Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611, USA.
Am J Physiol Cell Physiol. 2013 Feb 1;304(3):C257-62. doi: 10.1152/ajpcell.00336.2012. Epub 2012 Nov 21.
Iron deficiency decreases oxygen tension in the intestinal mucosa, leading to stabilization of hypoxia-inducible transcription factor 2α (Hif2α) 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 Hif2α 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 Hif2α-mediated transcriptional induction during iron deficiency.
缺铁会降低肠道黏膜中的氧张力，导致缺氧诱导转录因子 2α（Hif2α）的稳定，并随后上调涉及铁转运的基因[例如，二价金属转运蛋白（Dmt1）和铁蛋白 1（Fpn1）]。铁剥夺还会改变铜稳态，表现为肠道上皮细胞中铜的积累和细胞内铜结合蛋白[金属硫蛋白（Mt）]和铜输出蛋白[Menkes 铜 ATP 酶（Atp7a）]的诱导。重要的是，Atp7a 也是 Hif2α 的靶标。然而，先前的研究表明，缺铁大鼠十二指肠中 Atp7a 蛋白的表达比 mRNA 诱导得更强，这表明存在其他调节机制。因此，本研究旨在使用已建立的哺乳动物肠道体外模型（大鼠肠上皮（IEC-6）细胞）来阐明 Atp7a 在铁剥夺期间的调节机制。用铁螯合剂和/或铜处理细胞，以模拟体内情况。暴露于铜的 IEC-6 细胞表现出 Mt 表达的剂量依赖性增加，证实了细胞内铜的积累。铁螯合与铜加载增加了 Atp7a mRNA 和蛋白水平；然而，与我们的预期相反，单独的铜仅增加了蛋白水平。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 Atp7a 蛋白稳定性是否受到影响，使用了翻译抑制剂环己酰亚胺。IEC-6 细胞中的实验表明，Atp7a 蛋白的半衰期约为 41 小时，此外，细胞内铜积累增加了 Atp7a 蛋白的稳态水平。因此，这项研究揭示了 Atp7a 调节的一种新机制，其中铜稳定了蛋白，可能在缺铁时补充 Hif2α 介导的转录诱导。
