title: Thyroid hormone regulation of Na,K-ATPase alpha 2 gene expression in cardiac myocytes.
abstract: Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and beta 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-ATPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac myocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and alpha 2 mRNA levels were measured by Northern blot hybridization analysis. There was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatment, 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 of the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Transient transfection experiments utilizing this chimeric gene showed no T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 receptor (T3R). In contrast, cotransfection of T3R facilitated a strong stimulation of luciferase activity driven by a construct containing a single copy of a palindromic T3 response element (TRE). Nuclear run-on analysis indicated that the rate of transcription of the endogenous alpha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not regulated at either 1 or 6 d. These results indicate that the T3-dependent increase in alpha 2 mRNA content at 6 d is mediated at a post-transcriptional level. Unexpectedly, we observed a T3-dependent three-to sixfold 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.
authors: Department of Biochemistry, State University of New York Health Science Center at Brooklyn 11203.
authors: F
authors: F
authors: Huang
authors: H
authors: H
authors: He
authors: G
authors: G
authors: Gick
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D008156:Luciferases; D000254:Sodium-Potassium-Exchanging ATPase