scholarly journals Glucose and thyroid hormone co-regulate the expression of the intestinal fructose transporter GLUT5

1999 ◽  
Vol 339 (2) ◽  
pp. 233-239 ◽  
Author(s):  
Miriam MATOSIN-MATEKALO ◽  
José E. MESONERO ◽  
Thibaut J. LAROCHE ◽  
Michel LACASA ◽  
Edith BROT-LAROCHE
Keyword(s):  
Thyroid Hormone ◽  
Reporter Gene ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Culture Media ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Glucose Responsiveness

Expression of the fructose transporter GLUT5 in Caco-2 cells is controlled by the carbohydrate content of the culture media [Mesonero, Matosin, Cambier, Rodriguez-Yoldi and Brot-Laroche (1995) Biochem. J. 312, 757–762] and by the metabolic status of the cells [Mahraoui, Takeda, Mesonero, Chantret, Dussaulx, Bell, and Brot-Laroche (1994) Biochem. J. 301, 169–175]. In this study we show that, in fully differentiated Caco-2/TC7 cells, thyroid hormone and glucose increase GLUT5 mRNA abundance in a dose-dependent manner. Using Caco-2/TC7 cells stably transformed with various fragments of the GLUT5 promoter inserted upstream of the luciferase reporter gene, we localized the sequences that confer 3,3´,5-l-tri-iodothyronine (T3)- and/or glucose-sensitivity to the gene. Glucose responsiveness is conferred by the -272/+41 fragment of the promoter, but it is only with the -338/+41 region that transcription of the luciferase reporter gene is stimulated by T3. This 70 bp fragment from position -338 to -272 of the GLUT5 gene is able to confer T3/glucose-responsiveness to the heterologous thymidine kinase promoter. Electrophoretic-mobility-shift assays demonstrate that thyroid hormone receptors α and β are expressed in Caco-2/TC7 cells. They further show that the -308/-290 region of the GLUT5 promoter binds thyroid hormone receptor/retinoid X receptor heterodimers, and that glucose and/or T3 exert a deleterious effect on the binding of the nuclear protein complex.

In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

1999 ◽  
Vol 276 (4) ◽  
pp. C883-C891 ◽  
Author(s):  
Carola E. Wright ◽  
F. Haddad ◽  
A. X. Qin ◽  
P. W. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Reporter Gene ◽  
Normal Control ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Construct ◽  
Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

scholarly journals Inhibition of Estrogen Receptor Action by the Orphan Receptor SHP (Short Heterodimer Partner)

1998 ◽  
Vol 12 (10) ◽  
pp. 1551-1557 ◽  
Author(s):  
Wongi Seol ◽  
Bettina Hanstein ◽  
Myles Brown ◽  
David D. Moore
Keyword(s):  
Estrogen Receptor ◽  
Thyroid Hormone ◽  
Transcriptional Activation ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Orphan Receptor ◽  
Estrogen Signaling ◽  
Dependent Manner ◽  
Interaction Domain ◽  
Short Heterodimer Partner

Abstract SHP (short heterodimer partner) is an unusual orphan receptor that lacks a conventional DNA-binding domain. Previous results have shown that it interacts with several other nuclear hormone receptors, including the retinoid and thyroid hormone receptors, and inhibits their ligand-dependent transcriptional activation. Here we show that SHP also interacts with estrogen receptors and inhibits their function. In mammalian and yeast two-hybrid systems as well as glutathione-S-transferase pull-down assays, SHP interacts specifically with estrogen receptor-α (ERα) in an agonist-dependent manner. The same assay systems using various deletion mutants of SHP map the interaction domain with ERα to the same SHP sequences required for interaction with the nonsteroid hormone receptors such as retinoid X receptor and thyroid hormone receptor. In transient cotransfection assays, SHP inhibits estradiol -dependent activation by ERα by about 5-fold. In contrast, SHP interacts with ERβ independent of ligand and reduces its ability to activate transcription by only 50%. These data suggest that SHP functions to regulate estrogen signaling through a direct interaction with ERα.

scholarly journals Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

2010 ◽  
Vol 78 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
Chen Zhang ◽  
Shao-Hung Wang ◽  
Chung-Ping Liao ◽  
Shoujin Shao ◽  
Mark E. Lasbury ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Mrna Level ◽  
Gene Promoter ◽  
Pneumocystis Pneumonia ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

Abstract 09: Mtor Inhibition Decreases Ligand-induced Mineralocorticoid Receptor Activation

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Yusuf Ali ◽  
Elise P Gomez-sanchez ◽  
Celso E Gomez-sanchez
Keyword(s):  
Reporter Gene ◽  
Transcriptional Activity ◽  
Collecting Duct ◽  
Receptor Activation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mtor Inhibition ◽  
Positive Effects ◽  
Mtor Phosphorylation

Introduction: ULK1 phosphorylates the MR at S843, decreasing its ligand binding and transcriptional activity. Angiotensin II-induced mTOR phosphorylation of ULK1 inactivates ULK1, preventing its phosphorylation of MR. Aim: Further elucidate the role of mTOR in the regulation of MR transcriptional activity. Methods: M1 mouse cortical collecting duct cells stably transduced with the rat MR cDNA and a MMTV- Gaussia luciferase reporter gene, were incubated with an mTOR activator and several inhibitors, +/- aldosterone or corticosterone. Similar studies were done after lentiviral transduction of CRISPR/gRNA for raptor and rictor genes or mutated MR (mu/S843A) cDNA. Results: mTOR inhibition significantly decreased ligand activation of the MR reporter gene, while the mTOR activator MHY1485 had no effect suggesting that mTOR is tonically active. MR activation induced by aldosterone and corticosterone was also decreased by CRISPR/gRNA gene knockdown of raptor and rictor, the adaptors of mTOR complex 1 and 2, respectively, supporting a role for mTOR. The mTOR inhibitor AZD8055 (AZD) reduced phospho-ULK1 and attenuated ligand-mediated MR transactivation in a dose-dependent manner. The ULK1 inhibitor MRT68921 increased MR transactivation. We speculated that mTOR decreased ULK1 activity by phosphorylating it, thereby preventing ULK1 phosphorylation of the MR at Serine (S843). However, when M1 cells were transduced with an MR cDNA in which S843 was replaced with Alanine that cannot be phosphorylated, ligand-induced activation of the mu/S843A MR was still decreased by AZD, but unchanged by MRT68921. This suggests that mTOR has an additional effect on MR activity unrelated to ULK1 activity. AZD also decreased P70S6K and AKT phosphorylation in these cells. Conclusions: mTOR phosphorylation of ULK1 prevents its phosphorylation of the MR and reduction of MR transcriptional activity. mTOR inhibitors and deletion of raptor and rictor decreased MR transcriptional activity. mTOR has additional positive effects on MR activity possibly related to its phosphorylation of AKT and P70S6K. Inhibition of mTOR action may be a useful target for mitigating excessive MR activation.

scholarly journals Regulation of thyroid hormone activation via the liver X-receptor/retinoid X-receptor pathway

2010 ◽  
Vol 205 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Marcelo A Christoffolete ◽  
Márton Doleschall ◽  
Péter Egri ◽  
Zsolt Liposits ◽  
Ann Marie Zavacki ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Thyroid Hormone Receptor ◽  
Liver X Receptor ◽  
Retinoid X Receptor ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Diet Induced Obesity ◽  
Ectopic Liver ◽  
Dose Dependent

Thyroid hormone receptor (TR) and liver X-receptor (LXR) are the master regulators of lipid metabolism. Remarkably, a mouse with a targeted deletion of both LXRα and LXRβ is resistant to western diet-induced obesity, and exhibits ectopic liver expression of the thyroid hormone activating type 2 deiodinase (D2). We hypothesized that LXR/retinoid X-receptor (RXR) signaling inhibits hepatic D2 expression, and studied this using a luciferase reporter containing the human DIO2 (hDIO2) promoter in HepG2 cells. Given that, in contrast to mammals, the chicken liver normally expresses D2, the chicken DIO2 (cDIO2) promoter was also studied. 22(R)-OH-cholesterol negatively regulated hDIO2 in a dose-dependent manner (100 μM, approximately twofold), while it failed to affect the cDIO2 promoter. Truncations in the hDIO2 promoter identified the region −901 to −584 bp as critical for negative regulation. We also investigated if 9-cis retinoic acid (9-cis RA), the ligand for the heterodimeric partner of TR and LXR, RXR, could regulate the hDIO2 promoter. Notably, 9-cis RA repressed the hDIO2 luciferase reporter (1 μM, approximately fourfold) in a dose-dependent manner, while coexpression of an inactive mutant RXR abolished this effect. However, it is unlikely that RXR homodimers mediate the repression of hDIO2 since mutagenesis of a DR-1 at −506 bp did not interfere with 9-cis RA-mediated repression. Our data indicate that hDIO2 transcription is negatively regulated by both 22(R)-OH-cholesterol and 9-cis RA, which is consistent with LXR/RXR involvement. In vivo, the inhibition of D2-mediated tri-iodothyronine (T3) production by cholesterol/9-cis RA could function as a feedback loop, given that T3 decreases hepatic cholesterol levels.

scholarly journals Loss of thyroid hormone receptor activity in primary cultured rat hepatocytes is reversed by 2-mercaptoethanol

1992 ◽  
Vol 281 (3) ◽  
pp. 669-673 ◽  
Author(s):  
N Yamamoto ◽  
A Inoue ◽  
K P Takahashi ◽  
Q Li ◽  
H Nakamura ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Hormone Receptor ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Binding Activity ◽  
Dependent Manner ◽  
Hormone Binding ◽  
Level Of Activity

In primary cultures of rat hepatocytes, specific thyroid-hormone-binding activity diminished with time and was hardly detectable at 24 h. In accordance with the loss of 3,5,3′-tri-iodothyronine (T3) binding, responses to the hormone disappeared, as indicated by low induction of the thyroid-hormone-responsive gene S14. In contrast, thyroid hormone receptor proteins were present, as determined by immunostaining with a specific antibody against the receptor. Thus the loss of T3 binding was due to receptor inactivation. After various attempts to restore the T3-binding activity, we found that 2-mercaptoethanol, a reducing agent, when added to the culture medium restored the hormone binding activity in a dose- and time-dependent manner. The observed kinetics and experiments using cycloheximide suggested that mercaptoethanol prevented inactivation of the newly synthesized receptors. Oxidoreductive conditions within cells may have a role in determining the level of activity of thyroid hormone receptors.

scholarly journals Antithyroid Drugs Inhibit Thyroid Hormone Receptor-Mediated Transcription

2007 ◽  
Vol 92 (3) ◽  
pp. 1066-1072 ◽  
Author(s):  
Kenji Moriyama ◽  
Tetsuya Tagami ◽  
Takeshi Usui ◽  
Mitsuhide Naruse ◽  
Takuo Nambu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Transient Gene Expression ◽  
Antithyroid Drugs ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Peripheral Tissues

Abstract Context: Methimazole (MMI) and propylthiouracil (PTU) are widely used as antithyroid drugs (ATDs) for the treatment of Graves’ disease. Both MMI and PTU reduce thyroid hormone levels by several mechanisms, including inhibition of thyroid hormone synthesis and secretion. In addition, PTU decreases 5′-deiodination of T4 in peripheral tissues. ATDs may also interfere with T3 binding to nuclear thyroid hormone receptors (TRs). However, the effect of ATDs on the transcriptional activities of T3 mediated by TRs has not been studied. Objective: The present study was undertaken to determine whether ATDs have an effect on the gene transcription regulated by T3 and TRs in vitro. Methods: Transient gene expression experiments and GH secretion assays were performed. To elucidate possible mechanisms of the antagonistic action of ATDs, the interaction between TR and nuclear cofactors was examined. Results: In the transient gene expression experiments, both MMI and PTU significantly suppressed transcriptional activities mediated by the TR and T3 in a dose-dependent manner. In mammalian two-hybrid assays, both drugs recruited one of the nuclear corepressors, nuclear receptor corepressor, to the TR in the absence of T3. In addition, PTU dissociated nuclear coactivators, such as steroid receptor coactivator-1 and glucocorticoid receptor interacting protein-1, from the TR in the presence of T3. Finally, MMI decreased the GH release that was stimulated by T3. Conclusions: ATDs inhibit T3 action by recruitment of transcriptional corepressors and/or dissociation of coactivators. This is the first report to show that ATDs can modulate T3 action at the transcriptional level.

scholarly journals RORα Augments Thyroid Hormone Receptor-Mediated Transcriptional Activation*

Endocrinology ◽  
1999 ◽  
Vol 140 (3) ◽  
pp. 1356-1364 ◽  
Author(s):  
Noriyuki Koibuchi ◽  
Ying Liu ◽  
Harumi Fukuda ◽  
Akira Takeshita ◽  
Paul M. Yen ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Transcriptional Activation ◽  
Hormone Receptor ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Critical Role ◽  
Nuclear Hormone Receptor ◽  
Dominant Negative ◽  
Mobility Shift ◽  
Response Element

Abstract 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 TRE to exert dominant negative function, RORα1 augmented TR action. These results indicate that RORα1 may modify the effect of liganded TR on TH-responsive genes. Because TR and RORα are coexpressed in cerebellar Purkinje cells, and perinatal hypothyroid animals and RORα-disrupted animals show similar abnormalities of this cell type, cross-talk between these two receptors may play a critical role in Purkinje cell differentiation.

Effects of diabetes on rodent cardiac thyroid hormone receptor and isomyosin expression

1997 ◽  
Vol 272 (5) ◽  
pp. E856-E863 ◽  
Author(s):  
F. Haddad ◽  
P. W. Bodell ◽  
S. A. McCue ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Binding Capacity ◽  
Nuclear Extract ◽  
Female Rats ◽  
Binding Studies ◽  
Mobility Shift ◽  
Mrna Isoforms

Previous studies show that diabetes induces marked transformations in cardiac myosin heavy chain (MHC) gene expression that are somehow linked to the cellular action of thyroid hormone 3,5,3'-triiodothyronine (T3). In this study, we tested the hypothesis that diabetes induces a reduced expression of thyroid hormone receptors (TRs), which are known to be important transcription factors interacting with thyroid response elements (TREs) in the promoter region of both alpha- and beta-MHC genes. Adult female rats were randomly assigned to either a normal control (NC) or diabetic (D) group. Three and/or six weeks after induction of diabetes via streptozotocin injection, the hearts of the animals were analyzed for MHC and TR mRNA isoforms expression, nuclear T3 binding, and nuclear extract interaction with a palindromic TRE. Results showed that diabetes induced significant alteration in alpha- and beta-MHC expression. Northern blot analyses indicated no diabetes-associated differences in TR isoform mRNA signals. Cardiac nuclear T3 binding studies suggested no differences in either the binding capacity or the equilibrium binding constant among the two groups, indicating no changes in either the number of nuclear TRs or their affinity for T3. Furthermore, gel mobility shift assays detected no difference between NC and D groups for cardiac nuclear extract binding to palindromic TRE. Collectively, these findings suggest that, whereas diabetes exerts a profound effect on cardiac isomyosin gene expression, the underlying mechanism, although dependent on factors linked to T3 function, does not involve alterations in TR expression.

scholarly journals Luciferase Measurements in High Throughput Screening

1996 ◽  
Vol 1 (2) ◽  
pp. 85-88 ◽  
Author(s):  
Alfred J. Kolb ◽  
Kenneth Neumann
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Culture Media ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cell Culture Media ◽  
Luminescence Signal ◽  
Temperature Controlled

Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

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