wild-type TR. One
acid substitutions in the ninth heptad of TRb-1 mutants study showed that mutant homodimers could not be
decreased heterodimerization and dominant negative ac- readily dissociated by T3, suggesting that these receptors
tivity of TRb-1 mutants (21, 94, 344). These observations may have reduced T3-binding affinity after binding to DNA
have been used to support functional roles for either (571). Additionally, some of these mutants may have im-
mutant homo- or heterodimers. However, it may turn out paired corepressor release or interaction with coactiva-
that transcriptionally inactive homo- and heterodimers tors (97, 435).
both can mediate dominant negative activity as long as Yoh et al. (593) have studied SMRT corepressor in-
they compete effectively with wild-type TRs for binding to teraction with a battery of mutant TRs. They observed
TREs. that mutant TRs had defective dissociation from this core-
Dominant negative activity in cotransfection studies pressor. In general, the release of SMRT correlated with
and severity of clinical phenotype correlate well with the T3-binding affinity of the mutant receptors. However,
impairment of in vitro T3 binding by mutant TRbs (327, two mutants that had only mildly impaired T3 binding
415), although some exceptions have been reported (196, affinity were unable to dissociate SMRT even at 1 mM T3.
343). Recent studies on TRb mutations in the AF-2 region Additionally, two mutants, D432M and D432G, have en-
and hinge region have shed some light on this issue. TRb hanced association with SMRT. In general, the amount of
mutations in the AF-2 region have normal T3 binding, dominant negative activity correlated with the impair-
DNA binding, and heterodimerization but are transcrip- ment of SMRT dissociation in the presence of T3. The
tionally inactive in the presence of ligand due to their authors also showed that hinge region mutants that abro-
inability to interact with coactivators (521). Recently, sev- gate SMRT interaction also decrease the dominant nega-
eral groups have described RTH patients who have TRb tive activity. Tagami and Jameson (499) also found similar
mutations in the AF-2 region (96, 498, 549). Furthermore, results when they studied NCoR interaction with a battery
AF-2 mutants had potent dominant negative activity on of TR mutants. They also observed that a hinge region
wild-type TRs in cotransfection studies (96, 311, 506). mutant that abrogated NCoR interaction decreased basal
Recently, several mutations in the third hot spot also have repression of transcription of positively regulated genes
been characterized. Two mutants (R243Q and R243W) and ligand-independent activation of negatively regulated
had normal T3-binding affinity but transactivated poorly in genes. These findings suggest that NCoR could play a role
1124 PAUL M. YEN Volume 81

in mediating dominant negative activity of mutant TRs in dominant negative activity on wild-type TR. In this con-
positively and negatively regulated target genes. Addition- nection, a transgenic mouse that overexpressed v-erbA
ally, mutant TRbs located between the two major hot also developed hepatomas (32). A somatic mutation of
spots or near the AF-2 region also had impaired corepres- TRb has been identified in a thyrotropin-secreting tumor
sor release (92, 436, 498). and may cause the defective negative regulation observed
Liu et al. (311) studied the interaction of mutant TRs in these pituitary tumors (18).
with the corepressor NCoR and coactivator SRC-1. The There have been two reported cases of hypersensi-
G345H mutant, which has minimal T3 binding affinity, tivity to TH (222, 320). One patient was euthyroid on the
remained bound to NCoR in the presence of T3 and was basis of thyroid function tests but had a marked tachy-
unable to bind SRC-1. An AF-2 mutant could release cardia. Analyses of T3 binding in lymphocytes showed
NCoR in the presence of T3 but was unable to bind SRC-1. normal binding affinity but increased binding capacity.
Both mutant receptors had potent dominant negative ac- Family history showed several family members with par-
tivity. A mutant receptor (R320H) with threefold lower T3 oxysmal tachycardia and elevated T3 binding in lympho-
binding affinity than wild-type TR was able to release cytes. Sequencing of exons 9 and 10 of TRb showed no
NCoR at higher T3 concentrations and recruit SRC-1 at mutations. Another patient had serum thyroid function
higher T3 concentrations. Interestingly, this mutant had tests suggestive of hypothyroidism but exhibited no clin-
potent dominant negative activity at low T3 concentra- ical symptoms suggestive of hypothyroidism. This patient
tions but weaker dominant negative activity at higher had a V444A substitution in TRb resulting in increased
concentrations. Similarly, Chatterjee and co-workers (96, affinity for T3. A constitutively active estrogen receptor
97) have identified helix 3 and helix 12 mutants from with a mutation in the AF-2 domain has been described
patients with RTH that have impaired recruitment of co- (546), so it is theoretically possible that mutations that
activators. These findings and those showing impaired enhance interaction with coactivators could generate a
corepressor release by mutant TRbs (435, 498, 593) rein- TR with constitutive activity or increased ligand-mediated
force the notion that inability to release corepressors transcription.
and/or the inability to recruit coactivators (regardless of
the mechanism) can result in TR complexes that cannot
be activated by ligand and can mediate dominant negative XI. GENETICALLY ENGINEERED MOUSE
activity. MODELS OF THYROID HORMONE ACTION
Refetoff and co-workers (401, 548) recently have re-
ported families with RTH that did not contain TRb muta- Transgenic expression of dominant-negative mutant
tions. Additionally, linkage analyses suggested that RTH TRs, and the targeted gene inactivation or knockout of TR
was not associated with TRa. Nuclear extracts from af- isoforms, have been used to study TH action in mice.
fected patients showed an additional TR-associated band These mouse models have provided new information on
on electrophoretic mobility shift assay (548). These find- the developmental and physiological effects of TH.
ings suggest the possibility that mutations in cofactors or Two groups have examined the effects of ubiquitous
dysregulation of their expression may be involved in the expression of v-erbA and a natural mutant TRb-1 in trans-
RTH phenotype of these families. Currently, there is no genic mice (32, 564). These transgene products have dom-
direct evidence for such postreceptor defects. In one inant negative activity on the actions of wild-type TR. The
example of a coactivator mutation associated with human mice that expressed v-erbA had multiple abnormalities
disease, mutations in CBP were found in patients with including hypothyroidism (due to follicular disorganiza-
Rubinstein-Taybi syndrome (393), an autosomal dominant tion in the thyroid), inappropriate TSH response, enlarged
disorder in which patients have mental retardation, short seminal vesicles, hepatomas, decreased fertility, and re-
stature, and craniofacial abnormalities. However, thyroid duced adipose tissue. Because v-erbA has dominant neg-
function tests in these patients were surprisingly normal, ative activity on retinoic acid-mediated transcription, it is
suggesting one normal allele is sufficient for TH signaling possible that some of the observed effects may not be due
(367). Ando et al. (18) recently observed an intraexonic solely to blockade of T3-mediated transcription. The
splice variant of TRb in a TSH-secreting adenoma that transgenic mice that expressed mutant TRb-1 had ele-
may account for pituitary resistance to TH in that tumor. vated T3, inappropriately normal TSH, behavioral abnor-
Thus it is possible that nongenomic mechanisms may malities, decreased fertility, and decreased weight. These
account for RTH in some of these families. findings resembled the clinical phenotype observed in
There also have been several somatic TR mutations RTH patients harboring this mutation.
described in tumors. Lin et al. (309) have described so- Pituitary-specific targeting has been undertaken us-
matic mutations in TRa and TRb from a human hepatoma ing natural dominant negative mutants (TRb G345R,
cell line. It is not known whether these mutant TRs con- D337T) under the regulation of the TSHb or glycoprotein
tributed to oncogenesis, although they both exhibited hormone a-subunit promoters (4, 197). These transgenic
July 2001 THYROID HORMONE ACTION 1125

mice had slightly elevated T4, elevated TSH, inability to genic mice that lack only TRa-1 (TRa-1 2/2) have a
suppress TSH after T3 administration, and decreased cho- milder phenotype with decreased body temperature and
lesterol. These findings are consistent with TH resistance heart and prolonged Q-T interval on EKG (552). These
in the pituitary and TH sensitivity in the liver. Interest- findings suggest a major role for TRa-1 in regulating car-
ingly, one line (D337T) had a blunted rise in TSH com- diac function. The differences between the two pheno-
pared with littermate controls, suggesting the mutant TR types could be due to specific functions of c-erbAa-2,
may block the ligand-independent activation of TSH (4). although specific knockout of c-erbAa-2 did not affect
In contrast to these two studies, another transgenic line in survival of the pups (151). Samarut and co-workers (75)
which a frameshift mutant TR (448 frameshift) was tar- have reported generation of short TR isoforms from in-
geted to the pituitary with the glycoprotein hormone ternal translation start sites that can block the actions of
a-subunit did not show any abnormalities in the HPT axis TR. It is likely that these TR isoforms may be responsible
(606). It is possible that differences in the expression for the more severe phenotype of the TRa 2/2 knockout
levels of the mutant TRs may account for this difference mice. Indeed, a TRa knockout that lacks both TRa-1 and
in phenotype. c-erbAa-2 and does not express these isoforms (TRa o/o)
Cardiac-specific targeting of TRb D337T has resulted has a milder phenotype than TRa 2/2 and increased T3
in transgenic mice with abnormal papillary muscle con- sensitivity in tissues expressing TRb (316a).
traction and a prolonged QRS on EKG (166). a-Myosin Ablation of TRb by homologous recombination in
heavy chain and sarcoplasmic reticulum Ca21-ATPase mice (TRb 2/2) produced modest changes in phenotype
mRNA were decreased, whereas b-myosin heavy chain with elevated TSH and T4, thyroid hyperplasia, as well as
mRNA was increased in transgenic mice. These findings hearing defects as the major findings (149, 150). These
are similar to those observed in the hypothyroid heart; findings involving the HPT axis resemble those seen in
however, they contrast with the situation in patients with patients with RTH. Deafness was observed in the index
RTH who often have resting sinus tachycardia. The heart cases of RTH which had deletion of TRb (414). Taken
normally expresses mostly TRa and little TRb, so the together, these findings suggest a critical role for TRb in
finding in RTH patients may be due to the relatively low the development of the auditory system. Interestingly,
amount of dominant negative TRb in the context of ele- ligand-independent elevation of TSH is normal in hypo-
vated circulating TH levels (415). Another study in which thyroid TRb 2/2 mice, but the suppression of TSH by TH
the same mutant TRb was targeted to the heart showed is impaired (547). Recently, TRb-2 has been selectively
decreased contractility in isolated heart preparations knocked out (2). These mice had elevated levels of TH
(386). and TSH, implicating TRb-2 as the major regulator of TSH.
Hayashi et al. (195) have used an adenovirus-based Interestingly, these mice did not have any hearing defects.
expression system encoding a dominant negative TRb-1 These findings suggest that TRb-2 may not be critical for
mutant, G345R, to create a liver-specific model of RTH auditory development, or its function may be compen-
(195). They utilized the selective clearing and uptake of sated by TRb-1. Finally, recent studies in TRb 2/2 mice
injected adenovirus vector by the liver and enabled them suggest TRb2 may be involved in retinal development
to efficiently transfect hepatocytes with either TRb or (353a).
G345R. They demonstrated resistance to T3 in G345R- Given the relatively mild phenotypes of the TRa-1
transfected mice by measuring T3 regulation of several and TRb knockout mice, it is likely that the two isoforms
liver enzymes. Interestingly, serum cholesterol increased may have redundant transcriptional activity and can com-
more in the TRb and G345R transfected mice compared pensate for each other in most target genes. To examine
with controls. These findings suggest that unliganded the effects of abolishing all TR isoforms, TRa-1 2/2 TRb
wild-type TRb and G345R both may have effects on target 2/2 knockouts have been generated (171). These mice
genes in the cholesterol synthesis pathway. lack TRa-1 and TRb, but still express c-erbAa-2. These
Two different groups generated TRa knockout mice mice have markedly elevated T4, T3, and TSH with large
that have different phenotypes (152, 552). The structure of goiters. These mice also have growth retardation and
the TRa gene is complex because it encodes TRa-1, decreased fertility, as well as impaired bone development
c-erbAa-2 (which cannot bind T3), and rev-erbA (generat- and reduced bone mineral content. These mice also have
ed from the opposite strand encoding TRa) so the locus of reduced heart rate and impaired control of body temper-
homologous recombination will determine which iso- ature, similar to the TRa-1 2/2 mice. Although these mice
forms will be knocked out (293). Transgenic mice in have many symptoms of hypothyroidism, these mice did
which both TRa-1 and c-erbAa-2 have been deleted (TRa not have any reduction in activity level. There was no
2/2) have a more severe phenotype with hypothyroid- measurable T3 binding in liver and brain nuclear extracts,
ism, intestinal malformation, growth retardation, and confirming the absence of any TR in these mice.
early death shortly after weaning (152). The early death TRa 2/2//TRb 2/2 knockout mice which lack
can be partially rescued by T3 injection of pups. Trans- TRa-1, c-erbAa-2, and TRb, but express short TRa iso-
1126 PAUL M. YEN Volume 81

forms have been generated (157). They have a similar elevated T4, T3, and moderately elevated TSH levels. In-
phenotype as the TRa 2/2 mice except for markedly terestingly, suppression of TSH by T3 was impaired in
elevated T4, T3, and TSH, and more pronounced malfor- knockout mice, suggesting that SRC-1 may be needed for
mation in the ileum. TRa o/o//TRb 2/2, which lack the the negative regulation of TSH production. These findings
TRa-1, c-erbAa-2, and TRb-1 as well as the shorter TRa have clinical implications because several familes with
isoforms, survive until 6 mo of age and have a similar RTH have been described that do not have mutations in
phenotype similar to the TRa-1 2/2//TRb 2/2 mice TRa or TRb genes. It is possible that mutations in cofac-
(171). However, the females are completely sterile, and tors may account for their RTH phenotype. Additionally,
the males have decreased fertility. They also have mark- as knockout mice for various coactivators are generated,
edly elevated T4, T3, and TSH. Comparison of the the they will be useful tools in studying TH action on both
latter two double-knockout strains in addition to the positively and negatively regulated genes and the effects
c-erbAa-2 knockout mice (151) should provide informa- of coactivators on the function of the HPT axis.
tion on the specific role of c-erbAa-2 on development and
thyroid function.
The preceding double-knockout mouse studies dem- XII. CONCLUSION
onstrate that absence of TRs is not incompatible with life
and that mice without TRs can survive suprisingly well. Just as our early knowledge of TH action was inter-
Many of the effects on peripheral tissues were milder in woven with many of the key developments in biomedical
the double-knockout mice than those seen in congenital science, so it has been the case for the recent study of TH
hypothyroidism. The reason(s) for these observations is action. Even more than before, our knowledge has bene-
not known. Given the lack of T3 binding in nuclear ex- fited from the contributions of outstanding investigators
tracts of the TRa 2/2//TRb 2/2 mice, it is unlikely there from many different fields and countries. With the recent
is an additional TR isoform unless it is expressed during sequencing of the human genome and the development of
embryogenesis or in very limited subset of cells. It is new technologies such as microarrays, proteomics, and
possible that nongenomic effects of TH may be active in genetically engineered mouse models, we will have pow-
the double-knockout mice but not in congenital hypothy- erful new tools to study the complexities of TH action in
roidism. It also is possible that lack of TRs is less delete- the future. It is with eagerness that we look forward to an
rious than having TRs present during hypothyroidism. even deeper understanding of TH action.
Given the role of unliganded TRs and corepressors in
repressing basal transcription, it is possible that critical I thank Drs. Sheue-Yann Cheng (National Cancer Institute),
target genes may be shut down in the hypothyroid mice, Edward Rall [National Institute of Diabetes and Digestive and
whereas a certain degree of leaky basal transcription Kidney Diseases (NIDDK), National Institutes of Health], Leo-
occurs in the double-knockout mice. nard Kohn (NIDDK), Samuel Refetoff (Univ. of Chicago), Nich-
olas Sarlis (NIDDK), and Roy Weiss (Univ. of Chicago) for their
Recently, Cheng and co-workers (236) have gener-
helpful discussions and criticisms. I also thank members of my
ated a knock-in mouse model in which a mutant TR was laboratory: Drs. Stephen Angeloni, Shinichiro Ando, Xu Feng,
introduced into the endogenous TRb gene locus. Similar Padma Maruvada, and Pnina Rotman for their constructive com-
to patients with RTH, the heterozygous mice had elevated ments.
serum T4 and TSH, mild goiter, hypercholesterolemia, Address for reprint requests and other correspondence:
impaired weight gain, and abnormal bone development. P. M. Yen, Molecular Regulation and Neuroendocrinology Sec-
Homozygous mice had markedly elevated serum T4 and tion, Clinical Endocrinology Branch, NIDDK, NIH, Bethesda,
TSH and a much more severe phenotype. It thus appears MD 20892 (E-mail: pauly@intra.niddk.nih.gov).
that the mutant TR has dominant negative activity on TR
function in this mouse model of RTH. It also will be
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