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

转铁蛋白受体 2 在造血系统中的表达和功能。

• 批准号: 13671088
• 负责人: KAWABATA Hiroshi
• 金额: $ 1.6万
• 依托单位: KANAZAWA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671088/
• 关键词: Transferrin; Transferrin receptor; Iron metabolism; Erythroid cells; Hematopoiesis

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.

背景资料：在本项目期间，其他人报道转铁蛋白受体2（TfR 2）的纯合突变导致3型血色病。与Philip Koeffler（加州大学洛杉矶分校/Cedars-Sinai医学中心）和Robert Fleming（圣刘易斯大学）合作创建的纯合子TfR 2突变小鼠显示出肝脏中的铁积累，这与血色素沉着症相似。这些发现表明TfR 2参与铁代谢。本研究主要研究TfR 2在造血细胞中的表达和功能。方法和结果：首先，以细菌产生的TfR 2蛋白和TfR 2合成肽为抗原，制备了两株TfR 2多克隆抗体。这些抗体的特异性和灵敏度使用稳定转染有TfR 2或转铁蛋白受体1表达质粒的CHO-TRVb细胞进行测试。这些抗体可用于免疫组织化学和Western印迹分析。此外，我建立了一个夹心ELISA系统，用于检测可溶性形式的TfR 3。在人血清样品中未检测到显著水平的TfR 2，表明血清中可溶性TfR 2的量可能较低。接下来，我通过免疫组织化学检测了TfR 2在骨髓（BM）细胞中的表达。多数红系细胞呈阳性染色，而多数髓系细胞呈阴性染色。一个红白血病样品显示出非常强的TfR 2染色。我分析了纯合子TfR 2突变小鼠。骨髓和外周血细胞的细胞计数和形态均正常。这些小鼠骨髓的CFU-GM、BFU-E和CFU-Meg的集落计数也是正常的。讨论：最近的报道表明，铁调素，一种肝肽激素，在铁稳态中起着至关重要的作用。我现在正在研究铁调素和TfR 2之间的关系，TfR 2可能是肝脏铁转铁蛋白的传感器。此外，TfR 2在红白血病细胞中表达的意义仍有待确定。

项目成果

Kawabata H, Nakamaki T, Ikonomi P, Smith RD, Germain RS, Koeffler HP: "Expression of transferrin receptor 2 in normal and neoplastic hematopoietic cells"BLOOD. 68. 2712-2717 (2001)

Kawabata H、Nakamaki T、Ikonomi P、Smith RD、Germain RS、Koeffler HP：“转铁蛋白受体 2 在正常和肿瘤造血细胞中的表达”BLOOD。

Fleming RE, Ahmann JR, Migas MC, Waheed A, Koeffler HP, Kawabata H, Britton RS, Bacon BR, Sly WS: "Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis"Proc Natl Acad Sci USA. 99. 10653-10658 (2002)

Fleming RE、Ahmann JR、Migas MC、Waheed A、Koeffler HP、Kawabata H、Britton RS、Bacon BR、Sly WS：“鼠转铁蛋白受体 2 基因的定向诱变产生血色素沉着症”Proc Natl Acad Sci USA。

Kawabata H, Nakamaki T, Ikonomi P, Smith RD, Germain RS, Koeffler HP: "Expression of transferrin receptor 2 in normal and neoplastic hematopoietic cells"BLOOD. 98. 2712-2717 (2001)

Kawabata H、Nakamaki T、Ikonomi P、Smith RD、Germain RS、Koeffler HP：“转铁蛋白受体 2 在正常和肿瘤造血细胞中的表达”BLOOD。