Bogazzi F, Bartalena L, Brogioni S, Burelli A, Grasso L, Dell'Unto E, Manetti L, Martino E
Istituto di Endocrinologia, Università di Pisa, Presidio Ospedaliero di Cisanello, Italy.
Mol Cell Endocrinol. 1997 Oct 31;134(1):23-31. doi: 10.1016/s0303-7207(97)00156-1.
L-thyroxine (T4) has been considered mainly a prohormone, the hormonal action of which is related to its conversion to 3,5,3'-triiodothyronine (T3) in peripheral tissues. In this study we investigated in transient transfection assays whether T4 might directly affect the expression of thyroid hormone (TH) sensitive genes. The reporter construct ME-TRE-TK-CAT or TSH-TRE-TK-CAT containing the nucleotide sequence of the TH response element (TRE) of either malic enzyme (ME) or TSHbeta genes, was transfected with either TH receptor (TR) alpha alone or in combination with retinoid X receptor (RXR) beta into NIH3T3 cells. Addition of 100 nM T4 to the culture medium in the presence of TRalpha increased the basal level of ME-TRE-TK-CAT expression by 4.5-fold. T4 action was due to a direct interaction with TRalpha and not to its conversion to T3, since T4 effect persisted in the presence of 5'-deiodinase inhibitors (propylthiouracil, iopanoic acid) effectively preventing T3 generation, as assessed by the absence of T3 by HPLC in the cellular extracts of transfected cells. In a dose-response study half-maximal stimulation by T4 was achieved at a concentration of 100 nM, whereas 50% of maximal induction was produced by 1 nM T3 and 6 nM triiodothyroacetic acid (TRIAC). Coexpression of RXRbeta greatly enhanced the transcriptional activity of the ME-TRE-TK-CAT gene when either T3, T4 or TRIAC was added to the culture medium of NIH3T3 cells, but established a hormonal hierarchy in the reporter activation different than that observed in the presence of TRalpha alone (TRIAC > T3 > or = T4, instead of T3 > TRIAC > T4). T4 at a concentration of 100 nM could activate the TH/TR-dependent down-regulation mediated by the negative TSH-TRE, although at a lower level than that obtained with similar concentrations of T3 (35 and 55% inhibition, respectively). Our results demonstrate that, in addition to the action mediated through its monodeiodination to T3, T4 exerts a direct effect on genes that are either positively or negatively regulated by TH. Moreover, RXRbeta, forming heterodimers with TRs, appeared to exert a central role in modulating the sensitivity of TH-responsive genes to different iodothyronines.
左旋甲状腺素（T4）一直被主要视为一种前体激素，其激素作用与其在外周组织中转化为3,5,3'-三碘甲状腺原氨酸（T3）有关。在本研究中，我们通过瞬时转染实验研究了T4是否可能直接影响甲状腺激素（TH）敏感基因的表达。将含有苹果酸酶（ME）或促甲状腺激素β（TSHβ）基因的甲状腺激素反应元件（TRE）核苷酸序列的报告基因构建体ME-TRE-TK-CAT或TSH-TRE-TK-CAT，单独与甲状腺激素受体（TR）α或与视黄酸X受体（RXR）β一起转染到NIH3T3细胞中。在存在TRα的情况下，向培养基中添加100 nM T4可使ME-TRE-TK-CAT的基础表达水平提高4.5倍。T4的作用是由于其与TRα的直接相互作用，而不是其转化为T3，因为在存在5'-脱碘酶抑制剂（丙硫氧嘧啶、碘番酸）的情况下T4的作用仍然存在，这些抑制剂可有效阻止T3的生成，通过高效液相色谱法（HPLC）检测转染细胞的细胞提取物中不存在T3来评估。在剂量反应研究中，T4在100 nM浓度时达到半数最大刺激，而1 nM T3和6 nM三碘甲状腺乙酸（TRIAC）产生50%的最大诱导作用。当向NIH3T3细胞培养基中添加T3、T4或TRIAC时，RXRβ的共表达极大地增强了ME-TRE-TK-CAT基因的转录活性，但在报告基因激活中建立了一种与单独存在TRα时观察到的不同的激素层级关系（TRIAC > T3 > 或 = T4，而不是T3 > TRIAC > T4）。100 nM浓度的T4可以激活由负性TSH-TRE介导的TH/TR依赖性下调，尽管其水平低于用相似浓度T3获得的水平（分别为35%和55%的抑制）。我们的结果表明，除了通过单脱碘转化为T3介导的作用外，T4对受TH正向或负向调节的基因也有直接作用。此外，与TR形成异二聚体的RXRβ似乎在调节TH反应性基因对不同碘甲状腺原氨酸的敏感性方面发挥核心作用。
