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.

The thyroid hormone 3,3 0 ,5-triiodo-L-thyronine (T 3 ) 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 T 3 treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T 3 . However, the underlying mechanism and the physiological role of T 3 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 T 3 treatment in the HepG2-TRa1#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 T 3 response element on the spondin 2 promoter was localized in the K1104/K1034 or K984/K925 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 T 3 , has an important role in cell invasion, cell migration, and tumor progression.

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