Thyroid hormone receptors promote metastasis of human hepatoma cells via regulation of TRAIL

Although accumulating evidence has confirmed the important roles of thyroid hormone (T₃) and its receptors (TRs) in tumor progression, the specific functions of TRs in carcinogenesis remain unclear. In the present study, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was directly upregulated by T₃ in TR-overexpressing hepatoma cell lines. TRAIL is an apoptotic inducer, but it can nonetheless trigger non-apoptotic signals favoring tumorigenesis in apoptosis-resistant cancer cells. We found that TR-overexpressing hepatoma cells treated with T₃ were apoptosis resistant, even when TRAIL was upregulated. This apoptotic resistance may be attributable to simultaneous upregulation of Bcl-xL by T₃, because (1) knockdown of T₃-induced Bcl-xL expression suppressed T₃-mediated protection against apoptosis, and (2) overexpression of Bcl-xL further protected hepatoma cells from TRAIL-induced apoptotic death, consequently leading to TRAIL-promoted metastasis of hepatoma cells. Moreover, T₃-enhanced metastasis in vivo was repressed by the treatment of TRAIL-blocking antibody. Notably, TRAIL was highly expressed in a subset of hepatocellular carcinoma (HCC) patients, and this high-level expression was significantly correlated with that of TRs in these HCC tissues. Together, our findings provide evidence for the existence of a novel mechanistic link between increased TR and TRAIL levels in HCC. Thus, TRs induce TRAIL expression, and TRAIL thus synthesized acts in concert with simultaneously synthesized Bcl-xL to promote metastasis, but not apoptosis.     

Thyroid Hormone Receptor Sumoylation Is Required for Preadipocyte Differentiation and Proliferation

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.     

Gene Specific Actions of Thyroid Hormone Receptor Subtypes

There are two homologous thyroid hormone (TH) receptors (TRs α and β), which are members of the nuclear hormone receptor (NR) family. While TRs regulate different processes in vivo and other highly related NRs regulate distinct gene sets, initial studies of TR action revealed near complete overlaps in their actions at the level of individual genes. Here, we assessed the extent that TRα and TRβ differ in target gene regulation by comparing effects of equal levels of stably expressed exogenous TRs +/− T₃ in two cell backgrounds (HepG2 and HeLa). We find that hundreds of genes respond to T₃ or to unliganded TRs in both cell types, but were not able to detect verifiable examples of completely TR subtype-specific gene regulation. TR actions are, however, far from identical and we detect TR subtype-specific effects on global T₃ response kinetics in HepG2 cells and many examples of TR subtype specificity at the level of individual genes, including effects on magnitude of response to TR +/− T₃, TR regulation patterns and T₃ dose response. Cycloheximide (CHX) treatment confirms that at least some differential effects involve verifiable direct TR target genes. TR subtype/gene-specific effects emerge in the context of widespread variation in target gene response and we suggest that gene-selective effects on mechanism of TR action highlight differences in TR subtype function that emerge in the environment of specific genes. We propose that differential TR actions could influence physiologic and pharmacologic responses to THs and selective TR modulators (STRMs).     

Identification of thyroid regulatory elements in the Na-K-ATPase α3 gene promoter

A –1027 bp to +108 bp region of Na-K-ATPase ₃ gene promoter has been searched for the presence of thyroid response elements (TRE). Computer analysis of this sequence using a consensus TRE sequence revealed the presence of four putative TRE rich regions referred to as regions I (–636 to –457 bp), II (–218 to –106 bp), III (–106 to –6 bp) and IV (–6 to +108 bp). Cotransfection of the luciferase linked full length construct as well as constructs progressively devoid of the TRE rich regions in Cos1 cells revealed that regions I and III are positively regulated by T ₃ whereas there are some sequences in region II which can suppress the positive regulatory effect of region III but not of region I, TRE IV seems to have no functional role. EMSA of the three functional TRE rich regions (I, II and III) showed strong and specific interaction with thyroid hormone receptor (TR) cloned and expressed in baculovirus. The overall results suggest the regulation of Na-K-ATPase ₃ gene by T ₃ is complex involving several thyroidal regulatory elements.     

Thyroid hormone receptor subtype specificity for hormone-dependent neurogenesis in Xenopus laevis

Thyroid hormone (T₃) influences cell proliferation, death and differentiation during development of the central nervous system (CNS). Hormone action is mediated by T₃ receptors (TR) of which there are two subtypes, TRα and TRβ. Specific roles for TR subtypes in CNS development are poorly understood. We analyzed involvement of TRα and TRβ in neural cell proliferation during metamorphosis of Xenopus laevis. Cell proliferation in the ventricular/subventricular neurogenic zones of the tadpole brain increased dramatically during metamorphosis. This increase was dependent on T₃ until mid-prometamorphosis, after which cell proliferation decreased and became refractory to T₃. Using double labeling fluorescent histochemistry with confocal microscopy we found TRα expressed throughout the tadpole brain, with strongest expression in proliferating cells. By contrast, TRβ was expressed predominantly outside of neurogenic zones. To corroborate the histochemical results we transfected living tadpole brain with a Xenopus TRβ promoter-EGFP plasmid and found that most EGFP expressing cells were not dividing. Lastly, treatment with the TRα selective agonist CO23 increased brain cell proliferation; whereas, treatment with the TRβ-selective agonists GC1 or GC24 did not. Our findings support the view that T₃ acts to induce cell proliferation in the tadpole brain predominantly, if not exclusively, via TRα.     

3,5,3'-triiodothyronine receptor auxiliary protein (TRAP) enhances receptor binding by interactions within the thyroid hormone response element.

We have previously demonstrated that binding of in vitro synthesized thyroid hormone receptor (TR) to thyroid hormone response elements (TREs) is enhanced by the addition of nuclear extracts from several different cell types, suggesting that binding of TR is partially dependent on a T3 receptor auxiliary protein (TRAP). We have used the avidin-biotin complex DNA-binding assay to discriminate between regions of TREs that bind TR alone and sites that are influenced by interactions with TRAP. Mutations in the TREs from rat GH and glycoprotein hormone alpha-subunit genes show that a specific DNA sequence is required for TRAP-mediated enhancement of TR binding. Mutations in the B half-site of the rat GH TRE or in similar sequences [(T/A)GGGA] in the alpha-subunit TRE ablate the enhancement of TR binding by TRAP. Furthermore, binding of TR to a natural half-site in the TSH beta-subunit gene (bases -16 to 6), which lacks an additional AGGGA-like sequence, is not enhanced by the addition of TRAP. Binding of TR to TREs was also tested at physiological salt concentrations in the avidin-biotin complex DNA-binding assay. Binding of human TR beta to TREs decreases dramatically at 140 mM KCl compared to binding at 50 mM KCl; however, the addition of TRAP enhances the binding to almost 4-fold of basal binding, suggesting that TRAP may be important for stabilization of TR binding to TREs in the cell.     

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP_M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.     

Short-term effects of thyroid hormones during development: Focus on signal transduction

Extranuclear or nongenomic effects of thyroid hormones are mediated by receptors located at the plasma membrane or inside cells, and are independent of protein synthesis. Recently the αVβ3 integrin was identified as a cell membrane receptor for thyroid hormones, and a wide variety of nongenomic effects have now been shown to be induced through binding of thyroid hormones to this receptor. However, also other thyroid hormone receptors can produce nongenomic effects, including the cytoplasmic TRα and TRβ receptors and probably also a G protein-coupled membrane receptor, and increasing importance is now given to thyroid hormone metabolites like 3,5-diiodothyronine and reverse T₃ that can mimick some nongenomic effects of T₃ and T₄. Signal transduction from the αVβ3 integrin may proceed through at least three independent pathways (protein kinase C, Src or mitogen-activated kinases) but the details are still unknown. Thyroid hormones induce nongenomic effects on at least three important Na⁺-dependent transport systems, the Na⁺/K⁺-ATPase, the Na⁺/H⁺ exchanger, and amino acid transport System A, leading to a mitogenic response in embryo cells; but modulation of the same transport systems may have different roles in other cells and at different developmental stages. It seems that thyroid hormones in many cases can modulate nongenomically the same targets affected by the nuclear receptors through long-term mechanisms. Recent results on nongenomic effects confirm the old theory that the primary role of thyroid hormones is to keep the steady-state level of functioning of the cell, but more and more mechanisms are discovered by which this goal can be achieved.     

Effects of excess thyroid hormone on cell death,cell proliferation,and new neuron incorporation in the adult zebra finch telencephalon

Widespread telencephalic neuronal replacement occurs throughout life in birds. We explored the potential relationship between thyroxine (T₄) and cell turnover in the adult male zebra finch. We found that many cells in the zebra finch brain, including long‐projection neurons in the high vocal center (HVC), stained positively with an antibody to thyroid hormone receptors (TR). Labeling was generally weak in the ventricular zone (VZ) that gives rise to new neurons but some proliferative VZ cells and/or their progeny, identified by [³H]‐thymidine labeling, co‐labeled with anti‐TR antibody. Acute T₄ treatment dramatically increased the number of pyknotic and TUNEL‐positive cells in HVC and other telencephalic regions. In contrast, degenerating cells were never observed in the archistriatum or sub‐telencephalic regions, suggesting that excess T₄ augments cell death selectively in regions that show naturally occurring neuronal turnover. VZ mitotic activity was not altered shortly after acute T₄ treatment at a dosage that stimulated cell death, although [³H]‐labeling intensity per cell was slightly reduced. Moreover, the incorporation rates for neurons formed shortly before or after acute hormone treatment were no different from control values. Chronic T₄ treatment resulted in a reduction in the total number of HVC neurons. Thus, hyperthyroidism augmented neuronal death, which was not compensated for by neuronal replacement. Collectively, these results indicate that excess T₄ affects adult neuronal turnover in birds, and raises the possibility that thyroxine plays an important role in the postnatal development of the avian brain and vocal behavior. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 323–341, 2002