Zheng Dongling, Feeney Graham P, Kille Peter, Hogstrand Christer
Nutritional Sciences Division, King's College London, London, United Kingdom.
Physiol Genomics. 2008 Jul 15;34(2):205-14. doi: 10.1152/physiolgenomics.90206.2008. Epub 2008 May 13.
Resolving the mechanisms underlying the temporal and spatial profile of zinc transporter expression in response to zinc availability is key to understanding zinc homeostasis. The mRNA expression of seven zinc transporters was studied in zebrafish gills when treated with zinc deficiency/excess over a 14-day period. Of these, ZnT1, ZnT5, ZIP3, and ZIP10 were differentially expressed in response to changed zinc status. The mRNA level of zinc exporter, ZnT1, was upregulated in fish subjected to excess zinc and downregulated by zinc deprivation. This response was similar to that of metallothionein-2 (MT2). Zinc deficiency caused an increased abundance of mRNA for zinc importers ZnT5, ZIP3, and ZIP10. Expression of ZnT5 and ZIP10, but not ZIP3, was inhibited by excess zinc. Zinc influx function of ZIP10 was demonstrated by (65)Zn transport assays in Xenopus oocyte expression experiments, suggesting that the inverse relationship between zinc availability and ZIP10 expression serves to maintain zinc homeostasis. Two distinct transcription start sites (TSS) for ZIP10 were found in gill and kidney. Luciferase assays and mutation/deletion analysis of DNA fragments proximal to the respective TSS revealed that ZIP10 has two alternative promoters (P1 and P2) displaying opposite regulatory control in response to zinc status. Positive as well as negative regulation by zinc involves MRE clusters in the respective promoters. These results provide experimental evidence for MREs functioning as repressor elements, implicating MTF1 involvement in the negative regulation of ZIP10. This is in contrast to the well-established positive regulation by MTF1 of other genes, such as MT2 and ZnT1.
解析锌转运蛋白表达的时空分布模式响应锌可利用性的潜在机制是理解锌稳态的关键。在14天的时间里，用锌缺乏/过量处理斑马鱼鳃，研究了7种锌转运蛋白的mRNA表达。其中，ZnT1、ZnT5、ZIP3和ZIP10在锌状态改变时差异表达。锌输出蛋白ZnT1的mRNA水平在锌过量的鱼中上调，而在锌缺乏时下调。这种反应与金属硫蛋白-2（MT2）相似。锌缺乏导致锌输入蛋白ZnT5、ZIP3和ZIP10的mRNA丰度增加。过量的锌抑制了ZnT5和ZIP10的表达，但不影响ZIP3。在非洲爪蟾卵母细胞表达实验中，通过（65）Zn转运试验证明了ZIP10的锌内流功能，这表明锌可利用性与ZIP10表达之间的反向关系有助于维持锌稳态。在鳃和肾脏中发现了ZIP10的两个不同转录起始位点（TSS）。荧光素酶试验以及对各个TSS近端DNA片段的突变/缺失分析表明，ZIP10有两个替代启动子（P1和P2），它们在响应锌状态时表现出相反的调控作用。锌的正负调控都涉及各自启动子中的金属反应元件（MRE）簇。这些结果为MRE作为阻遏元件发挥作用提供了实验证据，暗示金属反应元件结合转录因子1（MTF1）参与ZIP10的负调控。这与MTF1对其他基因（如MT2和ZnT1）的既定正调控形成对比。
