Characterization of a negative thyroid hormone response element in the rat sodium, potassium-adenosine triphosphatase α3 gene promoter

The thyroid hormone L-T, elicits either a stimulatory or an inhibitory effect on expression of the Na,K-adenosine triphosphatase alpha 3-subunit gene in primary cultures of neonatal rat cardiac myocytes. The present study was undertaken to characterize a negative thyroid hormone response element present within the rat Na,K-adenosine triphosphatase alpha 3-subunit gene proximal promoter. Transient transfection assays indicated that the DNA-binding domain of thyroid hormone receptor was essential for mediating repression of alpha 3 gene transcription by thyroid hormone. This negative effect of thyroid hormone was enhanced in the presence of cotransfected retinoid X receptor and its ligand 9-cis-retinoic acid. Inhibition of alpha 3 chimeric gene expression by thyroid hormone was dependent on the initial cell plating density. The negative thyroid hormone response element was localized to a region between nucleotides -68 to -6 of the alpha 3 gene. Electrophoretic mobility shift assays showed that thyroid hormone receptor binds in a synergistic manner as a heterodimer with retinoid X receptor to two sites at positions -62 to -41 and -39 to -17 of the alpha 3 gene promoter. The upstream and downstream heterodimer binding sites coexist with CAAT and TATA elements, respectively.