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

背景：在本项目期间，有人报道了转铁蛋白受体2(TfR2)纯合子突变导致3型血色素沉着症。与加州大学洛杉矶分校/雪松西奈医学中心的Philip Koeffler和圣路易斯大学的Robert Fleming合作培育的TfR2纯合子突变小鼠显示出肝脏中铁的积累，与血色素沉着症的表型相似。这些发现表明TfR2参与了铁的代谢。本研究主要研究TfR2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TfR2蛋白和TfR2合成肽为抗原，制备了两株抗TfR2的多克隆抗体。用稳定表达TfR2或转铁蛋白受体1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，我还建立了检测可溶性TfR3的夹心ELISA法。在人血清标本中未检测到TfR2的显著水平，提示血清中可溶性TfR2的含量可能较低。接下来，我用免疫组织化学方法检测了TfR2在骨髓细胞中的表达。红系细胞多为阳性，髓系细胞多为阴性。一例红白血病标本TfR2染色很强。我分析了纯合子TfR2突变的小鼠。骨髓和外周血细胞计数和形态均正常。这些小鼠骨髓中CFU-GM、BFU-E和CFU-Meg的集落计数也正常。讨论：最近的报道表明，肝肽类激素海普西丁在铁稳态中起着重要作用。我现在正在研究海普西丁和TfR2之间的关系，TfR2可能是肝脏中铁转铁蛋白的传感器。此外，TfR2在红白血病细胞中表达的意义还有待确定。

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.