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.

A circulating, biologically inactive thyrotropin caused by a mutation in the beta subunit gene.

Mutation of a critical carboxy-terminal cysteine residue (C105V) in the thyrotropin-beta (TSH-beta) subunit gene was found in two related families with central hypothyroidism. Affected patients had low thyroid hormone levels and radioactive iodine uptake in the thyroid gland associated with measurable serum TSH. Thyrotropin-releasing hormone-stimulated TSH secretion did not increase thyroid hormone production in these patients as compared to their unaffected siblings, suggesting that the mutant TSH was biologically inactive in vivo. Recombinant TSH harboring this mutation was confirmed to be biologically inactive in an in vitro bioassay. Based on crystallographic structure of chorionic gonadotropin, a disulfide bond between C19 and C105 in the TSH-beta subunit is predicted to form the "buckle" of a "seat belt" that surrounds the common alpha subunit and maintains the conformation and bioactivity of the hormone. This natural mutation of the TSH-beta subunit confirms the importance of the seat belt in the family of pituitary and placental glycoprotein hormones.

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.

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.

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.

The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements.

TRH is the principal positive regulator of TSH synthesis and secretion in man. T3 is able to control TRH synthesis through feedback inhibition at the transcriptional level, presumably by binding to its receptor which interacts with one or more negative thyroid hormone response elements (TREs) present within the human TRH promoter. In the present study we have identified the specific negative TREs within the TRH promoter and characterized their ability to interact with thyroid hormone receptors (TRs), and the retinoid X receptor (RXR). Our analysis demonstrates that ligand-independent and dependent regulation of the human TRH promoter is restricted to the TR beta 1 isoform. Deletional analysis of the TRH promoter identified two discrete regions that are responsible for mediating ligand-dependent negative regulation of the TRH promoter. Mutagenesis of potential TR binding half-sites within these regions identified three separate half-sites (site 4 from -55 to -60 base pairs (bp); site 5, +14 to +19 bp; and site 6, +37 to +42 bp) which act in combination to allow for negative regulation. Mutation and/or deletion of each of these sites leads to a loss of negative regulation of the TRH promoter by T3. Gel-mobility shift assays of site 4 and its surrounding nucleotides revealed that this region of the promoter is capable of binding TR monomers, homodimers, and TR-RXR heterodimers. Mutagenesis of site 4 leads to a loss of all binding to this region. The region encompassing sites 5 and 6 binds only TR monomer, and the addition of RXR to the binding reaction leads to a loss of specific monomeric binding. To assess the functional importance of site 4 and its surrounding nucleotides we cotransfected RXR isoforms along with TR beta with TRH promoter constructs containing either site 4 or its mutant. In the presence of wild type site 4 sequence, cotransfected RXR enhanced negative regulation of the TRH promoter. Mutation and or deletion of site 4 leads to a loss of this enhancement. These data demonstrate that two structurally different negative TREs cooperate to allow for negative regulation of the human TRH promoter and that negative regulation is TR isoform-specific and modulated by the RXR-signaling pathway through a novel negative TRE.

Isoform variable action among thyroid hormone receptor mutants provides insight into pituitary resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is due to mutations in the beta-isoform of the thyroid hormone receptor (TR-beta). The mutant TR interferes with the action of normal TR to cause the clinical syndrome. Selective pituitary resistance to thyroid hormone (PRTH) results in inappropriate TSH secretion and peripheral sensitivity to elevated thyroid hormone levels. Association of the PRTH phenotype with in vitro behavior of the mutant TR has proved elusive. Alternative exon utilization results in two TR-beta isoforms, TR-beta 1 and TR-beta 2, which differ only in their amino termini. Although the TR-beta 1 isoform is ubiquitous, the TR-beta 2 isoform is found predominantly in the anterior pituitary and brain. To date, in vitro evaluation of RTH mutations has focused on the TR-beta 1 isoform. Site-directed mutagenesis was used to create several PRTH (R338L, R338W, V349M, R429Q, I431T) and generalized RTH (delta 337T, P453H) mutations in both TR-beta isoforms. The ability of mutant TRs to act as dominant negative inhibitors of wild type TR-beta function on positive and negative thyroid hormone response elements (pTREs and nTREs, respectively) was evaluated in transient transfection assays. PRTH mutants had no significant dominant negative activity as TR-beta 1 isoforms on pTREs found in peripheral tissues or on nTREs found on genes regulating TSH synthesis. PRTH mutants, in contrast, had strong dominant negative activity on these same nTREs as TR-beta 2 isoforms. Cotransfected retinoid X receptor-alpha was required for negative T3 regulation via the TR-beta 1 isoform but was not necessary for negative regulation via the TR-beta 2 isoform in CV-1 cells. The differing need for retinoid X receptor cotransfection demonstrates two distinct negative T3-regulatory pathways, one mediated by the TR-beta 1 and the other mediated by TR-beta 2. The selective effect of PRTH mutations on the TR-beta 2 isoform found in the hypothalamus and pituitary vs. the TR-beta 1 isoform found in peripheral tissues suggests a molecular mechanism for the PRTH disorder.

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.

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.

Dynamic regulation of gonadotropin-releasing hormone receptor mRNA levels in the anterior pituitary gland during the rat estrous cycle.

The number of gonadotropin-releasing hormone (GnRH) receptors on pituitary gonadotropes varies substantially during the rat estrous cycle and may modulate pituitary responsiveness to GnRH. The present studies were undertaken to determine to what extent these changes in GnRH receptor number reflect a change in GnRH receptor mRNA expression in the anterior pituitary gland. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), pituitary GnRH receptor mRNA levels were measured at various timepoints throughout the rat estrous cycle. There was a three-fold increase in GnRH receptor mRNA levels on the afternoon of proestrus (PRO) when compared to levels observed on the morning of metestrus (MET). This rise preceded the onset of the LH surge by 6h (1200h). GnRH receptor mRNA levels remained elevated through 2100h PRO, after which they dropped dramatically, and by 2400h PRO were not significantly different from levels observed at 0900h MET. A two-fold increase in GnRH receptor mRNA expression was also observed during the early stages of the estrous cycle (0900h to 1800h MET), and this increase was sustained until 1800h on diestrus, at which time mRNA levels decreased to levels observed at 0900h MET. These results demonstrate that pituitary GnRH receptor mRNAs are dynamically regulated during the rat estrous cycle, with receptor mRNA expression being greatest on the afternoon of PRO, the time of the estrous cycle at which gonadotropes are most sensitive to GnRH stimulation.

Hypercalcemia with low-normal serum intact PTH: a novel presentation of primary hyperparathyroidism.

The diagnosis of primary hyperparathyroidism has rested on the finding of hypercalcemia coupled with an elevated serum parathyroid hormone (PTH) level. Over 300 consecutive patients with primary hyperparathyroidism have had elevated or high-normal serum PTH levels using a specific immunoradiometric assay. Here we present a patient who proved to have surgically documented primary hyperparathyroidism in whom PTH levels were completely normal in all assays used. In the immunoradiometric assay, his normal result was unprecedentedly low (17 to 28 pg/mL; normal, 10 to 60 pg/mL) for this condition, and in a range consistent with non-PTH-dependent hypercalcemia or familial hypocalciuric hypercalcemia. This rare biochemical presentation of primary hyperparathyroidism should be considered in the differential diagnosis of hypercalcemia.

Multiple promoter elements in the human chorionic gonadotropin beta subunit genes distinguish their expression from the luteinizing hormone beta gene.

The beta subunit of human chorionic gonadotropin (CG beta) is encoded by a cluster of six genes, which have developed through gene duplication from an ancestral LH beta gene. Despite approximately 90% sequence homology between the CG beta and LH beta promoters, the CG beta gene is expressed in the placenta, whereas the LH beta promoter is active only in the pituitary. The CG beta gene uses a TATA-less promoter that is located upstream of the transcriptional start site used by the homologous LH beta gene. The purpose of this study was to use the high degree of homology among members of the CG beta gene cluster and between the CG beta and LH beta promoters to localize regulatory elements that confer CG beta expression in the placenta. The 5'-flanking regions of the different CG beta genes were cloned and expressed in JEG-3 placental cells. Naturally occurring sequence variations were correlated with promoter activity and used to identify candidate regulatory elements. Exchanges of homologous sequences in the CG beta 5 and LH beta proximal identified three separate regions between -362 and +104 that are necessary for full basal expression of the CG beta promoter. Site-directed mutagenesis of four evolutionarily divergent sequences near the CG beta transcription start site confirmed the importance of multiple distinct regulatory elements as each of these mutations resulted in an 80% decrease in promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.