Iron/IRP-1-dependent regulation of mRNA expression for transferrin receptor, DMT1 and ferritin during human erythroid differentiation
Iron/IRP-1-dependent regulation of mRNA expression for transferrin receptor, DMT1 and ferritin during human erythroid differentiation
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DOI：
10.1016/j.exphem.2007.03.005
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发表时间：
2007-06-01
影响因子：
2.6
通讯作者：
Niitsu, Yoshiro
中科院分区：
文献类型：
--
作者：
Kato, Junji;Kobune, Masayoshi;Niitsu, Yoshiro
Objective.采用体外三相液体培养技术，研究了转铁蛋白受体（TfR）、二价金属转运蛋白-1（DMT 1）和铁蛋白在红系分化过程中的铁调节蛋白（IRP）依赖性表达。外周血造血祖细胞与白细胞介素-3和干细胞因子（SCF）一起培养7天（第一阶段），随后与SCF、促红细胞生成素（EPO）和胰岛素样生长因子-I（IGF-I）一起培养5天（第二阶段），最后与EPO和IGF-I一起培养3天（第三阶段）。采用集落形成实验、流式细胞仪分析、mRNA检测、电泳迁移率变动实验、免疫印迹和免疫沉淀等方法检测细胞的增殖情况。在第二/第三阶段，红系细胞连续分化。在3 '-UTR处具有铁响应元件（IRE）的TfR和DMT 1 mRNA的表达在第二时相达到最大值，此后下降，而在5'-UTR处具有IRE的铁蛋白mRNA的表达在第二时相下降。多核糖体沉淀后，胞浆中的IRP在第二时相下降，表明IRP与多核糖体中这些mRNA的IRE结合。当细胞与（FeCl 3）-Fe-59共同孵育时，在第一相和第三相可检测到与抗IRP-1抗体免疫沉淀的Fe-59结合的IRP-1，但在第二相未检测到。这些结果表明，IRP-1/IRE的相互作用，这是推测感应细胞内非血红素铁水平的降低后诱导，发挥了至关重要的作用，转录后调节的TfR，DMT 1，和铁蛋白mRNA在正常人红细胞分化。(c)2007年国际实验血液学学会。爱思唯尔公司出版
Objective. We investigated iron regulatory protein (IRP)-dependent expression of transferrin receptor (TfR), divalent metal transpoter-1 (DMT1) and ferritin during erythroid differentiation system using an in vitro three-phase liquid culture.Method. Peripheral blood hematopoietic progenitor cells were cultured with interleukin-3 and stem cell factor (SCF) for 7 days (first phase), subsequently with SCF, erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) for 5 days (second phase), and finally with EPO and IGF-I for 3 days (third phase). Cells were subjected to colony assay, flow-cytometric analysis, mRNA assessment, electrophoretic mobility shift assay (EMSA), immunoblotting, and immunoprecipitation.Results. In the second/third phases, erythroid cells serially differentiated. Expression of TfR and DMT1 mRNA, which have iron-responsive elements (IREs) at 3'-UTR, reached a maximum on second phase, and thereafter decreased, while expression of ferritin mRNA, which has an IRE at the 5'-UTR, decreased reciprocally on second phase. IRP in the cytosol after precipitation of polysome decreased on second phase, suggesting that IRP bound to IREs of these mRNAs in the polysome. When cells were incubated with (FeCl3)-Fe-59, Fe-59-bound IRP-1 immunoprecipitated with anti-IRP-1 antibodies was detected on first phase and third phase, but was not detected on second phase.Conclusion. These results suggest that IRP-1/IRE interactions, which are supposedly induced after sensing a decrease of the intracellular non-Heme iron levels, play a crucial role on the posttranscriptional regulation of TfR, DMT1, and ferritin mRNAs during differentiation of normal human erythropoietic cells. (c) 2007 International Society for Experimental Hematology. Published by Elsevier Inc.