Iron-loaded transferrin potentiates erythropoietin effects on erythroblast proliferation and survival: a novel role through transferrin receptors

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) glycoprotein cytokine plays major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration Epo bone marrow hypothesized to be suboptimal, and the survival cells has been suggested depend on However, key factors control sensitivity remain unknown. Two types transferrin receptors (TfRs), TfR1 TfR2, are known play iron uptake cells. Here, we TfRs may additionally modulate during modulating receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) human progenitor models, report iron-loaded transferrin, is, holo-transferrin (holo-Tf), synergizes with suboptimal levels improve survival, proliferation, differentiation. This accomplished via signaling pathways which include signal transducer activator transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, found this cooperation improved by, but does not require, internalization TfR1. Interestingly, observed loss TfR2 stabilizes EpoR abolishes beneficial effects holo-Tf. Overall, these data reveal novel properties TfRs, involve regulation through Graphical abstract Erythropoiesis tightly regulated red production. Multiple factors, including microenvironmental growth cytokines, positively yield proliferation progenitors. factor required for [1Goldwasser E. its mode action.Blood Cells. 1984; 10: 147-162PubMed Google Scholar,2Goldwasser E Krantz SB Wang FF. differentiation.Symp Fundam Cancer Res. 37: 103-107Google Scholar]. As such, deletion can induce embryonic lethality mice [3Wu H Liu X Jaenisch R Lodish HF. 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To test involvement holo-Tf cells, UT7/Epo were starved treated concentrations (holo-Tf, charged iron), apo-transferrin (apo-Tf, empty various time points. illustrated Figure 1A–E www.exphem.org), more efficiently Epo-mediated phosphorylation ERK1/2, AKT, STAT5 than apo-Tf. result, was selected use subsequent experiments. next examined combinatorial survival. cultured without (No Epo), alone + holo-Tf), (Epo 0.1 U/mL), U/mL saturating holo-Tf). In absence failed support after days vitro (DIV). tended increase number DIV significantly increased 7 DIV, compared (Figure 1A). Next, quantified 0 60 min stimulation and/or earlier (Supplementary 1F). Phosphorylation ERK1/2 AKT induced further (0.2 mg/mL) within 10 treatment 1B). number, did when combined then stimulated increasing either 1C) 1D) min. 1C, 0.2 mg/mL phosphorylation. little concentration-dependent phosphorylation, 1D). 1E). suggest synergistic STAT5. Intravenous decreases erythropoiesis. 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At CD71 CD235a, comparable continuously 7, 14 percentages CD71+CD235a+-expressing 50.4% 53.9% holo-Tf, those alone, 2.88% 6.35%, respectively 2C). indicate holo-Tf-mediated dosages promote investigate dependency potentiation entry endocytosis/internalization, Dynasore, potent GTPase-mediated endocytosis [21Macia Ehrlich Massol Boucrot Brunner Kirchhausen T. cell-permeable dynamin.Dev Cell. 2006; 839-850Abstract (1488) internalization, A24, monoclonal antibody specifically directed against [22Moura Lepelletier Arnulf B neutralizing (mAb A24) induces apoptosis tumor lymphocytes ATL patients.Blood. 103: 1838-1845Crossref (89) By immunofluorescence microscopy, localization (CD71) A24 untreated visualized. massively internalized prevented recycling, CD71, intense localized cytosol 3A). fluorescence-activated sorting (FACS) decrease treatment. Dynasore mostly blocked 80 µmol/L 3B); therefore, reduced holo-Tf–induced ERK, 3C), thereby suggesting endocytosis. even presence still cooperative indicating partially endocytosis-dependent activation. Janus (JAK2), pathway, JAK2 (ruxolitinib). ruxolitinib 3D). Collectively, sensitizing endocytosis, activity. 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(0.1 4B). explain why sensitive analyzed Western blot level WT stabilized 4C). whereas 3C). proteasome inhibitor, MG132 4D), levels, negating decreased degradation 4E, culture, numbers relative numbers, suggests particularly (red line vs red-dashed line).Supplementary 1Transferrin (Tf) stimulate pathway. (A) Representative showing comparative apo-Tf supplemented phospho-AKT, phospho-ERK1/2, (B) phospho-STAT5 expression. 0.05, 0.1, harvested immunoblotting anti-phospho-AKT, anti-phospho-ERK1/2, anti- antibodies. Bar graphs show mean normalized densitometry values (C) (D) (E) phospho-STAT5, corresponding standard deviations independent All mean±S.E. (n=3), **p < 0.01 (Student's t-test). (F) time-dependent different amounts times (1-60 min) anti-phospho-STAT5 antibodies.View Large Image ViewerDownload Hi-res image Download (PPT)Supplementary 2The (TfR2−/−) TfR2−/− transfected Cas9-mediated vector gene anti-TfR2 anti-actin TfR2−/−UT7/Epo represent MG132. 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Hematopoietic leads block iron-deficient anemia.Am 91: 812-818Crossref (28) discrepancy owing differences additional events. stronger TfR2–/– Taken together, improves Here functional differentiation, PI3K/AKT, Specifically, distinct induction co-treatment (physiological) seems reinforced EpoR–JAK2 upregulate stabilization proliferation. knowledge, first describing believe help establish erythropoiesis-stimulating Epo-refractory anemia.