Identification of naturally occurring dominant negative mutants of thyroid hormone α1 and β1 receptors in a human hepatocellular carcinoma cell line

To understand the function of thyroid hormone nuclear receptors (TRs) in human hepatocellular carcinoma cells (HCC), we characterized the hormone binding and transactivational activity of TRs in a HCC cell line, J7. TRα1 (J7-TRα1) and TRβ1 (J7-TRβ1) complementary DNAs were cloned from this cell line, and the binding activity to the hormone response elements (TREs) and to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T₃) of the expressed TR proteins were evaluated. J7-TRα1 and J7-TRβ1 bound to TREs similarly as the TRs isolated from other tissues. However, J7-TRα1 did not bind to T₃, and J7-TRβ1 bound to T₃ with only about 10% the affinity of the wild-type TRβ1. Sequencing of the complementary DNAs shows a single Met²⁵⁹Ile mutation in J7-TRα1 and Met³³⁴Val in J7-TRβ1. Using reporters containing TREs, we found that J7-TRα1 and J7-TRβ1 had virtually lost their transactivational activity. Moreover, these two mutants inhibited the transactivational activity of the wild-type TRs by a dominant negative effect not only on the transfected TRs, but also on endogenous TRs in other two HCC cell lines, SK-Hep-1 and HepG2. The potency of the dominant negative effect of these two mutants inversely correlated with the expression level of endogenous TRs. The present studies identified two novel naturally occurring TR mutants that have potent dominant negative action. The identification of both the α and β dominant negative mutants in J7 made this cell line a useful model system to further understand the molecular mechanism of the dominant negative action of TR mutants.