William M. Wood

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

The molecular determinants governing cell-specific expression of the thyrotropin (TSH) β-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHβ promoter (−133 to −88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSHβ promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.

Signaling Pathways Responsible for Fetal Gene Induction in the Failing Human Heart Evidence for Altered Thyroid Hormone Receptor Gene Expression

Background —We have previously demonstrated that changes in myosin heavy chain (MHC) isoforms that occur in failing human hearts resemble the pattern produced in rodent myocardium in response to hypothyroidism. Because thyroid hormone status is usually within normal limits in these patients, we hypothesized that failing/hypertrophied human myocardium might have a defect in thyroid hormone signaling due to alterations in expression of thyroid hormone receptors (TRs). Methods and Results —To examine this hypothesis, we used RNase protection assay to measure mRNA levels of TRs in failing left ventricles that exhibited a fetal pattern of gene expression, ie, decreased expression of &agr;-MHC with increased &bgr;-MHC expression compared with left ventricles from age-matched controls. We detected expression of TR-&agr;1, -&agr;2, and -&bgr;1 isoforms in human left ventricles. In failing left ventricles, TR-&agr;1 was downregulated, whereas TR-&agr;2, a splice variant that does not bind thyroid hormone but inhibits responses to liganded TRs, was increased. Expression levels of TR-&bgr;1 did not differ significantly between the 2 groups. According to linear regression analysis, expression levels of TR-&agr;1 and -&agr;2 were positively and negatively correlated with those of &agr;-MHC, respectively. Conclusions —We conclude that decreases in TR-&agr;1 and increases in TR-&agr;2 may lead to local attenuation of thyroid hormone signaling in the failing human heart and that the resulting tissue-specific hypothyroidism is a candidate for the molecular mechanism that induces fetal gene expression in the failing human ventricle.

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

ABSTRACT 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.

Methamphetamine preconditioning alters midbrain transcriptional responses to methamphetamine-induced injury in the rat striatum.

Methamphetamine (METH) is an illicit drug which is neurotoxic to the mammalian brain. Numerous studies have revealed significant decreases in dopamine and serotonin levels in the brains of animals exposed to moderate-to-large METH doses given within short intervals of time. In contrast, repeated injections of small nontoxic doses of the drug followed by a challenge with toxic METH doses afford significant protection against monoamine depletion. The present study was undertaken to test the possibility that repeated injections of the drug might be accompanied by transcriptional changes involved in rendering the nigrostriatal dopaminergic system refractory to METH toxicity. Our results confirm that METH preconditioning can provide significant protection against METH-induced striatal dopamine depletion. In addition, the presence and absence of METH preconditioning were associated with substantial differences in the identity of the genes whose expression was affected by a toxic METH challenge. Quantitative PCR confirmed METH-induced changes in genes of interest and identified additional genes that were differentially impacted by the toxic METH challenge in the presence of METH preconditioning. These genes include small heat shock 27 kD 27 protein 2 (HspB2), thyrotropin-releasing hormone (TRH), brain derived neurotrophic factor (BDNF), c-fos, and some encoding antioxidant proteins including CuZn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx)-1, and heme oxygenase-1 (Hmox-1). These observations are consistent, in part, with the transcriptional alterations reported in models of lethal ischemic injuries which are preceded by ischemic or pharmacological preconditioning. Our findings suggest that multiple molecular pathways might work in tandem to protect the nigrostriatal dopaminergic pathway against the deleterious effects of the toxic psychostimulant. Further analysis of the molecular and cellular pathways regulated by these genes should help to provide some insight into the neuroadaptive potentials of the brain when repeatedly exposed to drugs of abuse.

Analysis of Pit-1 in regulating mouse TSH β promoter activity in thyrotropes

Abstract TSHβ gene expression is restricted to pituitary thyrotropes. Since Pit-1 is present in these cells, we characterized Pit-1 RNA and protein in thyrotropes, and tested its function in regulating TSHβ promoter activity. We demonstrate that both TtT-97 thyrotropic tumors and pituitaries contain four Pit-1 transcripts of 3.2, 2.6, 2.4, and 1.9 kb, respectively. Only two transcripts of 2.7 and 2.1 kb were detected in αTSH cells, a thyrotrope derived cell that no longer expresses TSHβ. Western analysis revealed Pit-1 protein in TtT-97 cells but not in αTSH cells. DNase I protection assays localized Pit-1 binding to three areas of the mouse TSHβ promoter. However, basal TSHβ promoter activity was minimally stimulated when αTSH cells or TtT-97 thyrotropes were co-transfected with mouse Pit-1 and a mTSHβ luciferase construct. These studies suggest that Pit-1 is not limiting for cell-specific expression of the TSHβ gene in thyrotrope-derived cells and implies that additional thyrotropic factors are likely required.

Determinants of thyrotrope-specific thyrotropin beta promoter activation. Cooperation of Pit-1 with another factor.

Thyrotropin (TSH) β is a subunit of TSH, the expression of which is limited to the thyrotrope cells of the anterior pituitary gland. We have utilized the thyrotrope-derived TtT-97 thyrotropic tumors to investigate tissue-specific expression of the TSHβ promoter. TSHβ promoter activity in thyrotropes is conferred by sequences between −270 and −80 of the 5′-flanking region. We have recently reported that the proximal region from −133 to −100 (P1) is required for promoter expression in thyrotropes. This region interacts with the pituitary-specific transcription factor Pit-1. While Pit-1 appears necessary for TSHβ promoter activity in thyrotropes, this transcription factor is not alone sufficient for promoter activity in pituitary-derived cells. In this report, we have generated a series of promoter mutations in the P1 region to identify additional protein-DNA interactions and determine their functional significance. We have found that Pit-1 interacts with the distal portion of the P1 region, and a second protein interacts with the proximal segment of this region. Each protein is able to independently interact with the TSHβ promoter, but neither alone can maintain promoter activity. Both proteins appear to be necessary for full promoter activity in thyrotropes. Southwestern analysis with the proximal segment of the P1 region (−117 to −88) reveals interaction with a 50-kDa protein. Interestingly, this protein is not found in the pituitary-derived GH3 cells and may represent a thyrotrope-specific transcription factor. Further characterization of this newly identified DNA-binding protein will further our understanding of the tissue-specific expression of the TSHβ gene.

Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer

BackgroundThyroid cancer is the most common endocrine malignancy, and many patients with metastatic differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) fail to respond to conventional therapies, resulting in morbidity and mortality. Additional therapeutic targets and treatment options are needed for these patients. We recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ATC and confers an aggressive phenotype when overexpressed in DTC cells.MethodsMicroarray analysis was used to identify downstream targets of PPARγ in ATC cells. Western blot analysis and immunohistochemistry (IHC) were used to assess thioredoxin interacting protein (TXNIP) expression in thyroid cancer cell lines and primary tumor specimens. Retroviral transduction was used to generate ATC cell lines that overexpress TXNIP, and assays that assess glucose uptake, viable cell proliferation, and invasion were used to characterize the in vitro properties of these cells. An orthotopic thyroid cancer mouse model was used to assess the effect of TXNIP overexpression in ATC cell lines in vivo.ResultsUsing microarray analysis, we show that TXNIP is highly upregulated when PPARγ is depleted from ATC cells. Using Western blot analysis and IHC, we show that DTC and ATC cells exhibit differential TXNIP expression patterns. DTC cell lines and patient tumors have high TXNIP expression in contrast to low or absent expression in ATC cell lines and tumors. Overexpression of TXNIP decreases the growth of HTh74 cells compared to vector controls and inhibits glucose uptake in the ATC cell lines HTh74 and T238. Importantly, TXNIP overexpression in T238 cells results in attenuated tumor growth and decreased metastasis in an orthotopic thyroid cancer mouse model.ConclusionsOur findings indicate that TXNIP functions as a tumor suppressor in thyroid cells, and its downregulation is likely important in the transition from differentiated to advanced thyroid cancer. These studies underscore the potential of TXNIP as a novel therapeutic target and prognostic indicator in advanced thyroid cancer.

Growth Factor Signals in Neural Cells COHERENT PATTERNS OF INTERACTION CONTROL MULTIPLE LEVELS OF MOLECULAR AND PHENOTYPIC RESPONSES

Individual neurons express receptors for several different growth factors that influence the survival, growth, neurotransmitter phenotype, and other properties of the cell. Although there has been considerable progress in elucidating the molecular signal transduction pathways and physiological responses of neurons and other cells to individual growth factors, little is known about if and how signals from different growth factors are integrated within a neuron. In this study, we determined the interactive effects of nerve growth factor, insulin-like growth factor 1, and epidermal growth factor on the activation status of downstream kinase cascades and transcription factors, cell survival, and neurotransmitter production in neural cells that express receptors for all three growth factors. We document considerable differences in the quality and quantity of intracellular signaling and eventual phenotypic responses that are dependent on whether cells are exposed to a single or multiple growth factors. Dual stimulations that generated the greatest antagonistic or synergistic actions, compared with a theoretically neutral summation of their two activities, yielded the largest eventual change of neuronal phenotype indicated by the ability of the cell to produce norepinephrine or resist oxidative stress. Combined activation of insulin-like growth factor 1 and epidermal growth factor receptors was particularly notable for antagonistic interactions at some levels of signal transduction and norepinephrine production, but potentiation at other levels of signaling and cytoprotection. Our findings suggest that in true physiological settings where multiple growth factors are present, activation of one receptor type may result in molecular and phenotypic responses that are different from that observed in typical experimental paradigms in which cells are exposed to only a single growth factor at a time.

Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSHβ 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.

Human progesterone A-receptors can be synthesized intracellularly and are biologically functional

In order to investigate the origin and functional independence of the human progesterone receptor A binding protein, we have expressed a truncated human progesterone receptor cDNA in both gene transfer and in vitro translation assays. Proteins identical in size and antigenicity to the A-receptors found naturally in human progesterone target cells are synthesized from this cDNA that lacks the putative B receptor initiator methionine codon of the complete cDNA. The functional independence of A-receptors is suggested by their ability to bind hormone and to stimulate transcription from the progestin responsive mouse mammary tumor virus promoter.