Thyroid hormone response element sequence and the recruitment of retinoid X receptors for thyroid hormone responsiveness.

Thyroid hormone receptors (TRs) are transcription factors that bind to thyroid hormone response elements (TREs) in the regulatory regions of target genes. TRs are thought to activate transcription primarily as heterodimers with retinoid X receptors (RXRs), with RXR binding upstream to the two directly repeated half-sites in a typical TRE. However, given that TRs and RXRs prefer to bind to different DNA sequences (T(A/G)AGGTCA and GGGGTCA), we postulate that only certain TREs require RXR-TR heterodimerization, depending on the TRE sequence. We have tested this hypothesis by comparing in Saccharomyces cerevisiae the functional activity of TR +/- RXR on 10 naturally occurring mammalian TREs. S. cerevisiae was used as a model system because yeast lack endogenous nuclear receptors and thus can be manipulated to express TRs and/or RXRs. We first studied ligand-independent reporter gene activation, which reflects the activity of the activator function 1 (AF-1) domain. The 10 TREs formed a continuous spectrum from being fully dependent on RXR for TR AF-1 activity to being essentially independent of RXR. Relative independence of RXR generally was seen when the TRE upstream half-site has a TA or TG 5' to the core hexamer. Gel mobility shift assays revealed that functional independence of RXR correlates with the strong binding of TR alone, whereas more RXR dependence correlates with higher binding of RXR-TR heterodimers. Restoration of ligand-dependent (AF-2 domain) reporter gene activation was achieved by expression of the coactivator TIF2. This ligand-induced stimulation was stronger in the presence of TR alone than with RXR plus TR, suggesting a preference for TIF2 activation of TR homodimers. Overall the data support the notion that the TRE sequence plays an important role in determining the nuclear hormone receptor and coactivator requirements for TR action.

Regulation of hepatocyte thyroxine 5'-deiodinase by T3 and nuclear receptor coactivators as a model of the sick euthyroid syndrome.

The syndrome of nonthyroidal illness, also known as the sick euthyroid syndrome, is characterized by a low plasma T3 and an "inappropriately normal" plasma thyrotropin in the absence of intrinsic disease of the hypothalamic-pituitary-thyroid axis. The syndrome is due in part to decreased activity of type I iodothyronine 5'-deiodinase (5' D-I), the hepatic enzyme that converts thyroxine to T3 and that is induced at the transcriptional level by T3. The hypothesis tested is that cytokines decrease T3 induction of 5' D-I, resulting in decreased T3 production and hence a further decrease in 5' D-I. The proposed mechanism is competition for limiting amounts of nuclear receptor coactivators between the 5' D-I promoter and the promoters of cytokine-induced genes. Using primary cultures of rat hepatocytes, we demonstrate that interleukins 1 and 6 inhibit the T3 induction of 5' D-I RNA and enzyme activity. This effect is at the level of transcription and can be partially overcome by exogenous steroid receptor coactivator-1 (SRC-1). The physical mass of endogenous SRC-1 is not affected by cytokine exposure, and exogenous SRC-1 does not affect 5' D-I in the absence of cytokines. The data support the hypothesis that cytokine-induced competition for limiting amounts of coactivators decreases hepatic 5' D-I expression, contributing to the etiology of the sick euthyroid syndrome.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis.

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17beta-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.

Gene regulation by thyroid hormone.

Regulation of gene expression by thyroid hormones (T3, T4) is mediated via thyroid hormone receptors (TRs). TRs are DNA-binding transcription factors that function as molecular switches in response to ligand. TRs can activate or repress gene transcription depending on the promoter context and ligand-binding status. In most cases, in the absence of ligand, TRs interact with a corepressor complex containing histone deacetylase activity, which actively inhibits transcription. The binding of ligand triggers a conformational change in the TR that results in the replacement of the corepressor complex by a coactivator complex containing histone acetyltransferase activity, through which the chromatin structure is remodeled, thereby leading to activation of transcription. In addition, the finding that several TR-interacting coregulators act more directly on the basal transcriptional machinery suggests that mechanisms independent of histone acetylation and deacetylation also are involved in TR action.

Dominant negative activity of thyroid hormone receptor variant alpha2 and interaction with nuclear corepressors.

The splicing variant of the thyroid hormone receptor alpha (TRalpha) gene, TR variant alpha2 (TRv alpha2), lacks the second half of the ninth heptad, a domain thought to be important for heterodimerization with retinoid X receptors (RXRs). In transient transfection studies, TRv alpha2 exhibits weak dominant negative inhibition of TRalpha1-mediated transcription. In contrast, a TRv alpha2 mutant in which the ninth heptad was restored (alpha2 + 9H), exhibits very strong dominant negative activity. We have examined the role of nuclear corepressors (CoRs) in the dominant negative activity of TRv alpha2 and alpha2 + 9H. Glutathione S-transferase pull down experiments revealed that TRv alpha2 barely interacts with CoRs, whereas alpha2 + 9H interaction with CoRs is as strong as that of TRalpha1. A P160R CoR box mutation was introduced in the context of TRv alpha2 and alpha2 + 9H, which nearly abolishes the ability of these receptors to interact with CoRs. In transient transfection the dominant negative activity of TRv alpha2 was only marginally impaired by the P160R mutation. In contrast, alpha2 + 9H-P160R had approximately 66% less dominant negative activity than alpha2 + 9H. These results suggest that the weak dominant negative activity of TRv alpha2 is due in part to its lack of interaction with CoRs, and that restoration of the ninth heptad restores CoR interaction and strong dominant negative activity. Further, the data reveal aspects of the dominant negative action that are dependent on the orientation of the TRE.

1Alpha,25-dihydroxyvitamin D3 up-regulates Bcl-2 expression and protects normal human thyrocytes from programmed cell death.

Apoptosis is thought to play an important role in the pathogenesis of autoimmune thyroid disease. 1alpha,25-dihydroxyvitamin D3 (VD3) has been shown to suppress several autoimmune diseases. However, the mechanism by which VD3 has these effects is not known. We evaluated the alterations in apoptosis, induced by VD3. Thyrocytes were treated with VD3, and the expression of the Bcl-2 family molecules was studied at both the messenger RNA and protein levels. It was found that VD3 significantly induced the expression of Bcl-2 messenger RNA and protein in thyrocytes but had no effect on the expression of Bcl-xl and Bax. The increase in Bcl-2 expression, mediated by VD3, correlated with protection of thyrocytes against the induction of apoptosis by either staurosporine or UV irradiation. VD3-induced increases in the expression of Bcl-2 could be mimicked by VD3 analogs with high nuclear receptor affinity, but not by analogs only with nongenomic actions. These data indicate a role for Bcl-2 in the regulation of apoptosis in thyrocytes and raise the possibility that VD3 or its agonists may have therapeutic benefit in thyroid disorders.

Protein-protein interaction domains and the heterodimerization of thyroid hormone receptor variant alpha2 with retinoid X receptors.

Heterodimerization between thyroid hormone receptors (TRs) and retinoid X receptors (RXRs) is mediated by a weak dimerization interface within the DNA- binding domains (DBDs) and a strong interface within the C-terminal ligand- binding domains of the receptors. Previous studies have shown that the conserved ninth heptad in the TR ligand-binding domain appears to play a critical role in heterodimerization with RXR. However, despite lacking the full ninth heptad, TR variant alpha2 (TRv alpha2) can heterodimerize with RXR on specific direct repeat response elements, but not on palindromic elements or in solution. Two possibilities may account for TRv alpha2-RXR heterodimerization on direct repeats. First, the DBD of TRv alpha2 may play a critical role in heterodimerization with RXR. Second, a specific sequence within the unique C terminus of TRv alpha2 may promote the formation of TRv alpha2-RXR heterodimers. In this study, we used receptor chimeras in which the DBD of RXR was replaced by either the TR DBD or an unrelated DBD from the metalloregulatory transcription factor AMT1 to address the role of the DBD dimerization interface in TRv alpha2-RXR heterodimerization. Gel mobility shift analyses showed that whereas TR alpha1 formed heterodimers with these chimeras, TRv alpha2 failed to do so. Deletion of the unique C terminus of TRv alpha2 had only a marginal effect on heterodimerization with RXR. Mutations within the DBD dimerization interface abolished heterodimerization of full-length TRv alpha2 with RXR but only marginally affected heterodimerization of full-length TR alpha1 with RXR. These data support the hypothesis that the TR-RXR DBD dimerization interface plays a critical role in TRv alpha2-RXR heterodimerization. Additional data show that the amino acid residues that make direct TR-RXR contacts within the DBDs also may play a role in receptor monomer binding to DNA, since mutations within these residues severely impair this interaction.

Thyroid hormone receptor coactivators and corepressors.

In the absence of triiodothyronine (T3), thyroid hormone receptors (TRs) repress transcription of many genes; in the presence of T3, TRs activate transcription of those same genes. Both of these events are dependent on interactions between TRs and other nuclear proteins. TRs bind to specific DNA sequences, generally found in the 5' flanking regions of target genes. In the unliganded state, TRs interact with one of several corepressor proteins. These proteins, in turn, interact with a series of other proteins, which includes histone deacetylases. Histone deacetylation tightens chromatin structure, thus impairing access of critical transcription factors and thereby repressing transcription. In addition, corepressors may invoke mechanisms of gene repression independent of histone deacetylation. The binding of T3 causes a conformational change in the TR that results in release of the corepressor and recruitment of coactivator proteins. Several coactivator proteins appear to bind the ligand-occupied TR as a multiprotein complex. Opposite to corepressors, coactivators acetylate histones, thereby loosening chromatin structure and facilitating access of key transcription factors. Again, mechanisms independent of histone acetylation also may be involved. Overall, gene activation by T3 is a two-step process; removal of active repression, and induction of transcription to levels above the "neutral" state.

Characterization of the DNA-binding and dominant negative activity of v-erbA homodimers.

The oncoprotein v-erbA is a mutated form of thyroid hormone receptor alpha1 that is virtually incapable of binding T3. V-erbA is a dominant repressor of transcription induced by thyroid hormone receptors and retinoic acid receptors; however, the genetic targets of v-erbA that lead to oncogenesis are not known. Although v-erbA can bind as monomers and dimers to DNA containing the consensus sequence AGGTCA arranged as direct, inverted, or everted repeats, it is not known which sequence represents the optimal v-erbA-binding site. Determination of the DNA recognition properties of v-erbA would allow a better understanding of the repressor activity of this oncoprotein. The current studies, by using a random DNA selection strategy, have determined that the imperfect everted repeat 5'-TGACC(T/C)NT(A/G)AGGTCAC is the optimal v-erbA homodimer-binding site, where N represents any di- or trinucleotide. Functional studies show that everted repeats containing this sequence are substantially more potent v-erbA response elements than direct or inverted repeats, even though many classic T3 response elements are direct repeats. Thus, v-erbA represses only a subset of T3 response elements. In a similar fashion, v-erbA was found to repress a subset of vitamin D response elements. Of general interest, the data indicate that the two molecules of a transcription factor homodimer do not necessarily have identical DNA-binding specificities.

Thyroid hormone response element architecture affects corepressor release from thyroid hormone receptor dimers.

Thyroid hormone receptors are ligand-modulated transcription factors that can repress or activate transcription depending upon the absence or presence of thyroid hormone and the nature of the hormone response element to which the receptors are bound. The ability of thyroid hormone receptors to repress transcription in the absence of ligand is thought to be due to associations with nuclear hormone receptor corepressors. Ligand binding by the thyroid hormone receptor is believed to dissociate these corepressors and recruit coactivators to promote transcription from target promoters. We hypothesize that variations in response element architecture may influence both the association and dissociation of corepressors from DNA-bound thyroid hormone receptors. Using a chimeric corepressor, we find that ligand alone does not fully relieve corepressor-mediated repression, particularly in the presence of thyroid hormone receptor and its heterodimerization partner, the retinoid X receptor. Interestingly, the steroid receptor coactivator 1 together with ligand is able to mediate full release of corepression, but this relief is dependent upon the architecture of the response element to which the nuclear receptor dimer-corepressor complex is bound. These studies suggest that other cellular factors in addition to ligand may be required for the release of corepressors from thyroid hormone receptor dimers.

Thyroid function in Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RTS) is a genetic syndrome characterized by broad thumbs and halluces, growth retardation, mental retardation, and craniofacial abnormalities. This condition recently was found to be caused by mutations in the gene encoding cAMP response element-binding protein (CREB)-binding protein. As CREB-binding protein has been shown to be a critical coactivator for thyroid hormone receptors, it is plausible that RTS would be characterized by thyroid hormone resistance. In fact, features of RTS, such as mental retardation and short stature, are consistent with thyroid hormone deficiency or resistance. To assess the function of the thyroid axis in RTS, free T4 and TSH were measured in 12 subjects with this syndrome. The free T4 level was normal in all 12 (mean +/- SD, 0.97 +/- 0.20 ng/dL; normal range, 0.73-1.79), as was the TSH level (2.24 +/- 0.87 microU/mL; normal range, 0.3-6.5). Thus, overt thyroid hormone resistance does not appear to be a typical feature of RTS.

Effect of glyoxylate on the function of the calcitriol receptor and vitamin D metabolism.

The biological action of calcitriol is mostly mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs) of target genes. These interactions produce special proteins that carry out the biological activities of calcitriol. Recently, we showed that the interaction of VDRs with VDREs is inhibited by uremic toxins. We hypothesize that uremic toxins that contain aldehyde or ketone groups potentially could form Schiff bases with lysine residues of the VDR DNA binding domain and inhibit VDR interaction with VDREs. We therefore chose glyoxylate, a compound which has an aldehyde group, to test this hypothesis. In vitro glyoxylate inhibited VDR binding to the osteocalcin and osteopontin VDREs as assessed by electrophoretic mobility shift assay and the inhibition was reversed when glyoxylate was preincubated with lysine. Further, this chemical compound also blocked the induction of chloramphenicol acetyltransferase (CAT) enzyme induced by calcitriol in cells transfected with a calcitriol responsive CAT reporter gene. Since induction of 24-hydroxylase synthesis is a VDR regulated process, we also studied the effect of glyoxylate on the activity of intestinal 24-hydroxylase in rats. This enzyme activity was suppressed in rats infused with glyoxylate. Taken together, our study suggests that glyoxylate could inhibit the interaction of VDR with VDREs and alter the biological action of calcitriol.

Effect of glucose on the function of the calcitriol receptor and vitamin D metabolism.

The genomic action of calcitriol is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs) of the target genes. It has been proposed that chemicals capable of Schiff base formation with the VDR potentially could alter the physiological function of VDR and calcitriol metabolism. Since glucose has been shown to form Schiff bases with proteins, we tested the hypothesis that glucose could influence the function of VDR and thereby alter calcitriol metabolism. Glucose 6-phosphate inhibited VDR binding to the osteocalcin VDRE and chemically modified the DNA binding domain or the dimerization domain of the VDR in vitro. Further, glucose also blocked the production of chloramphenicol acetyltransferase (CAT) enzyme induced by calcitriol in cells transfected with a constructed VDRE attached to a CAT reporter gene. Hyperglycemia induced by glucose infusion or by streptozotocin in normal rats significantly reduced intestinal 1 alpha, 25-dihydroxyvitamin D-24-hydroxylase activity. Taken together, these findings are consistent with the hypothesis that glucose could interact with the VDR to impair its DNA binding and function within cells.

5'-flanking sequences in thyroid hormone response element half-sites determine the requirement of retinoid X receptor for receptor-mediated gene expression.

Thyroid hormone receptors are ligand-inducible transcription factors that can potentially interact with thyroid hormone response elements as homodimers or heterodimers with the retinoid X receptor. It has generally been felt, however, that the heterodimer is responsible for induction of gene expression. We have demonstrated previously that the optimal thyroid hormone receptor binding sequence is not the consensus hexamer half-site AGGTCA but is an octamer, TAAGGTCA. Based upon these findings, we hypothesize that thyroid hormone response elements composed of optimal half-sites (TAAGGTCA) will bind thyroid hormone receptors readily and activate gene expression independently of the retinoid X receptor. In contrast, response elements composed of suboptimal half-sites (e.g. GCAGGTCA) will require the retinoid X receptor to facilitate thyroid hormone receptor-mediated gene expression. To test this hypothesis, we have reconstituted thyroid hormone receptor-mediated gene expression in yeast. Our studies confirm the hypothesis that the retinoid X receptor is required for gene expression from response elements composed of suboptimal half-sites, whereas thyroid hormone receptors are sufficient to activate gene expression maximally from response elements containing optimal half-sites. Furthermore, coexpression of steroid receptor coactivator-1 is required for ligand-dependent gene activation from single response elements. Surprisingly, however, coexpression of the retinoid X receptor decreases the steroid receptor coactivator-1-dependent thyroid hormone induction. Overall these data demonstrate that the architecture of the thyroid hormone response element dictates the nuclear receptor requirements for gene activation. The studies suggest that different coactivators may be required for gene activation depending upon the response element architecture and the nature of the bound thyroid hormone receptor complex (homo- versus heterodimer).

Effect of Schiff base formation on the function of the calcitriol receptor.

The genomic action of calcitriol is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs) of the target genes. We have shown that the interaction of VDRs with VDREs is inhibited by uremic toxins. We hypothesize that uremic toxins form Schiff bases with the lysine residues of the VDR DNA binding domain and inhibit the VDR interaction with the VDRE. In this study, pyridoxal 5'-phosphate was used as a probe to test Schiff base formation as the inhibitory mechanism, since it forms Schiff bases with steroid receptors. Pyridoxal 5'-phosphate inhibited the VDR binding to the VDREs and chemically modified the DNA binding domain of the VDR in vitro. The inhibition was reversed when pyridoxal 5'-phosphate was preincubated with lysine. Further, this chemical agent also blocked the production of chloramphenicol acetyltransferase (CAT) enzyme induced by calcitriol in cells transfected with a constructed VDRE attached to a CAT reporter gene. This finding is consistent with the hypothesis that pyridoxal 5'-phosphate could interact with the VDR and impair its DNA binding within cells. Since induction of 24-hydroxylase synthesis is a receptor mediated process, we studied the effect of pyridoxal 5'-phosphate on the synthesis of renal 24-hydroxylase in rats. When pyridoxal 5'-phosphate was infused to rats, renal 24-hydroxylase activity was suppressed, consequently, degradation of calcitriol was also reduced in these animals. Thus, chemicals capable of Schiff base formation potentially could alter the physiological function of VDR and calcitriol.

Thyroid hormone receptor variant alpha2. Role of the ninth heptad in dna binding, heterodimerization with retinoid X receptors, and dominant negative activity.

Thyroid hormone receptors bind DNA with highest affinity as heterodimers with retinoid X receptors, and such heterodimers generally are thought to be the biological mediators of thyroid hormone action. An alternative splice product of the thyroid hormone receptor alpha gene, thyroid hormone receptor variant alpha2, does not bind thyroid hormone and functions as a weak dominant negative inhibitor of thyroid hormone action. Thyroid hormone receptor variant alpha2 is missing one-half of the ninth heptad, a region of the bona fide receptor thought to be important for heterodimerization with retinoid X receptors. The role of the ninth heptad in heterodimerization has been evaluated further. Thyroid hormone receptor variant alpha2-retinoid X receptor heterodimers form on a subset of direct repeat response elements but not on palindromic or inverted palindromic elements. Restoration of the missing ninth heptad sequence is critical for restoring heterodimerization on the palindromic DNA, but either the ninth heptad amino acids or a stretch of alanines is equally able to restore heterodimerization on the inverted palindrome. Thus, the role of the ninth heptad in heterodimerization differs on direct repeat, palindromic, and inverted palindromic response elements, suggesting that the protein-protein interactions differ on each of these elements. The dominant negative activity of thyroid hormone receptor variant alpha2 requires DNA binding, but the relatively weak nature of the dominant negative activity is only partially explained by the weak DNA binding.

Unique autoantibody epitopes in an immunodominant region of thyroid peroxidase.

To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme-linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response.

Effects of hydrogen peroxide, nitric oxide and antioxidants on NF-κB.

The NF-κB complex consists of a family of transcription factors which bind to specific sequences present in the regulatory regions of mammalian genes and in the human immunodeficiency virus (HIV) long terminal repeat. It has been suggested that free radicals may play a role in NF-κB activation and in the activation of HIV expression. The effects of H2O2 and nitric oxide (NO(•)) on NF-κB deoxyribonucleic acid (DNA) binding were examined using the electrophoretic mobility shift assay. When nuclear protein extracts containing NF-κB are treated with H2O2 in vitro, DNA binding to the κB consensus element is inhibited, although the NF-κB heterodimer remains intact. This inhibitory effect is concentration- and temperature-dependent and can be reversed by the reducing agent dithiothreitol (DTT). Co-incubation with reduced glutathione protects nuclear extracts from H2O2, while other antioxidants such as vitamin C and the chelators deferoxamine and diethyldithiocarbamate provide no such protection. The thiol blocker iodoacetate also inhibits DNA binding similar to H2O2, suggesting that protein thiols are involved. The nitric oxide generating compound diethylamine NONOate inhibits the binding of NF-κB to DNA in vitro. This DNA binding inhibition may also be due to an interaction with protein thiols, since it is also reversible with DTT. Thus, although H2O2 and NO(•) activate NF-κB in vivo, they inhibit DNA binding in a cell-free system. This paradox suggests the involvement of other factors in the activation of NF-κB mediated transcription. A better understanding of this process will aid in an understanding of the pathogenesis of acquired immune deficiency syndrome (AIDS).