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

1)甲状腺激素反应性大鼠肝脏t蛋白是一种过氧化物酶体双功能酶。甲状腺切除术后t蛋白和t蛋白mRNA的量增加，并在给予少量三碘甲腺原氨酸（T3）（0.3-1.0 μ g/100 g体重）后24小时内减少。CAT分析表明，t蛋白基因5'端侧翼区含有负性T3反应元件，表明甲状腺激素对t蛋白的负性调控作用是在转录水平上进行的。2）用抗体探针法分离了大鼠肝细胞核n蛋白的cDNA片段。结果表明，T3诱导的n蛋白表达是由于mRNA水平的增加，但我们分离的cDNA片段是否真的编码n蛋白的观点尚未得到证实。为了这个目的，我们计划证明从cDNA的核苷酸序列推导的氨基酸序列和从蛋白质测序获得的氨基酸序列是相同的。3）我们表明载体介导的和能量依赖的细胞摄取T3是核T3的重要来源。我们发现，T3进入细胞核不仅仅是通过被动扩散，而且在细胞中受到某种程度的调节。T_3的细胞转运与T_4不同。与T4不同，T3转运随着细胞周期的进行而改变。当细胞核T3受体数量增加时，T3在S期转运最活跃。此外，T3通过缩短S期对dRLh-84细胞产生增殖作用，表明由于受体数量增加和T3摄取增强，T3作用在S期增强。

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.