Noriyuki Koibuchi

Promoter-specific regulation of the brain-derived neurotropic factor gene by thyroid hormone in the developing rat cerebellum.

Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I–IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still ...

Thyroid Hormone Response Elements Differentially Modulate the Interactions of Thyroid Hormone Receptors with Two Receptor Binding Domains in the Steroid Receptor Coactivator-1

Ligand-dependent transcriptional activation by nuclear receptors is mediated by interactions with coactivators. Recently, a consensus interaction motif (LXXLL) has been identified in a number of coactivators such as steroid receptor coactivator-1 (SRC-1). SRC-1 contains three such motifs in the central (nuclear receptor binding domain-1, NBD-1) and a single one in the C-terminal (NBD-2) regions. To define the nature and role of the two NBDs in SRC-1, interaction studies between the two NBDs and thyroid hormone receptor (TR) were performed. Although NBD-1 and NBD-2 showed similar ligand- and AF-2-dependent interactions with TR in solution, these two NBDs possessed distinct interaction properties with TR when TR is bound to a thyroid hormone-response element (TRE). Both in vitro and in vivo interaction studies demonstrate that NBD-1, but not NBD-2, exhibits ligand-dependent interaction with TR in the presence of TREs. In addition, a natural isoform of SRC-1, SRC-1E, which lacks NBD-2, preserved TR as well as progesterone receptor-mediated coactivator function on reporter gene expression. Finally, we found that NBD-1 failed to interact with a TR and retinoid X receptor heterodimer complex on a transcriptionally inactive direct repeat +4 TRE in electrophoretic mobility shift assays. These observations indicate that DNA-induced, as well as ligand-induced, conformational change(s) of TR may influence the nature of its binding to SRC-1, and that the two NBDs of SRC-1 may play different roles to regulate ligand-dependent transactivation of TRs.

RORα Gene Expression in the Perinatal Rat Cerebellum: Ontogeny and Thyroid Hormone Regulation1

Deficiency of thyroid hormone (TH) during the perinatal period results in severe neurological abnormalities in rodent cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. Of note, a mutant mouse, staggerer, in which the orphan nuclear hormone receptor RORα gene is disrupted, exhibits cerebellar abnormalities similar to those seen in the hypothyroid animals, despite normal thyroid function. We, therefore, speculated that TH (tetraiodo-l-thyronine; T4) may regulate RORα gene expression, which then may regulate genes essential for normal brain development. To test this hypothesis, we studied the changes in RORα gene expression in perinatal hypothyroid rat cerebellum and the effect of TH replacement using Northern blot analysis, ribonuclease protection assay and in situ hybridization histochemistry. During cerebellar development, an approximately 3-fold increase in the cerebellar content of RORα messenger RNA (mRNA) was seen in both prop...

Differential crosstalk between estrogen receptor (ER)α and ERβ and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE

Abstract Crosstalk between nuclear receptors is important for conversion of external and internal stimuli to a physiologically meaningful response by cells. Previous studies from this laboratory have demonstrated crosstalk between the estrogen (ER) and thyroid hormone receptors (TR) on two estrogen responsive physiological promoters, the preproenkephalin and oxytocin receptor gene promoter. Since ERα and ERβ are isoforms possessing overlapping and distinct transactivation properties, we hypothesized that the interaction of ERα and β with the various TR isoforms would not be equivalent. To explore this hypothesis, the consensus estrogen response element (ERE) derived from the Xenopus vitellogenin gene is used to investigate the differences in interaction between ERα and β isoforms and the different TR isoforms in fibroblast cells. Both the ER isoforms transactivate from the consensus ERE, though ERα transactivates to a greater extent than ERβ. Although neither of the TRβ isoforms have an effect on ERα transactivation from the consensus ERE, the liganded TRα1 inhibits the ERα transactivation from the consensus ERE. In contrast, the liganded TRα1 facilitates ERβ-mediated transactivation. The crosstalk between the TRβ isoforms with the ERα isoform, on the consensus ERE, is different from that with the ERβ isoform. The use of a TRα1 mutant, which is unable to bind DNA, abolishes the ability of the TRα1 isoform to interact with either of the ER isoforms. These differences in nuclear receptor crosstalk reveal an important functional difference between isoforms, which provides a novel mechanism for neuroendocrine integration.

Coactivator and Corepressor Gene Expression in Rat Cerebellum during Postnatal Development and the Effect of Altered Thyroid Status1

Thyroid hormone (TH) plays an important role in the postnatal development of the rodent cerebellum, particularly within the first 2 weeks of postnatal life. This action is exerted through the regulation of specific genes during development and is mediated by coactivator and corepressor proteins that determine transcriptional repression or activation, respectively. Thus, we hypothesized that the effect of TH on rodent cerebellar development could be influenced by the relative amounts of coactivator and corepressor proteins in vivo. These ratios might be modulated in an age-specific manner and/or by hormones to generate the critical period of TH action. To examine this hypothesis, we cloned rat complementary DNA fragments corresponding to coactivators (SRC1, TIF2 and TRAM1) and corepressors (N-CoR and SMRT), and studied the ontogenic changes in their corresponding messenger RNAs in rat cerebellum of normal and hypothyroid rats during postnatal development, using a RNase protection assay. We found an increased expression of SRC1 and TIF2, as well as of N-CoR, during rat cerebellar development but no change in the expression of SMRT and TRAM1 genes. However, thyroid hormone status did not affect the expression of coactivator and corepressor genes in the cerebellum. These results indicate that coactivator and corepressor messenger RNAs exhibit differential expression through cerebellar development but are not regulated by TH during this period.

Differential Interaction of Estrogen Receptor and Thyroid Hormone Receptor Isoforms on the Rat Oxytocin Receptor Promoter Leads to Differences in Transcriptional Regulation

Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, α and β, exist. The TRα and β isoforms are products of two distinct genes that are further differentially spliced to give TRα1 and α2, TRβ1 and β2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17β-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERα could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERβ was incapable of inducing the gene in either cell line. ERα is therefore not equivalent to ERβ on this physiological promoter. Indeed, in the neural cell line, ERβ can inhibit ERα-mediated induction from the OTR promoter. While the TRα1 isoform inhibited ERα-mediated induction in the neural cell line, the TRβ1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERα-mediated induction of the rat OTR gene promoter by the TRα1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRα1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRα-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.

Crosstalk Between Oestrogen Receptors and Thyroid Hormone Receptor Isoforms Results in Differential Regulation of the Preproenkephalin Gene

Nuclear receptors are ligand‐activated transcription factors, which have the potential to integrate internal metabolic events in an organism, with consequences for control of behaviour. Previous studies from this laboratory have shown that thyroid hormone receptor (TR) isoforms can inhibit oestrogen receptor (ER)α‐mediated induction of preproenkephalin (PPE) gene expression in the hypothalamus. Also, thyroid hormone administration inhibits lordosis, a behaviour facilitated by PPE expression. We have examined the effect of multiple ligand‐binding TR isoforms on the ER‐mediated induction of the PPE gene in transient transfection assays in CV‐1 cells. On a natural PPE gene promoter fragment containing two putative oestrogen response elements (EREs), both ERα and β isoforms mediate a four to five‐fold induction by oestrogen. Cotransfection of TRα1 along with ERα inhibited the ERα transactivation of PPE by approximately 50%. However, cotransfection with either TRβ1 or TRβ2 expression plasmids produced no effect on the ERα or ERβ mediated induction of PPE. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. Transfection with a TRα1 DNA‐binding mutant could also inhibit ERα transactivation, suggesting that competition for binding on the ERE may not be the exclusive mechanism for inhibition. Data with the coactivator, SRC‐1, suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ERβ is cotransfected, TRα1 stimulated ERβ‐mediated transactivation of PPE by approximately eight‐fold over control levels. This is the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggest that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the service of neuroendocrine integration.

RORα Augments Thyroid Hormone Receptor-Mediated Transcriptional Activation1

This study is designed to clarify the role of an orphan nuclear hormone receptor, RORα, on thyroid hormone (TH) receptor (TR)-mediated transcription on a TH-response element (TRE). A transient transfection study using various TREs [i.e., F2 (chick lysozyme TRE), DR4 (direct repeat), and palindrome TRE] and TR and RORα1 was performed. When RORα1 and TR were cotransfected into CV1 cells, RORα1 enhanced the transactivation by liganded-TR on all TREs tested without an effect on basal repression by unliganded TR. By electrophoretic mobility shift assay, on the other hand, although RORα bound to all TREs tested as a monomer, no (or weak) TR and RORα1 heterodimer formation was observed on various TREs except when a putative ROR-response element was present. The transactivation by RORα1 on a ROR-response element, which does not contain a TRE, was not enhanced by TR. The effect of RORα1 on the TREs is unique, because, whereas other nuclear hormone receptors (such as vitamin D receptor) may competitively bind to TR...

Co-expression of estrogen and thyroid hormone receptors in individual hypothalamic neurons.

Estrogen receptors (ER) and thyroid hormone receptors (TR) are members of the nuclear receptor family of transcription factors that induce or repress the expression of target genes. Previous behavioral studies in female rodents have demonstrated that thyroid hormones can antagonize the effects of estrogen in the central nervous system (CNS), particularly by attenuating estrogen’s ability to facilitate reproductive behaviors. Additional molecular studies have suggested a mechanism for this antagonism by showing that ligand‐activated ERα and TRs have the potential to interact in their transcriptional controls. Although the expression patterns of ERα and TRs in the rodent brain appear to overlap in behaviorally relevant areas, it remained to be determined whether these two classes of proteins coexist in vivo at the level of single neurons. To address this possibility, we employed a highly sensitive double‐label in situ hybridization technique using digoxigenin and 35S‐labeled cRNA probes to analyze, in detail, the expression of ERα mRNA with TRα1 and TRα2 mRNAs in the same neurons of the ovariectomized (OVX) adult mouse brain. Our results demonstrate that a large majority of the ERα‐positive neurons also expresses TRα1 and TRα2 mRNAs. Quantitative examination of the cellular expression in the ventromedial and arcuate nuclei of the hypothalamus (VMH and Arc) showed that 81.5% and 80.5% of the neurons endowed with ERα mRNA also contain TRα1 and TRα2 mRNAs, respectively. In the amygdala, more than 60.5% and 67% of ERα‐positive cells also contain TRα1 and TRα2 mRNAs, respectively. These findings provide the first anatomical evidence that ER and TR can be found in the same neurons, including hypothalamic neurons. This coexpression of ERα and TR provides the cellular basis for a new level of neuronal integration in a brain region where estrogens control female reproductive behaviors. J. Comp. Neurol. 437:286–295, 2001.

Cell type-specific roles of histone deacetylase in TR ligand-independent transcriptional repression

Recent evidence indicates that corepressor protein with histone deacetylase (HDAC) activity mediates thyroid hormone receptor (TR) transcriptional repression. In order to examine the physiological relevance of HDAC in ligand-independent TR-mediated repression, we studied the effect of trichostatin A (TSA), a specific HDAC inhibitor, in transient transfection studies with natural reporters, and assessed the expression of TR-regulated endogenous genes. Luciferase-coupled DR4-, F2-, PAL- or GH-TREs and TRbeta1 expression vectors were cotransfected in CV-1 and GH(3) cells. We did not observe any effect of TSA on TR-induced basal repression in CV-1 cells. Instead, TSA was able to induce an increase in transcription without T(3) on all TREs tested in GH(3) cells. This increase was >7-fold on F2-, >4-fold on DR4-, and 3-fold on GH-TREs. The cotransfection of a TRbeta1 mutant that exhibits decreased affinity with N-CoR (AHT) reduced the TSA effect in GH(3) cells, demonstrating a primary role for TR/N-CoR/Sin3/HDAC complex. Next, we examined the effects of TSA on endogenous GH mRNA production in GH(3) cells by Northern blot analysis. We observed an increase of 50-70% of GH mRNA in cells treated with TSA in hypothyroid medium, and an increase of GH mRNA in T(3)-treated cells after TSA treatment. Our results show that TSA can increase the expression of endogenous genes that are susceptible to TR regulation. These results support an active role of HDAC in transcriptional repression by ligand-independent TR.