The thyroid hormone L-T3 elicits either a stimulatory or an inhibitory effect on expression of the Na,K-adenosine triphosphatase alpha3-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 alpha3-subunit gene proximal promoter. Transient transfection assays indicated that the DNA-binding domain of thyroid hormone receptor was essential for mediating repression of alpha3 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 alpha3 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 alpha3 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 alpha3 gene promoter. The upstream and downstream heterodimer binding sites coexist with CAAT and TATA elements, respectively.
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