Cardiac specific effects of thyroid hormone analogues.

There is significant interest in development of thyroid hormone analogues to harness specific properties as therapeutic agents for a variety of clinical indications including obesity, hypercholesterolemia, heart failure, and thyrotoxicosis. To date, most analogues have been designed to target liver specific effects, which can promote weight loss and lipid lowering through either tissue specific uptake or thyroid hormone receptor (TR) ß isoform selectivity at the same time minimizing the unwanted cardiac and bone effects. We have developed a molecular biomarker assay to study the induction of the transcription of the cardiac specific α-myosin heavy chain (MHC) gene as a more sensitive and specific measure of thyroid hormone action on cardiac myocytes. We tested 5 TRß and 1 TRα selective agonists as well as 2 putative TR antagonists in our α-MHC hnRNA assay. Using reverse transcription and polymerase chain reaction, we measured the induction of the α-MHC primary transcript in response to administration of drug. The TRα and only 2 of the TRß agonists were highly active, when compared to the effect of T3, at the level of the cardiac myocyte. In addition, our data suggests that the reason that the antagonist NH-3 is not able to block the T3-mediated induction of α-MHC is that it does not get transported into the cardiac myocyte. Our data suggest that this assay will be useful in preclinical studies of the potential cardiac specific effects of thyroid hormone analogues and that predictions of function based on structure are not necessarily accurate or complete.

Thyroid hormone transporters in health and disease: advances in thyroid hormone deiodination.

Thyroid hormone metabolism by the three deiodinase selenoproteins -- DIO1, DIO2, and DIO3 -- regulates the local availability of various iodothyronine metabolites and thus mediates their effects on gene expression, thermoregulation, energy metabolism, and many key reactions during the development and maintenance of an adult organism. Circulating serum levels of thyroid hormone and thyroid-stimulating hormone, used as a combined indicator of thyroid hormone status, reflect a composite picture of: thyroid secretion; tissue-specific production of T(3) by DIO1 and DIO2 activity, which both contribute to circulating levels of T(3); and degradation of the prohormone T4, of the thyromimetically active T(3), of the inactive rT(3), of other iodothyronines metabolites with a lower iodine content and of thyroid hormone conjugates. Degradation reactions are catalyzed by either DIO1 or DIO3. Aberrant expression of individual deiodinases in disease, single nucleotide polymorphisms in their genes, and novel regulators of DIO gene expression (such as bile acids) provide a more complex picture of the fine tuning and the adaptation of systemic and local bioavailability of thyroid hormones.

Changes in the content of steroid receptor coactivator-1 and silencing mediator for retinoid and thyroid hormone receptors in the rat brain during the estrous cycle.

In this work, we determined the variations in the content of the steroid receptor coactivator (SRC-1) and the silencing mediator for retinoic acid and thyroid hormone receptors corepressor (SMRT) in the hypothalamus, the preoptic area, and the hippocampus of adult intact rats during the estrous cycle by Western blot. SRC-1 content changed only in the hypothalamus where its lowest content was found on diestrus day with a significant increase at proestrus. This increase was maintained on estrus day. In contrast, SMRT content changed only in the preoptic area where it diminished at metestrus in comparison with the other days of the cycle. SRC-1 content was higher than that of SMRT in the hypothalamus throughout the estrous cycle, whereas SMRT content was higher in the preoptic area. In the hippocampus, there were no significant differences in the content of any cofactor. These results demonstrate that SRC-1 and SMRT content change in a tissue-specific manner in the rat brain during the estrous cycle, and suggest that the transcriptional activity of steroid hormone receptors in the rat brain in physiological conditions is regulated by changes in SRC-1 and SMRT content.

General receptor for phosphoinositides 1, a novel repressor of thyroid hormone receptor action that prevents deoxyribonucleic acid binding.

Thyroid hormone receptors (TRs) bind to response elements (TREs) located in the promoter region of target genes and modulate their transcription. The effects of TRs require the presence of coregulators that act as adaptor molecules between TRs and complexes that are involved in chromatin remodeling or that directly contact the basal transcription machinery. Using the yeast two-hybrid system, we identified a new interacting partner for TRs: GRP1 (general receptor for phosphoinositides-1), a nucleotide exchange factor, which had never been shown to interact with nuclear receptors. We reconfirmed the interaction between TRs and GRP1 in yeast and glutathione-S-transferase pull-down assays, and determined the areas of TRs and GRP1 involved in the interaction. Coimmunoprecipitation studies demonstrated that the interaction between GRP1 and TRs takes place in the cytoplasm and the nucleus of mammalian cells. To assess functional consequences of the interaction, we used transient transfection of CV-1 cells with TR and GRP1 expression vectors and luciferase reporter genes. On positive TREs, GRP1 decreased activation by 45-60%. On the negative TREs it increased repression by blunting the activation in the absence of T3, except for TRbeta2, which was not affected. Using EMSA, we have determined that addition of GRP1 diminishes the formation of TR/TR homodimers and TR/retinoid X receptor heterodimers on TREs, which could explain the effect of GRP1 on transcription. Furthermore, protein interaction assays using increasing concentrations of double-stranded TREs show a dose-dependent decrease of the interaction between GRP1 and TRs. The homo/heterodimers formed by TRs and retinoic X receptor-alpha were not influenced by the presence of GRP1, also suggesting that GRP1 interferes directly with DNA binding. Taken together, these data provide evidence that GRP1 is a new corepressor for TRs, which modulates both positive and negative regulation by T3 by decreasing TR-complex formation on TREs.

Discovery of diverse thyroid hormone receptor antagonists by high-throughput docking.

Treatment of hyperthyroidism, a common clinical condition that can have serious manifestations in the elderly, has remained essentially unchanged for >30 years. Directly antagonizing the effect of the thyroid hormone at the receptor level may be a significant improvement for the treatment of hyperthyroid patients. We built a computer model of the thyroid hormone receptor (TR) ligand-binding domain in its predicted antagonist-bound conformation and used a virtual screening algorithm to select 100 TR antagonist candidates out of a library of >250,000 compounds. We were able to obtain 75 of the compounds selected in silico and studied their ability to act as antagonists by using cultured cells that express TR. Fourteen of these compounds were found to antagonize the effect of T3 on TR with IC50s ranging from 1.5 to 30 microM. A small virtual library of compounds, derived from the highest affinity antagonist (1-850) that could be rapidly synthesized, was generated. A second round of virtual screening identified new compounds with predicted increased antagonist activity. These second generation compounds were synthesized, and their ability to act as TR antagonists was confirmed by transfection and receptor binding experiments. The extreme structural diversity of the antagonist compounds shows how receptor-based virtual screening can identify diverse chemistries that comply with the structural rules of TR antagonism.

The human peroxisome proliferator-activated receptor (PPAR) subtype NUC1 represses the activation of hPPAR alpha and thyroid hormone receptors.

We have cloned two human peroxisome proliferator-activated receptor (PPAR) subtypes, hPPAR alpha and hNUC1. hPPAR alpha is activated by clofibric acid and other PPAR activators. hNUC1 is not activated by these compounds acting instead as a repressor of hPPAR alpha and human thyroid hormone receptor transcriptional activation. Repression is specific since hNUC1 does not significantly repress activation by the progesterone or retinoic acid receptors. We demonstrate co-operative binding of hNUC1 and hRXR alpha to a PPAR-responsive element and show that in the presence of hRXR alpha, the affinity of hNUC1 for the peroxisome proliferator is comparable to that of hPPAR alpha. Furthermore, repression of hPPAR alpha can be overcome by transfecting excess hPPAR alpha. We propose that hNUC1 represses the activity of hPPAR alpha by titrating out a factor required for activation. Our data further suggests convergence of thyroid hormone- and peroxisome-mediated fatty acid metabolism pathways. Overcoming hNUC1 repression could be a means of increasing the activity of these receptors.