Higher T3 Concentrations Research Articles

Defective Release of Corepressor by Hinge Mutants of the Thyroid Hormone Receptor Found in Patients with Resistance to Thyroid Hormone

On positive thyroid hormone response elements (pTREs), thyroid hormone receptor (TR) binding to DNA in the absence of ligand (thyroid hormone, T3) decreases transcription (silencing). Silencing is due to a family of recently described nuclear corepressor proteins (NCoR and SMRT) which bind to the CoR box in the hinge region of TR. Ligand-dependent activation of TR is associated with displacement of corepressors and recruitment of coactivating proteins. Resistance to thyroid hormone (RTH) is due to mutations in the beta isoform of the thyroid hormone receptor (TR-beta). To date, three RTH mutations reportedly with near-normal T3 binding (A234T, R243Q, and R243W) have been described in or near the CoR box. To determine the mechanism of RTH caused by these mutants, the interaction of wild type (wt) and mutant TRs with the corepressor, NCoR, and the coactivator, SRC-1, was tested in gel-shift assays. As expected, NCoR bound wt TR in the absence of T3 and dissociated from TR with increasing T3 concentration. SRC-1 failed to bind wt TR in the absence of T3, but bound to TR with increasing avidity as T3 concentrations rose. At no T3 concentration did both NCoR and SRC-1 bind to wt TR, indicating that their binding to TR was mutually exclusive. Hinge mutants bound NCoR normally in the absence of T3; however, dissociation of NCoR and recruitment of SRC-1 was markedly impaired except at very high T3 concentrations. Importantly, hinge mutant TRs when complexed to DNA bound T3 poorly despite their near-normal T3 binding in solution. These binding studies correlated with functional assays showing defective transactivation of pTREs by hinge mutants except at high T3 concentrations. Thus, we describe a novel mechanism of RTH whereby TR hinge mutants selectively affect T3 binding when complexed to DNA, and prevent NCoR dissociation from TR. Our data also suggest that solution T3 binding by RTH mutants may not accurately reflect physiologically relevant T3 binding by TR when bound to DNA.

Ligand- and nuclear factor-dependent change in hydrophobicity of thyroid hormone beta1 receptor.

An aqueous two-phase partitioning assay was performed using in vitro translated human thyroid hormone beta1 receptor (TRbeta1). Wild-type TRbeta1 was less hydrophobic in the presence of both triiodothyronine (T3) and nuclear extract. This reflects a conformational change, or change in electrostatic properties, of the TRbeta1-nuclear factor complex as a result of T3 binding. Mutant TRbeta1s with reduced T3 binding affinity required a higher concentration of T3 for the shift of hydrophobicity, and a mutant without T3 binding activity did not show any shift, even in the presence of 1 mM T3. The unique mutant receptor, R243Q, has impaired transcriptional function despite virtually normal binding affinity for T3. When this mutant was examined in this assay, the shift of hydrophobicity was significantly impaired even in the presence of both nuclear extract and a high concentration of T3. Nuclear extract of COS1 cells did not affect the T3-binding affinity of R243Q. These results indicate that the R243Q mutant has impaired a ligand-dependent conformational change and interaction with nuclear factor(s). Inability of R243Q to interact normally with nuclear factor(s) may explain, in part, the molecular mechanism of discordance between ligand binding and transactivation function of this mutant.

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.

Regulation of uncoupling protein messenger ribonucleic acid and 5'-deiodinase activity by thyroid hormones in fetal brown adipose tissue.

We studied the regulation of type II 5'-deiodinase (5'D-II) activity and uncoupling protein (UCP) messenger RNA (mRNA) by thyroid hormones in fetal brown adipose tissue (BAT). Fetuses were obtained from hypothyroid pregnant rats infused with increasing doses of T4 or T3. Infusion of T4 into hypothyroid pregnant rats increased T4 and T3 concentrations and inhibited 5'D-II activity in fetal BAT. In contrast, infusion of T3 increased BAT 5'D-II activities at low, normal, or high BAT T3 concentrations. The relationship between thyroid hormone concentrations in fetal BAT and plasma showed that BAT T3 concentrations are relatively stable, increasing less than 2-fold over a wide range of circulating T4 (3-fold) or T3 (8-fold) concentrations. Most T3 in fetal BAT are locally derived from T4 and not from plasma T3. UCP mRNA expression decreased to 30% of control values in hypothyroid fetuses. UCP mRNA levels were restored to normal in parallel with BAT T3 concentrations after the infusion of either T4 or T3. UCP mRNA levels correlate well with BAT T3 concentrations. Supraphysiological doses of T4 did not further increase either BAT T3 or UCP mRNA levels. T3 might regulate basal UCP mRNA expression during fetal life.

Structural features of thyroid hormone response elements that increase susceptibility to inhibition by an RTH mutant thyroid hormone receptor.

The chicken lysozyme silencer F2 (F2) thyroid hormone response element (TRE) contains an unusual everted palindromic arrangement, has a high affinity for thyroid hormone receptor (TR) homodimers, and is especially sensitive to dominant negative inhibition by, the T3 resistance (RTH) mutant TR beta P453H. We used various TREs and TR mutations to determine the mechanisms for this sensitivity. Changing the F2 orientation from an everted palindrome to a direct repeat with a 4-bp gap (DR+4) (F2-DR) decreased the sensitivity to inhibition at high T3 concentrations, while a loss of this sensitivity occurred with a palindromic arrangement of these same half-sites. F2 contains the dinucleotide TG 5' to each consensus half-site conforming to the optimal TR-binding octamer, YRRGGTCA. A T to A change in position 1 of both F2 half-sites markedly reduced T3-induction, yet only slightly reduced TR homodimer or TR-retinoid X receptor (RXR) heterodimer binding. The TR beta ninth heptad mutation, L428R, prevents TR heterodimerization with RXR and eliminates the inhibitory effect of the P453H mutant TR on the F2-DR, but not the F2 element. Structural features of a TRE that favor strong TR binding of both TR homodimers and TR-RXR heterodimers containing the mutant TR, such as the everted palindromic conformation or the optimal TR-binding consensus octamer, enhance the sensitivity of a TRE to inhibition by the mutant TR. Thus, both half-site orientation and sequence contribute to the sensitivity of a given TRE to dominant negative inhibition by a mutant TR.

Thyroid hormone control of thermogenesis and energy balance.

The mechanisms whereby thyroid hormone increases heat production have been analyzed with emphasis in more recent developments. Thyroid hormone increases obligatory thermogenesis as a result of the stimulation of numerous metabolic pathways involved in development, remodeling, and delivery of energy to the tissues. In addition, thyroid hormone may specifically stimulate some thermogenic mechanisms selected during evolution of homeotherms (e.g., Na/K-ATPase, Ca2+ cycling in muscle). Thyroid hormone also plays an essential role in facultative thermogenesis interacting with the sympathetic nervous system (SNS) at various levels. Peripherally, thyroid hormone potentiates the effects of the SNS at the level of the adrenergic receptor and adenylyl cyclase complex as well as distal from this point. Synergistic interactions between T3 and cAMP on the regulation of gene expression have been described. Brown adipose tissue (BAT) T4-5'-deiodinase plays a central role in controlling heat production. When this enzyme is stimulated by norepinephrine in the euthyroid and hypothyroid condition, it provides high concentrations of T3 to BAT; inhibition by T4 in hyperthyroidism may limit brown fat thermogenic responses. Also, thyrotoxicosis uniquely reduces the expression of beta 3-adrenergic receptors in brown adipose tissue, and the increased obligatory thermogenesis of this condition, via afferent neural pathways, may reduce the hypothalamic stimulation of brown fat, providing additional mechanisms to limit brown adipose tissue thermogenesis in hyperthyroidism.

Tri-iodothyronine (T3) and dexamethasone interact to modulate osteoprogenitor cell differentiation in fetal rat calvaria cell cultures

We investigated the role of 3,5,3'-tri-iodothyronine (T3) in regulating differentiation of osteoprogenitor cells and also studied the effects of the glucocorticoid hormone dexamethasone (Dex) on the T3-induced effects on osteoprogenitor populations. This was done by determining the effects of either hormone alone, or of combinations of the two hormones, on the number of bone nodules formed in long-term cultures of rat calvaria cells. In this system, Dex has been shown to increase bone nodule formation, the maximal effective dose being 10 nM (Bellows et al. Endocrinology 121: 1985-1992; 1987). In standard culture medium containing 15% fetal bovine serum FBS), low concentrations of T3 (0.001-0.1 nM) had no effect on the number of bone nodules, while higher concentrations of 1-100 nM inhibited. However, in culture medium containing 10 nM Dex, the lower concentrations of T3 markedly increased the number of nodules. Short term pulse experiments with these low concentrations of T3 in the presence of Dex indicated that stimulation of nodule formation occurred only when T3 was present prior to confluency. Higher concentrations of T3 (1-100 nM) decreased nodule number whether or not Dex was added. We then cultured cells in medium containing FBS from which T3 and T4 were removed by treatment with AG-1 chi-10 resin. In both + or - Dex conditions, bone nodule formulation was increased 1.5 to 2-fold in T3, T4-depleted medium when compared with cultures maintained in standard culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotrophin-releasing hormone (TRH) and lung maturation.

Clinical trials of thyrotrophin-releasing hormone (TRH) in conjunction with antepartum glucocorticoid treatment in the prevention of respiratory distress syndrome is based on experimental evidence that fetal lung maturation is accelerated by exposure to raised concentrations of triiodothyronine (T3) in fetal plasma. Studies of fetal rat and rabbit lung in vitro show an inconsistent increase in surfactant synthesis in response to T3 and potentiation of the response to corticosteroid. Experiments with fetal rodents in vivo are difficult to interpret because of confounding effects of the procedures and the responses to T3 are variable. In fetal sheep, very high concentrations of T3 are without effect on lung maturation. These observations suggest that the action of TRH on the lung may be mediated at least in part by one of the numerous, non-hormonal pathways known to be stimulated by TRH, particularly the autonomic nervous system. Experiments in rats and sheep lend support to this possibility. It is concluded that available evidence is inadequate to determine the mechanism of action of TRH.

Roles of v-erbA homodimers and heterodimers in mediating dominant negative activity by v-erbA.

v-erbA, a viral oncogenic homolog of thyroid hormone receptor (TR), blocks the effect of T3 in TR-mediated transcription. The mechanism(s) for this dominant negative effect by v-erbA on TRs is unknown but may involve competition between v-erbA and TR-containing complexes for binding to thyroid hormone response elements (TREs) and/or protein-protein interactions between v-erbA and TR. To investigate these potential mechanisms, we used the electrophoretic mobility shift assay to compare in vitro translated v-erbA and TR alpha binding to two TREs-chick lysozyme TRE (F2) and direct repeat TRE (DR4). v-erbA bound as a homodimer to these TREs, whereas TR alpha bound as a homodimer and monomer. T3 decreased TR alpha homodimer binding to the TREs as we reported previously; however, surprisingly, high concentrations of T3 (10(-6) M) also decreased v-erbA homodimer binding to the TREs. Additionally, v-erbA formed heterodimers with nuclear proteins such as retinoid X receptor and T3 receptor auxiliary protein as well as with TR alpha. These dimers remained bound to DNA in the presence of T3. Finally, v-erbA could not mediate ligand-dependent transcriptional activation even at 10(-6) M T3 but could block ligand-dependent TR-mediated transactivation in co-transfection experiments. v-erbA also exhibited differential dominant negative activity on F2 and DR4 suggesting that half-site sequence and/or orientation may influence v-erbA-dominant negative activity. In sum, there are multiple v-erbA complexes that bind to TREs in the presence of T3, which all may contribute to v-erbA's dominant negative effect on TR-mediated transcription by competing with TR-containing complexes for binding to TREs.

Control of intracellular creatine concentration in a mouse myoblast cell line.

The muscle cell creatine concentration is an important bioenergetic parameter. Pathological alterations in muscle [creatine] have been found in e.g. muscular dystrophy, and dietary creatine supplementation has been shown to raise the muscle [creatine] and increase the strength of maximal contraction [ l ] . We studied factors influencing intracellular [creatine] in the mouse muscle cell line G8. G8 cells (passage 8-16) were cultured to confluence in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 10% horse serum and 2mM glutamine. Myoblast cells were also fused to myotubes using DMEM containing 1% fetal calf serum and 1% horse serum. Total intracellular [creatine] (i.e. creatine plus phosphocreatine) was measured in NaOH cell extracts using a ninhydrin based fluorimetric technique, and expressed relative to total cell protein determined by the Lowry method. In addition, cell water volume was determined by incubating the cells with Vitiated methylglucose for 90 minutes. In 48h incubations at altered creatine concentrations, intracellular [creatine] was relatively independent of extracellular [creatine], the relationship between them being steepest over the physiological range of extracellular [creatine] (Figure 1). This would be partly consistent with a simple model in which intracellular [creatine] is determined by the balance between active carrier-mediated influx and passive efflux [2]. In accordance with this, Na-creatine cotransport has been described in the rat muscle cell line L6 [3]. However, this model would predict that complete creatine depletion should be possible during prolonged incubation without creatine. In fact, cellular (creatine] decreased slowly by only 30% over 6 days (Figure 2). This suggests either that passive efflux permeability is very low, or that a large component of total cell creatine is not available for efflux under these conditions. Furthermore, the effects of creatine-free incubation may be complicated by an upregulation in the synthesis of Na-creatine carrier protein, such as is seen in L6 [3]; this should reduce further the dependence of cell [creatine] on extracellular [creatine] [2]. Following differentiation of myotubes to myoblasts the creatine content of the cells (expressed per microgram of cell protein) increased by 50-100%. However, as the cell water content of myoblasts was approximately 1.9 pllmg cell protein, while that of the fused cells was approximately 3.9 pl/mg cell protein, it is possible to interpret this as an attempt to maintain myoblast [creatine] levels in cytosolic water. In the hyperthyroid heart. both active uptake and passive efflux of creatine are enhanced [4]. We exposed myoblasts to triiodothyronine (T3) in the presence of 0.5mM creatine simultaneously for 48 h. As Figure 3 shows, this increased cellular [creatine] by up to 200%. suggesting that active creatine uptake was increased. Interestingly, it has been shown that thyroid hormones increase the amount and activity of the Na,K-ATPase in rat myotubes and make the membrane potential more negative [5]. The effect on creatine uptake that we describe here may be related to this in several ways: first, an increased negative membrane potential would be expected to enhance the (presumably) electrogenic cotransport of neutral creatine and Na cations; second, Na/K pump-mediated local increases in cytosolic [Na] might enhance Na-creatine cotransport; thirdly, a primary effect on Na-creatine cotransport might tend to increase cell [Na] and so contribute to a secondary increase in the Na,KATPase. The decreasing effect at higher T3 concentration is consistent with an decreased stability in the membrane leading to increased passive efflux.

New insights on the mechanism(s) of the dominant negative effect of mutant thyroid hormone receptor in generalized resistance to thyroid hormone.

Generalized resistance to thyroid hormone (GRTH) is a syndrome of hyposensitivity to triiodothyronine (T3) that displays autosomal dominant inheritance. The genetic defect commonly lies in the ligand-binding domain of one of the TR beta alleles. Since there are two major thyroid hormone receptor (TR) isoforms, TR alpha and TR beta, it is not known how the mutant receptor mediates a dominant negative effect. Previously, we showed that T3 caused dissociation of TR homodimers and TR alpha/TR beta dimers from several thyroid hormone response elements (TREs). Hence, we used the electrophoretic mobility shift assay to compare the effect of T3 on the DNA binding of mutant TR beta-1 (Mf-1) from a kindred with GRTH with normal TR beta. Mf-1 bound better as a homodimer than TR beta, but dissociated from DNA only at high T3 concentrations. Both receptors heterodimerized with nuclear auxiliary proteins. They also dimerized with TR alpha and with each other. Surprisingly, T3 disrupted the DNA binding of the Mf-1/TR isoform dimers. Thus, mechanisms for the dominant negative effect by mutant TRs likely involve either increased binding to TREs by mutant homodimers that cannot bind T3 (hence cannot dissociate from DNA) and/or the formation of inactive mutant TR/nuclear protein heterodimers.

Adenosine 3',5'-monophosphate and thyroid hormone control of uncoupling protein messenger ribonucleic acid in freshly dispersed brown adipocytes

We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.

Adenosine 3',5'-monophosphate and thyroid hormone control of uncoupling protein messenger ribonucleic acid in freshly dispersed brown adipocytes.

We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.

Thyroid hormone responsiveness of the L1210 murine leukemia cell line.

The presence of saturable and high affinity 3,5,3'-triiodothyronine (T3) binding sites was demonstrated in L1210 murine leukemia cell nuclei. Scatchard analysis revealed one class of receptors for T3 with Ka = 2.187 x 10(9) l/mol and a maximum binding capacity (Bmax) of 3.96 fmol/10(6) cells. The effects of T3 on protein phosphorylation and growth rate of L1210 cells were investigated in a medium containing T3-depleted fetal calf serum. T3 was observed to be effective in enhancing protein phosphorylation (153.06% +/- 5.99 SD) compared to cells grown in the absence of T3 (81.49% +/- 13.50 SD). Moreover, in the presence of high T3 concentration (11.15 nmol/l) T3 was found to significantly increase the cell growth rate. In addition, the T3 receptor-associated alterations during the cell cycle, as measured by flow cytometry, suggest that the presence of T3 receptors becomes evident during the late G1 phase of the cell cycle, and T3 receptor numbers increase during the S phase. These results suggest that in in vitro conditions representing high T3 concentration, the number of L1210 leukemia cells may be increased by T3 via nuclear receptors. The L1210 leukemia cell line may serve as a convenient tool for in vitro studies of nuclear receptors and/or mechanism of action of T3. The binding affinity of T3 receptors is similar to that found in rat hepatocytes or human lymphocytes.

Variable transcriptional activity and ligand binding of mutant beta 1 3,5,3'-triiodothyronine receptors from four families with generalized resistance to thyroid hormone.

Mutations in the gene encoding the human beta 1 T3 receptor (hTR beta 1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR beta 1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T3 affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T3 affinity required 5- and 15-fold higher T3 concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTR beta 1s were able to inhibit the function of transfected normal hTR beta 1 and endogenous retinoic acid receptor in activating a palindromic positive T3 response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T3 concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTR beta 1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTR beta 1 was assessed by immunocytochemistry. The hTR beta 1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells. These data suggest that different mutations in the ligand-binding domain of the human hTR beta 1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T3 receptor dimers.

Triiodothyronine stimulates growth of peripheral blood mononuclear cells in serum-free cultures in uremic patients.

The effect of triiodothyronine (T3) on the responses to mitogens and on the production of prostaglandin E2 and interleukin 2 were studied in serum-free cultures of peripheral blood mononuclear cells (PBMC) in 20 patients undergoing hemodialysis and in 30 control subjects. T3 increased the growth of PMBC induced by phytohemagglutinin and pokeweed mitogen in both groups. PBMC reached growth maximum at 0.5 nM T3 when stimulated by phytohemagglutinin in both groups. At higher concentrations of T3 the effect declined in the control group, but the response of uremic PBMC was constant. The response to T3 of pokeweed mitogen stimulated PBMC was lower in the uremic patients. The production of prostaglandin E2 by PBMC was higher in the uremic patients than in the controls. T3 had no effect on prostaglandin E2 production. Indomethacin alone and in combination with T3 had a stimulatory effect on cell growth in the patient group. T3 had no effect on the release of interleukin 2 by PBMC. An additive effect of interleukin 2 and T3 was observed in cultures stimulated by suboptimal concentrations of the mitogens. In conclusion, the impaired growth of PBMC in serum-free cultures from uremic patients was enhanced, however, not normalized, by external addition of T3, inhibition of prostaglandin E2 synthesis, and addition of interleukin 2.

The Role of Thyroid Hormone in the Regulation of Hepatic Carbamyl Phosphate Synthetase Activity inRana catesbeiana*

Both spontaneous and thyroid hormone (TH)-induced metamorphosis of Rana catesbeiana are accompanied by a marked increase in the activity of the urea cycle enzyme carbamyl phosphate synthetase (CPS). The increase induced by exogenous TH is de novo synthesis of enzyme and appears to be secondary to an increase in the CPS mRNA level resulting from the elevated plasma TH. Since endogenous TH levels rise sharply during spontaneous metamorphosis, a similar sequence of events would be anticipated. However, after midclimax, CPS activity continues to increase, while plasma TH levels steadily decline, suggesting that other factors are involved. To obtain insight into this problem, the changes in CPS mRNA level during spontaneous development were determined using a mammalian CPS cDNA probe and correlated with changes in CPS activity and plasma T3 concentration. CPS mRNA level and CPS activity were barely detectable until midprometamorphosis, but both increased rapidly during the latter half of this phase. CPS activity continued to rise, reaching a maximum in the adult frog. The CPS mRNA level, however, was highest during the first half of climax, but declined after midclimax and was relatively low in the adult frog. Studies were also performed in which the rise and fall in the plasma T3 concentration typical of metamorphic climax were induced by exposure of premetamorphic tadpoles to T3, followed by its withdrawal. Both CPS activity and CPS mRNA level were induced by T3, but when plasma T3 levels fell after removal of the exogenous T3, CPS mRNA level, but not CPS activity, also decreased. Additional studies indicated that the TH-induced increase in CPS mRNA was evident within 24 h, could be prevented by simultaneous injection of actinomycin-D, and could not be induced in tadpoles undergoing climax; in this phase the T3 receptors are fully occupied with endogenous TH. When premetamorphic tadpoles were immersed in T3-containing water (0-500 nM) for 6 days, CPS mRNA, CPS activity, and plasma T3 concentration increased in parallel, reaching a maximum at 50 nM. At 50 nM T3, the plasma T3 level was sufficient to saturate the receptors, and no additional increase in CPS mRNA level or CPS activity was obtained at higher concentrations of T3. These studies indicate that the CPS mRNA level during spontaneous development correlates with the plasma T3 concentration and suggest that it is a function of T3 receptor occupancy. The data are also consistent with an effect of TH on transcription of the CPS gene and with a relatively long half-life of its protein product, the CPS enzyme.

Cardiac changes in experimental hyperthyroidism in dogs

Injection of triiodothyronine (T3) (1 mg/kg per day subcutaneously for 14 days) to 10 healthy dogs produced a hyperthyroid state characterised by high serum T3 concentrations, hypokalaemia, hyperactivity, loss of weight, diarrhoea and thirst. Electrocardiographic measurements showed that these dogs had an increase in heart rate of 63 +/- 11 beats/min with a significantly increased T wave amplitude without changes in R wave amplitude. Echocardiographic measurements showed no changes in fractional shortening and no evidence of ventricular hypertrophy, in contrast to reports in humans, cats and rats. However, the smooth muscle of the coronary arteries was markedly hypertrophied, which may cause a decrease in myocardial perfusion.

Thyroid hormone resistance syndrome. Inhibition of normal receptor function by mutant thyroid hormone receptors.

Thyroid hormone (T3) resistance is inherited in most cases in an autosomal dominant manner. The disorder is characterized by elevated free thyroid hormone levels and partial resistance to thyroid hormone at the cellular level. Distinct single amino acid substitutions in the ligand binding domain of the beta form of the thyroid hormone receptor have been described in two kindreds with this disorder. We used transient expression assays to characterize the functional properties of these receptor mutants, one containing a Gly to Arg change at amino acid 340 (G340R) and the other a Pro to His change at amino acid 448 (P448H). A nine amino acid carboxy terminal deletion (delta 448-456), analogous to an alteration that occurs in v-erbA, was also studied for comparison with the mutations that occur in the T3 resistance syndrome. None of the receptor mutants were able to mediate thyroid hormone dependent activation (TreTKCAT) or repression (TSH alpha CAT) of reporter genes when compared with the wild type receptor. In addition, the mutants inhibited the activity of normal alpha and beta receptor isoforms when examined in coexpression assays. This activity, referred to as dominant negative inhibition, was manifest with respect to both the positively and negatively regulated reporter genes. Although mutant receptor binding to DNA was unaffected, ligand binding studies showed that the G340R and delta 448-456 mutants failed to bind T3, whereas the P448H mutant bound hormone with reduced affinity (approximately 10% of normal) compared to the wild type receptor. Consistent with this finding, the P448H mutant receptor was partially active at higher T3 concentrations. Furthermore, the dominant negative inhibition elicited by the P448H receptor mutant at higher T3 concentrations was reversed in the presence of high doses of T3. These findings indicate that mutant beta receptors in patients with thyroid hormone resistance have reduced affinity for T3 and are functionally deficient, but impair the activity of normal receptors, thereby providing a mechanism for the dominant mode of inheritance in this disorder.

Transthyretin Receptors on Human Astrocytoma Cells*

Transthyretin (TTR), a transport protein for T4 and retinol-binding protein, is the principal T4-binding protein of cerebrospinal fluid. Its function in regard to the delivery of its ligands and in other respects is unclear. The binding of [125I] TTR to cultured human astrocytoma cells was studied to determine whether these cells carry receptors for TTR. Scatchard analysis was consistent with a single class of binding sites with a Kd of 3 nM. No significant cross-reactivity with transferrin or serum albumin was observed. Internalization of TTR was temperature dependent and proportional to receptor occupancy. Dilutions of cerebrospinal fluid displaced [125I]TTR in proportion to their content of radioimmunoassayable TTR and in parallel with purified TTR. The uptake and internalization of TTR increased in the presence of high T4 or T3 concentrations. These results demonstrate that TTR binds to specific high affinity receptors on human astrocytoma cells. Receptor binding of TTR provides a potential mechanism for the delivery of its ligands within the central nervous system.