Thyroid hormone alters the DNA binding properties of chicken thyroid hormone receptors alpha and beta.

The effects of thyroid hormone agonists on thyroid hormone receptor (TR)/DNA complex formation was investigated to elucidate the mechanism by which TRs transactivate genes in response to ligand. The data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of TRs bound to DNA, irrespective of if the element is occupied by monomeric TR, homodimeric TR/TR, or heterodimeric complexes with the retinoid receptors RAR or RXR. Furthermore, triiodo-thyronine (T3) prevents 2 TR molecules from binding to oligonucleotides containing direct repeats or inverted palindromes of the consensus AGGTCA motif, an effect that was not detected with palindromic elements. Heterodimers bound to direct repeats were less affected: RXR/TR were fully and RAR/TR complexes partially resistant to thyroid hormone. The data suggest that a ligand-induced conformational change in TR prevents double TR occupancy of a response element containing 2 direct repeats of the consensus binding motif, possibly by steric hindrance, whereas such an event does not prevent TR/RXR heterodimers from binding to DNA. Finally, our data show that a monomeric, liganded TR bound preferentially to the second half site in a AGGTCActcaAGGTCA element, and therefore indicate that nucleotides adjacent to the consensus half site contribute to binding specificity.     

Differential binding of IL-1 alpha and IL-1 beta to receptors on B and T cells

The interleukin 1 receptors (IL-1R) on the human B lymphoma RAJI and on the murine thymoma EL4-6.1 have been characterized. Equilibrium binding analysis using both 125I-labeled IL-1 alpha and IL-1 beta showed that RAJI cells have a higher number of binding sites/cell for IL-1 beta (2400, Kd 2.2 nM) than for IL-1 alpha (316, Kd 0.13 nM). On the other hand, EL4-6.1 cells have more receptors/cell for IL-1 alpha (22 656, Kd 1 nM) than for IL-1 beta (2988, Kd 0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time-dependent increase in binding sites for both IL-1 beta and IL-1 alpha without affecting their binding affinities. However, while receptor-bound 125I-IL-1 alpha was displaced with equal efficiency by both IL-1 forms, only unlabeled IL-1 beta could effectively displace 125I-IL-1 beta. Cross-linking experiments indicated that RAJI cells have a predominant IL-1R of about 68 kDa, while EL4-6.1 cells have an IL-1-binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL-1R with distinct binding properties for IL-1 alpha and IL-1 beta.     

Plasma free fatty acids, inhibitor of extrathyroidal conversion of T4 to T3 and thyroid hormone binding inhibitor in patients with various nonthyroidal illnesses.

In order to clarify the role of free fatty acid (FFA) in thyroid hormone abnormalities in patients with nonthyroidal illness, thyroid function, FFA, inhibitor of extrathyroidal conversion of T4 to T3 (IEC) and thyroid hormone binding inhibitor (THBI) were studied in 99 patients with various nonthyroidal illnesses including diabetes mellitus (DM) (n = 35), liver cirrhosis (LC) (n = 33), chronic obstructive pulmonary disease (COPD) (n = 17) and chronic heart failure (CHF) (n = 14). Patients were divided into three groups based on the level of serum T3: Group I (T3 < 50 ng/dl), Group II (50 < or = T3 < 80) and Group III (80 < or = T3). Serum T4, FT3 and the T3/T4 ratio decreased significantly in the order Group III, Group II and Group I (Group III > II > I). The plasma FFA level was 0.91 +/- 0.12 mmol/l in Group I (P < 0.05, vs. Group III), 0.65 +/- 0.06 in Group II and 0.54 +/- 0.04 in Group III, respectively. The incidence of positive IEC was 80.0% in Group I (P < 0.05, vs. Group III), 53.7% in Group II (P < 0.05, vs. Group III) and 34.2% in Group III. However, IEC was not correlated with the serum T3 concentration. The incidence of positive THBI was 80% in Group I (P < 0.05, vs. Group III), 68.3% in Group II and 47.4% in Group III, but THBI was not correlated with the serum T4 level. Positive correlations were observed among FFA, IEC and THBI (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA binding of thyroid hormone receptors.

Thyroid hormone receptors, isolated from rat liver nuclei, bind to purified DNA. By contrast, free triiodothyronine and plasma proteins which bind thyroid hormone do not associate with DNA. Thus, the nuclear localization of thyroid hormone in target tissues may be explained by the association of its receptors with DNA.     

Differential modulation of Toll-like receptors by fatty acids: preferential inhibition by n-3 polyunsaturated fatty acids

Human subjects consuming fish oil showed a significant suppression of cyclooxygenase-2 (COX-2) expression in blood monocytes when stimulated in vitro with lipopolysaccharide (LPS), an agonist for Toll-like receptor 4 (TLR4). Results with a murine monocytic cell line (RAW 264.7) stably transfected with COX-2 promoter reporter gene also demonstrated that LPS-induced COX-2 expression was preferentially inhibited by docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3), the major n-3 polyunsaturated fatty acids (PUFAs) present in fish oil. Additionally, DHA and EPA significantly suppressed COX-2 expression induced by a synthetic lipopeptide, a TLR2 agonist. These results correlated with the preferential suppression of LPS- or lipopeptide-induced NF kappa B activation by DHA and EPA. The target of inhibition by DHA is TLR itself or its associated molecules, but not downstream signaling components. In contrast, COX-2 expression by TLR2 or TRL4 agonist was potentiated by lauric acid, a saturated fatty acid. These results demonstrate that inhibition of COX-2 expression by n-3 PUFAs is mediated through the modulation of TLR-mediated signaling pathways. Thus, the beneficial or detrimental effects of different types of dietary fatty acids on the risk of the development of many chronic inflammatory diseases may be in part mediated through the modulation of TLRs.     

The binding of thyroid hormone receptors to DNA

The behaviour of tri-iodothyronine (T3)- and thyroxine (T4)-receptor complexes when bound to native DNA-cellulose is reported. Equal and large proportions of both T3- and T4-receptor complexes bind to DNA but although T3-receptor complexes are 99% recoverable by 0.5 M NaCl buffer elution, only 60-70% of the T4-receptor complexes are regained. The balance appears as free T4, apparently released as the T4-receptor complexes bind to the DNA whilst the corresponding receptor remains bound. This effect is independent of T4-receptor complex/DNA ratio up to ca. 4 fmol/micrograms DNA, of the presence of an equal amount of unoccupied receptor and of an eight-fold concentration range of both T4-receptor complex and DNA at a fixed ratio, in the cellulose matrix. Pre-formed receptor-DNA material, likewise, only accepts some 60% of the expected quantity of T4 whereas the capacity for T3 appears to be similar to that of free receptors.     

The mechanism of SR95531 inhibition at GABA receptors examined in human alpha1beta1 and alpha1beta1gamma2S receptors

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.     

Distinct functions for thyroid hormone receptors alpha and beta in brain development indicated by differential expression of receptor genes.

Thyroid hormones are essential for correct brain development, and since vertebrates express two thyroid hormone receptor genes (TR alpha and beta), we investigated TR gene expression during chick brain ontogenesis. In situ hybridization analyses showed that TR alpha mRNA was widely expressed from early embryonic stages, whereas TR beta was sharply induced after embryonic day 19 (E19), coinciding with the known hormone-sensitive period. Differential expression of TR mRNAs was striking in the cerebellum: TR beta mRNA was induced in white matter and granule cells after the migratory phase, suggesting a main TR beta function in late, hormone-dependent glial and neuronal maturation. In contrast, TR alpha mRNA was expressed in the earlier proliferating and migrating granule cells, and in the more mature granular and Purkinje cell layers after hatching, indicating a role for TR alpha in both immature and mature neural cells. Surprisingly, both TR genes were expressed in early cerebellar outgrowth at E9, before known hormone requirements, with TR beta mRNA restricted to the ventricular epithelium of the metencephalon and TR alpha expressed in migrating cells and the early granular layer. The results implicate TRs with distinct functions in the early embryonic brain as well as in the late phase of hormone requirement.     

An assay for thyroid hormone binding to chromatin receptors

I have measured the interaction of T₃ with highly soluble, expanded, rat liver chromatin using a new assay for the study of hormone binding to nucleoprotein. Bound hormone and free hormone were rapidly and quantitatively separated by the adsorption of the hormone-nucleoprotein complex onto hydroxylapatite. This procedure satisfies several criteria for a successful binding assay: (1) The binding capacity is stable throughout the time required to reach equilibrium, (2) the ratio of specific to nonspecific binding (signal/noise) is at least 20:1, (3) large numbers of samples can be handled easily, (4) the amount of bound hormone is directly proportional to the quantity of chromatin employed, (5) the hormone and its analogs display a range of affinities for the binding site, and (6) the binding occurs to a limited number of sites, over a free hormone concentration range which is similar to the hormone concentrations found in vivo.     

A nuclear factor that enhances binding of thyroid hormone receptors to thyroid hormone response elements

Recent studies from this laboratory have demonstrated the presence of thyroid hormone response elements (TREs) in the 5'-flanking region of the rat alpha and TSH beta subunit genes. Using an avidin-biotin complex DNA binding assay, we have shown that these TREs bind the thyroid hormone (T3) receptor present in nuclear extracts of GH3 cells, as well as the in vitro synthesized Hc-erbA beta, which has been identified as a member of the family of T3 receptors. The binding of Hc-erbA beta to the alpha subunit TRE can be enhanced 3-4-fold by including GH3 nuclear extract in the binding assay. Binding to the TRE present in the TSH beta gene or the rat growth hormone gene was similarly enhanced, although to a lesser degree. The enhanced binding activity is trypsin-sensitive and heat labile, and is not reproduced by the addition of histones, bovine serum albumin, or cytosol instead of nuclear extract. Gel exclusion chromatography suggests a molecular size of approximately 65,000 Da. This protein, which is present in several different cell types, is also able to complement binding of the rat erbA alpha-1 and the pituitary-specific erbA beta-2 forms of the receptor. These data suggest that the binding of the T3 receptor to a TRE is augmented by another nuclear protein, which may be involved in the mechanism of action of thyroid hormone.     

Resistance to thyroid hormone mediated by defective thyroid hormone receptor alpha

Background

Thyroid hormone acts via receptor subtypes (TRα1, TRβ1, TRβ2) with differing tissue distributions, encoded by distinct genes (THRA, THRB). THRB mutations cause a disorder with central (hypothalamic–pituitary) resistance to thyroid hormone action with markedly elevated thyroid hormone and normal TSH levels.

Scope of review

This review describes the clinical features, genetic and molecular pathogenesis of a homologous human disorder mediated by defective THRA. Clinical features include growth retardation, skeletal dysplasia and constipation associated with low-normal T4 and high-normal T3 levels and a low T4/T3 ratio, together with subnormal reverse T3 levels. Heterozygous TRa1 mutations in affected individuals generate defective mutant receptors which inhibit wild-type receptor action in a dominant negative manner.

Major conclusions

Mutations in human TRα1 mediate RTH with features of hypothyroidism in particular tissues (e.g. skeleton, gastrointestinal tract), but are not associated with a markedly dysregulated pituitary–thyroid axis.

General significance

Human THRA mutations could be more common but may have eluded discovery due to the absence of overt thyroid dysfunction. Nevertheless, in the appropriate clinical context, a thyroid biochemical signature (low T4/T3 ratio, subnormal reverse T3 levels), may enable future identification of cases.This article is part of a Special Issue entitled Thyroid hormone signalling.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relations between fatty acids and oestrogen binding properties of pure rat alpha 1-foetoprotein.

A delipidation procedure based on treatment with charcoal at pH 3 has been applied to highly purified rat alpha 1-foetoprotein preparations. The oestrogen binding properties of the delipidated proteins have been studied with an equilibrium dialysis technique, and compared with the properties of the untreated foetal protein, as well as those of preparations reconstituted from the defatted alpha 1-foetoprotein and the removed lipids. An important increase has been evidenced for the binding levels of oestrone, oestradiol-17 beta and diethylstilboestrol by the delipidated alpha 1-foetoprotein. A reversal of this effect has been obtained by incubating the delipidated protein either with the lipids extracted from the purified alpha 1-foetoprotein or with a potent competitor of the rat alpha 1-foetoprotein-oestrogen interaction, designated as 'L', previously demonstrated and isolated from whole rat sera, and tentatively characterized as a mixture of fatty acids. Scatchard analysis of the oestrone and oestradiol-17 beta binding parameters show that the enhanced fixation of the hormones after defatting is primarily due to a two-fold increase of the apparent number of binding sites/mol alpha 1-foetoprotein. The results are interpreted in terms of the probable, at least partial, identity between the lipids closely associated with the pure alpha 1-foetoprotein and the fatty acid mixture 'L' isolated from whole sera. The possible biological role of complex interplay between oestrophilic alpha 1-foetoproteins, phenolsteroids and fatty acids in the control of oestrogen levels during development is discussed briefly.     

Modulation of thyroid hormone nuclear receptors by short-chain fatty acids in glial C6 cells. Role of histone acetylation

We have studied the effect of butyrate and other short-chain fatty acids on thyroid hormone nuclear receptors in C6 cells, a rat glioma cell line. Exposure of C6 cells to butyrate leads to increased levels of L-triiodothyronine (T3) in the nuclear and extranuclear compartments. The rise in nuclear binding is not merely a reflection of the higher cellular hormone content, and Scatchard analysis of T3 binding to isolated nuclei reveals that butyrate increases receptor number without changing affinity. The effect on the receptor is quantitatively important: a 48-h incubation with 2 mM butyrate increases nuclear binding by 2-3-fold, and 5 mM butyrate by 3-5-fold. Other short-chain fatty acids were found to similarly influence both nuclear receptor and extranuclear T3 levels with the following potency: butyrate greater than valerate greater than propionate greater than acetate. On the contrary, ketone bodies were ineffective. Butyrate increases receptor levels by decreasing receptor degradation, since the apparent t1/2 of receptor disappearance increased by approximately 3-fold in cells incubated with 2 mM butyrate for 48 h. The regulation of receptor number might be secondary to an action of butyrate on regions of the chromatin to which the receptor associates. We then examined the effect of butyrate on histone acetylation. The fatty acid had little effect in increasing the level of multiacetylated forms of H3 and H4 histone when studied in acid-urea gels, but it markedly inhibited the turnover of [3H] acetate from the histone fraction. There was a striking similarity in the dose-response of butyrate for increasing receptor levels and inhibiting histone deacetylation. Furthermore, a very close correlation between receptor levels and [3H]acetate release was also found when different short-chain fatty acids were used. We thus conclude that the effect of butyrate on the receptor could be explained by a modification of the chromatin structure of C6 cells secondary to acetylation.     

The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production.

The diverse functions of thyroid hormones are thought to be mediated by two nuclear receptors, T3R alpha1 and T3R beta, encoded by the genes T3R alpha and T3R beta respectively. The T3R alpha gene also produces a non-ligand-binding protein T3R alpha2. The in vivo functions of these receptors are still unclear. We describe here the homozygous inactivation of the T3R alpha gene which abrogates the production of both T3R alpha1 and T3R alpha2 isoforms and that leads to death in mice within 5 weeks after birth. After 2 weeks of life, the homozygous mice become progressively hypothyroidic and exhibit a growth arrest. Small intestine and bones showed a strongly delayed maturation. In contrast to the negative regulatory function of the T3R beta gene on thyroid hormone production, our data show that the T3R alpha gene products are involved in up-regulation of thyroid hormone production at weaning time. Thus, thyroid hormone production might be balanced through a positive T3R alpha and a negative T3R beta pathway. The abnormal phenotypes observed on the homozygous mutant mice strongly suggest that the T3R alpha gene is essential for the transformation of a mother-dependent pup to an 'adult' mouse. These data define crucial in vivo functions for thyroid hormones through a T3R alpha pathway during post-natal development.     

Molecular modeling of the thyroid hormone interactions with alpha v beta 3 integrin

A cell surface receptor for thyroid hormone has recently been identified on the extracellular domain of integrin alphavbeta3. In a variety of human and animal cell lines this hormone receptor mediates activation by thyroid hormone of the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. An arginine-glycine-aspartate (RGD) recognition site on the heterodimeric integrin is essential to the binding of a variety of extracellular matrix proteins. Recent competition data reveal that RGD peptides block hormone-binding by the integrin and consequent MAPK activation, suggesting that the hormone interaction site is located at or near the RGD recognition site on integrin alphavbeta3. A deaminated thyroid hormone (l-thyroxine, T4) analogue, tetraiodothyroacetic acid (tetrac, T4ac), inhibits binding of T4 and 3,5,3'-triiodo-l-thyronine (T3) to alphavbeta3, but does not activate MAPK. Structural data show that the RGD cyclic peptide binds at the interface of the propeller of the alphav and the B domains on the integrin head [Xiong JP, Stehle T, Zhang R, Joachimiack A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin alphavbeta3 in complexing with an Arg-Gly-Asp ligand. Science 2002;296:151-5]. To model potential interactions of thyroid hormone analogues with integrin, we mapped T4 and T4ac to the binding site of the RGD peptide. Modeling studies indicate that there is sufficient space in the cavity for the thyroid hormone to bind. Since the hormone is smaller in overall length than the RGD peptide, the hormone does not interact with the Arg recognition site in the propeller domain from alphav. In this model, most of the hormone interactions are with betaA domain of the integrin. Mutagenic studies can be carried out to validate the role of these residues in directing hormone interactions.     

Protein kinase inhibition by omega-3 fatty acids

Recent data suggest that omega-3 fatty acids may be effective in epilepsy, cardiovascular disorders, arthritis, and as mood stabilizers for bipolar disorder; however, the mechanism of action of these compounds is unknown. Based on earlier studies implicating omega-3 fatty acids as inhibitors of protein kinase C activity in intact cells, we hypothesized that omega-3 fatty acids may act through direct inhibition of second messenger-regulated kinases and sought to determine whether the omega-3 double bond might uniquely confer pharmacologic efficacy and potency for fatty acids of this type. In our studies we observed that omega-3 fatty acids inhibited the in vitro activities of cAMP-dependent protein kinase, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinase (MAPK). Our results with a series of long-chain fatty acid structural homologs suggest an important role for the omega-3 double bond in conferring inhibitory efficacy. To assess whether omega-3 fatty acids were capable of inhibiting protein kinases in living neurons, we evaluated their effect on signal transduction pathways in the hippocampus. We found that omega-3 fatty acids could prevent serotonin receptor-induced MAPK activation in hippocampal slice preparations. In addition, we evaluated the effect of omega-3 fatty acids on hippocampal long-term potentiation, a form of synaptic plasticity known to be dependent on protein kinase activation. We observed that omega-3 fatty acids blocked long-term potentiation induction without inhibiting basal synaptic transmission. Overall, our results from both in vitro and live cell preparations suggest that inhibition of second messenger-regulated protein kinases is one locus of action of omega-3 fatty acids.