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Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Douglas Forrest, Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York 10029. E-mail: . douglas.forrest{at}mssm.edu
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Introduction |
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 Top Introduction References |
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The nuclear thyroid hormone receptors (TRs) act as ligand-activated transcription factors and occupy a central position in mediating the functions of thyroid hormone. Related TR
1 and TRß receptors are found across vertebrate species suggesting that both receptor types are fundamental components of the thyroid hormone signaling mechanism. The genes encoding TR1 and TRß (THRA and THRB, respectively, in humans, or Thra and Thrb in mice) are both expressed in the CNS with profiles that are consistent with functions in the known periods of hormone sensitivity in CNS maturation (4). However, the receptor genes are also expressed in early embryogenesis, raising questions about additional functions in the early formation of the brain. Several approaches have been taken to determine the function of TRs in the CNS including studies of the differentiation in culture of neuronal cell lines carrying transfected TRs (5, 6), use of antisense oligonucleotides to block TR expression (7), transgenic methods to inhibit TR pathways in vivo (8, 9), and targeted mutagenesis to delete receptors in mice (10). The report by Liu, Tachiki, and Brent in this issue of Endocrinology (11) describes another novel approach that indicates a role for T3 and the TR1 receptor in the neuronal differentiation of mouse embryonic stem (ES) cells in culture. Cells in culture lack the physiological relevance of an animal model, but offer instead the benefit of allowing investigation of a defined cell type under controlled conditions. This may reveal functions that are difficult to unravel in the multicellular context of the brain in vivo. Liu et al. (11) also targeted a point mutation into Thra with results that have interesting implications for potential CNS phenotypes that may arise with mutations in the human THRA gene. To date, only THRB and not THRA mutations have been found in inherited disease.
ES cells have remarkable multipotential properties that enable them to differentiate into all of the other cell types that constitute an organism. This property has led to the popularity of mouse ES cells as vehicles for making knockout or targeted mutations in specific genes. The manipulated but still undifferentiated cells in culture can be transferred back into embryos to generate mice that carry the mutation. ES cells in culture can also be induced to differentiate into a variety of cell types. Neuronal characteristics are acquired upon treatment with retinoic acid (RA) (12). Liu et al. (11) have shown that not only RA but also T3, the major active form of thyroid hormone, stimulates neuronal differentiation with the appearance of neurite outgrowths and expression of the neuronal markers nestin and neurofilament. Moreover, two known T3-response genes in the brain, neurogranin (RC3) and Ca2+/calmodulin-dependent kinase (CaMKIV), are induced in the differentiated cells.
Retinoids, or vitamin A derivatives, are known to be important in early CNS formation and RA deficiency or excess can cause brain malformations (13, 14). There is less evidence suggesting that T3 has such potent actions on the early structures of the CNS. Rather, many studies suggest major actions for T3 on the maturation of the CNS. For example, in the cochlea, RA exposure modifies the numbers of sensory hair cells formed, whereas the major actions of T3 are in later maturation of cochlear and hair cell function (4, 15). To some extent, the lack of detection of gross actions of T3 in the early CNS may be because the fetal brain is partly shielded from thyroid hormone fluctuations by the thyroid hormone-metabolizing enzymes, the iodothyronine deiodinases. Type 2 deiodinase converts thyroxine into T3, whereas type 3 deiodinase inactivates thyroid hormone. Deiodinases are strategically expressed for such a protective role in the immature CNS and in the placenta and uterus supporting the fetus (16, 17, 18, 19). The finding that T3 induces neuronal differentiation of ES cells in culture, however, may support a role for T3 at earlier stages in the CNS than has been hitherto obvious. It has been reported that maternal hypothyroidism during pregnancy is subsequently associated with impaired neuropsychological development of the child (20), which may be another indicator of T3 actions in the early CNS. These might be independent of, and more subtle than, the more obvious, known functions of T3 in postnatal rodent brain development (1). Conceivably, T3 sets in motion an internal chain of events that is not necessary for gross development but rather facilitates the eventual, correct function of neurons. Further studies of this area may require new assays in animal models for corresponding defects in attention, learning or other forms of behavior (21).
Liu et al. (11) also tested the receptor requirement for the neuronal differentiation of the ES cells. A point mutation (P398H) was targeted into an exon encoding the C-terminal T3-binding domain of TR1. This abrogated T3 binding by TR1 and reduced the nuclear T3 binding capacity of the cells by 50%, as expected for a mutation at one Thra allele. These data also imply that TR1 is the major form of TR in the undifferentiated ES cells, with little TRß present, agreeing with the expression of TR1 in the early embryonic brain. Although T3 still induced differentiation of the mutant ES cells into a neuronal-like morphology, induction of the T3-response gene RC3 was blocked and CaMKIV was partly blocked, indicating impairment of an underlying part of the neuronal differentiation process. In addition, in wild-type ES cells, RA increased the frequency of cell death, whereas this response was lessened when T3 was added with RA. The mutant ES cells showed resistance to this action of T3, suggesting that TR1 was involved. The molecular basis of the interaction between RA and T3 in apoptosis is unknown, although RA receptors and TRs are closely related and may interact at the same target DNA response elements or through protein-protein interactions (13).
How might the TR1 P398H mutation inhibit neuronal gene expression in the ES cells? A 50% reduction (haploinsufficiency) in TR1 may be enough to impair a subset of T3-inducible events, namely, induction of certain target genes and the modulation of RA-dependent apoptotic pathways. However, because T3 still induces a gross neuronal morphology in the mutant ES cells, the remaining 50% level of TR1 would be adequate for most other differentiation events. It is noteworthy, however, that no phenotypes attributable to haploinsufficiency of either TR1 or TRß have been reported to date in knockout mouse strains (10). Alternatively, it seems more likely that the mutation changes rather than abolishes the function of TR1, creating a dominant inhibitory protein. The TR1 P398H mutation was based on a known THRB mutation in the human syndrome of resistance to thyroid hormone (RTH). RTH is inherited dominantly through heterozygous, change-of-function mutations in the TRß C terminus (22, 23). These changes impair T3 binding and cause aberrant interactions with transcription cofactors such that in cell transfection assays, transactivation is impaired. Moreover, the proteins inhibit transactivation by normal receptors, suggesting a mechanism for the dominant manifestation of the disease. Thus, the TR1 P398H mutation may exert a partly dominant inhibition over normal TR pathways in differentiating ES cells.
THRA mutations have so far not been associated with inherited human diseases, although many THRB mutations have been found in RTH syndrome. This may be because THRA mutations are innocuous, or at the other extreme, they may be deleterious in early development. Alternatively, any disease phenotypes may arise in unpredicted areas, with few clues suggesting a link to a TR. Potential phenotypes have been suggested in mice deleted for TR1, which display defects in thermoregulation, basal heart rate (24, 25), and female mating behavior (21). However, change-of-function mutations in Thra in mice may yield more valuable clues of phenotypes associated with any human THRA mutations, given that recessive diseases caused by homozygous gene deletions are rarer than dominant disorders. Indeed, there are often distinct consequences for the complete loss of a gene vs. a change-of-function mutation in the gene product. This is illustrated, for example, for TRß, where the absence of TRß causes dysregulaton of the pituitary-thyroid axis, severe deafness, and color blindness (4). Although the change-of-function mutations in TRß in RTH cause dysregulation of the pituitary-thyroid axis and a variety of other symptoms, they have relatively little impact on hearing and color vision (22).
A recent study of an RTH-like mutation introduced into the C terminus of TR1 in mice described a dominant phenotype with dwarfism, infertility (26), and reduced CNS glucose utilization suggestive of defective synaptic activity (27). Interestingly, the equivalent mutation in TRß does not produce this phenotype in brain, suggesting a specific role for TR1 in synaptic function. A distinct RTH-like mutation in TR1 in mice retards growth transiently and alters heart function (Tinnikov, A., V. K. Chatterjee, and B. Vennström, personal communication). Both of the above TR1 mutations cause only marginal changes in thyroid hormone levels and suggest instead that clearer indicators of TR1 mutations may be observed in growth, cardiac, and neurological parameters.
The THRA/Thra gene is complex and encodes splice variants such that different types of mutation could yield distinct phenotypes. A C-terminal variant, TR2, that lacks T3 binding ability is widely coexpressed with the TR1 T3 receptor (28). The role of this nonreceptor product has been enigmatic, however, the deletion specifically of TR2 results in viable mice with modest phenotypes in growth, heart rate, and slightly reduced thyroid hormone levels (29). Thus, the balance of splicing between TR1 and TR2 is important in determining the T3 response status in several tissues. Another mutation in Thra that deleted both TR1 and TR2 and that also generated partial C-terminal products, was lethal after weaning. A further deletion that removed the partial products gave viable mice with modest phenotypes and indicated that the lethality was somehow caused by the residual Thra products (30). Evidently, this potential for complex secondary changes should be taken into account in the interpretation of any Thra mutation. These complexities were neatly bypassed by Liu et al. (11), whose gene targeting strategy left only the desired point mutation and no other change in the Thra gene.
TRs bind target DNA elements in the presence or absence of T3, and it has long been a subject of conjecture that TRs may therefore have physiological functions that reflect both T3-dependent and T3-independent activities. Recent evidence supports such T3-independent actions for TRs in the CNS. The cerebellum provides a classical model of retarded neuronal migration and differentiation in hypothyroidism. Somewhat surprisingly, however, the deletion of TRß or TR1 does not produce major hypothyroid-like CNS phenotypes in mice (10). These findings may be explained if a receptor acting as a constitutive T3-independent repressor is required for causing the hypothyroid defects. One RTH-like mutation targeted into Thrb resulted in a TRß protein that fails to bind T3 and which chronically associates with transcription corepressors. Mice carrying this mutation have retarded cerebellar development, despite the fact that they do not have low levels of thyroid hormones (31). Thus, these results support the view that T3-independent repression by TRs may explain some of the CNS damage that occurs in hypothyroidism. Another study showed that hypothyroidism failed to produce the characteristic cerebellar abnormalities in TR1-deficient mice, providing strong evidence of the need for TR1, presumably acting as a T3-independent repressor, in causing some CNS defects (32). This work and that of Liu et al. (11) indicate an emerging role for TR1 in the CNS.
Much remains to be resolved about the specific contributions of TRß and TR1 to CNS development and about genotype-phenotype correlations of particular mutations. Based on known THRB mutations in RTH, it is clear that phenotypic variation arises, some of which may be explained by modification by the genetic background (22). The additional possibility that distinct mutation types specify some phenotypic outcomes may be investigated by the targeting of different mutations into mice on uniform genetic backgrounds. Indeed, different subdomains of the receptor C terminus interact not only with T3, but also with corepressors and coactivators and, conceivably, these may determine certain tissue-specific functions of TR1 and TRß (23).
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Acknowledgments |
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Footnotes |
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Abbreviations: CNS, Central nervous system; ES, embryonic stem; RA, retinoic acid; RTH, resistance to thyroid hormone; THRA and THRB, the genes encoding TR1 and TRß; TR, thyroid hormone receptor.
Received April 30, 2002.
Accepted for publication May 1, 2002.
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References |
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TopIntroductionReferences |
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thyroid hormone receptor oligonucleotides. J Biol Chem 270:12100–12108 gene dominant-negative mutation (P398H) selectively impairs gene expression in differentiated embryonic stem cells. Endocrinology 143:2664–2672 inhibits retinoic acid-responsive gene expression and modulates retinoic acid-stimulated neural differentiation in mouse embryonic stem cells. Mol Endocrinol 8:746–7561. EMBO J 17:455–461[CrossRef][Medline]
or ß. Endocrinology 142:544–550 or ß receptor gene. Proc Natl Acad Sci USA 98:9913–9918-related protein which binds deoxyribonucleic acid but does not bind thyroid hormone. Mol Endocrinol 2:893–9012 and a concomitant overexpression of 1 yields a mixed hypo- and hyperthyroid phenotype in mice. Mol Endocrinol 15:2115–2128 locus. Mol Cell Biol 21:4748–4760 1 prevents the structural alterations of the cerebellum induced by hypothyroidism. Proc Natl Acad Sci USA 99:3985–3989
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