Wang Wei, Di Xiumin, D'Agostino Ralph B, Torti Suzy V, Torti Frank M
Department of Cancer Biology, Public Health Sciences, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, USA.
J Biol Chem. 2007 Aug 24;282(34):24650-9. doi: 10.1074/jbc.M703167200. Epub 2007 Jun 28.
Iron regulatory proteins (IRP1 and IRP2) are master regulators of cellular iron metabolism. IRPs bind to iron-responsive elements (IREs) present in the untranslated regions of mRNAs encoding proteins of iron storage, uptake, transport, and export. Because simultaneous knockout of IRP1 and IRP2 is embryonically lethal, it has not been possible to use dual knockouts to explore the consequences of loss of both IRP1 and IRP2 in mammalian cells. In this report, we describe the use of small interfering RNA to assess the relative contributions of IRP1 and IRP2 in epithelial cells. Stable cell lines were created in which either IRP1, IRP2, or both were knocked down. Knockdown of IRP1 decreased IRE binding activity but did not affect ferritin H and transferrin receptor 1 (TfR1) expression, whereas knockdown of IRP2 marginally affected IRE binding activity but caused an increase in ferritin H and a decrease in TfR1. Knockdown of both IRPs resulted in a greater reduction of IRE binding activity and more severe perturbation of ferritin H and TfR1 expression compared with single IRP knockdown. Even though the knockdown of IRP-1, IRP-2, or both was efficient, resulting in nondetectable protein and under 5% of wild type levels of mRNA, all stable knockdowns retained an ability to modulate ferritin H and TfR1 appropriately in response to iron challenge. However, further knockdown of IRPs accomplished by transient transfection of small interfering RNA in stable knockdown cells completely abolished the response of ferritin H and TfR1 to iron challenge, demonstrating an extensive excess capacity of the IRP system.
铁调节蛋白（IRP1和IRP2）是细胞铁代谢的主要调节因子。IRP与存在于编码铁储存、摄取、运输和输出蛋白的mRNA非翻译区的铁反应元件（IRE）结合。由于IRP1和IRP2的同时敲除在胚胎期是致死性的，因此无法使用双敲除来探究IRP1和IRP2在哺乳动物细胞中缺失的后果。在本报告中，我们描述了使用小干扰RNA来评估IRP1和IRP2在上皮细胞中的相对作用。构建了稳定细胞系，其中IRP1、IRP2或两者均被敲低。IRP1的敲低降低了IRE结合活性，但不影响铁蛋白H和转铁蛋白受体1（TfR1）的表达，而IRP2的敲低对IRE结合活性影响较小，但导致铁蛋白H增加和TfR1减少。与单个IRP敲低相比，两者均敲低导致IRE结合活性更大程度的降低以及铁蛋白H和TfR1表达更严重的紊乱。尽管IRP-1、IRP-2或两者的敲低都是有效的，导致无法检测到蛋白质且mRNA水平低于野生型水平的5%，但所有稳定敲低的细胞系在受到铁刺激时仍保留了适当调节铁蛋白H和TfR1的能力。然而，通过在稳定敲低细胞中瞬时转染小干扰RNA进一步敲低IRP，完全消除了铁蛋白H和TfR1对铁刺激的反应，表明IRP系统具有广泛的过剩能力。
