Ronald N. Cohen

1,25-Dihydroxyvitamin D3 suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin gene promoter

We have shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from –2725 to –2647 (distal fragment) and from –117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)2D3 inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)2D3-induced suppression. EMSA revealed that 1,25(OH)2D3 markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)2D3 suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.

An unliganded thyroid hormone receptor causes severe neurological dysfunction.

Congenital hypothyroidism and the thyroid hormone (T3) resistance syndrome are associated with severe central nervous system (CNS) dysfunction. Because thyroid hormones are thought to act principally by binding to their nuclear receptors (TRs), it is unexplained why TR knock-out animals are reported to have normal CNS structure and function. To investigate this discrepancy further, a T3 binding mutation was introduced into the mouse TR-β locus by homologous recombination. Because of this T3 binding defect, the mutant TR constitutively interacts with corepressor proteins and mimics the hypothyroid state, regardless of the circulating thyroid hormone concentrations. Severe abnormalities in cerebellar development and function and abnormal hippocampal gene expression and learning were found. These findings demonstrate the specific and deleterious action of unliganded TR in the brain and suggest the importance of corepressors bound to TR in the pathogenesis of hypothyroidism.

The Nuclear Receptor Corepressors NCoR and SMRT Decrease Peroxisome Proliferator-activated Receptor γ Transcriptional Activity and Repress 3T3-L1 Adipogenesis

The peroxisome proliferator-activated receptor γ (PPARγ) is a central regulator of adipogenesis and recruits coactivator proteins in response to ligand. However, the role of another class of nuclear cofactors, the nuclear receptor corepressors, in modulating PPARγ transcriptional activity is less clear. Such corepressors include the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT). Our data suggest that PPARγ recruits SMRT and NCoR in the absence of ligand and that these corepressors are capable of down-regulating PPARγ-mediated transcriptional activity. The addition of the PPARγ ligand pioglitazone results in dissociation of the PPARγ-corepressor complex. To define the role of SMRT and NCoR in PPARγ action, 3T3-L1 cells deficient in SMRT or NCoR were generated by RNA interference. When these cells are exposed to differentiation media, they exhibit increased expression of adipocyte-specific genes and increased production of lipid droplets, as compared with control cells. These data suggest that the nuclear receptor corepressors decrease PPARγ transcriptional activity and repress the adipogenic program in 3T3-L1 cells.

Partial deficiency of Thyroid transcription factor 1 produces predominantly neurological defects in humans and mice

Three genes, TTF1, TTF2, and PAX8, involved in thyroid gland development and migration have been identified. Yet systematic screening for defects in these genes in thyroid dysgenesis gave essentially negative results. In particular, no TTF1 gene defects were found in 76 individuals with thyroid dysgenesis even though a deletion of this gene in the mouse results in thyroid and lung agenesis and defective diencephalon. We report a 6-year-old boy with predominant dyskinesia, neonatal respiratory distress, and mild hyperthyrotropinemia. One allele of his TTF1 gene had a guanidine inserted into codon 86 producing a nonsense protein of 407, rather than 371, amino acids. The mutant TTF1 did not bind to its canonical cis-element or transactivate a reporter gene driven by the thyroglobulin promoter, a natural target of TTF1. Failure of the mutant TTF1 to interfere with binding and transactivation functions of the wild-type TTF1 suggested that the syndrome was caused by haploinsufficiency. This was confirmed in mice heterozygous for Ttf1 gene deletion, heretofore considered to be normal. Compared with wild-type littermates, Ttf1(+/-) mice had poor coordination and a significant elevation of serum thyrotropin. Therefore, haploinsufficiency of the TTF1 gene results in a predominantly neurological phenotype and secondary hyperthyrotropinemia.

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 β isoform of the thyroid hormone receptor (TR-β). To date, three RTH mutations reportedly with near-normal T3binding (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 T3concentrations. 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.

Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo.

Thyroid hormone action is mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. DNA-binding is presumed to be essential for all nuclear actions of thyroid hormone. To test this hypothesis in vivo, the DNA-binding domain of TR-beta was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-beta DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homozygous mutant (TR-betaGS/GS) mice displayed abnormal T3 regulation of the hypothalamic-pituitary-thyroid axis and retina identical to abnormalities previously observed in TR-beta KO (TR-beta-/-) mice. However, TR-betaGS/GS mutant mice maintained normal hearing at certain frequencies and did not display significant outer hair cell loss, in contrast to TR-beta-/- mice. DNA-binding, therefore, is essential for many functions of the TR, including retinal development and negative feedback regulation by thyroid hormone of the hypothalamic-pituitary-thyroid axis. Inner ear development, although not completely normal, can occur in the absence of TR DNA-binding, suggesting that an alternative and perhaps novel thyroid hormone-signaling pathway may mediate these effects.

Thyroid Hormone-independent Interaction between the Thyroid Hormone Receptor β2 Amino Terminus and Coactivators

Thyroid hormone receptors (TRs) mediate hormone action by binding to DNA response elements (TREs) and either activating or repressing gene expression in the presence of ligand, T3. Coactivator recruitment to the AF-2 region of TR in the presence of T3 is central to this process. The different TR isoforms, TR-β1, TR-β2, and TR-α1, share strong homology in their DNA- and ligand-binding domains but differ in their amino-terminal domains. Because TR-β2 exhibits greater T3-independent activation on TREs than other TR isoforms, we wanted to determine whether coactivators bound to TR-β2 in the absence of ligand. Our results show that TR-β2, unlike TR-β1 or TR-α1, is able to bind certain coactivators (CBP, SRC-1, and pCIP) in the absence of T3 through a domain which maps to the amino-terminal half of its A/B domain. This interaction is specific for certain coactivators, as TR-β2 does not interact with other co-factors (p120 or the CBP-associated factor (pCAF)) in the absence of T3. The minimal TR-β2 domain for coactivator binding is aa 21–50, although aa 1–50 are required for the full functional response. Thus, isoform-specific regulation by TRs may involve T3-independent coactivator recruitment to the transcription complex via the AF-1 domain.

Negative regulation by thyroid hormone receptor requires an intact coactivator-binding surface

Thyroid hormone (TH) action is mediated by TH receptors (TRs), which are members of the nuclear hormone receptor superfamily. In vitro studies have demonstrated that TR activity is regulated by interactions with corepressor and coactivator proteins (CoRs and CoAs, respectively). TH stimulation is thought to involve dissociation of CoRs and recruitment of CoAs to the liganded TR. In contrast, negative regulation by TH is thought to occur via recruitment of CoRs to the liganded TR. The physiological role of CoAs bound to TRs, however, has yet to be defined. In this study, we used gene-targeting techniques to mutate the TR-beta locus within its activation function-2 (AF-2) domain (E457A). This mutation was chosen because it completely abolished CoA recruitment in vitro, while preserving normal triiodothyronine (T3) binding and CoR interactions. As expected, TH-stimulated gene expression was reduced in homozygous E457A mice. However, these animals also displayed abnormal regulation of the hypothalamic-pituitary-thyroid axis. Serum thyroxine, T3, and thyroid-stimulating hormone (TSH) levels and pituitary Tshb mRNA levels were inappropriately elevated compared with those of WT animals, and L-T3 treatment failed to suppress serum TSH and pituitary Tshb mRNA levels. Therefore, the AF-2 domain of TR-beta is required for positive and, paradoxically, for negative regulation by TH in vivo.

Cross-talk between Thyroid Hormone Receptor and Liver X Receptor Regulatory Pathways Is Revealed in a Thyroid Hormone Resistance Mouse Model

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor β (TR-β) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-β knock-out mice, generalized expression of a non-ligand binding TR-β protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7α-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-β and liver X receptor α (LXR-α), which both bind to a direct repeat + 4(DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-β antagonized induction of this promoter by LXR-α. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-β. A mechanism for cross-talk, and potential antagonism, between TR-β and LXR-α is proposed.

Impact of subclinical hypothyroidism in women with recurrent early pregnancy loss

OBJECTIVE To assess the impact of subclinical hypothyroidism (SCH) in women with recurrent early pregnancy loss (REPL). DESIGN Observational cohort study. SETTING REPL program in an academic medical center. PATIENT(S) 286 women with a history of ≥2 pregnancy losses <10 weeks. INTERVENTION(S) From 2004-2007, no treatment for women with SCH (thyroid-stimulating hormone [TSH] >2.5 mIU/L with a normal free thyroxine or free thyroxine index); from 2008 onward, levothyroxine treatment prepregnancy to maintain TSH ≤2.5 mIU/L. MAIN OUTCOME MEASURE(S) Live-birth rate (LBR). RESULT(S) The prevalence of SCH was 55 (19%) of 286 in this REPL cohort. The cumulative LBR was 27 (69%) of 39 for women with SCH versus 104 (74%) of 141 for euthyroid women. The per-pregnancy LBR was 34 (49%) of 69 for SCH versus 129 (58%) of 221 for euthyroid women. When the LBR was compared between treated and untreated SCH, the cumulative LBR was 17 (71%) of 24 versus 10 (67%) of 15, respectively. The per-pregnancy LBR for SCH treated versus untreated women was 22 (48%) of 46 versus 12 (52%) of 23, respectively. CONCLUSION(S) Although there was a high prevalence of SCH in the REPL cohort, there was no statistically significant difference in the subsequent live-birth rate when comparing women with SCH and euthyroid women, or treated and untreated SCH.