Huang Wen, Xu Fei, Qu Tao, Zhang Rui, Li Li, Que Huayong, Zhang Guofan
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
University of Chinese Academy of Sciences, Beijing, China.
PLoS One. 2015 Dec 28;10(12):e0144991. doi: 10.1371/journal.pone.0144991. eCollection 2015.
Thyroid hormones (THs) play important roles in development, metamorphosis, and metabolism in vertebrates. During the past century, TH functions were regarded as a synapomorphy of vertebrates. More recently, accumulating evidence has gradually convinced us that TH functions also occur in invertebrate chordates. To date, however, TH-related studies in non-chordate invertebrates have been limited. In this study, THs were qualitatively detected by two reliable methods (HPLC and LC/MS) in a well-studied molluscan species, the Pacific oyster Crassostrea gigas. Quantitative measurement of THs during the development of C. gigas showed high TH contents during embryogenesis and that oyster embryos may synthesize THs endogenously. As a first step in elucidating the TH signaling cascade, an ortholog of vertebrate TH receptor (TR), the most critical gene mediating TH effects, was cloned in C. gigas. The sequence of CgTR has conserved DNA-binding and ligand-binding domains that normally characterize these receptors. Experimental results demonstrated that CgTR can repress gene expression through binding to promoters of target genes and can interact with oyster retinoid X receptor. Moreover, CgTR mRNA expression was activated by T4 and the transcriptional activity of CgTR promoter was repressed by unliganded CgTR protein. An atypical thyroid hormone response element (CgDR5) was found in the promoter of CgTR, which was verified by electrophoretic mobility shift assay (EMSA). These results indicated that some of the CgTR function is conserved. However, the EMSA assay showed that DNA binding specificity of CgTR was different from that of the vertebrate TR and experiments with two dual-luciferase reporter systems indicated that l-thyroxine, 3,3',5-triiodothyronine, and triiodothyroacetic acid failed to activate the transcriptional activity of CgTR. This is the first study to functionally characterize TR in mollusks. The presence of THs and the functions of CgTR in mollusks contribute to better understanding of the evolution of the TH system.
甲状腺激素（THs）在脊椎动物的发育、变态和新陈代谢中发挥着重要作用。在过去的一个世纪里，TH的功能被视为脊椎动物的一个共有衍征。最近，越来越多的证据逐渐使我们相信TH的功能也存在于无脊椎脊索动物中。然而，迄今为止，非脊索动物无脊椎动物中与TH相关的研究一直很有限。在本研究中，通过两种可靠的方法（高效液相色谱法和液相色谱/质谱法）在一种研究充分的软体动物——太平洋牡蛎（Crassostrea gigas）中对THs进行了定性检测。对太平洋牡蛎发育过程中THs的定量测量表明，胚胎发生期间TH含量很高，并且牡蛎胚胎可能内源性合成THs。作为阐明TH信号级联反应的第一步，在太平洋牡蛎中克隆了脊椎动物甲状腺激素受体（TR）的一个直系同源物，TR是介导TH效应的最关键基因。太平洋牡蛎TR（CgTR）的序列具有保守的DNA结合和配体结合结构域，这些结构域是这些受体的典型特征。实验结果表明，CgTR可以通过与靶基因的启动子结合来抑制基因表达，并且可以与牡蛎视黄酸X受体相互作用。此外，CgTR mRNA表达受T4激活，而未结合配体的CgTR蛋白可抑制CgTR启动子的转录活性。在CgTR的启动子中发现了一个非典型甲状腺激素反应元件（CgDR5），这通过电泳迁移率变动分析（EMSA）得到了验证。这些结果表明CgTR的一些功能是保守的。然而，EMSA分析表明CgTR的DNA结合特异性与脊椎动物的TR不同，并且用两种双荧光素酶报告系统进行的实验表明，L-甲状腺素、3,3',5-三碘甲状腺原氨酸和三碘甲状腺乙酸均未能激活CgTR的转录活性。这是第一项对软体动物中TR进行功能表征的研究。THs的存在以及CgTR在软体动物中的功能有助于更好地理解TH系统的进化。
