ISL1 Is Necessary for Maximal Thyrotrope Response to Hypothyroidism.

ISLET1 is a homeodomain transcription factor necessary for development of the pituitary, retina, motor neurons, heart, and pancreas. Isl1-deficient mice (Isl1(-/-)) die early during embryogenesis at embryonic day 10.5 due to heart defects, and at that time, they have an undersized pituitary primordium. ISL1 is expressed in differentiating pituitary cells in early embryogenesis. Here, we report the cell-specific expression of ISL1 and assessment of its role in gonadotropes and thyrotropes. Isl1 expression is elevated in pituitaries of Cga(-/-) mice, a model of hypothyroidism with thyrotrope hypertrophy and hyperplasia. Thyrotrope-specific disruption of Isl1 with Tshb-cre is permissive for normal serum TSH, but T4 levels are decreased, suggesting decreased thyrotrope function. Inducing hypothyroidism in normal mice causes a reduction in T4 levels and dramatically elevated TSH response, but mice with thyrotrope-specific disruption of Isl1 have a blunted TSH response. In contrast, deletion of Isl1 in gonadotropes with an Lhb-cre transgene has no obvious effect on gonadotrope function or fertility. These results show that ISL1 is necessary for maximal thyrotrope response to hypothyroidism, in addition to its role in development of Rathke's pouch.

The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains.

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors [NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)] in the absence of ligand to silence transcription. NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT. Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains. An analogous domain is not present in SMRT. This domain is specific for TR and interacts poorly with RAR. Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells. Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions. In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1. Additional regions distal to the CoRNR box sequence are needed for optimal binding. Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1. These preferences are likely to be important in corepressor action in vivo.

Transcriptional regulation of the leptin gene promoter in rat GH3 pituitary and C6 glioma cells.

Leptin was originally believed to be an exclusively adipocyte-derived hormone regulating appetite and energy balance. It has recently become apparent that leptin is actively expressed in a number of other tissues including the CNS and pituitary, as well as brain- and pituitary-derived cell lines. However, the factors controlling leptin expression in cells of neuroectodermal origin are unknown. The mouse leptin gene 5'-flanking DNA contains multiple AP-1 and SRF-1 binding sites as well as a consensus CRE site at -491 to -482 bp. In addition, a number of potential PIT1 and Oct-1 binding sites may contribute to leptin gene transcription in pituitary and brain. We have used leptin promoter-luciferase reporter constructs to examine the regulation of the leptin promoter in 3T3-L1 preadipocytes, C6 glioma cells, and GH3 pituitary cells in response to serum and hormonal stimuli. Cells were transiently transfected with reporter constructs containing either the proximal 500 bp of the leptin promoter (-500-luc) or 6000 bp of the leptin gene 5' flanking region (-6000-luc). Functional analysis indicates that the leptin promoter is constitutively active in all 3 cell lines. Transcriptional activity was significantly higher with a -500 to +9 promoter than with a construct containing -6000 to +9 bp of 5' flanking DNA, indicating the presence of repressor elements which may contribute to the tissue-specific regulation of leptin expression. However, qualitatively similar results were observed with both constructs in response to serum and hormonal manipulation. Leptin promoter activity was significantly stimulated by serum in all cell lines, although to varying extents. In contrast, the response of the leptin promoter to insulin, IGF-1 and dibutyryl cAMP was cell-type specific and dependent on the presence or absence of FBS in the culture medium. Insulin, IGF-1 and dibutyryl cAMP each caused an approximately two-fold stimulation of leptin promoter activity in 3T3-L1 cells under serum-free conditions, but had no significant effect in the presence of 10% FBS. In contrast, dibutyryl cAMP markedly stimulated leptin promoter activity (5-8-fold) in C6 or GH3 cells in the presence or absence of FBS, whereas insulin or IGF-1 had minimal effects. These findings support our previous studies on the regulation of leptin steady state mRNA levels in C6 cells and demonstrate tissue-specific differences in the regulation of leptin gene transcription in adipose vs. neuroectodermal tissues.

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling.

Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, alpha-melanocyte stimulating hormone (alpha-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). Indeed, a novel Stat-response element in the TRH promoter is necessary for leptin's effect. Thus, the TRH promoter is an ideal target for further characterizing the integration of transcriptional pathways through which leptin acts.

cAMP response element-binding protein-binding protein mediates thyrotropin-releasing hormone signaling on thyrotropin subunit genes.

Transcription of pituitary alpha-glycoprotein hormone subunit (alpha-GSU) and thyrotropin beta subunit (TSH-beta) genes is stimulated by thyrotropin-releasing hormone (TRH). Since cAMP response element-binding protein (CREB)-binding protein (CBP) integrates a number of cell signaling pathways, we investigated whether CBP is important for TRH stimulation of the TSH subunit genes. Cotransfection of E1A in GH(3) cells completely blocked TRH stimulation of the TSH subunit genes, suggesting that CBP is a key factor for TRH signaling in the pituitary. CBP and Pit-1 acted synergistically in TRH stimulation of the TSH-beta promoter, and amino acids 1-450 of CBP were sufficient for the TRH effect. In contrast, on the human alpha-GSU promoter, CREB and P-Lim mediated TRH signaling. Intriguingly, CREB was phosphorylated upon TRH stimulation, leading to CBP recruitment to the alpha-GSU promoter. CBP also interacted with P-Lim in a TRH-dependent manner, suggesting that P-Lim is an important factor for non-cAMP response element-mediated TRH stimulation of this promoter. Distinct domains of CBP were required for TRH signaling by CREB and P-Lim on the alpha-GSU promoter, amino acids 450-700 and 1-450, respectively. Thus, the amino terminus of CBP plays a critical role in TRH signaling in the anterior pituitary via both Pit-1-dependent and -independent pathways, yielding differential regulation of pituitary gene products.

The nuclear corepressors recognize distinct nuclear receptor complexes.

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms have the capacity to silence gene expression in the absence of their ligands on target response elements. This active repression is mediated by the ability of the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to recruit a complex containing histone deacetylase activity. Interestingly, NCoR and SMRT share significant differences in the their two nuclear receptor-interacting domains (IDs), suggesting that they may recruit receptors with different affinities. In addition, the role of the receptor complex bound to a response element has not been fully evaluated in its ability to recruit separate corepressors. We demonstrate in this report that the proximal ID in NCoR and SMRT, which share only 23% homology, allows preferential recognition of nuclear receptors, such that TR prefers to recruit NCoR, and RAR prefers to recruit SMRT, to DNA response elements. However, mutations in the TR found in the syndromes of resistance to thyroid hormone can change the corepressor recruited by changing the complex (homodimer or heterodimer) formed on the TRE. These results demonstrate that the corepressor complex recruited can be both nuclear receptor- and receptor complex-specific.

Thyroid hormone-independent interaction between the thyroid hormone receptor beta2 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, T(3). Coactivator recruitment to the AF-2 region of TR in the presence of T(3) is central to this process. The different TR isoforms, TR-beta1, TR-beta2, and TR-alpha1, share strong homology in their DNA- and ligand-binding domains but differ in their amino-terminal domains. Because TR-beta2 exhibits greater T(3)-independent activation on TREs than other TR isoforms, we wanted to determine whether coactivators bound to TR-beta2 in the absence of ligand. Our results show that TR-beta2, unlike TR-beta1 or TR-alpha1, is able to bind certain coactivators (CBP, SRC-1, and pCIP) in the absence of T(3) through a domain which maps to the amino-terminal half of its A/B domain. This interaction is specific for certain coactivators, as TR-beta2 does not interact with other co-factors (p120 or the CBP-associated factor (pCAF)) in the absence of T(3). The minimal TR-beta2 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 T(3)-independent coactivator recruitment to the transcription complex via the AF-1 domain.

p120 acts as a specific coactivator for 9-cis-retinoic acid receptor (RXR) on peroxisome proliferator-activated receptor-gamma/RXR heterodimers.

p120 was originally isolated as a novel nuclear co-activator for thyroid hormone receptor. In this study, we characterized its interaction and transactivation of peroxisome proliferator-activated receptor-gamma (PPARgamma) and 9-cis-retinoic acid receptor (RXR) heterodimers. Transient transfection study revealed that p120 enhanced the transcriptional activation of PPARgamma/RXR induced by PPARgamma- or RXR-specific ligands. In the glutathione-S-transferase pull-down assay, while steroid receptor coactivator-1 showed apparent interactions with both RXR and PPARgamma, p120 bound only to RXR in a 9-cis-retinoic acid (RA)-dependent manner and also did not bind to PPARgamma even in the presence of thiazolidinediones. The yeast two-hybrid analysis showed no interaction of p120 with PPARgamma under any conditions, and electophoretic mobility shift assay showed apparent DNA-PPARgamma/RXR/p120 complex formation only in the presence of 9-cis-RA. Furthermore, the yeast three-hybrid assay clearly revealed a significant interaction between p120 and PPARgamma via RXR of PPARgamma/RXR heterodimer only in the presence of 9-cis-RA. These findings indicate that p120 acts as a specific co-activator for the RXR of PPARgamma/RXR heterodimer in a 9-cis-RA-dependent manner.

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 beta isoform of the thyroid hormone receptor (TR-beta). To date, three RTH mutations reportedly with near-normal T3 binding (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 T3 concentrations. 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.

Two separate NCoR (nuclear receptor corepressor) interaction domains mediate corepressor action on thyroid hormone response elements.

The nuclear corepressor (NCoR) binds to the thyroid hormone receptor (TR) in the absence of ligand. NCoR-TR interactions are mediated by two interaction domains in the C-terminal portion of NCoR. Binding of NCoR to TR results in ligand-independent repression on positive thyroid hormone response elements. The interactions between NCoR interaction domains and TR on DNA response elements, however, have not been well characterized. We have found that both interaction domains are capable of binding TR on thyroid hormone response elements. In addition, the NCoR interaction domains interact much more strongly with the TR than those present in the silencing mediator of retinoic acid and TRs (SMRT). Furthermore, deletion of either NCoR interaction domain does not significantly impair ligand-independent effects on positive or negative thyroid hormone response elements. Finally, both NCoR interaction domains appear to preferentially bind TR homodimer over TR-retinoid X receptor heterodimer in electrophoretic mobility shift assays. These data suggest that either NCoR interaction domain is capable of mediating the ligand-independent effects of TR on positive and negative thyroid hormone response elements.

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 unique role of the beta-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-beta2, 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-beta2 achieved significantly greater ligand-independent activation on the thyrotropin-releasing hormone (TRH) and common glycoprotein alpha-subunit genes than either TR-beta1 or TR-alpha1. A chimeric TR-beta isoform containing the TR-beta2 amino terminus linked to the TR-alpha1 DNA- and ligand-binding domains functioned like the TR-beta2 isoform on these promoters, confirming that the amino terminus of TR-beta2 was both necessary and sufficient to mediate this effect. By constructing deletion mutants of the TR-beta2 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-beta2. We conclude that the central nervous system-restricted TR-beta2 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-beta2.