Tsuyoshi Monden

A Unique Role of the β-2 Thyroid Hormone Receptor Isoform in Negative Regulation by Thyroid Hormone MAPPING OF A NOVEL AMINO-TERMINAL DOMAIN IMPORTANT FOR LIGAND-INDEPENDENT ACTIVATION

Negative regulation by thyroid hormone is mediated by nuclear thyroid hormone receptors (TRs) acting on thyroid hormone response elements (TREs). We examine here the role of human TR-β2, a TR isoform with central nervous system-restricted expression, in the regulation of target genes whose expression are decreased by triiodothyronine (T3). Using transient transfection studies, we found that TR-β2 achieved significantly greater ligand-independent activation on the thyrotropin-releasing hormone (TRH) and common glycoprotein α-subunit genes than either TR-β1 or TR-α1. A chimeric TR-β isoform containing the TR-β2 amino terminus linked to the TR-α1 DNA- and ligand-binding domains functioned like the TR-β2 isoform on these promoters, confirming that the amino terminus of TR-β2 was both necessary and sufficient to mediate this effect. By constructing deletion mutants of the TR-β2 amino terminus, we demonstrate that amino acids 89–116 mediate this function. This domain, important in ligand-independent activation on negative TREs, is discrete from a previously described activation domain in the amino-terminal portion of TR-β2. We conclude that the central nervous system-restricted TR-β2 isoform has a unique effect on negative regulation by T3 that can be mapped to amino acids 89–116 of the amino terminus of the human TR-β2.

Function of Nuclear Co-repressor Protein on Thyroid Hormone Response Elements Is Regulated by the Receptor A/B Domain

Recently, a family of nuclear co-repressor proteins (TRACs) have been identified that interact with thyroid hormone (TR) and retinoic acid receptors to mediate ligand-independent repression of gene transcription. In this report, we have cloned and characterized a human TRAC, which when expressed as a truncated protein lacking its repressing domains, can abolish endogenous cellular TRAC activity. Use of this inhibitor has uncovered a differential function of TRACs on negative versus positive thyroid hormone response elements and has demonstrated the importance of the TR A/B domain in modulating TRAC function. Thus, isoform-specific functions of the TR may be mediated by their functional interaction with co-repressor proteins.

Isolation and characterization of a novel ligand-dependent thyroid hormone receptor-coactivating protein.

The thyroid hormone receptor (TR) regulates the expression of target genes upon binding to triiodothyronine (T3) response elements. In the presence of T3, the TR recruits coactivating proteins that both modulate and integrate the ligand response. We report here the cloning of a novel protein using the TR ligand-binding domain as bait in the yeast two-hybrid system. Analysis of a putative full-length clone demonstrates a cDNA sequence that encodes a protein of 920 amino acids with a size of 120 kDa (p120). Alignment with known sequences shows homology to a previously identified protein of unknown function, termed skeletal muscle abundant protein. Interaction studies demonstrate that p120 interacts with the TR AF-2 domain in the presence of ligand through a 111-amino acid region. Northern analysis demonstrates widespread expression in human tissues. Cotransfection assays in CV-1 cells demonstrate that p120 enhances TR-mediated transactivation on multiple T3 response elements in the presence of T3. In addition, CREB-binding protein synergizes with p120 to enhance this effect. When linked to the GAL4 DNA-binding domain, p120 is an activator of transcription alone. Thus, p120 satisfies a number of important criteria as a nuclear receptor coactivator.

A liver X receptor (LXR)-β alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-β

We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-beta LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-beta gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-beta. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

Leucine at Codon 428 in the Ninth Heptad of Thyroid Hormone Receptor β1 is Necessary for Interactions with the Transcriptional Cofactors and Functions Regardless of Dimer Formations

Structure/function studies of the thyroid hormone receptor (TR) beta(1) have demonstrated that single amino acid substitutions in either position 428 or 429 in the ligand-binding domain (LBD) can alter heterodimerizations and homodimerizations, respectively. A leucine at 428 is located in a highly conserved region corresponding to the putative ninth heptad repeat of a leucine-zipper-like motif in the LBD of TRbeta(1). To investigate how the side chain of amino acids at 428 affect receptor characteristics, gel-shift mobility shift assays and yeast two-hybrid assays were analyzed. The neutral status amino acids such as a leucine (wild-type) or a glutamine at 428 preferred heterodimerization with RXR. Furthermore, a positively charged side chain of amino acids at 428 such as an arginine or a lysine, preserved homodimer formation. Irrespective of charge, ninth heptad mutant receptors did not bind the ligand and were not able to interact with either corepressor or coactivating proteins. Limited trypsinization assays revealed no major conformational change in the ninth heptad mutant receptors. Together, these findings suggested that a leucine at 428 was a critical amino acid for both interaction with the thyroid hormone receptor associated proteins and ligand-independent and -dependent functions regardless of dimer formations.

Severe Hypoglycemia Accompanied with Thyroid Crisis

We report a 32-year-old Japanese women with severe hypoglycemia accompanied with thyroid crisis. She complained of dyspnea, general fatigue, and leg edema. She was diagnosed with hyperthyroidism with congestive heart failure and liver dysfunction. Soon after admission, sudden cardiopulmonary arrest occurred. She was then transferred to the intensive care unit. Her serum glucose level was 7u2009mg/dl. Intravenous glucose, hydrocortisone, diuretics, and continuous hemodiafiltration (CHDF) saved her. We considered that hypoglycemia occurred due to heart failure and liver dysfunction due to thyroid crisis.