Functional Clarification Of Transferrin Receptor 1 In Hematopoiesis

Objective:Iron,one of the most abundant essential trace elements in human,plays an important physiological function in life processes.Hematopoiesis is sensitive to iron deficiency,with an insufficiency of available iron in the body being readily reflected as iron-deficiency anemia.Maternal anemia during pregnancy has higher risk of low birth weight and preterm birth.Moreover,experimental preclinical data converge to indicate the promoting role of iron on leukemia development,and conversely,the role of iron chelation/deprivation to counteract cancer.So,it will be of clinical importance to explore the regulatory mechanism of iron homeostasis in hematopoiesis.Transferrin receptor 1?Tfr1?facilitates the uptake of iron on the cell surface by binding to holo-transferrin.Tfr1is ubiquitously expressed in mammalian tissues and has been called“cellular iron gate”.However,ongoing studies have not directly addressed the specific regulatory role of Tfr1in HSCs.In this study,we aim to further investigate the function and mechanism of Tfr1in hematopoiesis,and explore the significance of iron homeostasis in hematopoietic system.Methods:1)The expression of Tfr1 in the hematopoietic system was analyzed via GEO and Bloodspot database and flow cytometry.2)Hematopoietic specific Tfr1 knockout mice(Tfr1^fl/fl;Vav-Cre,cKO)were generated,and hematopoiesis in bone marrow and fetal liver were analyzed by flow cytometry,HE staining and hematological parameters.3)Competitive and non-competitive transplantation assays were performed to explore the self-renewal and differentiation function of hematopoietic stem cells.4)Annexin-V and Brdu staining were used to detect the apoptosis and proliferation of hematopoietic stem/progenitor cells?HSPCs?,respectively.5)Inductively coupled plasma mass spectrometry?ICP-MS?and Calcein-AM probe was used to determine tissue iron concentrations and intracellular iron levels,respectively.6)Experiments on low-iron diet treatment in vivo and treated HSPCs in vitro with iron chelating agent?DFO?were designed to mimic the dysfunction of cKO HSPCs.7)Besides,HSPCs were treated with5?M holo-transferrin?holo-Tf?,10?M ferric ammonium citrate?FAC?or 10?M hemin.8)Moreover,exogenous Tfr1^R654A or Tfr1^L622Awere overexpressed in HSPCs through lentiviral overexpression system,then the colony-forming ability was analyzed.9)cKit⁺HSPCs were sorted by immunomagnetic beads,and the m RNA levels of several critical transcription factors for cells development were measured by real-time fluorescent quantitative PCR.Results:1)The Tfr1^fl/fl;Vav-Cre newborns were paler than control(Tfr1^fl/fl),failed to thrive and died within the first postnatal week.Hematological analysis of pups showed that the red blood cells[2.3±0.7(10¹²/L)]of cKO mice were significantly lower than that of Tfr1^fl/fl control mice[3.6±0.4(10¹²/L)]?P<0.001?,white blood cells[5.1±0.9?10⁹/L?]were also significantly reduced compared with control[8.4±2.2?10⁹/L?]?P<0.001?.Similarly,in E14.5,E16.5,E18.5 fetal liver,cKO showed defected erythropoiesis.In addition,although the development of CD11b⁺Gr1⁺myeloid cells and CD3⁺T lymphocytes was severely impaired,CD19⁺B cell development was rarely affected.Histomorphology and flow cytometry analysis showed that the number and proportion of hematopoietic stem cells(Lin^-cKit⁺Sca1^hi)and hematopoietic progenitor cells?Lin^-cKit⁺Sca1^-?in cKO bone marrow were significantly decreased.Moreover,the apoptosis ratio of cKO hematopoietic stem cells[?43.40±1.82?%]was significantly higher than control[?16.89±0.65?%]?P<0.001?.However,the number of cKO HSCs remain unchanged in fetal liver.The number of granulocyte/monocyte progenitor cells?GMPs?increased[Tfr1^fl/fl:4.45±1.19×10⁵;cKO:6.54±1.25×10⁵,?p<0.05?],Common myeloid progenitor cells?CMPs?[Tfr1^fl/fl:4.38±0.25×10⁵;cKO:3.34±0.62×10⁵,?p<0.05?]and megakaryocytes/erythrocyte progenitor cells?MEPs?[Tfr1^fl/fl:1.44±0.11×10⁶;cKO:1.07±0.24×10⁶,?p<0.05?]reduced compared with control.2)Serum iron of cKO pups[?297.92±31.17??g/d L]was significantly higher than control[?136.63±50.66??g/d L]?P<0.001?,and liver iron[?338.95±72.99?g/g wet weight]was also increased compared with control[?72.84±28.81?g/g wet weight]?P<0.001?.Conversely,the intracellular iron of HSPCs and mature lineage?Lin⁺?was significantly lower than that of control group?P<0.05?.In E16.5 fetal liver,the intracellular iron of cKO HSPCs was similar to control,while the intracellular iron of mature lineage?Lin⁺?was significantly decreased?P<0.05?.3)Tfr1^fl/fl fetal liver cells can form colony units?BFU-E,CFU-GM,CFU-GEMM?,while cKO cells cannot.Competitive and non-competitive bone marrow transplantation experiments showed that Tfr1^fl/fl HSCs regenerated hematopoietic system in recipient mice,while cKO HSCs cannot.This indicates that reduced regenerative capacity in Tfr1-deficient HSCs is due to a cell-autonomous mechanism.4)Wild-type HSCs treated with 5?M DFO resulted in a significant decrease of CFU colony units[Ctrl:46.5±2.12;5?M DFO:7.5±2.12,?p<0.05?].cKO HSCs added with 10?M FAC or 5?M holo-Tf still failed to form any CFU colony unit.While added with hemin,cKO HSCs formed BFU-E,CFU-GM,and CFU-GEMM colony units in a dose-dependent manner.In addition,exogenous Tfr1^L622A partially rescued colony-forming ability of cKO HSPCs,while Tfr1^R654Acannot.Finally,compared with control,the expression of Notch1,Gata3,Gata2,PU.1,Fli,Gfi1 and C/EBP?in cKO cKit⁺HSPCs was significantly declined.Conclusion:These results provide direct in vivo evidence that Tfr1 plays an essential role in hematopoiesis.In particular,this research shows that Tfr1 is required for the differentiation of hematopoietic progenitor/precursor cells into a range of mature cell types.Importantly,our results suggest that iron uptake appears to be the principal mechanism by which Tfr1 mediates the differentiation and survival of hematopoietic cells,thereby uncovering the important role of intracellular iron homeostasis in hematopoiesis.