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

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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.