THYROID-HORMONE REGULATION OF NA,K-ATPASE ALPHA-2 GENE-EXPRESSION IN CARDIAC MYOCYTES

Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and bet a subunit mRNA abundances in myocardial cells in vivo and in vitro. In this study, we used transient transfection and nuclear run-on assays to determine whether T3 regulates the transcription rate of the Na,K-A TPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac m yocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and (alpha 2 mRNA levels were measured by Northern blot hybridization analysis. The re was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatme nt, whereas a two- and threefold increase in alpha 2 mRNA was evident when cells were exposed to T3 for 3 and 6 d, respectively. A portion o f the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Tr ansient transfection experiments utilizing this chimeric gene showed n o T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 rece ptor (T3R). In contrast, cotransfection of T3R facilitated a strong st imulation of luciferase activity driven by a construct containing a si ngle copy of a palindromic T3 response element (TRE). Nuclear run-on a nalysis indicated that the rate of transcription of the endogenous alp ha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not re gulated at either 1 or 6 d. These results indicate that the T3-depende nt increase in alpha 2 mRNA content at 6 d is mediated at a post-trans criptional level. Unexpectedly, we observed a T3-dependent three-to si xfold repression of alpha 2/luciferase expression in cardiac myocytes cotransfected with T3R. Deletion analysis of the 5' end of the alpha 2 gene revealed a negative TRE between nucleotides -354 and -100.