Cloning and characterization of a tilapia (Oreochromis aureus) metallothionein gene promoter in Hepa-T1 cells following the administration of various heavy metal ions

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• 作者：William Wai Lun Chan ; King Ming Chan
• 关键词：LC50 values of metal ions ; Gene transcription ; Gel-shift assay ; Metal induction ; Transient gene expression
• 刊名：Aquatic Toxicology
• 出版年：2008
• 期刊代码：19_0166445x
• 类别：abs
• 出版时间：20 January 2008
• 卷：86
• 期：1
• 页码：59-75
• 文件大小：2785 K

摘要

Metallothioneins (MTs) are highly conserved intracellular metal-binding proteins that contribute to the homeostasis of essential metals and the detoxification of non-essential heavy metals. MT gene expression is induced by various heavy metal ions, and Zn²⁺ is able to bind and activate a transcription factor associated with the MT gene that is known as the metal responsive element (MRE) binding transcription factor-1 (MTF-1). Heavy metals other than Zn²⁺, such as Cd²⁺ and Cu²⁺, fail to activate the binding of MTF-1 to MREs despite their ability to induce the transcription of the MT gene. To study how different metal ions regulate MT gene expression, a tilapia (ti)-MT gene promoter was cloned and its responses to activation by various metal ions measured using a Hepa T1 cell culture model. The tiMT gene promoter contains six functional MREs within 2118 bp 5′ of the translational start site. A transient gene expression study showed the tiMT gene promoter fragment to be responsive to Cd²⁺, Cu²⁺, Hg²⁺, Pb²⁺, and Zn²⁺. Deletions from the 5′ end and the site-directed mutagenesis of individual MREs in the tiMT gene promoter confirmed that both proximal and distal clusters of MREs were required for the maximal metal induction of the tiMT gene. The distal cluster of MREs greatly enhanced the induction of tiMT gene expression by several of the heavy metal ions, and especially the non-Zn²⁺ ions. Individual MREs showed a different responsiveness to metal ions, with MREe being the most potent, MREb being responsive to Zn²⁺ but not to other metal ions, and MREa being mainly for the basal expression of the tiMT gene. Electrophoretic mobility shift assay (EMSA) identified a transcription factor that was able to bind most of the MREs, with the exception of MREd, but the binding was only activated by the in vivo administration of Zn²⁺, not the administration of Cd²⁺ or Cu²⁺. In conclusion, the results of this study on a Hepa T1 cell model suggest that the mechanism of MT gene activation by non-Zn²⁺ metal ions is different from that of activation by Zn²⁺, and that different MREs may be involved in the activation of the tiMT gene by different metal ions without enhancing the binding of MTF-1 to MREs.