Absence Of T3 Research Articles

Auto-regulation of thyroid hormone receptor genes during metamorphosis: roles in apoptosis and cell proliferation.

Amphibian metamorphosis is an excellent model system for studying postembryonic development in vertebrates. It involves specific degeneration of larval cells through programmed cell death with apoptotic morphology and selective proliferation and differentiation of adult cell types. Thyroid hormone (T3) plays a causative role in this process and the effects of T3 is presumed to be mediated by T3 receptors (TRs). Studies in other systems have suggested that TRs function as heterodimers formed with RXRs (9-cis retinoic acid receptors) and require the presence of various cofactor in transcriptional activation and repression in the presence and absence of T3, respectively. The T3-induced transcriptional activation leads to chromatin remodeling which may involve some of the cofactors. Recent investigation on receptor expression has implicated a role of TRs in T3-induced apoptosis in larval tissues and proliferation of adult cell types. Functional studies in tadpoles and developing embryos have provide strong support for such a role and further demonstrate the importance of RXR in mediating the effect of T3.

Zinc chelation enhances thyroid hormone induction of growth hormone mRNA in GH3 cells

The effects of restriction and addition of zinc on thyroid hormone responsiveness of the growth hormone gene were investigated in GH3, rat pituitary tumor cells. Addition of diethylenetriaminepenta-acetic acid (DTPA), a membrane-impermeable chelator, resulted in up to 10-fold increases in GH mRNA in the presence of 10 nM T3, with half-maximal induction at 50 μM DTPA. Only minor effects were seen in the absence of T3. Addition of zinc inhibited the stimulatory effect of DTPA in a dose-dependent manner. Equimolar concentrations of other divalent cations could not substitute for zinc, though inhibitions of the DTPA effect were observed at higher concentrations. In the absence of DTPA, exogenous zinc (100 μM) inhibited T3-induced GH mRNA by ≈33%. Addition of DTPA or zinc did not affect T3 binding to its nuclear receptor. DTPA also enhanced the stimulatory effect of dexamethasone on GH mRNA. The results demonstrate that restricted zinc availability positively affects T3 induction of the GH gene in GH3 cells.

An inhibitory region of the DNA-binding domain of thyroid hormone receptor blocks hormone-dependent transactivation.

We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor beta ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-beta (TRbeta), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRbeta derepressed transcription to basal level. Using this system and a battery of TRbeta mutants, we found that TRbeta/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRbeta ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRbeta DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRbeta from achieving transcriptional activation above basal level in this chimeric receptor system.

L-thyroxine directly affects expression of thyroid hormone-sensitive genes: regulatory effect of RXR β

l-thyroxine (T4) has been considered mainly a prohormone, the hormonal action of which is related to its conversion to 3,5,3′-triiodothyronine (T3) in peripheral tissues. In this study we investigated in transient transfection assays whether T4 might directly affect the expression of thyroid hormone (TH) sensitive genes. The reporter construct ME-TRE-TK-CAT or TSH-TRE-TK-CAT containing the nucleotide sequence of the TH response element (TRE) of either malic enzyme (ME) or TSH β genes, was transfected with either TH receptor (TR) α alone or in combination with retinoid X receptor (RXR) β into NIH3T3 cells. Addition of 100 nM T4 to the culture medium in the presence of TR α increased the basal level of ME-TRE-TK-CAT expression by 4.5-fold. T4 action was due to a direct interaction with TR α and not to its conversion to T3, since T4 effect persisted in the presence of 5′-deiodinase inhibitors (propylthiouracil, iopanoic acid) effectively preventing T3 generation, as assessed by the absence of T3 by HPLC in the cellular extracts of transfected cells. In a dose-response study half-maximal stimulation by T4 was achieved at a concentration of 100 nM, whereas 50% of maximal induction was produced by 1 nM T3 and 6 nM triiodothyroacetic acid (TRIAC). Coexpression of RXR β greatly enhanced the transcriptional activity of the ME-TRE-TK-CAT gene when either T3, T4 or TRIAC was added to the culture medium of NIH3T3 cells, but established a hormonal hierarchy in the reporter activation different than that observed in the presence of TR α alone (TRIAC>T3≥T4, instead of T3>TRIAC>T4). T4 at a concentration of 100 nM could activate the TH/TR-dependent down-regulation mediated by the negative TSH-TRE, although at a lower level than that obtained with similar concentrations of T3 (35 and 55% inhibition, respectively). Our results demonstrate that, in addition to the action mediated through its monodeiodination to T3, T4 exerts a direct effect on genes that are either positively or negatively regulated by TH. Moreover, RXR β, forming heterodimers with TRs, appeared to exert a central role in modulating the sensitivity of TH-responsive genes to different iodothyronines.

Both Thyroid Hormone and 9-cis Retinoic Acid Receptors Are Required To Efficiently Mediate the Effects of Thyroid Hormone on Embryonic Development and Specific Gene Regulation in Xenopus laevis

Tissue culture transfection and in vitro biochemical studies have suggested that heterodimers of thyroid hormone receptors (TRs) and 9-cis retinoic acid receptors (RXRs) are the likely in vivo complexes that mediate the biological effects of thyroid hormone, 3,5,3'-triiodothyronine (T3). However, direct in vivo evidence for such a hypothesis has been lacking. We have previously reported a close correlation between the coordinated expression of TR and RXR genes and tissue-dependent temporal regulation of organ transformations during Xenopus laevis metamorphosis. By introducing TRs and RXRs either individually or together into developing Xenopus embryos, we demonstrate here that RXRs are critical for the developmental function of TRs. Precocious expression of TRs and RXRs together but not individually leads to drastic, distinct embryonic abnormalities, depending upon the presence or absence of T3, and these developmental effects require the same receptor domains as those required for transcriptional regulation by TR-RXR heterodimers. More importantly, the overexpressed TR-RXR heterodimers faithfully regulate endogenous T3 response genes that are normally regulated by T3 only during metamorphosis. That is, they repress the genes in the absence of T3 and activate them in the presence of the hormone. On the other hand, the receptors have no effect on a retinoic acid (RA) response gene. Thus, RA- and T3 receptor-mediated teratogenic effects in Xenopus embryos occur through distinct molecular pathways, even though the resulting phenotypes have similarities.

Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix.

The purpose of our studies was to identify factors which regulate the composition of airway secretions produced by normal human tracheobronchial epithelial (NHTBE) cells. Individual factors were removed from the culture media of NHTBE cells grown in air-liquid interface (ALI) cultures (which support mucociliary differentiation) and the effects on mucin, lysozyme (LZ), and secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined. Deletion of hydrocortisone, epinephrine, transferrin, or gentamycin-amphotericin from the media had no reproducible effects; deletion of insulin was incompatible with culture growth. We identified 3 factors, namely retinoic acid (RA), triiodothyronine (T3) and collagen gel substratum, which had a major impact on the profile of NHTBE secretions. Removal of RA from the media caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes MUC2 and MUC5AC.LZ and SLPI secretions were increased in these cultures. Paradoxically LZ mRNA was decreased, while SLPI mRNA levels were increased. Removal of T3 selectively increased mucin secretion, MUC2 gene expression was not affected, but MUC5AC mRNA levels reproducibly increased, suggesting that the expression of these two mucin genes is differentially regulated. LZ and SLPI secretion levels were not significantly affected by deletion of T3 from the culture media; however, LZ mRNA levels were increased in the absence of T3 while SLPI transcript levels were not affected. Omission of the attachment substratum, type I collagen gel, resulted in significant increases in all 3 secretory products. MUC2 and MUC5AC steady state mRNA levels were not consistently affected. In contrast LZ and SLPI gene expression were reproducibly increased. Our studies show that individual factors in the epithelial environment can regulate expression of specific secretory cell gene products in a highly selective manner.

Molecular cloning and functional characterization of the human cytosolic malic enzyme promoter: thyroid hormone responsiveness.

We report the structural and functional features of the 5'-flanking region of the human cytosolic malic enzyme (ME) gene. A 2.2-kb subclone, comprising 1.5 kb upstream of the translation initiation codon, the first exon, and 0.7 kb of flanking intronic region, was sequenced and mapped to chromosome 6. The proximal promoter region is rich in G + C, lacks TATA or CCAAT boxes, and shows multiple transcription start sites, the major one 106 nucleotides upstream the ATG codon. Sequences -59/-13 and -137/-103 conferred maximal promoter activity. Deletional analysis revealed the presence of two regions positively regulated by 3,5,3'-triiodo-L-thyronine (T3). The proximal region confers the strongest T3 inducibility to the human ME as well as to a heterologous promoter. Thyroid hormone receptor beta (TRbeta) binds to an inverted palindromic T3 response element (TRE) at position -105/-87 in a manner that is prevented by T3. Nuclear extracts or in vitro-translated retinoid acid receptor alpha (RXR alpha) shifted the TRbeta retarded band to slower-mobility complexes, which are unaffected by T3. In the absence of T3, overexpression of TRbeta repressed the ME promoter activity, most probably, through binding of TRbeta homodimers to the TRE. Thus, T3 seems to control ME transcription by inducing the dissociation of TRbeta homodimers and the functional activation of liganded heterodimers.

Critical role of glutamine 252 in the hormone-dependent transcriptional activity of the thyroid hormone beta1 nuclear receptor.

To understand the molecular basis of the ligand-dependent transcriptional activity of thyroid hormone nuclear receptors (TRs), we investigated the effect of mutation of glutamine 252 (Q252) on the function of human TR subtype beta1 (wTRbeta1). Q252 is conserved in TRs in all species and is located in a region of the hormone binding domain that has been shown to undergo 3,3',5-triiodo-L-thyronine (T3) induced conformational changes. Q252 was mutated to Gly (Q252G) or Asn (Q252N) and their immunoreactivity, hormone, and DNA binding activities were characterized. Mutants Q252G and Q252N bound to T3 with similar affinity as the wTRbeta1. However, they failed to interact with a monoclonal anti-wTRbeta1 antibody whose epitope is located in the region of amino acids 248-256, suggesting that mutation of Q252 to Gly or Asn resulted in local structural alteration in TRbeta1. In addition, mutation of Glu to Gly or Asn led to increases in their binding to the thyroid hormone response elements (TREs) as homodimers and as heterodimers with the retinoid X receptor. Mutants Q252G and Q252N were more effective as repressors in the absence of T3, while both had a 1.5-2-fold higher T3-dependent transcriptional activity mediated by three TREs than the wTRbeta1. The increases in the transcriptional activity were not due to an increase in the expression of the mutant receptor proteins because the in vivo expression level of the mutant receptor proteins was identical to that of the wTRbeta1. Our data indicate that the T3-dependent transcriptional activity is not entirely dependent on the T3 binding activity of the receptor. The interplay of ligand and DNA binding domains plays a pivotal role in the transcriptional activity of the TRs.

Transient stimulation of myelin basic protein gene expression in differentiating cultured oligodendrocytes: a model for 3,5,3'-triiodothyronine-induced brain development.

We compared the regulation of myelin basic protein (MBP) gene expression by T3 in differentiating oligodendrocytes in culture with that previously observed by us in the neonatal rat brain. As in intact brain, expression of the T3R alpha gene preceded that of the T3R beta gene. Although the absence of T3 retarded the rate of accumulation of MBP messenger RNA, the level ultimately attained was similar to that reached in the presence of T3. This relationship mirrored the pattern observed in the neonatal brain. Transient transfection experiments showed that T3 regulates MBP expression at the transcriptional level, but only for a limited period during differentiation. These observations imply that the early rise of MBP messenger RNA is T3 dependent, whereas the terminal levels are maintained independently of T3. Both the T3-dependent and, surprisingly, the T3-independent expression of MBP require the presence of an intact T3 response element. T3 receptor may regulate MBP expression in a ligand-independent manner, or a nuclear factor other than T3 receptor may bind to the T3 response element of MBP to regulate terminal gene expression. These findings support the use of differentiating oligodendrocytes as a model of T3-induced brain development.

Triiodothyronine Stimulates and Glucagon Inhibits Transcription of the Acetyl-CoA Carboxylase Gene in Chick Embryo Hepatocytes: Glucose and Insulin Amplify the Effect of Triiodothyronine

The mechanisms by which triiodothyronine (T3), glucose, insulin, and glucagon regulate acetyl-CoA carboxylase expression in primary cultures of chick embryo hepatocytes have been investigated. Incubating hepatocytes with T3 in the absence of glucose caused a fourfold increase in acetyl-CoA carboxylase activity. Addition of glucose (20 mm) enhanced the T3-induced increase in acetyl-CoA carboxylase activity by threefold but had no effect on enzyme activity in the absence of T3. The effects of T3 and glucose on acetyl-CoA carboxylase activity were accompanied by similar changes in acetyl-CoA carboxylase mRNA levels, indicating that regulation occurred at a pretranslational step. Xylitol mimicked the effect of glucose on acetyl-CoA carboxylase mRNA abundance, suggesting that an intermediate(s) of the nonoxidative branch of the pentose phosphate pathway may be involved in mediating this response. Insulin accelerated the accumulation of acetyl-CoA carboxylase mRNA abundance caused by T3 and glucose but had no effect on steady-state levels of acetyl-CoA carboxylase mRNA in the absence or presence of T3. Glucagon caused a 65% decrease in the accumulation of acetyl-CoA carboxylase mRNA in hepatocytes incubated with T3 and glucose. The effects of T3, glucose, insulin, and glucagon on the abundance of acetyl-CoA carboxylase mRNA were accounted for by changes in the transcription rate of the acetyl-CoA carboxylase gene. These data support the hypothesis that T3, glucose, insulin, and glucagon play a role in mediating the effects of nutritional manipulation on transcription of acetyl-CoA carboxylase in liver.

Transcription of the rat sarcoplasmic reticulum Ca2+ adenosine triphosphatase gene is increased by 3,5,3'-triiodothyronine receptor isoform-specific interactions with the myocyte-specific enhancer factor-2a.

Thyroid hormone (T3) increases the transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) gene (SERCA 2) through three thyroid hormone response elements. The existence of repetitive cis elements with different configurations is likely to serve specific functions such as interactions with nuclear transcription factors. In addition, the presence of different T3 receptor isoforms (T3Rs) may contribute to another level of complexity in providing specificity for T3 action. In this study, we investigated T3R alpha 1-vs. T3R beta 1-specific interactions with the myocyte enhancer-specific factor-2 (MEF-2) on the expression of the SERCA 2 gene in transient transfection assays in embryonal heart-derived H9c2 cells. MEF-2a in combination with either T3R alpha 1 or T3R beta 1 isoforms resulted in a 2.5-fold increase in SERCA 2 transgene expression in the absence of T3. Addition of T3 did not induce any further increase in SERCA 2 expression when T3R alpha 1 and MEF-2a expression vectors were cotransfected. In contrast, in the presence of T3R beta 1 and MEF-2, the addition of T3 increased chlorampenicol acetyltransferase activity by an additional 2.2-fold to a total 5.5-fold increase. The interaction between MEF-2a and T3R is transcription factor specific because another factor that binds to MEF-2 consensus sites (heart factor 1b) was not able to interact with T3R. In addition, MEF-2a failed to interact with other nuclear factors (cAMP response element-binding protein and Egr-1) that stimulate SERCA 2 gene transcription. In addition, we found that a single homologous thyroid hormone response element is not able to mediate the interactions between MEF-2a and T3Rs to increase SERCA 2 gene transcription. Our findings point to T3R isoform-specific interactions with a cell type-specific transcription factor (MEF-2) in the regulation of SERCA 2 gene expression.

Regulation of the human trh (htrh) gene by human thyroid hormone receptor β1 (hTRβ1) mutants

TRH is negatively regulated by T3 both in the hypothalamic paraventricular nucleus and transient transfection models. Mutations in hTRβ1 genes are associated with the syndrome of generalized resistance to thyroid hormone. To investigate potential effects of mutant TRs on T3 regulation of the hTRH gene, transient gene expression assays were performed in human neuroblastoma (HTB-11) cells with an hTRH promoter-luciferase construct, wild type (WT) hTRβ1, and three qualitatively distinct hTRβ1 mutant forms (ED, OK and PV). In the presence of T3 (10−9 M), liganded WT-hTRβ1 inhibited hTRH promoter activity significantly (40%). Co-transfection of each of the two mutants (ED and OK) achieved similar levels of inhibition only at 10 to 100 fold increased T3 concentrations. Of interest, a 10x excess of mutant ED or OK could also exert dominant negative effects upon WT hTRβ1-T3 mediated inhibitory actions on the hTRH promoter. In contrast, mutant TR-PV exerted neither inhibitory nor dominant negative effects at even higher concentrations of T3. Moreover, all three unliganded mutant forms stimulated TRH promoter activity significantly in the absence of T3, despite their different mutations in the ligand-binding domain (LBD). These data demonstrate that thyroid hormone resistance at the level of TRH gene regulation, due to reduced inhibitory actions of mutant TR-T3 complexes, as well as dominant negative effects upon WT hTRβ1 mediated inhibition, likely contribute to elevated TSH values observed in the syndrome of thyroid hormone resistance.

Fiber-specific regulation of Ca(2+)-ATPase isoform expression by thyroid hormone in rat skeletal muscle.

We studied the effect of thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) on the expression of sarcoplasmic reticulum (SR) fast- and slow-type Ca(2+)-ATPase isoforms, SERCA1 and SERCA2a, respectively, and total SR Ca(2+)-ATPase activity in rat skeletal muscle. Cross sections and homogenates of soleus and extensor digitorum longus muscles from hypo-, eu-, and hyperthyroid rats were examined, and expression of Ca(2+)-ATPase isoforms in individual fibers was compared with expression of fast (MHC II) and slow (MHC I) myosin heavy chain isoforms. In both muscles, T3 induced a coordinated and full conversion to a fast-twitch phenotype in one-half of the fibers that were slow twitch in the absence of T3. The conversion was partial in the other one-half of the fibers, giving rise to a mixed phenotype. The stimulation by T3 of total SERCA expression in all fibers was reflected by increased SR Ca(2+)-ATPase activity. The time course of the T3-induced changes of SERCA isoform expression was examined 1-14 days after the start of daily T3 treatment of euthyroid rats. SERCA1 expression was stimulated by T3 at a pretranslational level in all fibers. SERCA2a mRNA expression was transiently stimulated and disappeared in a subset of fibers. In these fibers SR Ca(2+)-ATPase activity was high because of high SERCA1 protein levels. These data suggest that the ultimate downregulation of SERCA2a expression, which is always associated with high SR Ca(2+)-ATPase activities, occurs at a pretranslational level.

Differential Effects of 3,5,3′-Triiodothyronine on Control andmdxMyoblasts and Fibroblasts: Analysis by Flow Cytometry

The possibility of differential effects of triiodothyronine (T3) treatmentin vivoon myoblast and fibroblast cell proliferation was examined in control andmdxmuscle cultures. Cell isolates were purified in a Percoll gradient, sorted by flow cytometry (light scatter), and characterized as myoblasts and fibroblasts using anti-skeletal muscle myosin fluorescence. The two cell types were grown separately or remixed (1:1). Cultures were incubated with or without T3 (10−9M) for 19 h. Cells were either exposed to [3H]thymidine for 1 h and DNA prepared for scintillation counts or stained with propidium iodide for cell cycle analysis by flow cytometry. Overall [3H]thymidine uptake per cell was greater inmdxthan control cells (mainly fibroblasts and mixed cells) and was decreased by T3 only in myoblast and mixed cultures. Cell cycle data showed that the effects of T3 originated primarily at the G0/G1phase. There were moremdxthan control myoblasts at G0/G1without T3. After T3 treatment, more control fibroblasts than myoblasts were at G0/G1, but moremdxmyoblasts than fibroblasts were at G0/G1. In the absence of T3, there were also fewermdxthan control myoblasts in S. After T3, only the proportion ofmdxmyoblasts in S phase was reduced. Results are consistent with distinct T3 effects on muscle regenerationin vivoand support the hypothesis that cycling and proliferation ofmdxand control myoblasts are differentially modulated by T3. As control andmdxfibroblasts also showed distinct responses to T3, muscle regeneration likely occurs by a complex regulation of gene expression endogenous to specific cell types as well as interactions between cells of different lineage.

Thyroid Hormone Activation of Transcription Is Potentiated by Activators of cAMP-dependent Protein Kinase

We characterized the cross-talk between activators of protein kinase A (PKA) and thyroid hormone (T3) in T3 receptor (TR)-mediated transcription. U937 cells were cotransfected with a plasmid expressing the TR and a reporter plasmid containing a T3 response element (TRE) oriented either as a direct repeat or as a palindrome upstream of the thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene. T3 activated transcription by 10-fold. T3 response was potentiated 2.5-3-fold by activators of PKA, but an activator of protein kinase C or of guanylate kinase was ineffective. In the absence of T3, activators of PKA had no effect on transcription. TR heterodimerization with the retinoid X receptor may facilitate T3/PKA cross-talk because coexpression of the retinoid X receptor potentiated cross-talk. Synergy was not observed in JEG-3, F9, CV-1, HeLa, L929, and HTC cells, indicating that it may require cell-specific factors. Synergy required the DNA- and ligand-binding domains, but not the amino-terminal domain, indicating that T3- and TRE-induced conformational changes on the TR are essential for cross-talk. PKA phosphorylated the TR in vitro, suggesting that, like other nuclear receptors, the TR is a target for PKA. These results imply that PKA cross-talks with T3 at the level of the TRE-bound TR, enhancing its transcriptional activity in a cell-specific manner.

In serum-free culture thyroid hormones can induce full expression of chondrocyte hypertrophy leading to matrix calcification.

A serum-free culture system has been developed to examine the biologic factors involved in the regulation of cellular maturation, extracellular matrix assembly, and calcification in the physis of the bovine fetal growth plate. Isolated prehypertrophic chondrocytes in high density culture undergo a process of cellular maturation whereby full expression of the hypertrophic phenotype is characterized first by type X collagen synthesis followed by matrix calcification. Using this culture system, we compared the capacity of tri-iodothyronine (T3) with thyroxine (T4) to stimulate expression of the hypertrophic phenotype and matrix calcification in three (B, C, and D) maturationally distinct prehypertrophic chondrocyte subpopulations. The B cell subpopulation was the most mature followed by C and D subpopulations in order of decreasing maturity. Comparisons were made to cultures in fetal calf serum (FCS). In Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, and selenium, both hormones (T3/T4) separately induced, in a dose-dependent manner, chondrocyte maturation to the hypertrophic phenotype characterized by increased type X collagen mRNA and induction of protein synthesis of this molecule, together with increased alkaline phosphatase activity, and eventually calcification of the extracellular matrix. Such cellular maturation to the hypertrophic phenotype was not observed in the absence of T3 or T4 with subpopulations C and D. Only in older fetuses (> 210 days) was this observed and then only in the B subpopulation. Furthermore, T3 was at least 50-fold more potent than T4. The effects of T3 were most pronounced with the most immature cells (subpopulations C and D) where, in the case of the subpopulation C, in contrast to 0.5 nM T3 50 nM T4 was unable to induce expression of the hypertrophic phenotype. Alkaline phosphatase activity was also increased in the C cell subpopulation treated with 1 nM T3 (35.5 U/micrograms of DNA) over that supplemented with 50 nM T4 (7.8 U/micrograms of DNA). Furthermore, matrix calcification, measured by the incorporation of 45Ca2+ into the cell layer, always occurred earlier in cells cultured with T3 compared with T4. Cellular maturation to the hypertrophic phenotype was not accompanied by significant changes in DNA content; this ordinarily increases during culture in the presence of serum. Compared with cells cultured in the presence of serum, either thyroid hormone more potently induced cellular maturation. This study demonstrates that the most immature chondrocytes at the prehypertrophic stage are direct targets for T3 and T4 and, to a much a lesser degree, that either hormone is able to induce full chondrocyte hypertrophy from an early maturational stage leading to matrix calcification. But T3 is much more potent than T4. These studies also offer a new serum-free chemically defined medium containing T3 or T4 for the culture of defined prehypertrophic chondrocytes that supports matrix assembly, hypertrophic expression, followed by matrix calcification.

A Unique Thyroid Hormone Response Element in the Human Immunodeficiency Virus Type 1 Long Terminal Repeat That Overlaps the Sp1 Binding Sites

Long terminal repeat (LTR) of human immunodeficiency virus (HIV) type 1 is activated by thyroid hormone (T3) receptor alpha (T3R alpha) in the absence of ligand. Addition of T3 reverses this effect. This activity is mediated by a high affinity T3 response element (T3RE) within the HIV-1 LTR, termed the HIV-T3RE (bases -74 to -50), which coincides with the Sp1 element as demonstrated by mobility shift, DNaseI footprinting, and methylation interference analyses. HIV-T3RE mediates ligand-independent activation of transcription by T3R alpha when linked to a heterologous promoter. In addition, the viral transactivator Tat synergizes with T3R alpha to activate the HIV-1 LTR in the absence of T3, which is relieved in its presence. These findings have implications for the possible control of HIV-1 LTR activity by T3.

Novel location and function of a thyroid hormone response element.

We describe a novel thyroid hormone response element (TRE)-containing sequence, clone 144, isolated by immunoprecipitation of nuclear thyroid hormone receptor (TR)-DNA complexes from the rat pituitary tumor cell line GH4. These cells express several mRNAs of approximately 10 kb that hybridize to the TRE-containing genomic clone 144. These mRNAs are up-regulated at the transcriptional level in the absence of thyroid hormone (T3) and repressed in its presence. The sequence protected from DNase I digestion by TR in clone 144 contains two consensus TRE half-sites arranged as inverted palindromes. The clone 144 TRE is located in the 3' untranslated region (UTR) of several related mRNAs. A reporter construct transfected into 293 cells was responsive to TR regulation when the clone 144 TRE was inserted in the 3' UTR but not when inserted upstream of the promoter. As found for the endogenous 144 mRNAs, the 144 TRE reporter construct is activated by TR in the absence of T3, but not in its presence. Deletion analysis showed that clone 144 sequences flanking the TRE were necessary for TR-mediated regulation, suggesting that the mechanism by which TR regulates transcription through a TRE in the 3' UTR is different from that through the TREs located in the promoter region.

Coordinated Regulation of and Transcriptional Activation by Xenopus Thyroid Hormone and Retinoid X Receptors

Thyroid hormone (T3) plays a causative role in amphibian metamorphosis. This regulation is thought to be mediated by heterodimers of T3 receptors (TRs) and retinoid X receptors (RXRs). We report here that Xenopus TRs can indeed form strong heterodimers with Xenopus RXRs on the T3 response element (TRE) present in Xenopus TR beta genes. Using a T3-responsive in vivo transcription system established by introducing TRs and RXRs into Xenopus oocytes, we demonstrated that TR-RXR heterodimers repressed TR beta gene promoter in the absence of T3 and activated the promoter in the presence of the hormone. Furthermore, by analyzing the expression of TR and RXR genes, we showed that TR and RXR genes were coordinately regulated in different tissues during metamorphosis. Thus high levels of their mRNAs are present in the limb during early stages of limb development when morphogenesis occurs and in the tail toward the end of metamorphosis when it is being resorbed. Such correlations coupled with our TRE-binding and in vivo transcriptional activation experiments provide strong evidence that TRs and RXRs function together to mediate the effects of T3 during metamorphosis. These results further suggest a possible molecular basis for the temporal regulation of tissue-specific metamorphosis.

Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor.

The thyroid hormone (T3) receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes. To help elucidate the mechanisms that underlie this transcriptional regulation and other potential TR activities, we used the yeast interaction trap to isolate clones encoding proteins that specifically interact with the ligand binding domain of the rat TR beta. Several such proteins, called Trips (TR-interacting proteins), were isolated from independent selections carried out either in the presence or absence of T3. Surprisingly, all of the Trips were dependent on hormone for interaction with the TR, with some interacting only when T3 is present and others only when it is absent. Nearly all of the Trips also show similar ligand-dependent interaction with the retinoid X receptor (RXR), but none interact with the glucocorticoid receptor under any conditions. The sequences of three of the Trips predict specific functional roles: one is an apparent human homolog of a yeast transcriptional coactivator, one is a new member of a class of nonhistone chromosomal proteins, and one contains a conserved domain associated with ubiquitination of specific target proteins. Consistent with the pleiotropic effects of TR and RXR, several other Trips show significant amino acid sequence similarity with proteins involved in various regulatory pathways. The inherent transcriptional activity of the Trips was tested in yeast, and a chimeric protein consisting of a fusion of Trip4 to the bacterial LexA repressor protein is a relatively strong transcriptional activator. Similar LexA fusions to Trip9 and Trip10 had no transcriptional activity on their own but, when coexpressed with both TR and RXR, conferred T3-dependent activation to a reporter gene controlled by LexA binding sites. We suggest that this indirect T3 response provides a novel mechanism for hormonal activation of gene expression, and that studies of the Trips will provide important insights into the specific mechanisms of action of TRs and other receptors.