Differential Effects of Monoferric Transferrin Forms on Erythropoiesis Are Mediated By TFR2

Abstract

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), monoferric iron bound to the N-lobe or C-lobe (mono-TF) or to both lobes (holo-TF). All TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TFR2 which is mainly expressed by hepatocytes and erythroid precursors to modulate cellular signaling events regulating hepcidin expression and erythropoiesis. We previously generated N-lobe blocked ( Tf N-bl) or C-lobe blocked ( Tf C-bl) TF mutant mice, and observed marked differences between them in iron parameters, RBC count and EPO sensitivity (Parrow et al., 2019). In this current work, we investigate the effects of the mutated TFs on ineffective erythropoiesis in murine β-thalassemia intermedia. We moreover examine the consequences of erythroid knockout of TFR2 in the TF N-blocked and C-blocked mice. Based on observations in the TF mutant mice, we hypothesized that Hbb th3/+ mice crossed with Tf C-bl would demonstrate improved erythropoietic and iron parameters compared with Hbb th3/+Tf N-bl. Hbb th3/+Tf C-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbb th3/+Tf C-bl relative to Hbb th3/+Tf +/+controls. By contrast, similar improvements in RBC counts and hemoglobin were not observed in Hbb th3/+Tf N-bl mice. Nonetheless, the Hbb th3/+Tf N-blmice displayed lower reticulocytes, increased platelets, decreased IE, decreased splenomegaly, and decreased serum ERFE compared to Hbb th3/+Tf +/+controls and thus displayed a phenotype intermediate between Hbb th3/+Tf C-bl and Hbb th3/+Tf +/+controls. Serum EPO was elevated in Hbb th3/+Tf N-blcompared to either Hbb th3/+Tf C-blor Hbb th3/+Tf +/+mice. Moreover, the increased hepcidin observed in Hbb th3/+Tf C-bl compared with Hbb th3/+Tf +/+was not observed in Hbb th3/+Tf N-bl mice. To examine the contribution of erythroid TFR2 to the different phenotypes of the N-blocked and C-blocked TF mice, we generated a mouse line expressing TFR2-3xFLAG that is flanked by loxp sites ( TFR2-3xFLAG fl/fl). These mice were used to generate mice with erythroid knockout of TFR2 in the TF mutant mice; i.e., EPOR Cre-tdtomTf N-blTFR2 cko and EPOR Cre-tdtomTf C-blTFR2 cko. N-blocked mice with conditional knockout of TFR2 demonstrated increases in RBC counts and Hb levels compared with the TF N-blocked mice ( EPOR Cre-tdtom Tf N-blTFR2 cko vs Tf N-bl/Tf N-blTFR2-3xFLAG fl/fl), and had values similar to the C-blocked mice ( Tf C-bl/Tf C-blTFR2-3xFLAG fl/fl or EPOR Cre-tdtomTf N-blTFR2 cko). Likewise, conditional KO of TFR2 in the N-blocked mice resulted in lower EPO levels, which were similar to those in the C-blocked mice. These data support the hypothesis that differences observed between the mono-TF forms at the erythroid level are mediated via TFR2. These data support a model by which lobe-specific iron occupancy of TF influences hematopoiesis via erythroid TFR2.