Analysis of thyroid hormone action in the cell ; Mechanism of induction and function of the hormone-responsive protein.

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1) Thyroid hormone-responsive rat hepatic t protein turned out to be a peroxisomal bifunctional enzyme. The amount of t Protein and the t Protein mRNA were increased after thyroidectomy and were decreased within 24 hrs after administration of small amount of triiodothyronine(T3) (0.3-1.0 mug/100 g body weight). By means of CAT assay, it was shown that 5' flanking region of the t protein gene contained negative T3 responsive element, indicating that negative regulation of the t protein by thyroid hormone was exerted at the transcriptional level.2) We isolated cDNA fragment of rat hepatic nuclear n protein using antibody probe. It was shown that the induction of n protein by T3 was due to the increase of mRNA.However the idea that the cDNA fragment we isolated really encodes the n protein was not yet verified. For this purpose, we are planning to show that the amino acid sequences deduced from nucleotide sequences of the cDNA and those obtained from protein sequencing are the same.3) We showed that carrier mediated and energy dependent cellular uptake of T3 is important source of nuclear T3. We showed that the nuclear entry of T3 is not solely via passive diffusion and is somehow regulated in the cell. It was also shown that the cellular transport of T3 is different from that of thyroxine(T4). Unlike T4, T3 transport is altered as the cells go through cell cycle. T3 is most actively transported into the cell at S phase when number of nuclear T3 receptor was increased. Additionally, T3 exerted proliferative effect on dRLh-84 cells through shortening of the S phase, suggesting that T3 action is enhanced in S phase due to increased number of receptor and enhanced uptake of T3.