Wardrop S L, Richardson D R
Department of Medicine, Royal Brisbane Hospital, Brisbane, Queensland, Australia.
Eur J Biochem. 1999 Jul;263(1):41-9. doi: 10.1046/j.1432-1327.1999.00447.x.
Recent studies have demonstrated that the protein product (natural resistance associated macrophage protein 2, Nramp2) encoded by the gene Nramp2 acts as an Fe transporter involved in the uptake of Fe from transferrin (Tf) and low Mr Fe complexes. Interestingly, there are two splice variants of Nramp2, one with a putative iron-responsive element (IRE) in its 3' untranslated region (UTR) and another without. Due to the importance of Nramp2 in Fe transport, and the presence of an IRE in its 3'-UTR, we have examined the effect of Fe-deprivation, Fe-loading, and nitrogen monoxide on the expression of Nramp2 mRNA. These results were compared to the expression of transferrin receptor (TfR) mRNA which also has IREs in its 3'-UTR and is regulated by Fe and NO via the binding of iron-regulatory proteins (IRPs) to its IREs. Our experiments show that the IRE in Nramp2 mRNA does bind the IRPs in lysates from a mouse fibroblast cell line (LMTK-). Moreover, reverse transcription-PCR (RT-PCR) demonstrated that both the IRE and non-IRE-containing transcripts were present within these cells. However, there was no change in Nramp2 mRNA expression in LMTK- cells after a 20-h incubation with either the Fe chelator, desferrioxamine (DFO), the Fe donor, ferric ammonium citrate (FAC), or the NO generator, S-nitroso-N-acetylpenicillamine (SNAP). In contrast, these agents caused a marked change in the RNA-binding activity of the IRPs and the expression of TfR mRNA. In addition, both FAC and DFO caused an appropriate change in [59Fe] uptake from [59Fe]Tf, viz., an increase in Fe uptake after exposure to DFO and a decrease after treatment with FAC. As Nramp2 can transport Fe from non-Tf-bound Fe, the effect of preincubation with DFO and FAC was also examined on Fe uptake from [59Fe]nitrilotriacetate and [59Fe]citrate. However, in contrast to the results found for [59Fe]Tf, incubation with DFO and FAC did not result in appropriate regulation of Fe uptake from [59Fe]nitrilotriacetate or [59Fe]citrate. These data demonstrate that non-Tf-bound Fe uptake was not under control of the IRP-IRE system in these cells. Collectively, the results indicate that in LMTK-fibroblasts Nramp2 mRNA expression was not regulated like TfR mRNA.
最近的研究表明，Nramp2基因编码的蛋白质产物（天然抗性相关巨噬细胞蛋白2，Nramp2）作为一种铁转运蛋白，参与从转铁蛋白（Tf）和低分子量铁复合物中摄取铁。有趣的是，Nramp2有两种剪接变体，一种在其3'非翻译区（UTR）有一个假定的铁反应元件（IRE），另一种则没有。由于Nramp2在铁转运中的重要性，以及其3'-UTR中存在IRE，我们研究了缺铁、铁负载和一氧化氮对Nramp2 mRNA表达的影响。这些结果与转铁蛋白受体（TfR）mRNA的表达进行了比较，TfR mRNA在其3'-UTR中也有IREs，并且通过铁调节蛋白（IRPs）与其IREs的结合受铁和一氧化氮的调节。我们的实验表明，Nramp2 mRNA中的IRE确实能与小鼠成纤维细胞系（LMTK-）裂解物中的IRPs结合。此外，逆转录聚合酶链反应（RT-PCR）表明，这些细胞中同时存在含IRE和不含IRE的转录本。然而，在用铁螯合剂去铁胺（DFO）、铁供体柠檬酸铁铵（FAC）或一氧化氮供体S-亚硝基-N-乙酰青霉胺（SNAP）孵育20小时后，LMTK-细胞中Nramp2 mRNA的表达没有变化。相反，这些试剂导致IRPs的RNA结合活性和TfR mRNA的表达发生显著变化。此外，FAC和DFO都引起了从[59Fe]Tf摄取[59Fe]的适当变化，即，暴露于DFO后铁摄取增加，用FAC处理后铁摄取减少。由于Nramp2可以从非Tf结合的铁中转运铁，因此还研究了用DFO和FAC预孵育对从[59Fe]次氮基三乙酸和[59Fe]柠檬酸盐摄取铁的影响。然而，与[59Fe]Tf的结果相反，用DFO和FAC孵育并没有导致对从[59Fe]次氮基三乙酸或[59Fe]柠檬酸盐摄取铁的适当调节。这些数据表明，在这些细胞中，非Tf结合的铁摄取不受IRP-IRE系统的控制。总的来说，结果表明在LMTK-成纤维细胞中，Nramp2 mRNA的表达不像TfR mRNA那样受到调节。
