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The thyroid hormone 3,3′,5-triiodo-l-thyronine (T3) regulates growth, development, and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). Microarray analyses were performed previously to study the mechanism of regulation triggered by T3 treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T3. However, the underlying mechanism and the physiological role of T3 in the regulation of spondin 2 are not clear. To verify the microarray results, spondin 2 was further investigated using semi-quantitative reverse transcription-PCR and western blotting. After 48 h of T3 treatment in the HepG2–TRα1#1 cell line, spondin 2 mRNA and protein levels increased by 3.9- to 5.7-fold. Similar results were observed in thyroidectomized rats. To localize the regulatory region in spondin 2, we performed serial deletions of the promoter and chromatin immunoprecipitation assays. The T3 response element on the spondin 2 promoter was localized in the −1104/−1034 or −984/−925 regions. To explore the effect of spondin 2 on cellular function, spondin 2 knockdown cell lines were established from Huh7 cells. Knockdown cells had higher migration ability and invasiveness compared with control cells. Conversely, spondin 2 overexpression in J7 cells led to lower migration ability and invasiveness compared with control cells. Furthermore, this study demonstrated that spondin 2 overexpression in some types of hepatocellular carcinomas is TR dependent. Together, these experimental findings suggest that spondin 2, which is regulated by T3, has an important role in cell invasion, cell migration, and tumor progression.

The thyroid hormone receptor (TR) and human testicular orphan receptor 4 (TR4) belong to the nuclear hormone receptor superfamily. They are ligand-dependent transcription factors. TR and TR4 bind to a similar thyroid response element (TRE), known as a direct repeat with four nucleotide spacing (DR4). This study examined the possible interaction or cross-talking between those two receptors. We hypothesized that protein-protein interaction between TR4 and TR may promote TR-mediated transcriptional activity. Glutathione S-transferase pull-down and immunoprecipitation assays showed direct interaction between TR and TR4. Electrophoretic mobility-shift assay demonstrated that TR and TR4 could co-occupy the same TRE. The interaction between TR4 and TR may enhance regulation of genes targeted by TR, such as furin, fibrinogen, cdk2 and p21 expression. We found that TR4 function is similar with TR as TR4 alone could regulate expression of some TR target genes, and could increase cell migration or inhibit cell proliferation. Importantly, the TR-dependent inhibition of cell proliferation and stimulation of cell migration are more enhanced in the HepG2-TR cells stably over-expressing TR4. Overall, TR4 not only has modulation abilities similar to TR but also can cross-talk with TR and promote the TR signaling pathway.

The objective of this study was to identify genes regulated by thyroid hormone (T(3)) and associated with tumor invasion. The gene encoding furin, as previously identified by cDNA microarray, is known to be up-regulated by T(3) treatment, and stimulated furin production occurs in thyroidectomized rats after administration of T(3). Presently, by using serial deletion of the promoter and EMSAs, the T(3) response element on the furin promoter was localized to the -6317/-6302 region. T(3)-mediated furin up-regulation was cooperative with TGF-beta because T(3) induction increased after Smad3/4 addition. Furthermore, the invasiveness of HepG2-thyroid hormone receptor (TR) cells was significantly increased by T(3) treatment, perhaps due to furin processing of matrix metalloproteinase-2 and -9. In addition, furin up-regulation either by stable overexpression or T(3) and/or TGF-beta induction was evident in severe-combined immune-deficient mice inoculated with HepG2-TRalpha1 cells. The HepG2-furin mice displayed a higher metastasis index and tumor size than HepG2-neo mice. Notably, the increased liver and lung tumor number or size in the hyperthyroid severe-combined immune-deficient mice as well as TGF-beta mice was attributed specifically to furin overexpression in the HepG2-TRalpha1 cells. Furthermore, this study demonstrated that furin overexpression in some types of hepatocellular carcinomas is TR dependent and might play a crucial role in the development of hepatocellular carcinoma. Thus, T(3) regulates furin gene expression via a novel mechanism or in cooperation with TGF-beta to enhance tumor metastasis in vitro and in vivo.

Thyroid hormone (T3) regulates multiple physiological processes during development, growth, differentiation, and metabolism. Most T3 actions are mediated via thyroid hormone receptors (TRs) that are members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors. The effects of T3 treatment on target gene regulation was previously examined in TRalpha1-overexpressing hepatoma cell lines (HepG2-TRalpha1). Androgen receptor (AR)-associated protein 70 (ARA70) was one gene found to be up-regulated by T3. The ARA70 is a ligand-dependent coactivator for the AR and was significantly increased by 4- to 5-fold after T3 treatment by Northern blot analyses in the HepG2-TRalpha1 stable cell line. T3 induced a 1- to 2-fold increase in the HepG2-TRbeta1 stable cell line. Both stable cell lines attained the highest fold expression after 24 h treatment with 10 nM T3. The ARA70 protein was increased up to 1.9-fold after T3 treatment in HepG2-TRalpha1 cells. Similar findings were obtained in thyroidectomized rats after T3 application. Cycloheximide treatment did not suppress induction of ARA70 transcription by T3, suggesting that this regulation is direct. A series of deletion mutants of ARA70 promoter fragments in pGL2 plasmid were generated to localize the thyroid hormone response element (TRE). The DNA fragments (-234/-190 or +56/+119) gave 1.55- or 2-fold enhanced promoter activity by T3. Thus, two TRE sites exist in the upstream-regulatory region of ARA70. The TR-TRE interaction was further confirmed with EMSAs. Additionally, ARA70 could interfere with TR/TRE complex formation. Therefore, the data indicated that ARA70 suppresses T3 signaling in a TRE-dependent manner. These experimental results suggest that T3 directly up-regulates ARA70 gene expression. Subsequently, ARA70 negatively regulates T3 signaling.

The objective of this study was to identify genes regulated by thyroid hormone (T(3)) mediated by its receptor (TR) and associated with tumorigenesis. The gene encoding aldo-keto reductase family 1, member B1 (AKR1B1), as previously identified by c-DNA microarray, is known to be up-regulated by T(3) treatment. Enzyme AKR1B1 was elevated roughly 3-fold in HepG2-TRalpha1 cells at the protein level and 4.6-fold increase at the mRNA level after 48 h T(3) treatment. Similar findings were obtained from thyroidectomized rats after T(3) application. To identify and localize the critical TR element (TRE), series deletion of the promoter mutant were constructed and electrophoretic mobility shift assays were carried out. The TRE on the AKR1B1 promoter was localized to the -1099/-1028 region. Further, this study demonstrated that AKR1B1 over-expression in some types of hepatocellular carcinomas (HCCs) is TR-dependent and might play a crucial role in the development of HCC. Thus, T(3) regulates AKR1B1 gene expression via a TRE-dependant mechanism and associates liver cancer.

Pituitary tumor transforming gene 1 (PTTG1) is expressed in most tumors. However, whether thyroid hormone (T3) and its receptors (TR) regulate PTTG1 in human hepatocellular carcinomas (HCC) remains unclear. Previous cDNA microarrays revealed PTTG1 is down-regulated by T3/TR. This study investigated the significance of PTTG1 regulation by T3 in HCC cells. The PTTG1 mRNA and protein expression were repressed by T3 in HCC cell lines overexpressing TR. However, after knockdown of TRs expression by RNA interference, PTTG1 repression by T3 was abolished. Similar results were observed in thyroidectomized rats. To localize the regulatory region in the PTTG1 promoter, serial deletions within the PTTG1 promoter region were constructed. The promoter activity of the PTTG1 gene was repressed (25–51%) by T3. Additionally, these findings indicate that PTTG1 may be regulated by Sp1. The critical role of the −594 and −520 Sp1 binding sites was confirmed by electrophoretic mobility shift assay. Transfection with Sp1 expression vector enhanced the activity of the PTTG1 promoter fragment reporter. Also, Sp1 was down-regulated in HCC cells and in thyroidectomized rat after T3 treatment. Additionally, ectopic expression of PTTG1 promotes cell proliferation in Hep3B hepatoma cells. Conversely, knockdown of PTTG1 or Sp1 expression reduced cell proliferation in HepG2 cells. Notably, the expression of PTTG1 and Sp1 was inversely correlated with the expression of TR proteins in HCC. Together, these findings indicate that PTTG1 gene expression is mediated by Sp1 and is indirectly down-regulated by T3. Finally, overexpression of PTTG1 or SP1 in HCCs is TR-dependent and crucial in the development of HCC. [Cancer Res 2008;68(6):1697–1706]

Thyroid hormone, T(3), regulates cell metabolism, differentiation, and development. cDNA microarrays were performed to study the mechanism of target gene regulation after T(3) treatment in a thyroid hormone receptor-alpha (TRalpha)-overexpressing hepatoma cell line (HepG2-TRalpha). The differentially expressed target genes are several metabolic enzymes, including dehydroepiandrosterone-sulfotransferase family 1A member 2 (SULT2A1). Enzyme SULT2A1 was elevated roughly 5-fold at the protein level and 9-fold increase at the mRNA level after 48 h T(3) treatment in HepG2-TRalpha cells. Cycloheximide inhibited T(3)-induced SULT2A1 expression, suggesting that regulation was indirect. SULT2A1 has been reported to be regulated by the two transcription factors, steroidogenic factor 1 (SF1) and GATA, in the human adrenal gland. T(3) induced a 2.5- to 3.5-fold elevation of SF1 at the protein level and a 6.2-fold increase at the RNA level in HepG2-TRalpha cells. About seven SF1 binding sites exist on the SULT2A1 gene. To identify and localize the critical SF1 binding site, series of deletion mutants of SULT2A1 promoter fragments in pGL2 plasmid were constructed. The promoter activity of the SULT2A1 gene was enhanced about 2.8- to 7.1-fold by T(3). The -228 SF1 binding site was identified as the most critical site because deleting this region reduced T(3)-induced expression. Transcription factor SF1 application enhanced the -228 but not -117 reporter plasmid activities. SULT2A1 and SF1 up-regulation at protein and RNA levels in thyroidectomized rats occurred after T(3) application. In summary, this work demonstrated that the SULT2A1 gene was mediated by SF1 and indirectly regulated by T(3). Further study is required to elucidate the physiological importance of SULT2A1 induction mediated by T(3).