Expression and Function of Transferrin Receptor 2 in the Hematopoietic Systems.

背景：本项目期间，有文献报道转铁蛋白受体2 （TfR2）纯合突变导致3型血色素沉着症。与Philip Koeffler（加州大学洛杉矶分校/雪松-西奈医学中心）和Robert Fleming（圣路易斯大学）合作创建的纯合子tfr2突变小鼠在肝脏中表现出铁积累，与血色素沉着症相似。这些发现表明TfR2参与铁代谢。在本研究中，我重点研究了TfR2在造血细胞中的表达和功能。方法与结果：第一；我制作了两种针对TfR2的多克隆抗体，使用细菌中产生的TfR2蛋白或TfR2合成肽作为抗原。使用稳定转染TfR2或转铁蛋白受体1表达质粒的CHO-TRVb细胞检测这些抗体的特异性和敏感性。这些抗体可用于免疫组织化学和免疫印迹分析。此外，我建立了一个夹心ELISA系统来检测TfR3的可溶性形式。人血清样品中未检测到显著水平的TfR2，表明血清中可溶性TfR2的含量可能较低。接下来，我通过免疫组织化学检测骨髓（BM）细胞中TfR2的表达。多数红系细胞呈阳性，髓系细胞呈阴性。一个红细胞白血病样本显示TfR2染色非常强。我分析了纯合子tfr2突变小鼠。骨髓及外周血细胞计数及形态均正常。小鼠骨髓中CFU-GM、BFU-E、CFU-Meg菌落计数均正常。讨论：最近的报道表明，hepcidin，一种肝肽激素，在铁稳态中起重要作用。我现在正在研究hepcidin和TfR2之间的关系，TfR2可能是肝脏铁转铁蛋白的传感器。此外，TfR2在红白血病细胞中的表达意义仍有待确定。

Background : During the term of this project, it was reported by others that homozygous mutations of transferrin receptor 2 (TfR2) cause hemochromatosis type 3. Homozygous TfR2-mutated mice created in collaboration with Philip Koeffler (UCLA/Cedars-Sinai Medical Center) and Robert Fleming (Saint Louis University) exhibited iron accumulation in the liver, a similar phenotype to hemochromatosis. These findings demonstrated that TfR2 is involved in iron metabolism. In this study, I focused on the expression and function of TfR2 in hematopoietic cells.Methods and Results : First; I made two polyclonal antibodies against TfR2, using either TfR2 protein produced in bacteria or a TfR2 synthetic peptide as antigens. Specificities and sensitivities of these antibodies were tested using CHO-TRVb cells stably transfected with TfR2 or transferrin receptor 1 expression plasmids. These antibodies were useful for immunohistochemistry and Western blot analysis. Additionally, I established a sandwich ELISA system for detecting soluble forms of TfR3. Significant levels of TfR2 in the human serum samples could not be detected, indicating that the quantity of soluble TfR2 in the serum may be low. Next, I examined expression of TfR2 in bone marrow (BM) cells by immunohistochemistry. Most erythroid cells showed positive staining, while most myeloid cells were negative. One erythroleukemia sample showed very strong staining for TfR2. I analyzed the homozygous TfR2-mutated mice. Cell counts and morphologies of the both BM and peripheral blood cells were normal. Colony counts of CFU-GM, BFU-E and CFU-Meg of the BM of these mice were also normal.Discussion : Recent reports indicate that hepcidin, a hepatic peptide hormone, plays an essential role in iron homeostasis. I am now working on the relationship between hepcidin and TfR2, which may be the sensor for iron-transferrin in liver. Also, the significance of expression of TfR2 in erythroleukemia cells remains to be determined.