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首页> 外文期刊>American Journal of Physiology >Copper stabilizes the Menkes copper-transporting ATPase (Atp7a) protein expressed in rat intestinal epithelial cells
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Copper stabilizes the Menkes copper-transporting ATPase (Atp7a) protein expressed in rat intestinal epithelial cells

机译：铜可稳定在大鼠肠上皮细胞中表达的Menkes铜转运ATPase（Atp7a）蛋白

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摘要

Iron deficiency decreases oxygen tension in the intestinal mucosa, leading to stabilization of hypoxia-inducible transcription factor 2alpha (Hif2alpha) and subsequent upregulation of genes involved in iron transport [e.g., divalent metal transporter (Dmt1) and ferroportin 1 (Fpn1)]. Iron deprivation also alters copper homeostasis, reflected by copper accumulation in the intestinal epithelium and induction of an intracellular copper-binding protein [metallothionein (Mt)] and a copper exporter [Menkes copper ATPase (Atp7a)]. Importantly, Atp7a is also a Hif2alpha target. It was, however, previously noted that Atp7a protein expression was induced more strongly than mRNA in the duodenum of iron-deprived rats, suggesting additional regulatory mechanisms. The current study was thus designed to decipher mechanistic aspects of Atp7a regulation during iron deprivation using an established in vitro model of the mammalian intestine, rat intestinal epithelial (IEC-6) cells. Cells were treated with an iron chelator and/or copper loaded to mimic the in vivo situation. IEC-6 cells exposed to copper showed a dose-dependent increase in Mt expression, confirming intracellular copper accumulation. Iron chelation with copper loading increased Atp7a mRNA and protein levels; however, contrary to our expectation, copper alone increased only protein levels. This suggested that copper increased Atp7a protein levels by a posttranscriptional regulatory mechanism. Therefore, to determine if Atp7a protein stability was affected, the translation inhibitor cycloheximide was utilized. Experiments in IEC-6 cells revealed that the half-life of the Atp7a protein was ~41 h and, furthermore, that intracellular copper accumulation increased steady-state Atp7a protein levels. This investigation thus reveals a novel mechanism of Atp7a regulation in which copper stabilizes the protein, possibly complementing Hif2alpha-mediated transcriptional induction during iron deficiency.
机译：铁缺乏症会降低肠粘膜中的氧气张力，从而导致缺氧诱导型转录因子2alpha（Hif2alpha）稳定，并随后上调参与铁转运的基因[例如，二价金属转运蛋白（Dmt1）和铁转运蛋白1（Fpn1）]。铁的剥夺还改变了铜的稳态，这反映在肠上皮中的铜积累以及细胞内铜结合蛋白[金属硫蛋白（Mt）]和铜输出蛋白[门克斯铜ATP酶（Atp7a）]的诱导。重要的是，Atp7a还是Hif2alpha目标。但是，以前曾指出，在缺铁大鼠的十二指肠中，Atp7a蛋白的表达比mRNA更强烈地被诱导，这提示了其他调控机制。因此，本研究旨在利用已建立的哺乳动物肠，大鼠肠上皮（IEC-6）细胞体外模型，来解释铁剥夺过程中Atp7a调控的机制。用铁螯合剂和/或负载铜以模拟体内情况处理细胞。暴露于铜的IEC-6细胞显示Mt表达呈剂量依赖性，这证实了细胞内铜的积累。铁螯合和铜负载增加了Atp7a mRNA和蛋白水平。但是，与我们的预期相反，铜本身只能增加蛋白质水平。这表明铜通过转录后调节机制增加了Atp7a蛋白水平。因此，为了确定Apt7a蛋白稳定性是否受到影响，使用了翻译抑制剂环己酰亚胺。在IEC-6细胞中进行的实验表明，Atp7a蛋白的半衰期约为41小时，此外，细胞内铜的积累增加了稳态Atp7a蛋白的水平。因此，这项研究揭示了Atp7a调控的新机制，其中铜稳定了蛋白质，可能在铁缺乏时补充了Hif2alpha介导的转录诱导。

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