GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

MED220/thyroid receptor-associated protein 220 functions as a transcriptional coactivator with Pit-1 and GATA-2 on the thyrotropin-beta promoter in thyrotropes.

Mediator (MED) 220/thyroid receptor-associated protein (TRAP) 220 is a transcriptional mediator that interacts with liganded thyroid/steroid hormone receptors. MED220 haploinsufficient heterozygotes exhibited hypothyroidism and reduced TSHbeta transcripts, suggesting a specific function for TSHbeta transcription. We previously demonstrated that Pit-1 and GATA-2 can bind to a composite element within the proximal TSHbeta promoter and synergistically activate transcription. We detected MED220 expression in TtT-97 thyrotropes by Northern and Western blot analysis. Cotransfections in CV-1 cells showed that Pit-1, GATA-2, or MED220 alone did not markedly stimulate the TSHbeta promoter. However, Pit-1 plus GATA-2 resulted in an 10-fold activation, demonstrating synergistic cooperativity. Titration of MED220 resulted in a further dose-dependent stimulation up to 25-fold that was promoter specific. Glutathione-S-transferase interaction studies showed that MED220 or GATA-2 each bound the homeodomain of Pit-1, whereas MED220 interacted independently with each zinc finger of GATA-2 but not with either terminus. MED220 interacted with GATA-2 and Pit-1 over a broad region of its N terminus. These regions of interaction were also important for maximal function. Coimmunoprecipitation confirmed that all three factors can interact in thyrotropes and chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSHbeta promoter. Thus, the TSHbeta gene is maximally activated by a combination of three thyrotrope transcription factors that act via both protein-DNA and protein-protein interactions.

Pituitary-specific Gata2 knockout: effects on gonadotrope and thyrotrope function.

GATA2 is expressed in the pituitary during development and in adult gonadotropes and thyrotropes. It is proposed to be important for gonadotrope and thyrotrope cell fate choice and for TSH production. To test this idea, we produced a pituitary-specific knockout of Gata2, designed so that the DNA-binding zinc-finger region is deleted in the presence of a pituitary-specific recombinase transgene. These mice have reduced secretion of gonadotropins basally and in response to castration challenge, although the mice are fertile. GATA2 deficiency also compromises thyrotrope function. Mutants have fewer thyrotrope cells at birth, male Gata2-deficient mice exhibit growth delay from 3-9 wk of age, and adult mutants produce less TSH in response to severe hypothyroidism after radiothyroidectomy. Therefore, Gata2 appears to be dispensable for gonadotrope and thyrotrope cell fate and maintenance, but important for optimal gonadotrope and thyrotrope function. Gata2-deficient mice exhibit elevated levels of Gata3 transcripts in the pituitary gland, suggesting that GATA3 can compensate for GATA2.

The proliferative status of thyrotropes is dependent on modulation of specific cell cycle regulators by thyroid hormone.

In this report we have examined changes in cell growth parameters, cell cycle effectors, and signaling pathways that accompany thyrotrope growth arrest by thyroid hormone (TH) and growth resumption after its withdrawal. Flow cytometry and immunohistochemistry of proliferation markers demonstrated that TH treatment of thyrotrope tumors resulted in a reduction in the fraction of cells in S-phase that is restored upon TH withdrawal. This is accompanied by dephosphorylation and rephosphorylation of retinoblastoma (Rb) protein. The expression levels of cyclin-dependent kinase 2 and cyclin A, as well as cyclin-dependent kinase 1 and cyclin B, were decreased by TH, and after withdrawal not only did these regulators of Rb phosphorylation and mitosis increase in their expression but so too did the D1 and D3 cyclins. We also noted a rapid induction and subsequent disappearance of the type 5 receptor for the growth inhibitor somatostatin with TH treatment and withdrawal, respectively. Because somatostatin can arrest growth by activating MAPK pathways, we examined these pathways in TtT-97 tumors and found that the ERK pathway and several of its upstream and downstream effectors, including cAMP response element binding protein, were activated with TH treatment and deactivated after its withdrawal. This led to the hypothesis that TH, acting through increased type 5 somatostatin receptor, could activate the ERK pathway leading to cAMP response element binding protein-dependent decreased expression of critical cell cycle proteins, specifically cyclin A, resulting in hypophosphorylation of Rb and its subsequent arrest of S-phase progression. These processes are reversed when TH is withdrawn, resulting in an increase in the fraction of S-phase cells.

Atypical expression of type 2 iodothyronine deiodinase in thyrotrophs explains the thyroxine-mediated pituitary thyrotropin feedback mechanism.

T(4), the main product of thyroid secretion, is a critical signal in plasma that mediates the TSH-negative feedback mechanism. As a prohormone, T(4) must be converted to T(3) to acquire biological activity; thus, type 2 iodothyronine deiodinase (D2) is expected to play a critical role in this feedback mechanism. However, the mechanistic details of this pathway are still missing because, counterintuitively, D2 activity is rapidly lost in the presence of T(4) by a ubiquitin-proteasomal mechanism. In the present study, we demonstrate that D2 and TSH are coexpressed in rat pituitary thyrotrophs and that hypothyroidism increases D2 expression in these cells. Studies using two murine-derived thyrotroph cells, TtT-97 and TalphaT1, demonstrate high expression of D2 in thyrotrophs and confirm its sensitivity to negative regulation by T(4)-induced proteasomal degradation of this enzyme. Despite this, expression of the Dio2 gene in TalphaT1 cells is higher than their T(4)-induced D2 ubiquitinating capacity. As a result, D2 activity and net T(3) production in these cells are sustained, even at free T(4) concentrations that are severalfold above the physiological range. In this system, free T(4) concentrations and net D2-mediated T(3) production correlated negatively with TSHbeta gene expression. These results resolve the apparent paradox between the homeostatic regulation of D2 and its role in mediating the critical mechanism by which T(4) triggers the TSH-negative feedback.

Growth arrest of thyrotropic tumors by thyroid hormone is correlated with novel changes in Wnt-10A.

The molecular mechanism underlying thyroid hormone inhibition of thyrotrope cell growth is poorly understood. A comprehensive screen for T3-regulated genes involved in thyrotrope cell regulation was performed by Affymetrix MGU74A Genechip microarray analyses, which compared total RNA from hypothyroid versus 24 h T3-treated TtT-97 tumors. Of the 13,000 genes screened, a number of novel, T3-responsive candidate genes were identified. Within the Wnt family of growth factors, only Wnt-10A transcripts were abundantly expressed in hypothyroid TtT-97 tumors, and were down-regulated with T3 by 6 h of treatment. In addition, nuclear beta-catenin, which is a downstream mediator of canonical Wnt signaling, was decreased at the protein and functional levels. TtT-97 growth suppression was associated with decreased cyclin A transcript levels. We conclude that treatment of thyrotropic TtT-97 tumors with T3 resulted in the decreased expression of Wnt-10A, and that thyroid hormone may inhibit growth via cyclin A regulation.

Mutational analysis of the mouse somatostatin receptor type 5 gene promoter.

We have previously characterized the structure of the murine somatostatin receptor type 5 gene (sst5). Initial transient transfection studies in pituitary somatolactotropes (GH(3)) mapped the promoter activity of this gene to a region 290 bp upstream of the transcription start site. The current study identifies the sst5 promoter region critical for basal activity. A series of deletions was generated, and promoter activity was localized to a region between -83 and -19. Similar promoter deletion patterns were evident in five pituitary cell types. Seven 10-bp transversion mutations encompassing the region between -83 and -19 were generated, and functional activity was assessed. Promoter activity was reduced by the mutations spanning -67 to -47 compared with the wild-type construct. Another mutation between -26 and -17 resulted in promoter activity reduction in GH(3) cells, but not TtT-97 thyrotropes. Deoxyribonuclease I protection analysis of the sst5 promoter region between -208/+47 was performed using GH(3) and TtT-97 nuclear extracts. The most striking protected regions, located between -61 and -41 and -25 and -3, correlated with functionally important regions identified by transfection studies. In summary, the mouse sst5 gene promoter has been characterized, and functional activity and nuclear factor interactions were mapped to two specific promoter regions. The region between -67 and -47 appears to contain a nucleotide sequence critical for basal transcriptional regulation of the mouse sst5 gene in pituitary cells.

Early gene expression changes preceding thyroid hormone-induced involution of a thyrotrope tumor.

Treatment with thyroid hormone (TH) results in shrinkage of a thyrotropic tumor grown in a hypothyroid host. We used microarray and Northern analysis to assess the changes in gene expression that preceded tumor involution. Of the 1,176 genes on the microarray, 7 were up-regulated, whereas 40 were decreased by TH. Many of these were neuroendocrine in nature and related to growth or apoptosis. When we examined transcripts for cell cycle regulators only cyclin-dependent kinase 2, cyclin A and p57 were down-regulated, whereas p15 was induced by TH. Retinoblastoma protein, c-myc, and mdm2 were unchanged, but E2F1 was down-regulated. TH also decreased expression of brain-derived neurotrophic factor, its receptor trkB, and the receptor for TRH. These, in addition to two other genes, neuronatin and PB cadherin, which were up- and down-regulated, respectively, showed a more rapid response to TH than the cell cycle regulators and may represent direct targets of TH. Finally, p19ARF was dramatically induced by TH, and although this protein can stabilize p53 by sequestering mdm2, we found no increase in p53 protein up to 48 h of treatment. In summary, we have described early changes in the expression of genes that may play a role in TH-induced growth arrest of a thyrotropic tumor. These include repression of specific growth factor and receptors and cell cycle genes as well as induction of other factors associated with growth arrest and apoptosis.

Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSH beta gene.

Previous studies showed that Pit-1 functionally cooperates with GATA-2 to stimulate transcription of the TSH beta gene. Pit-1 and GATA-2 are uniquely coexpressed in pituitary thyrotropes and activate transcription by binding to a composite promoter element. To define the domains of Pit-1 important for functional cooperativity with GATA-2, we cotransfected a set of Pit-1 deletions with an mTSH beta-luciferase reporter. Plasmids were titrated to express equivalent amounts of protein. A mutant containing a deletion of the hinge region between the POU and homeodomains retained the ability to fully synergize with GATA-2. In contrast, mutants containing deletions of amino acids 2-80 or 72-125 demonstrated 56 or 34% of the synergy found with the full-length protein, suggesting that these regions contributed to cooperativity. Mutants with deletions of the POU-specific or homeodomain further reduced the effect signifying the requirement for DNA binding. GST interaction studies demonstrated that only the homeodomain of Pit-1 interacted with GATA-2. Finally, several mutations between the Pit-1 and GATA-2 sites on the TSH beta promoter reduced binding for each factor and greatly reduced ternary complex formation. Thus multiple domains of Pit-1 are required for full synergy with GATA-2 and sequences between the two binding sites contribute to co-occupancy with both factors on the proximal TSH beta promoter.

Congenital isolated central hypothyroidism caused by a "hot spot" mutation in the thyrotropin-beta gene.

Two adult siblings presented to our practice with a known history of congenital central isolated hypothyroidism. Their growth, development, and general health had been normal. Although the disorder was known to result from thyrotropin (TSH) deficiency, providers in the past had made multiple adjustments in their levothyroxine replacement doses in attempts to normalize serum TSH levels. This suggests a need for better education of providers who care for patients with central hypothyroidism. We performed DNA sequencing of the TSHbeta gene and identified a homozygous single base deletion in codon 105, on exon 3, resulting in a frameshift and a premature termination signal at codon 114. This same mutation (C105FS114X) has been previously reported in South America and Europe and appears to be the most common genetic mutation associated with congenital isolated TSH deficiency. The identification of this mutation for the first time in the United States suggests that this disorder, now described in patients from countries on multiple continents, is more common than previously appreciated and may be a mutational "hot spot."

Pituitary tumors arising from glycoprotein hormone alpha-subunit-deficient mice contain transcription factors and receptors present in thyrotropes.

Glycoprotein-hormone alpha-subunit deficient (alphaSUnull) mice are hypothyroid and hypogonadal due to the absence of functional TSH, LH and FSH, despite normal production of the corresponding beta subunits. Pituitary tumors spontaneously developing in alphaSUnull mice were propagated in hypothyroid mice. The purpose of the current studies was to compare the gene expression profile of these alphaSUnull tumors with previously characterized TtT-97 thyrotropic tumors. A group of animals bearing each tumor type was treated with thyroid hormone (T4) prior to tumor removal. Both tumor types equally expressed TSHbeta mRNA, which significantly decreased when exposed to T4, whereas alpha-subunit mRNA was absent in alphaSUnull tumors. Northern blot analysis was performed using cDNA probes for the following transcription factors: Pit1, GATA2, pLIM, Msx1, Ptx1 and Ptx2. Both tumors were found to contain identical transcripts with similar responses to T4, with the exception of Pit1. In contrast to the signal pattern seen in TtT-97, only two bands were seen in alphaSUnull tumors, which were similar in size to those in alphaTSH cells, a thyrotropic cell line that lacks TSHbeta-subunit expression and Pit1 protein. However, western blot analysis revealed a protein band in the alphaSUnull tumors consistent with Pit1, while this signal was absent in alphaTSH cells. Northern blot analysis was also performed with specific cDNA probes for the following receptors: TRbeta1, TRbeta2, TRalpha1, non-T3 binding alpha2, RXRgamma and Sst5. Similarly-sized transcripts were found in both types of tumor, although the signal for Sst5 was seen in T4-treated alphaSUnull tumors only with a more sensitive RT-PCR analysis. The overall similarity between the two tumor types renders the alphaSUnull tumor as a suitable thyrotropic tumor model.

Isolation and functional analysis of the mouse RXRgamma1 gene promoter in anterior pituitary cells.

The retinoid X receptor (RXR) isoform RXRgamma has limited tissue expression, including brain, skeletal muscle, and anterior pituitary gland. Within the anterior pituitary gland, RXRgamma expression is limited primarily to the thyrotropes. In this report, we have isolated approximately 3 kb of 5'-flanking DNA of the mouse RXRgamma1 gene. We have identified the major transcription start site in the thyrotrope-derived TtT-97 cells. Transient transfection studies show that a 1.4-kb promoter fragment has full promoter activity in TtT-97 cells. This promoter has much less activity in thyrotrope-derived alphaTSH cells, pituitary-derived GH3 somatomammotropes, and non-pituitary CV-1 cells. None of these cell lines has detectable RXRgamma1 mRNA. A previous report has identified a non-consensus direct repeat (DR-1) element in the RXRgamma2 gene promoter region that mediates stimulation of promoter activity by 9-cis-retinoic acid (9-cis-RA). Inspection of the RXRgamma1 promoter region revealed a non-consensus DR-1 element at -232 bp from the transcription start site. Interestingly, RXRgamma1 promoter activity was suppressed 50% by 9-cis-RA in the TtT-97 thyrotropes. Further experiments in non-pituitary cells showed that suppression of RXRgamma1 promoter activity was RXR-dependent. Mutagenesis of the DR-1 element abrogated suppression of promoter activity by 9-cis-RA, suggesting that this negative regulation requires both RXR and this specific DR-1 element. In summary, we have isolated the mouse RXRgamma1 gene promoter region and identified the major start site in thyrotropes. Promoter activity is uniquely suppressed by 9-cis-RA through a DR-1 element. Isolation and characterization of the mouse RXRgamma1 promoter region provides a tool for further investigation focusing on thyrotrope-specific gene expression as well as negative regulation of genes by retinoic acid.