Ligand-mediated decrease of thyroid hormone receptor-α1in cardiomyocytes by proteosome-dependent degradation and altered mRNA stability

Abstract

Tri-iodo-l-thyronine (T3) is essential for maintaining normal cardiac contractile function by regulating transcription of numerous T3-responsive genes. Both hormone availability and relative amounts of nuclear thyroid hormone receptor isoforms (TRα1, TRβ1) determine T3effectiveness. Cultured neonatal rat ventricular myocytes grown in T3-depleted medium expressed predominantly TRα1 protein, but within 4 h of T3treatment, TRβ1 protein increased significantly, whereas TRα1 was decreased by 46 ± 5%. Using replication-defective adenoviruses to overexpress TRα1 in cardiomyocytes, we studied the mechanisms by which T3mediated the decrease in TRα1 protein. Inhibitors of the proteosome pathway resulted in an accumulation of ubiquitylated TRα1 in the nucleus and prevented T3-induced degradation of ubiquitylated TRα1, suggesting that T3induced proteosome-mediated degradation of TRα1; however, TR ubiquitylation was T3independent. TRα1 transcriptional activity, measured using transient transfection of a thyroid hormone-responsive element (TRE) reporter plasmid, was T3dose dependent and inversely proportional to nuclear TRα1 content, with 10 nM T3having maximum effect. Quantitative RT-PCR showed that both endogenous and adenovirus-expressed TRα1 mRNAs were significantly decreased to 54 ± 11 and 25 ± 5%, respectively, within 4 h of T3treatment. Measurements of TRα1 mRNA half-life in actinomycin D-treated cardiomyocytes showed that T3treatment significantly decreased TRα1 mRNA half-life from 4 h to less than 2 h, whereas it had no effect of TRβ1 mRNA half-life. These data support a role for both the proteosome degradation pathway and altered mRNA stability in T3-induced decrease of nuclear TRα1 in the cardiomyocyte and provide novel cellular targets for therapeutic development.