Yan-Yun Liu

A thyroid hormone receptor α gene mutation (P398H) Is associated with visceral adiposity and impaired catecholamine-stimulated lipolysis in mice

Thyroid hormone has profound effects on metabolic homeostasis, regulating both lipogenesis and lipolysis, primarily by modulating adrenergic activity. We generated mice with a point mutation in the thyroid hormone receptor α (TRα) gene producing a dominant-negative TRα mutant receptor with a proline to histidine substitution (P398H). The heterozygous P398H mutant mice had a 3.4-fold (p < 0.02) increase in serum thyrotropin (TSH) levels. Serum triiodothyronine (T3) and thyroxine (T4) concentrations were slightly elevated compared with wild-type mice. The P398H mice had a 4.4-fold increase in body fat (as a fraction of total body weight) (p < 0.001) and a 5-fold increase in serum leptin levels (p < 0.005) compared with wild-type mice. A 3-fold increase in serum fasting insulin levels (p < 0.002) and a 55% increase in fasting glucose levels (p < 0.01) were observed in P398H compared with wild-type mice. There was a marked reduction in norepinephrine-induced lipolysis, as reflected in reduced glycerol release from white adipose tissue isolated from P398H mice. Heart rate and cold-induced adaptive thermogenesis, mediated by thyroid hormone-catecholamine interaction, were also reduced in P398H mice. In conclusion, the TRα P398H mutation is associated with visceral adiposity and insulin resistance primarily due to a marked reduction in catecholamine-stimulated lipolysis. The observed phenotype in the TRα P398H mouse is likely due to interference with TRα action as well as influence on other metabolic signaling pathways. The physiologic significance of these findings will ultimately depend on understanding the full range of actions of this mutation.

Thyroid hormone crosstalk with nuclear receptor signaling in metabolic regulation

Thyroid hormone influences diverse metabolic pathways important in lipid and glucose metabolism, lipolysis and regulation of body weight. Recently, it has been recognized that thyroid hormone receptor interacts with transcription factors that predominantly respond to nutrient signals including the peroxisome proliferator-activated receptors, liver X receptor and others. Crosstalk between thyroid hormone signaling and these nutrient responsive factors occurs through a variety of mechanisms: competition for retinoid X receptor heterodimer partners, DNA binding sites and transcriptional cofactors. This review focuses on the mechanisms of interaction of thyroid hormone signaling with other metabolic pathways and the importance of understanding these interactions to develop therapeutic agents for treatment of metabolic disorders, such as dyslipidemias, obesity and diabetes.

Phosphoinositide-3-kinase inhibition induces sodium/iodide symporter expression in rat thyroid cells and human papillary thyroid cancer cells.

TSH stimulation of sodium iodide symporter (NIS) expression in thyroid cancer promotes radioiodine uptake and is required to deliver an effective treatment dose. Activation of the insulin/phosphoinositide-3-kinase (PI3K) signaling pathway in TSH-stimulated thyroid cells reduces NIS expression at the transcriptional level. We, therefore, investigated the effects of PI3K pathway inhibition on iodide uptake and NIS expression in rat thyroid cell lines and human papillary thyroid cancer cells. A PI3K inhibitor, LY294002, significantly enhanced iodide uptake in two rat thyroid cell lines, FRTL-5 and PCCL3. The induction of Nis mRNA by LY294002 occurred 6 h after treatment, and was abolished by a translation inhibitor, cycloheximide. Expression of the transcription factor, Pax8, which stimulates NIS expression, was significantly increased in PCCL3 cells after LY294002 treatment. Removal of insulin abrogated the stimulatory effects of LY294002 on NIS mRNA and protein expression, but not on iodide uptake. These findings suggest that PI3K pathway inhibition results in post-translational stimulation of NIS. Inhibition of the PI3K pathway also significantly increased iodide uptake ( approximately 3.5-fold) in BHP 2-7 papillary thyroid cancer cells (Ret/PTC1 positive), engineered to constitutively express NIS. Pharmacological inhibition of Akt, a factor stimulated by the PI3K pathway, increased exogenous NIS expression in BHP 2-7 as was seen with LY294002, but not increase the endogenous NIS expression in FRTL-5 cells. PI3K pathway inhibition increases functional NIS expression in rat thyroid cells and some papillary thyroid cancer cells by several mechanisms. PI3K inhibitors have the potential to increase radioiodide accumulation in some differentiated thyroid cancer.

A Targeted Thyroid Hormone Receptor α Gene Dominant-Negative Mutation (P398H) Selectively Impairs Gene Expression in Differentiated Embryonic Stem Cells

Thyroid hormone and retinoic acid (RA) are essential for normal neural development in vivo, yet all in vitro differentiation strategies of embryonic stem (ES) cells use only RA. We developed a novel differentiation strategy of mouse ES cells using T(3). A dominant-negative knock-in point mutation (P398H) was introduced into the thyroid hormone receptor alpha gene to determine the influence of T(3) on ES cell differentiation. Differentiation promoted by T(3) (1 nM), RA (1 microM), or combined T(3)/RA was assessed in wild-type (wt) and mutant (m) ES cells on the basis of neuronal-specific gene expression and cell cycle. T(3) alone stimulated neural differentiation in a similar fashion as that seen with RA in both wtES and mES cells. Expression of neurogranin and Ca(2+)/calmodulin-dependent kinase IV mRNA (identified in vivo as T(3)-regulated genes), however, was markedly reduced in mES, compared with wtES cells. RA treatment enhanced apoptosis, significantly greater than that seen with T(3) stimulation. T(3) treatment given with RA significantly reduced the apoptotic effects of RA, an effect not seen in mES cells. T(3)-induced ES cell neural differentiation of thyroid hormone alpha mutant and wtES cells provides an in vitro model to study T(3)-dependent gene regulation in neural development. This system could also be used to identify novel T(3)-regulated genes. The modulation of the apoptotic effects of RA by T(3) may have implications for stem cell therapy.

Thyroid hormone receptor isoform-specific modification by small ubiquitin-like modifier (SUMO) modulates thyroid hormone-dependent gene regulation.

Background: Thyroid hormone receptor (TR) isoforms α and β have distinct biological and physiological roles in development and in adult tissues. Results: TRα and β are posttranslationally modified by different SUMO isoforms and require specific SUMO E3 ligases. TR sumoylation influences corepressor and coactivator recruitment. Conclusion: TR-SUMO conjugation is important for thyroid hormone action. Significance: Identifying a mechanism contributing to TR isoform-specific action. Thyroid hormone receptor (TR) α and β mediate thyroid hormone action at target tissues. TR isoforms have specific roles in development and in adult tissues. The mechanisms underlying TR isoform-specific action, however, are not well understood. We demonstrate that posttranslational modification of TR by conjugation of small SUMO to TRα and TRβ plays an important role in triiodothyronine (T3) action and TR isoform specificity. TRα was sumoylated at lysines 283 and 389, and TRβ at lysines 50, 146, and 443. Sumoylation of TRβ was ligand-dependent, and sumoylation of TRα was ligand-independent. TRα-SUMO conjugation utilized the E3 ligase PIASxβ and TRβ-SUMO conjugation utilized predominantly PIAS1. SUMO1 and SUMO3 conjugation to TR was important for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The functional role of SUMO1 and SUMO3 in T3 induction in transient expression assays was closely matched to the pattern of TR and cofactor recruitment to thyroid hormone response elements (TREs) as determined by ChIP assays. SUMO1 was required for the T3-induced recruitment of the co-activator CREB-binding protein (CBP) and release of nuclear receptor co-repressor (NCoR) on a TRE but had no significant effect on TR DNA binding. SUMO1 was required for T3-mediated recruitment of NCoR and release of CBP from the TSHβ-negative TRE. SUMO3 was required for T3-stimulated TR binding to the TSHβ-negative TRE and recruitment of NCoR. These findings demonstrate that conjugation of SUMO to TR has a TR-isoform preference and is important for T3-dependent gene induction and repression.

Retinoic acid induces expression of the thyroid hormone transporter, monocarboxylate transporter 8 (Mct8)

Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. Mct8 (monocarboxylate transporter 8), expressed predominantly in the brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells toward neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All-trans-RA (tRA) and other RA receptor (RAR) agonists dramatically (>300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1- and 4.3-fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and 5′-rapid amplification of cDNA ends PCR analysis in F9 cells identified 11 transcription start sites and a proximal Sp1 site but no TATA box. tRA significantly enhanced Mct8 promoter activity through a consensus RA-responsive element located 6.6 kilobases upstream of the coding region. A chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid X receptor to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates cross-talk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.

Thyroid Hormone Receptor Sumoylation Is Required for Preadipocyte Differentiation and Proliferation

Background: Thyroid hormone receptor (TR) sumoylation is essential for thyroid hormone regulation of gene expression. Results: TR sumoylation mutants impair differentiation though down-regulation of C/EBPs, constitutive interaction with NCoR, interference with PPARγ signaling, and disruption of the Wnt canonical signaling pathway important for preadipocyte proliferation. Conclusion: TR sumoylation site mutations impair preadipocyte proliferation and differentiation. Significance: TR sumoylation is required for adipogenesis. Thyroid hormone and thyroid hormone receptor (TR) play an essential role in metabolic regulation. However, the role of TR in adipogenesis has not been established. We reported previously that TR sumoylation is essential for TR-mediated gene regulation and that mutation of either of the two sites in TRα or any of the three sites in TRβ reduces TR sumoylation. Here, we transfected TR sumoylation site mutants into human primary preadiocytes and the mouse 3T3L1 preadipocyte cell line to determine the role of TR sumoylation in adipogenesis. Reduced sumoylation of TRα or TRβ resulted in fewer and smaller lipid droplets and reduced proliferation of preadipocytes. TR sumoylation mutations, compared with wild-type TR, results in reduced C/EBP expression and reduced PPARγ2 mRNA and protein levels. TR sumoylation mutants recruited NCoR and disrupted PPARγ-mediated perilipin1 (Plin1) gene expression, associated with impaired lipid droplet formation. Expression of NCoRΔID, a mutant NCoR lacking the TR interaction domain, partially “rescued” the delayed adipogenesis and restored Plin1 gene expression and adipogenesis. TR sumoylation site mutants impaired Wnt/β-catenin signaling pathways and the proliferation of primary human preadipocytes. Expression of the TRβ K146Q sumoylation site mutant down-regulated the essential genes required for canonical Wnt signal-mediated proliferation, including Wnt ligands, Fzds, β-catenin, LEF1, and CCND1. Additionally, the TRβ K146Q mutant enhanced the canonical Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1). Our data demonstrate that TR sumoylation is required for activation of the Wnt canonical signaling pathway during preadipocyte proliferation and enhances the PPARγ signaling that promotes differentiation.

Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury

Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and β, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and β, increased myoblast differentiation whereas GC1, a selective TRβ agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/β-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRβ PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.

Regulation of Sodium Iodide Symporter Gene Expression by Rac1/p38β Mitogen-activated Protein Kinase Signaling Pathway in MCF-7 Breast Cancer Cells

Background: Induction of the iodide transporter in cancer cells confers targeted cytotoxicity with radioiodide. Results: Isoforms of p38 MAPK were identified that specifically promote iodide uptake in breast cancer cells. Conclusion: p38 isoform-specific stimulation may induce iodide uptake sufficient for radioiodide therapy in breast cancer. Significance: Study of p38 isoform-specific signaling improves understanding of cancer cell differentiation and identifies novel therapeutic targets. Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide 131I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of 131I concentration and treatment of breast cancer.

Stealth Sequences in Reporter Gene Vectors Confound Studies of T3-Regulated Negative Gene Expression

Firefly luciferase is a commonly used reporter gene that allows for rapid and precise quantitation of gene expression. It is utilized in a wide range of vectors to evaluate many aspects of transcription regulation. The native luciferase gene (luc), however, contains DNA sequences that are recognized binding sites for a large number of transcription factors (Table 1). In this issue of Thyroid, Chan et al. report that the pBi-L vector (Clontech, Mountain View, CA) is negatively regulated by triiodothyronine (T3) (1). In a series of transfection studies, utilizing several cell lines, they demonstrate that negative regulation is conferred by sequences in the luciferase gene and that expression of thyroid hormone receptor (TR) is required. The pBi-L vectors are tetracycline-regulated reporters. The luciferase gene sequence in pBi-L is 99% identical to that in the pGL-2 reporter series (Promega, Madison, WI), which carries a minimally modified native luc. The expression of the luciferase gene in the pGL-2 promoter vector was previously reported to be inhibited by T3 treatment in CV1 and JEG3 cells (2,3), thought to be mediated by a negative thyroid hormone response element (nTRE) in the luciferase gene. Luciferase gene expression in Caco-2 cells is inhibited by 1,25-dihydroxycholecalciferol when using the pGL-2 promoter vector, but not when using the pGL-3 promoter vector (4). In the pGL-3 reporter series, luciferase gene (lucþ) is modified from native luc in order to reduce anomalous expression and enhance Lucþ expression. Even with this modification, however, the pGL-3 series still carries numerous binding sites for transcription factors (Table 1). Examples of these include elements for CREB, CAR, SRF, PAR, RXR, HNF, Oct, CAR, CHOP, and other factors (Table 1). We have reported that lucþ expression is induced by alltrans retinoic acid up to fourfold in MCF-7 cells (5). The further modified version of the luciferase gene (luc2) used in the pGL-4 reporter vector series dramatically reduces the number of transcription factor binding sites, but binding sites for a few common transcription factors remain (Table 1). Despite several reports of T3-dependent repression mediated by sequences in the luciferase gene, the nTRE sequence in the luciferase gene has not been identified. The sequence configurations that confer positive regulation have been well defined (6), but the mechanism of negative regulation by T3 is more complex. The ability of an element to confer negative regulation is influenced by both sequence and position (7). In models involving TR directly binding to an nTRE for T3-dependent repression, a variety of sequences have been identified, including those regulating TSHb subunit, CD44, and CYP7A1 (8–10). These nTREs vary in DNA sequence, configuration, and position. No consensus nTRE sequence, however, has been established. Others argue that T3-dependent repression may not require direct TR binding to DNA. TR interacts with jun=fos bound to the AP1 site by a protein–protein interaction (11), and addition of T3 represses transcription. Although T3-dependent repression is an important biological function of T3, only a small number of nTREs that mediate this repression have been identified. Most transfection studies use a luciferase reporter vector to quantitate the influence of factors that modulate gene expression. There are many reporter vectors that use the same luciferase gene as that used by the pGL series vectors. Chan et al. made their observation of an unexpected nTRE in a reporter gene when developing a vector for a transgenic animal project (1). It is especially important in animal studies, which require a large investment of time and funds, that vector sequences do not confound the results. To be confident of the results of gene expression analysis, it is important to search the vector sequence for known binding sites relevant to the studies and include experimental conditions that assess the effect of any treatments on vector expression. Those studying gene expression with reporter vectors need to carefully consider the influence of unexpected ‘‘stealth’’ response sequences.