CLONING OF THE GENE RELATED TO MITOCHONDRIAL IRON DEPOSITION IN MYELODYSPLASTIC SYNDROME

为了研究细胞内铁超载的机制，我们分析了参与细胞内铁运输的功能分子。我们发现，铁转运最重要的细胞表面分子转铁蛋白受体（TfR）的表达在炎症细胞因子和酒精的作用下上调，而不依赖于细胞内铁浓度。然后，我们研究了HFE对TfR的功能调节。已知HFE与TfR相关，HFE突变可导致遗传性血色素沉着病。我们从人肝细胞系中分离出HFE cDNA，通过转染HFE基因建立了过表达HFE蛋白的人肝癌细胞。细胞表面TfR表达和转铁蛋白对TfR的亲和力显著降低，导致这些细胞中转铁蛋白结合的铁摄取减少。我们发现，在这些细胞中，HFE蛋白减缓了TfR循环的速度，特别是从内体到表面的循环。这些结果强烈表明，除了降低受体亲和力外，HFE还对转铁蛋白受体的再循环起着额外的作用，从而导致细胞铁的减少。最后，我们研究了Nramp2 （DMT-1）在tfr介导的铁转运中的作用。Nramp2是肠腔到肠上皮细胞的主要铁转运体，在多种细胞中，内体的游离铁通过Nramp2转运到细胞质。我们建立了转染人肝癌细胞的Nramp2基因，并分析了转铁蛋白- tfr系统对铁的摄取。Nramp2过表达不改变铁摄取和TfR再循环。由于我们没有像分析肠上皮细胞那样分析游离铁的摄取，因此Nramp2可能在这些细胞中具有从细胞表面到细胞质的游离铁转运体的功能。分析线粒体铁沉积的机制，有助于阐明细胞内铁超载的机制。

To investigate the mechanisms of intracellular iron overload, we analyzed the functional molecules involved in intracellular iron transport. We found that the expression of transferrin receptor (TfR), which is the most important cell surface molecule for iron transport, was upregulated by inflammatory cytokines and alcohol independent of intracellular iron concentration. We then examined the functional regulation of TfR by HFE.HFE is known to associate with TfR and the mutation of HFE causes hereditary hemochromatosis. We isolated the HFE cDNA from human hepatocyte cell line and established the human hepatoma cells overexpressing HFE protein by HFE gene transfection. Cell surface TfR expression and affinity of transferrin to TfR were significantly decreased and led to reduce the transferrin bound iron uptake in these cells. We found that the HFE protein slowed the rate of TfR recycling, especially return from endosome to surface, in these cells. These results strongly suggested an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron. Finally, we examined that the roles of Nramp2 (DMT-1) on TfR-mediated iron transport. Nramp2 is a major iron transporter from intestinal lumen to intestinal epithelial cells and is believed that free iron of endosome is transported to cytoplasm by Nramp2 in variety of cells. We established the Nramp2 gene transfected human hepatoma cells and analyzed the iron uptake by transferrin-TfR system. Nramp2 overexpression did not alter the iron uptake and TfR recycling. Because we did not analyzed the free iron uptake as analyzed by intestinal epithelial cells, it is possible that Nramp2 may have the function as a free iron transporter from cell surface to cytoplasm in these cells. To analyze the mechanisms of mitochondrial iron deposition, it would be useful to clarify the mechanisms of intracellular iron overload..